A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-04-01

MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector that is not prone to miR-9 regulation rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-01-01

Summary MicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation. PMID:19330006

Efficacy of just-in-time plerixafor rescue for Hodgkin's lymphoma patients with poor peripheral blood stem cell mobilization.

PubMed

Yuan, Shan; Nademanee, Auayporn; Kaniewski, Mark; Palmer, Joycelynne; Shayani, Sepideh; Wang, Shirong

2014-08-01

Plerixafor is a Food and Drug Administration-approved agent for improving peripheral blood stem cell (PBSC) mobilization in filgrastim (granulocyte-colony-stimulating factor [G-CSF])-stimulated patients with multiple myeloma and non-Hodgkin's lymphoma. Limited information is available on its use in Hodgkin's lymphoma (HL) patients. We describe our experience with plerixafor as an immediate rescue agent in HL patients with poor PBSC mobilization. We retrospectively reviewed the collection data of 27 consecutive HL patients at our center in whom plerixafor was added to rescue a failing PBSC collection after G-CSF and chemotherapy (26) or G-CSF alone (1). Plerixafor was added in 11 patients due to peripheral blood (PB) CD34+ counts that persisted below the threshold (>10 × 10(6) /L) to initiate collection (median, 1.47 × 10(6) ; range 0 × 10(6) -6.28 × 10(6) /L) and in 16 patients due to low collection yields, who had a median yield of 0.33 × 10(6) (0.14 × 10(6) -0.65 × 10(6) ) CD34+ cells/kg on the last collection before plerixafor administration. After a median of 2 (range, 2-4) collections with plerixafor, the patients collected a median of 1.82 × 10(6) (0.52 × 10(6) -11.14 × 10(6) ) CD34+ cells/kg. The addition of plerixafor enabled 20 patients (74.1%) to reach the 2.0 × 10(6) CD34+ cells/kg minimum required for autologous stem cell transplantation (ASCT) during the same collection cycle. Subsequent remobilization in three patients with plerixafor enabled all three to reach this goal. Plerixafor can be used in HL patients with poor mobilization as a rescue agent and boosts mobilization sufficiently in most patients in the same collection attempt, thus not only permitting ASCT, but also avoiding remobilization and the associated costs, treatment delays, and patient inconvenience. © 2014 AABB.

Poor stem cell harvest may not always be related to poor mobilization: lessons gained from a mobilization study in patients with β-thalassemia major.

PubMed

Constantinou, Varnavas C; Bouinta, Asimina; Karponi, Garyfalia; Zervou, Fani; Papayanni, Penelope-Georgia; Stamatoyannopoulos, George; Anagnostopoulos, Achilles; Yannaki, Evangelia

2017-04-01

Hematopoietic stem cell mobilization and leukapheresis in adult patients with β-thalassemia have recently been optimized in the context of clinical trials for obtaining hematopoietic stem cells for thalassemia gene therapy. In some patients, however, the yield of cluster of differentiation 34-positive (CD34+) cells was poor despite successful mobilization, and a modification of apheresis settings was mandatory for harvest rescue. Data were analyzed from 20 adult patients with β-thalassemia who were enrolled in a clinical trial of optimizing mobilization strategies for stem cell gene therapy. The aim of this post-hoc analysis was to assess how certain hematological and/or clinical parameters may correlate with low collection efficiency in the presence of adequate numbers of circulating stem cells after pharmacological mobilization and standard leukapheresis procedures. Among 19 patients who achieved optimal mobilization with Plerixafor, four who underwent splenectomy demonstrated disproportionately poor CD34+ cell harvests, as determined by their circulating CD34+ cell counts after mobilization. All four patients who underwent splenectomy presented at baseline and before first apheresis with lymphocytosis resulting in lymphocyte/neutrophil ratios well above 1 and marked reticulocytosis compared with patients who achieved optimal mobilization/CD34+ cell harvest. Such unexpected expansion of specific cell populations disrupted the normal cell layer separation and necessitated modification of the apheresis settings to rescue the harvests. By close examination of certain hematological and/or clinical parameters before leukapheresis, patients who, despite adequate mobilization, are at risk for poor CD34+ cell harvests may be identified, and harvest failure can be prevented by adjusting the apheresis settings. © 2016 AABB.

Childhood Central Nervous System Germ Cell Tumors Treatment (PDQ®)—Health Professional Version

Cancer.gov

Treatment for children with central nervous system germ cell tumors (GCT) depend upon the specific tumor type. Options include radiation therapy, chemotherapy, surgery (in various combinations) and stem cell rescue. Get detailed information about GCTs in this clinician summary.

Long-term follow up of high-dose chemotherapy with autologous stem cell rescue in adults with Ewing tumor.

PubMed

Laurence, Valérie; Pierga, Jean-Yves; Barthier, Sophie; Babinet, Antoine; Alapetite, Claire; Palangié, Thao; de Pinieux, Gonzagues; Anract, Philippe; Pouillart, Pierre

2005-06-01

Ewing tumors remain of poor prognosis, with 5-year overall survival of 55% to 65% in localized patients and not exceeding 25% in primarily metastatic disease. Several reports, mainly in children, have reported that some patients with poor-risk Ewing tumors may benefit from high-dose chemotherapy (HDCT) with autologous stem cell rescue. This retrospective study analyzed 46 patients treated in our institution between 1987 and 2000 for localized or primary metastatic Ewing tumors by HDCT followed by stem cell rescue. Median follow up was 7.1 years. Median age was 21 years (range, 15-46 years). Twenty-two percent of patients had metastases at diagnosis. The tumor site was axial in 56% of patients. Median tumor size was 9.5 cm. The treatment regimen consisted of induction chemotherapy, local treatment, maintenance chemotherapy, and consolidation HDCT based on alkylating agents. No toxic death was observed in the intensive therapy phase. Five-year overall survival and progression-free survival were 63 +/- 7.7% and 47 +/- 7.6%, respectively. Pejorative prognostic factors in this population were metastases at diagnosis (5-year overall survival 34% vs.71%, P = 0.017) and poor pathologic response (5-year overall survival 44% vs.77%, P = 0.03). This retrospective study shows a high long-term survival rate with high-dose chemotherapy in adults.

Retinoblastoma—Health Professional Version

Cancer.gov

Retinoblastoma is a pediatric cancer. For patients with extraocular retinoblastoma, intensive chemotherapy is required, including high-dose chemotherapy and autologous hematopoietic stem cell rescue. Find evidence-based information on retinoblastoma treatment.

Neural stem cells rescue nervous purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets.

PubMed

Li, Jianxue; Imitola, Jaime; Snyder, Evan Y; Sidman, Richard L

2006-07-26

Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and re-implanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells. In nervous (nr) mutant mice Purkinje neuron (PN) mitochondria become abnormal by the second postnatal week, and a majority of PNs die in the fourth to fifth weeks. We previously identified in nr cerebellum a 10-fold increase in tissue plasminogen activator (tPA) as a key component of the mechanism causing nr PN death. Here we report that undifferentiated wild-type murine NSCs, when transplanted into the newborn nr cerebellar cortex, do not replace host PNs but contact imperiled PNs and support their mitochondrial function, dendritic growth, and synaptogenesis, subsequently leading to the rescue of host PNs and restoration of motor coordination. This protection of nr PNs also is verified by an in vitro organotypic slice model in which nr cerebellar slices are cocultured with NSCs. Most importantly, the integrated NSCs in young nr cerebellum rectify excessive tPA mRNA and protein to close to normal levels and protect the mitochondrial voltage-dependent anion channel and neurotrophins, downstream targets of the tPA/plasmin proteolytic system. This report demonstrates for the first time that NSCs can rescue imperiled host neurons by rectifying their gene expression, elevating somatic stem cell therapeutic potential beyond solely cell replacement strategy.

The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

PubMed Central

Shields, Alicia R.; Spence, Allyson C.; Yamashita, Yukiko M.; Davies, Erin L.; Fuller, Margaret T.

2014-01-01

Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia. PMID:24346697

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-01-01

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation. PMID:20133835

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-02-02

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.

Mechanism of mesenchymal stem cell-induced neuron recovery and anti-inflammation.

PubMed

Huang, Peng; Gebhart, Nichole; Richelson, Elliott; Brott, Thomas G; Meschia, James F; Zubair, Abba C

2014-10-01

After ischemic or hemorrhagic stroke, neurons in the penumbra surrounding regions of irreversible injury are vulnerable to delayed but progressive damage as a result of ischemia and hemin-induced neurotoxicity. There is no effective treatment to rescue such dying neurons. Mesenchymal stem cells (MSCs) hold promise for rescue of these damaged neurons. In this study, we evaluated the efficacy and mechanism of MSC-induced neuro-regeneration and immune modulation. Oxygen-glucose deprivation (OGD) was used in our study. M17 neuronal cells were subjected to OGD stress then followed by co-culture with MSCs. Rescue effects were evaluated using proliferation and apoptosis assays. Cytokine assay and quantitative polymerase chain reaction were used to explore the underlying mechanism. Antibody and small molecule blocking experiments were also performed to further understand the mechanism. We showed that M17 proliferation was significantly decreased and the rate of apoptosis increased after exposure to OGD. These effects could be alleviated via co-culture with MSCs. Tumor necrosis factor-α was found elevated after OGD stress and was back to normal levels after co-culture with MSCs. We believe these effects involve interleukin-6 and vascular endothelial growth factor signaling pathways. Our studies have shown that MSCs have anti-inflammatory properties and the capacity to rescue injured neurons. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation*

PubMed Central

Meade, Kate A.; White, Kathryn J.; Pickford, Claire E.; Holley, Rebecca J.; Marson, Andrew; Tillotson, Donna; van Kuppevelt, Toin H.; Whittle, Jason D.; Day, Anthony J.; Merry, Catherine L. R.

2013-01-01

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells. PMID:23235146

Immobilization of heparan sulfate on electrospun meshes to support embryonic stem cell culture and differentiation.

PubMed

Meade, Kate A; White, Kathryn J; Pickford, Claire E; Holley, Rebecca J; Marson, Andrew; Tillotson, Donna; van Kuppevelt, Toin H; Whittle, Jason D; Day, Anthony J; Merry, Catherine L R

2013-02-22

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

PubMed

Xiao, Guan-Yu; Liu, I-Hsuan; Cheng, Chun-Chun; Chang, Chia-Chun; Lee, Yen-Hua; Cheng, Winston Teng-Kuei; Wu, Shinn-Chih

2014-01-01

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells.

PubMed

Sada, Aiko; Hasegawa, Kazuteru; Pin, Pui Han; Saga, Yumiko

2012-02-01

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. Copyright © 2011 AlphaMed Press.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

PubMed

Matsa, Elena; Dixon, James E; Medway, Christopher; Georgiou, Orestis; Patel, Minal J; Morgan, Kevin; Kemp, Paul J; Staniforth, Andrew; Mellor, Ian; Denning, Chris

2014-04-01

Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes

PubMed Central

Matsa, Elena; Dixon, James E.; Medway, Christopher; Georgiou, Orestis; Patel, Minal J.; Morgan, Kevin; Kemp, Paul J.; Staniforth, Andrew; Mellor, Ian; Denning, Chris

2014-01-01

Aims Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. Methods and results We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K+ currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). Conclusions These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart. PMID:23470493

Stem cell transplantation as rescue therapy for refractory Crohn's disease: a sytematic review.

PubMed

Labidi, Asma; Serghini, Meriem; Ben Mustapha, Nadia; Fekih, Monia; Boubaker, Jalel; Filali, Azza

2014-11-01

Crohn's disease is a chronic relapsing- remitting affection. It has a strong immunologic component which represent the target of standard therapies including immunosppressants and biological therapies. However, many patients remain refracory or intolerant to these therapies. The aim of this review is to determine the effects of stem cell transplantation in patients with refractory Crohn's disease. Systematic review of observational studies, clinical trials and case reports that focused on the effectiveness and safety of stem cell transplantation in patients with refractory Crohn's disease. Hematopoietic stem cell transplantation seems to be efficient in maintaining clinical and endoscopic remission in patients with Crohn's disease refractory or intolerant to current therapies. However, it has been associated to high morbidity and mortality due to chemotherapy. Mesenchymal stem cell transplantation could induce remission in patients with fistulising refractory Crohns disease with no severe side effects. Its impact on luminal Crohns disease is still controversial. Stem cell transplantation seems to hold promising in patients with refractory Crohn's disease. However, because of the high morbidity and mortality related to chemotherapy, hematopoietic stem cell transplantation should be used as last resort to control this disease. Effectiveness of mesenchymal stem cell transplantation in luminal Crohn's disease has yet to be proven.

Retinoblastoma Treatment (PDQ®)—Patient Version

Cancer.gov

Retinoblastoma treatment may include cryosurgery, laser therapy (thermotherapy), chemotherapy, radiation therapy, high-dose chemotherapy with stem cell rescue, and sometimes surgery. Learn more about newly diagnosed and recurrent retinoblastoma in this expert-reviewed summary.

Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells

PubMed Central

Vu, Ly P.; Prieto, Camila; Amin, Elianna M.; Chhangawala, Sagar; Krivtsov, Andrei; Calvo-Vidal, M. Nieves; Chou, Timothy; Chow, Arthur; Minuesa, Gerard; Park, Sun Mi; Barlowe, Trevor S.; Taggart, James; Tivnan, Patrick; Deering, Raquel P.; Chu, Lisa P; Kwon, Jeong-Ah; Meydan, Cem; Perales-Paton, Javier; Arshi, Arora; Gönen, Mithat; Famulare, Christopher; Patel, Minal; Paietta, Elisabeth; Tallman, Martin S.; Lu, Yuheng; Glass, Jacob; Garret-Bakelman, Francine; Melnick, Ari; Levine, Ross; Al-Shahrour, Fatima; Järås, Marcus; Hacohen, Nir; Hwang, Alexia; Garippa, Ralph; Lengner, Christopher J.; Armstrong, Scott A; Cerchietti, Leandro; Cowley, Glenn S; Root, David; Doench, John; Leslie, Christina; Ebert, Benjamin L; Kharas, Michael G.

2017-01-01

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia. PMID:28436985

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

PubMed

Lund, Raymond D; Wang, Shaomei; Klimanskaya, Irina; Holmes, Toby; Ramos-Kelsey, Rebeca; Lu, Bin; Girman, Sergej; Bischoff, N; Sauvé, Yves; Lanza, Robert

2006-01-01

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

Fertility rescue and ovarian follicle growth promotion by bone marrow stem cell infusion.

PubMed

Herraiz, Sonia; Buigues, Anna; Díaz-García, César; Romeu, Mónica; Martínez, Susana; Gómez-Seguí, Inés; Simón, Carlos; Hsueh, Aaron J; Pellicer, Antonio

2018-05-01

To assess if infusion of human bone marrow-derived stem cells (BMDSCs) could promote follicle development in patients with impaired ovarian functions. Experimental design. University research laboratories. Immunodeficient NOD/SCID female mice. Human BMDSCs were injected into mice with chemotherapy-induced ovarian damage and into immunodeficient mice xenografted with human cortex from poor-responder patients (PRs). Follicle development, ovulation, and offspring. Apoptosis, proliferation, and vascularization were evaluated in mouse and human ovarian stroma. Fertility rescue and spontaneous pregnancies were achieved in mice ovaries mimicking PRs and ovarian insufficiency, induced by chemotherapy, after BMDSC infusion. Furthermore, BMDSC treatment resulted in production of higher numbers of preovulatory follicles, metaphase II oocytes, 2-cell embryos, and healthy pups. Stem cells promoted ovarian vascularization and cell proliferation, along with reduced apoptosis. In xenografted human ovarian tissues from PRs, infusion of BMDSCs and their CD133+ fraction led to their engraftment close to follicles, resulting in promotion of follicular growth, increases in E 2 secretion, and enhanced local vascularization. Our results raised the possibility that promoting ovarian angiogenesis by BMDSC infusion could be an alternative approach to improve follicular development in women with impaired ovarian function. NCT02240342. Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Stem cell senescence. Effects of REAC technology on telomerase-independent and telomerase-dependent pathways.

PubMed

Rinaldi, S; Maioli, M; Pigliaru, G; Castagna, A; Santaniello, S; Basoli, V; Fontani, V; Ventura, C

2014-09-16

Decline in the gene expression of senescence repressor Bmi1, and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated β-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression.

Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β.

PubMed

Wu, Xiaoyang; Shen, Qing-Tao; Oristian, Daniel S; Lu, Catherine P; Zheng, Qinsi; Wang, Hong-Wei; Fuchs, Elaine

2011-02-04

Homeostasis and wound healing rely on stem cells (SCs) whose activity and directed migration are often governed by Wnt signaling. In dissecting how this pathway integrates with the necessary downstream cytoskeletal dynamics, we discovered that GSK3β, a kinase inhibited by Wnt signaling, directly phosphorylates ACF7, a > 500 kDa microtubule-actin crosslinking protein abundant in hair follicle stem cells (HF-SCs). We map ACF7's GSK3β sites to the microtubule-binding domain and show that phosphorylation uncouples ACF7 from microtubules. Phosphorylation-refractile ACF7 rescues overall microtubule architecture, but phosphorylation-constitutive mutants do not. Neither mutant rescues polarized movement, revealing that phospho-regulation must be dynamic. This circuitry is physiologically relevant and depends upon polarized GSK3β inhibition at the migrating front of SCs/progeny streaming from HFs during wound repair. Moreover, only ACF7 and not GSKβ-refractile-ACF7 restore polarized microtubule-growth and SC-migration to ACF7 null skin. Our findings provide insights into how this conserved spectraplakin integrates signaling, cytoskeletal dynamics, and polarized locomotion of somatic SCs. Copyright © 2011 Elsevier Inc. All rights reserved.

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis

PubMed Central

Jacob, Bindya; Yamashita, Namiko; Wang, Chelsia Qiuxia; Taniuchi, Ichiro; Littman, Dan R.; Asou, Norio

2010-01-01

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1−/− mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1−/− HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1−/− HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications. PMID:20008790

Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin.

PubMed

Barton, Lacy J; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2016-07-01

The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Bone marrow mesenchymal stem cells ameliorate inflammatory factor-induced dysfunction of INS-1 cells on chip.

PubMed

Sun, Yu; Yao, Zhina; Lin, Peng; Hou, Xinguo; Chen, Li

2014-05-01

Using a microfluidic chip, we have investigated whether bone marrow mesenchymal stem cells (BM-MSCs) could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. BM-MSCs were obtained from diabetes mellitus patients and their cell surface antigen expression profiles were analyzed by flow cytometric. INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium containing 1 ng/mL IL-1β and 2.5 U/mL IFN-γ for 72 h. BM-MSCs could partially rescue INS-1 cells from cytokine-induced dysfunction and ameliorate the expression of insulin and PDX-1 gene in INS-1 cells. Thus BM-MSCs can be viewed as a promising stem cell source to depress inflammatory factor-induced dysfunction of pancreatic β cells in diabetic patients. © 2014 International Federation for Cell Biology.

Coordinate Regulation of Stem Cell Competition by Slit-Robo and JAK-STAT Signaling in the Drosophila Testis

PubMed Central

Stine, Rachel R.; Greenspan, Leah J.; Ramachandran, Kapil V.; Matunis, Erika L.

2014-01-01

Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche. PMID:25375180

Childhood Central Nervous System Embryonal Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Childhood central nervous system embryonal tumors and pineal tumors are treated with surgery, radiation therapy, chemotherapy, high-dose chemotherapy with stem cell rescue and targeted therapy. Learn more in this expert-reviewed summary.

Advances in Bone Marrow Stem Cell Therapy for Retinal Dysfunction

PubMed Central

Park, Susanna S.; Moisseiev, Elad; Bauer, Gerhard; Anderson, Johnathon D.; Grant, Maria B.; Zam, Azhar; Zawadzki, Robert J.; Werner, John S.; Nolta, Jan A.

2016-01-01

The most common cause of untreatable vision loss is dysfunction of the retina. Conditions, such as age-related macular degeneration, diabetic retinopathy and glaucoma remain leading causes of untreatable blindness worldwide. Various stem cell approaches are being explored for treatment of retinal regeneration. The rationale for using bone marrow stem cells to treat retinal dysfunction is based on preclinical evidence showing that bone marrow stem cells can rescue degenerating and ischemic retina. These stem cells have primarily paracrine trophic effects although some cells can directly incorporate into damaged tissue. Since the paracrine trophic effects can have regenerative effects on multiple cells in the retina, the use of this cell therapy is not limited to a particular retinal condition. Autologous bone marrow-derived stem cells are being explored in early clinical trials as therapy for various retinal conditions. These bone marrow stem cells include mesenchymal stem cells, mononuclear cells and CD34+ cells. Autologous therapy requires no systemic immunosuppression or donor matching. Intravitreal delivery of CD34+ cells and mononuclear cells appears to be tolerated and is being explored since some of these cells can home into the damaged retina after intravitreal administration. The safety of intravitreal delivery of mesenchymal stem cells has not been well established. This review provides an update of the current evidence in support of the use of bone marrow stem cells as treatment for retinal dysfunction. The potential limitations and complications of using certain forms of bone marrow stem cells as therapy are discussed. Future directions of research include methods to optimize the therapeutic potential of these stem cells, non-cellular alternatives using extracellular vesicles, and in vivo high-resolution retinal imaging to detect cellular changes in the retina following cell therapy. PMID:27784628

Neuroblastoma Treatment (PDQ®)—Health Professional Version

Cancer.gov

Neuroblastoma treatment generally is based on whether the tumor is low, intermediate, or high risk. Treatment options include surgery, observation, radiation therapy, chemotherapy, stem cell rescue, and targeted therapy. Get detailed neuroblastoma treatment information in this summary for clinicians.

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia.

PubMed

Bär, Christian; Povedano, Juan Manuel; Serrano, Rosa; Benitez-Buelga, Carlos; Popkes, Miriam; Formentini, Ivan; Bobadilla, Maria; Bosch, Fatima; Blasco, Maria A

2016-04-07

Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres. © 2016 by The American Society of Hematology.

Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel.

PubMed

He, Li; Si, Guangwei; Huang, Jiuhong; Samuel, Aravinthan D T; Perrimon, Norbert

2018-03-01

Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca 2+ levels, and increases in cytosolic Ca 2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca 2+ signalling. Further studies suggest that Ca 2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca 2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells

PubMed Central

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2012-01-01

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 plays an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that histone demethylase LSD1, a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development. PMID:22068596

Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction

PubMed Central

Tulpule, Asmin; Kelley, James M.; Lensch, M. William; McPherson, Jade; Park, In Hyun; Hartung, Odelya; Nakamura, Tomoka; Schlaeger, Thorsten M.; Shimamura, Akiko; Daley, George Q.

2013-01-01

Summary Shwachman-Diamond syndrome (SDS), a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS by knock-down of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two SDS patients. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated auto-digestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease. PMID:23602541

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

PubMed

Rocca, Celine J; Kreymerman, Alexander; Ur, Sarah N; Frizzi, Katie E; Naphade, Swati; Lau, Athena; Tran, Tammy; Calcutt, Nigel A; Goldberg, Jeffrey L; Cherqui, Stephanie

2015-11-01

Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis. We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein. We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells. To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.

Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor

PubMed Central

Biehs, Brian; Hu, Jimmy Kuang-Hsien; Strauli, Nicolas B.; Sangiorgi, Eugenio; Jung, Heekyung; Heber, Ralf-Peter; Ho, Sunita; Goodwin, Alice F.; Dasen, Jeremy S.; Capecchi, Mario R.; Klein, Ophir D.

2013-01-01

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation. PMID:23728424

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

PubMed Central

Jones, Morgan; Osawa, Gail; Regal, Joshua A.; Weinberg, Daniel N.; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O.; Keegan, Catherine E.; Maillard, Ivan

2013-01-01

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors. PMID:24316971

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

PubMed Central

Starich, Todd A.; Hall, David H.; Greenstein, David

2014-01-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans. PMID:25195067

Childhood Atypical Teratoid/Rhabdoid Tumor Treatment (PDQ®)—Patient Version

Cancer.gov

Childhood central nervous system atypical teratoid/rhabdoid tumor treatment may include surgery, radiation therapy, chemotherapy, and high-dose chemotherapy with stem cell rescue. Learn more about newly diagnosed and recurrent childhood AT/RT in this expert-reviewed summary.

Subretinal transplantation of rat MSCs and erythropoietin gene modified rat MSCs for protecting and rescuing degenerative retina in rats.

PubMed

Guan, Y; Cui, L; Qu, Z; Lu, L; Wang, F; Wu, Y; Zhang, J; Gao, F; Tian, H; Xu, L; Xu, G; Li, W; Jin, Y; Xu, G-T

2013-11-01

For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cell based gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i.e., rat mesenchymal stem cells (rMSCs) alone, erythropoietin (EPO) gene modified rMSCs (EPO-rMSCs) or doxycycline (DOX) inducible EPO expression rMSCs (Tet-on EPO-rMSCs), were transplanted into the subretinal spaces of SI-treated rats. The rMSCs were prepared for transplantation after 3 to 5 passages or modified with EPO gene. During the 8 weeks after the transplantation, the rats treated with rMSCs alone or with two types of EPO-rMSCs were all monitored with fundus examination, fundus fluorescein angiography (FFA) and electroretinogram. The transplantation efficiency of donor cells was examined for their survival, integration and differentiation. Following the transplantation, labeled donor cells were observed in subretinal space and adopted RPE morphology. EPO concentration in vitreous and retina of SI-treated rats which were transplanted with EPO-rMSCs or Tet-on EPO-rMSCs was markedly increased, in parallel with the improvement of retinal morphology and function. These findings suggest that rMSCs transplantation could be a new therapy for degenerative retinal diseases since it can protect and rescue RPE and retinal neurons, while EPO gene modification to rMSCs could be an even better option.

A Review of Autologous Stem Cell Transplantation in Lymphoma.

PubMed

Zahid, Umar; Akbar, Faisal; Amaraneni, Akshay; Husnain, Muhammad; Chan, Onyee; Riaz, Irbaz Bin; McBride, Ali; Iftikhar, Ahmad; Anwer, Faiz

2017-06-01

Chemotherapy remains the first-line therapy for aggressive lymphomas. However, 20-30% of patients with non-Hodgkin lymphoma (NHL) and 15% with Hodgkin lymphoma (HL) recur after initial therapy. We want to explore the role of high-dose chemotherapy (HDT) and autologous stem cell transplant (ASCT) for these patients. There is some utility of upfront consolidation for-high risk/high-grade B-cell lymphoma, mantle cell lymphoma, and T-cell lymphoma, but there is no role of similar intervention for HL. New conditioning regimens are being investigated which have demonstrated an improved safety profile without compromising the myeloablative efficiency for relapsed or refractory HL. Salvage chemotherapy followed by HDT and rescue autologous stem cell transplant remains the standard of care for relapsed/refractory lymphoma. The role of novel agents to improve disease-related parameters remains to be elucidated in frontline induction, disease salvage, and high-dose consolidation or in the maintenance setting.

Orphan nuclear receptor TLX activates Wnt/β-catenin signalling to stimulate neural stem cell proliferation and self-renewal

PubMed Central

Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T.; Gage, Fred H.; Evans, Ronald M.; Shi, Yanhong

2010-01-01

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/β-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/β-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active β-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a β-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active β-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active β-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active β-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode. PMID:20010817

Orphan nuclear receptor TLX activates Wnt/beta-catenin signalling to stimulate neural stem cell proliferation and self-renewal.

PubMed

Qu, Qiuhao; Sun, Guoqiang; Li, Wenwu; Yang, Su; Ye, Peng; Zhao, Chunnian; Yu, Ruth T; Gage, Fred H; Evans, Ronald M; Shi, Yanhong

2010-01-01

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

RNA binding protein Lin28B confers gastric cancer cells stemness via directly binding to NRP-1.

PubMed

Wang, Xiaocong; Hu, Huihua; Liu, Hebo

2018-05-19

This work aims to explore the roles and related mechanisms of RNA binding protein Lin28B in gastric cancer cells stemness. We found that Lin28B expression was negatively correlated with the overall survival (OS) of gastric cancer patients, and significantly increased in gastric cancer cells compared with that in gastric epithelial cells. Lin28B overexpression increased spheroid formation, expression of gastric cancer stemness-related markers, and decreased cisplatin sensitivity in gastric cancer cells. Mechanistically, Lin28B could directly bind to NRP-1 3'UTR, thus increasing NRP-1 mRNA stability and expression, and activate the downstream Wnt/β-catenin signaling. Knockdown of NRP-1 or treatment with Wnt/β-catenin antagonist could rescue the promotive effects of Lin28B on gastric cancer stemness. Thus, thes results indicate that Lin28B could facilitate gastric cancer stemness via directly binding to NRP-1 3'UTR and activating the downstream Wnt/β-catenin signaling. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.

PubMed

Baghdadi, Meryem B; Castel, David; Machado, Léo; Fukada, So-Ichiro; Birk, David E; Relaix, Frederic; Tajbakhsh, Shahragim; Mourikis, Philippos

2018-05-01

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix 1-3 . Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells 4-9 , the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

PubMed Central

Kobari, Ladan; Yates, Frank; Oudrhiri, Noufissa; Francina, Alain; Kiger, Laurent; Mazurier, Christelle; Rouzbeh, Shaghayegh; El-Nemer, Wassim; Hebert, Nicolas; Giarratana, Marie-Catherine; François, Sabine; Chapel, Alain; Lapillonne, Hélène; Luton, Dominique; Bennaceur-Griscelli, Annelise; Douay, Luc

2012-01-01

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. PMID:22733021

Alk5-Mediated Transforming Growth Factor β Signaling Acts Upstream of Fibroblast Growth Factor 10 To Regulate the Proliferation and Maintenance of Dental Epithelial Stem Cells▿

PubMed Central

Zhao, Hu; Li, Sha; Han, Dong; Kaartinen, Vesa; Chai, Yang

2011-01-01

Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5fl/fl mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5fl/fl incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5fl/fl mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration. PMID:21402782

Stem cell fusion as an ultimate line of defense against xenobiotics.

PubMed

Padron Velazquez, Julio Lazaro

2006-01-01

There are several indications that the potential of stem cells to fuse with somatic cells is extremely high and, what's more exciting, in some instances goes as far as reprogramming and/or rescuing altered cells. It remains unclear, however, how frequent this mechanism is and what patho-physiological role it might play in nature. A plausible hypothesis, discussed in this paper, suggests that stem cell niches might provide a safeguard for the intact genome and epigenome. By fusing with somatic de-differentiated cells, stem cells might consent epigenetic reprogramming and/or genetic recovery of genes which otherwise could drive altered cells to malignancy. If the many sophisticated mechanisms of metabolism, cell repair, programmed cell death and tissue regeneration should fail, stem cells might represent a final attempt to recover dedifferentiated cells to avoid inflowing in cancer. In the current reappraisal of the different mechanisms of defense against xenobiotics, even the incidence of cancer itself is considered an evolving mechanism which, through a kind of programmed death of individuals exhibiting defective mutations, favors advancement of the phenotypes which adapt best. Additionally, with regard to the mechanisms of transmitting somatic mutations, based on stem cells' capacity to migrate and to fuse, here it is speculated that stem cells might be capable of carrying acquired somatic mutations from peripheral tissues to the gonads, and transmit that information into the germinal line. If appropriately demonstrated, these mechanisms might delineate a novel therapeutic area to be explored. The use of stem cells to reprogram/recover irreversibly damaged cells or to transmit beneficial mutations might be a valuable therapeutic approach in the future.

Wilms Tumor and Other Childhood Kidney Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Wilms tumor and other childhood kidney tumors treatment usually includes surgery and may be followed by radiation therapy or chemotherapy. Other treatments may include immunotherapy or high-dose chemotherapy with stem cell rescue. Learn more in this expert-reviewed summary.

Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia

PubMed Central

Denton, Kyle R.; Lei, Ling; Grenier, Jeremy; Rodionov, Vladimir; Blackstone, Craig; Li, Xue-Jun

2013-01-01

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP. PMID:24123785

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells.

PubMed

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2011-11-08

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 has an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that the histone lysine-specific demethylase 1 (LSD1), a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development.

The Emerging Role of Epigenetics in Stroke

PubMed Central

Qureshi, Irfan A.; Mehler, Mark F.

2013-01-01

The transplantation of exogenous stem cells and the activation of endogenous neural stem and progenitor cells (NSPCs) are promising treatments for stroke. These cells can modulate intrinsic responses to ischemic injury and may even integrate directly into damaged neural networks. However, the neuroprotective and neural regenerative effects that can be mediated by these cells are limited and may even be deleterious. Epigenetic reprogramming represents a novel strategy for enhancing the intrinsic potential of the brain to protect and repair itself by modulating pathologic neural gene expression and promoting the recapitulation of seminal neural developmental processes. In fact, recent evidence suggests that emerging epigenetic mechanisms are critical for orchestrating nearly every aspect of neural development and homeostasis, including brain patterning, neural stem cell maintenance, neurogenesis and gliogenesis, neural subtype specification, and synaptic and neural network connectivity and plasticity. In this review, we survey the therapeutic potential of exogenous stem cells and endogenous NSPCs and highlight innovative technological approaches for designing, developing, and delivering epigenetic therapies for targeted reprogramming of endogenous pools of NSPCs, neural cells at risk, and dysfunctional neural networks to rescue and restore neurologic function in the ischemic brain. PMID:21403016

Evidence of a cellular immune response against sialyl-Tn in breast and ovarian cancer patients after high-dose chemotherapy, stem cell rescue, and immunization with Theratope STn-KLH cancer vaccine.

PubMed

Sandmaier, B M; Oparin, D V; Holmberg, L A; Reddish, M A; MacLean, G D; Longenecker, B M

1999-01-01

Seven ovarian and 33 breast high-risk stage II/III and stage IV cancer patients received high-dose chemotherapy followed by stem cell rescue. Thirty to 151 days after stem cell transplantation, the patients received their first immunotherapy treatment with Theratope STn-KLH cancer vaccine. Most patients developed increasing IgG anti-STn titers to a sustained peak after the fourth or fifth immunizations. Only one patient had elevated CA27.29 (MUC1 mucin) serum levels at trial entry. Five of the seven patients with preimmunotherapy elevated serum CA125 levels demonstrated decreasing CA125 levels during immunotherapy, consistent with an antitumor response. Evidence of STn antigen-specific T-cell proliferation was obtained from 17 of the 27 evaluable patients who received at least three immunotherapy treatments. Eleven of the 26 patients tested had evidence of an anti-STn TH1 antigen-specific T-cell response as determined by interferon-gamma, but not interleukin (IL)-4, production. After immunization, lytic activity of peripheral blood lymphocytes (PBLs) tested against a lymphokine activated killer (LAK)-sensitive cell line, a natural killer (NK)-sensitive cell line, and an STn-expressing cancer cell line (OVCAR) increased significantly. In vitro IL-2 treatment of the PBLs after vaccination greatly enhanced killing of the STn+ cancer cell line. Evidence of the development of OVCAR specific killing activity, over and above that seen due to LAK or NK killing, is presented. These studies provide the strongest evidence in humans of the development of an antitumor T-cell response after immunization with a cancer-associated carbohydrate antigen.

Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

PubMed

Starich, Todd A; Hall, David H; Greenstein, David

2014-11-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma-germline interactions in C. elegans. Copyright © 2014 by the Genetics Society of America.

Six years' experience of tolerance induction in renal transplantation using stem cell therapy.

PubMed

Vanikar, Aruna V; Trivedi, Hargovind L; Thakkar, Umang G

2018-02-01

Tolerance induction (TI) has been attempted with chimerism/clonal deletion. We report results of TI protocol (TIP) using stem cell therapy (SCT) included adipose derived mesenchymal stem cells (AD-MSC) and hematopoietic stem cells (HSC) in 10 living-donor related renal transplantation (LDRT) patients under non-myeloablative conditioning with Bortezomib, Methylprednisone, rabbit-anti-thymoglobulin and Rituximab, without using conventional immunosuppression. Transplantation was performed following acceptable lymphocyte cross-match, flow cross-match, single antigen assay and negative mixed lymphocyte reaction (MLR). Monitoring included serum creatinine (SCr), donor specific antibodies (DSA) and MLR. Protocol biopsies were planned after 100days and yearly in willing patients. Rescue immunosuppression was planned for rejection/DSA/positive MLR. Over mean 6±0.37year follow-up patient survival was 80% and death-censored graft survival was 90%. Mean SCr was 1.44±0.41mg/dL. This is the first clinical report of sustained TI in LDRT for 6years using SCT. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

PubMed

Shi, Yanhong; Chichung Lie, D; Taupin, Philippe; Nakashima, Kinichi; Ray, Jasodhara; Yu, Ruth T; Gage, Fred H; Evans, Ronald M

2004-01-01

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

PubMed

Sun, Zhao; Yu, Wenjie; Sanz Navarro, Maria; Sweat, Mason; Eliason, Steven; Sharp, Thad; Liu, Huan; Seidel, Kerstin; Zhang, Li; Moreno, Myriam; Lynch, Thomas; Holton, Nathan E; Rogers, Laura; Neff, Traci; Goodheart, Michael J; Michon, Frederic; Klein, Ophir D; Chai, Yang; Dupuy, Adam; Engelhardt, John F; Chen, Zhi; Amendt, Brad A

2016-11-15

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development. © 2016. Published by The Company of Biologists Ltd.

Inhibition of HSP90 Promotes Neural Stem Cell Survival from Oxidative Stress through Attenuating NF-κB/p65 Activation

PubMed Central

Jiang, Wenkai; Zhou, Lin

2016-01-01

Stem cell survival after transplantation determines the efficiency of stem cell treatment, which develops as a novel potential therapy for several central nervous system (CNS) diseases in recent decades. The engrafted stem cells face the damage of oxidative stress, inflammation, and immune response at the lesion point in host. Among the damaging pathologies, oxidative stress directs stem cells to apoptosis and even death through several signalling pathways and DNA damage. However, the in-detail mechanism of stem cell survival from oxidative stress has not been revealed clearly. Here, in this study, we used hydrogen peroxide (H2O2) to induce the oxidative damage on neural stem cells (NSCs). The damage was in consequence demonstrated involving the activation of heat shock protein 90 (HSP90) and NF-κB/p65 signalling pathways. Further application of the pharmacological inhibitors, respectively, targeting at each signalling indicated an upper-stream role of HSP90 upon NF-κB/p65 on NSCs survival. Preinhibition of HSP90 with the specific inhibitor displayed a significant protection on NSCs against oxidative stress. In conclusion, inhibition of HSP90 would attenuate NF-κB/p65 activation by oxidative induction and promote NSCs survival from oxidative damage. The HSP90/NF-κB mechanism provides a new evidence on rescuing NSCs from oxidative stress and also promotes the stem cell application on CNS pathologies. PMID:27818721

Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia

PubMed Central

Fan, Yongjun; Wali, Gautam; Sutharsan, Ratneswary; Bellette, Bernadette; Crane, Denis I.; Sue, Carolyn M.; Mackay-Sim, Alan

2014-01-01

ABSTRACT Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS) cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine) that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials. PMID:24857849

Effect of essential amino acids on enteroids: Methionine deprivation suppresses proliferation and affects differentiation in enteroid stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Yuki; Iwatsuki, Ken; Hanyu, Hikaru

We investigated the effects of essential amino acids on intestinal stem cell proliferation and differentiation using murine small intestinal organoids (enteroids) from the jejunum. By selectively removing individual essential amino acids from culture medium, we found that 24 h of methionine (Met) deprivation markedly suppressed cell proliferation in enteroids. This effect was rescued when enteroids cultured in Met deprivation media for 12 h were transferred to complete medium, suggesting that Met plays an important role in enteroid cell proliferation. In addition, mRNA levels of the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) decreased in enteroids grown in Met deprivationmore » conditions. Consistent with this observation, Met deprivation also attenuated Lgr5-EGFP fluorescence intensity in enteroids. In contrast, Met deprivation enhanced mRNA levels of the enteroendocrine cell marker chromogranin A (ChgA) and markers of K cells, enterochromaffin cells, goblet cells, and Paneth cells. Immunofluorescence experiments demonstrated that Met deprivation led to an increase in the number of ChgA-positive cells. These results suggest that Met deprivation suppresses stem cell proliferation, thereby promoting differentiation. In conclusion, Met is an important nutrient in the maintenance of intestinal stem cells and Met deprivation potentially affects cell differentiation. - Highlights: • Met influences the proliferation of enteroids. • Met plays a crucial role in the maintenance of stem cells. • Met deprivation potentially promotes differentiation into secretory cells.« less

Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

PubMed

Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

2016-05-01

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models

PubMed Central

Lin, Tai-Chi; Zhu, Danhong; Hinton, David R.; Clegg, Dennis O.; Humayun, Mark S.

2017-01-01

Dysfunction and death of retinal pigment epithelium (RPE) and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula). Further, surgical placement of stem cell-derived grafts (RPE, retinal progenitors, and photoreceptor precursors) into the vitreous cavity or subretinal space has been well established. For preclinical tests, assessments of stem cell-derived graft survival and functionality are conducted in animal models by various noninvasive approaches and imaging modalities. In vivo experiments conducted in animal models based on replacing photoreceptors and/or RPE cells have shown survival and functionality of the transplanted cells, rescue of the host retina, and improvement of visual function. Based on the positive results obtained from these animal experiments, human clinical trials are being initiated. Despite such progress in stem cell research, ethical, regulatory, safety, and technical difficulties still remain a challenge for the transformation of this technique into a standard clinical approach. In this review, the current status of preclinical safety and efficacy studies for retinal cell replacement therapies conducted in animal models will be discussed. PMID:28928775

miR-125b Functions as a Key Mediator for Snail-induced Stem Cell Propagation and Chemoresistance*

PubMed Central

Liu, Zixing; Liu, Hao; Desai, Shruti; Schmitt, David C.; Zhou, Ming; Khong, Hung T.; Klos, Kristine S.; McClellan, Steven; Fodstad, Oystein; Tan, Ming

2013-01-01

Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/β-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy. PMID:23255607

Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.

PubMed

Liao, L; Yang, X; Su, X; Hu, C; Zhu, X; Yang, N; Chen, X; Shi, S; Shi, S; Jin, Y

2013-04-18

During the process of aging, especially for postmenopausal females, the cell lineage commitment of mesenchymal stem cells (MSCs) shift to adipocyte in bone marrow, resulting in osteoporosis. However, the cell-intrinsic mechanism of this cell lineage commitment switch is poorly understood. As the post-transcription regulation by microRNAs (miRNAs) has a critical role in MSCs differentiation and bone homeostasis, we performed comprehensive miRNAs profiling and found miR-705 and miR-3077-5p were significantly enhanced in MSCs from osteoporosis bone marrow. Both miR-705 and miR-3077-5p acted as inhibitors of MSCs osteoblast differentiation and promoters of adipocyte differentiation, by targeting on the 3'untranslated region (3'UTR) of HOXA10 and RUNX2 mRNA separately. Combined inhibition of miR-705 and miR-3077-5p rescued the cell lineage commitment disorder of MSCs through restoring HOXA10 and RUNX2 protein level. Furthermore, we found excessive TNFα and reactive oxygen species caused by estrogen deficiency led to the upregulation of both miRNAs through NF-κB pathway. In conclusion, our findings showed that redundant miR-705 and miR-3077-5p synergistically mediated the shift of MSCs cell lineage commitment to adipocyte in osteoporosis bone marrow, providing new insight into the etiology of osteoporosis at the post-transcriptional level. Moreover, the rescue of MSCs lineage commitment disorder by regulating miRNAs expression suggested a novel potential therapeutic target for osteoporosis as well as stem cell-mediated regenerative medicine.

PRMT7 Preserves Satellite Cell Regenerative Capacity.

PubMed

Blanc, Roméo Sébastien; Vogel, Gillian; Chen, Taiping; Crist, Colin; Richard, Stéphane

2016-02-16

Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries.

PubMed

Xiong, Jiaqiang; Lu, Zhiyong; Wu, Meng; Zhang, Jinjin; Cheng, Jing; Luo, Aiyue; Shen, Wei; Fang, Li; Zhou, Su; Wang, Shixuan

2015-01-01

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.

Nutrition and lifestyle in healthy aging: the telomerase challenge.

PubMed

Boccardi, Virginia; Paolisso, Giuseppe; Mecocci, Patrizia

2016-01-01

Nutrition and lifestyle, known to modulate aging process and age-related diseases, might also affect telomerase activity. Short and dysfunctional telomeres rather than average telomere length are associated with longevity in animal models, and their rescue by telomerase maybe sufficient to restore cell and organismal viability. Improving telomerase activation in stem cells and potentially in other cells by diet and lifestyle interventions may represent an intriguing way to promote health-span in humans.

Sodium Caseinate (CasNa) Induces Mobilization of Hematopoietic Stem Cells in a BALB/c Mouse Model

PubMed Central

Santiago-Osorio, Edelmiro; Ledesma-Martínez, Edgar; Aguiñiga-Sánchez, Itzen; Poblano-Pérez, Ignacio; Weiss-Steider, Benny; Montesinos-Montesinos, Juan José; de Lourdes Mora-García, María

2015-01-01

Background Hematopoietic stem cells transplantation has high clinical potential against a wide variety of hematologic, metabolic, and autoimmune diseases and solid tumors. Clinically, hematopoietic stem cells derived from peripheral blood are currently used more than those obtained from sources such as bone marrow. However, mobilizing agents used in the clinic tend to fail in high rates, making the number of mobilized cells insufficient for transplantation. We investigated whether sodium caseinate induces functional mobilization of hematopoietic stem cells into peripheral blood of Balb/c mice. Material/Methods Using a mouse model, we administrated sodium caseinate or Plerixafor, a commercial mobilizing agent, and analyzed counts of hematopoietic stem cells in peripheral blood, and then cells were transplanted into lethally irradiated mice to restore hematopoiesis. All assays were performed at least twice. Results We found that sodium caseinate increases the number of mononuclear cells in peripheral blood with the immunophenotype of hematopoietic stem cells (0.2 to 0.5% LSK cells), allowing them to form colonies of various cell lineages in semisolid medium (p<0.05). This effect is similar to that of Plerixafor, and cells transplanted into lethally irradiated mice can restore hematopoiesis at higher percentages than mononuclear cells mobilized by Plerixafor (40% vs. 20%, respectively). Further, a secondary transplant rescued a separate group of irradiated mice from death, proving definitive evidence of hematopoietic reconstitution after hematopoietic stem cells transplantation. Data are presented as mean ± standard deviation. To determine significant differences between the data, one-way ANOVA and the Tukey test were used. Conclusions Collectively these results show the utility of sodium caseinate as a mobilizer of hematopoietic stem cells and its potential clinical application in transplantation settings. PMID:26409928

Sodium Caseinate (CasNa) Induces Mobilization of Hematopoietic Stem Cells in a BALB/c Mouse Model.

PubMed

Santiago-Osorio, Edelmiro; Ledesma-Martínez, Edgar; Aguiñiga-Sánchez, Itzen; Poblano-Pérez, Ignacio; Weiss-Steider, Benny; Montesinos-Montesinos, Juan José; Mora-García, María de Lourdes

2015-09-25

BACKGROUND Hematopoietic stem cells transplantation has high clinical potential against a wide variety of hematologic, metabolic, and autoimmune diseases and solid tumors. Clinically, hematopoietic stem cells derived from peripheral blood are currently used more than those obtained from sources such as bone marrow. However, mobilizing agents used in the clinic tend to fail in high rates, making the number of mobilized cells insufficient for transplantation. We investigated whether sodium caseinate induces functional mobilization of hematopoietic stem cells into peripheral blood of Balb/c mice. MATERIAL AND METHODS Using a mouse model, we administrated sodium caseinate or Plerixafor, a commercial mobilizing agent, and analyzed counts of hematopoietic stem cells in peripheral blood, and then cells were transplanted into lethally irradiated mice to restore hematopoiesis. All assays were performed at least twice. RESULTS We found that sodium caseinate increases the number of mononuclear cells in peripheral blood with the immunophenotype of hematopoietic stem cells (0.2 to 0.5% LSK cells), allowing them to form colonies of various cell lineages in semisolid medium (p<0.05). This effect is similar to that of Plerixafor, and cells transplanted into lethally irradiated mice can restore hematopoiesis at higher percentages than mononuclear cells mobilized by Plerixafor (40% vs. 20%, respectively). Further, a secondary transplant rescued a separate group of irradiated mice from death, proving definitive evidence of hematopoietic reconstitution after hematopoietic stem cells transplantation. Data are presented as mean ± standard deviation. To determine significant differences between the data, one-way ANOVA and the Tukey test were used. CONCLUSIONS Collectively these results show the utility of sodium caseinate as a mobilizer of hematopoietic stem cells and its potential clinical application in transplantation settings.

IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozeki, Nobuaki; Hase, Naoko; Hiyama, Taiki

2014-10-15

We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3more » mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells. - Highlights: • IL-1β induces Wnt5, Lrp5/Fzd9 and MMP-3 in ES cell-derived odontoblast-like cells. • IL-1β-induced Wnt5 expression results in increased cell proliferation. • Exogenous Wnt5 increases MMP-3 activity and cell proliferation. • Exogenous Wnt5 rescues IL-1β-driven proliferation with anti-Wnt5 siRNA suppression. • IL-1β-induced cell proliferation involves Wnt5, Lrp5, and MMP-3 sequentially.« less

Human endothelial progenitor cells rescue cortical neurons from oxygen-glucose deprivation induced death.

PubMed

Bacigaluppi, Susanna; Donzelli, Elisabetta; De Cristofaro, Valentina; Bragazzi, Nicola Luigi; D'Amico, Giovanna; Scuteri, Arianna; Tredici, Giovanni

2016-09-19

Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

PubMed

Cleyrat, Cédric; Girard, Romain; Choi, Eun H; Jeziorski, Éric; Lavabre-Bertrand, Thierry; Hermouet, Sylvie; Carillo, Serge; Wilson, Bridget S

2017-09-26

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34 + cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

PubMed Central

Girard, Romain; Choi, Eun H.; Jeziorski, Éric; Lavabre-Bertrand, Thierry; Hermouet, Sylvie; Carillo, Serge; Wilson, Bridget S.

2017-01-01

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood–derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients. PMID:29296828

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

PubMed

Schraermeyer, U; Thumann, G; Luther, T; Kociok, N; Armhold, S; Kruttwig, K; Andressen, C; Addicks, K; Bartz-Schmidt, K U

2001-01-01

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Apelin: an endogenous peptide essential for cardiomyogenic differentiation of mesenchymal stem cells via activating extracellular signal-regulated kinase 1/2 and 5.

PubMed

Wang, Li; Zhu, Zhi-Ming; Zhang, Ning-Kun; Fang, Zhi-Rong; Xu, Xiao-Hong; Zheng, Nan; Gao, Lian-Ru

2016-05-01

Growing evidence has shown that apelin/APJ system functions as a critical mediator of cardiac development as well as cardiovascular function. Here, we investigated the role of apelin in the cardiomyogenic differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord in vitro. In this research, we used RNA interference methodology and gene transfection technique to regulate the expression of apelin in Wharton's jelly-derived mesenchymal stem cells and induced cells with a effective cardiac differentiation protocol including 5-azacytidine and bFGF. Four weeks after induction, induced cells assumed a stick-like morphology and myotube-like structures except apelin-silenced cells and the control group. The silencing expression of apelin in Wharton's jelly-derived mesenchymal stem cells decreased the expression of several critical cardiac progenitor transcription factors (Mesp1, Mef2c, NKX2.5) and cardiac phenotypes (cardiac α-actin, β-MHC, cTnT, and connexin-43). Meanwhile, endogenous compensation of apelin contributed to differentiating into cells with characteristics of cardiomyocytes in vitro. Further experiment showed that exogenous apelin peptide rescued the cardiomyogenic differentiation of apelin-silenced mesenchymal stem cells in the early stage (1-4 days) of induction. Remarkably, our experiment indicated that apelin up-regulated cardiac specific genes in Wharton's jelly-derived mesenchymal stem cells via activating extracellular signal-regulated kinase (ERK) 1/2 and 5. © 2016 International Federation for Cell Biology.

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures - a new donor for cell therapy.

PubMed

Wu, Wei; Zeng, Yuxiao; Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

2016-04-19

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

PubMed

Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

2016-01-01

Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

Usp16 contributes to somatic stem cell defects in Down syndrome

PubMed Central

Adorno, Maddalena; Sikandar, Shaheen; Mitra, Siddhartha S.; Kuo, Angera; Di Robilant, Benedetta Nicolis; Haro-Acosta, Veronica; Ouadah, Youcef; Quarta, Marco; Rodriguez, Jacqueline; Qian, Dalong; Reddy, Vadiyala M.; Cheshier, Samuel; Garner, Craig C.; Clarke, Michael F.

2013-01-01

SUMMARY Down syndrome (DS) results from full or partial trisomy of chromosome 21. However, the consequences of the underlying gene-dosage imbalance on adult tissues remain poorly understood. Here we show that in Ts65Dn mice, trisomic for 132 genes homologous to HSA21, triplication of Usp16 reduces self-renewal of hematopoietic stem cells and expansion of mammary epithelial cells, neural progenitors, and fibroblasts. Moreover, Usp16 is associated with decreased ubiquitination of Cdkn2a and accelerated senescence in Ts65Dn fibroblasts. Usp16 can remove ubiquitin from H2AK119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal USP16 allele or by shRNAs, largely rescues all these defects. Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and post-natal neural progenitors while downregulation of USP16 partially rescues the proliferation defects of DS fibroblasts. Taken together, these results suggest that USP16 plays an important role in antagonizing the self-renewal and/or senescence pathways in Down syndrome and could serve as an attractive target to ameliorate some of the associated pathologies. PMID:24025767

Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes.

PubMed

Jeong, Jaemin; Conboy, Michael J; Conboy, Irina M

2013-08-01

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice. Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses. The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling. The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.

miR-206 Inhibits Stemness and Metastasis of Breast Cancer by Targeting MKL1/IL11 Pathway.

PubMed

Samaeekia, Ravand; Adorno-Cruz, Valery; Bockhorn, Jessica; Chang, Ya-Fang; Huang, Simo; Prat, Aleix; Ha, Nahun; Kibria, Golam; Huo, Dezheng; Zheng, Hui; Dalton, Rachel; Wang, Yuhao; Moskalenko, Grigoriy Y; Liu, Huiping

2017-02-15

Purpose: Effective targeting of cancer stem cells is necessary and important for eradicating cancer and reducing metastasis-related mortality. Understanding of cancer stemness-related signaling pathways at the molecular level will help control cancer and stop metastasis in the clinic. Experimental Design: By analyzing miRNA profiles and functions in cancer development, we aimed to identify regulators of breast tumor stemness and metastasis in human xenograft models in vivo and examined their effects on self-renewal and invasion of breast cancer cells in vitro To discover the direct targets and essential signaling pathways responsible for miRNA functions in breast cancer progression, we performed microarray analysis and target gene prediction in combination with functional studies on candidate genes (overexpression rescues and pheno-copying knockdowns). Results: In this study, we report that hsa-miR-206 suppresses breast tumor stemness and metastasis by inhibiting both self-renewal and invasion. We identified that among the candidate targets, twinfilin ( TWF1 ) rescues the miR-206 phenotype in invasion by enhancing the actin cytoskeleton dynamics and the activity of the mesenchymal lineage transcription factors, megakaryoblastic leukemia (translocation) 1 (MKL1), and serum response factor (SRF). MKL1 and SRF were further demonstrated to promote the expression of IL11 , which is essential for miR-206's function in inhibiting both invasion and stemness of breast cancer. Conclusions: The identification of the miR-206/TWF1/MKL1-SRF/IL11 signaling pathway sheds lights on the understanding of breast cancer initiation and progression, unveils new therapeutic targets, and facilitates innovative drug development to control cancer and block metastasis. Clin Cancer Res; 23(4); 1091-103. ©2016 AACR . ©2016 American Association for Cancer Research.

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

PubMed

Cai, Jie; Xie, Xiaohong; Hu, Yi; Zhan, Yang; Yu, Wanting; Wang, Aibing; Wang, Naidong

2017-06-01

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

Mitochondrial transfer from Wharton's jelly-derived mesenchymal stem cells to mitochondria-defective cells recaptures impaired mitochondrial function.

PubMed

Lin, Hung-Yu; Liou, Chia-Wei; Chen, Shang-Der; Hsu, Te-Yao; Chuang, Jiin-Haur; Wang, Pei-Wen; Huang, Sheng-Teng; Tiao, Mao-Meng; Chen, Jin-Bor; Lin, Tsu-Kung; Chuang, Yao-Chung

2015-05-01

Adult mesenchymal stem cell (MSC)-conducted mitochondrial transfer has been recently shown to rescue cellular bioenergetics and prevent cell death caused by mitochondrial dysfunction. Wharton's jelly-derived MSCs (WJMSCs) harvested from postpartum umbilical cords are an accessible and abundant source of stem cells. This study aimed to determine the capability of WJMSCs to transfer their own mitochondria and rescue impaired oxidative phosphorylation (OXPHOS) and bioenergetics caused by mitochondrial DNA defects. To do this, WJMSCs were co-cultured with mitochondrial DNA (mtDNA)-depleted ρ(0) cells and the recapture of mitochondrial function was evaluated. WJMSCs were shown to be capable of transferring their own mitochondria into ρ(0) cells and underwent interorganellar mixture within these cells. Permissive culture media (BrdU-containing and pyruvate- and uridine-free) sieved out a survival cell population from the co-cultured WJMSCs (BrdU-sensitive) and ρ(0) cells (pyruvate/uridine-free). The survival cells had mtDNA identical to that of WJMSCs, whereas they expressed cellular markers identical to that of ρ(0) cells. Importantly, these ρ(0)-plus -WJMSC-mtDNA (ρ(+W)) cells recovered the expression of mtDNA-encoded proteins and exhibited functional oxygen consumption and respiratory control, as well as the activity of electron transport chain (ETC) complexes I, II, III and IV. In addition, ETC complex V-inhibitor-sensitive ATP production and metabolic shifting were also recovered. Furthermore, cellular behaviors including attachment-free proliferation, aerobic viability and OXPHOS-reliant cellular motility were also regained after mitochondrial transfer by WJMSCs. The therapeutic effect of WJMSCs-derived mitochondrial transfer was able to stably sustain for at least 45 passages. In conclusion, this study suggests that WJMSCs may serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction through the donation of healthy mitochondria to cells with genetic mitochondrial defects. Copyright © 2015 Elsevier B.V. All rights reserved.

Mesenchymal stromal cell derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

PubMed Central

Wen, Sicheng; Dooner, Mark; Cheng, Yan; Papa, Elaine; Del Tatto, Michael; Pereira, Mandy; Deng, Yanhui; Goldberg, Laura; Aliotta, Jason; Chatterjee, Devasis; Stewart, Connor; Carpanetto, Andrea; Collino, Federica; Bruno, Stefania; Camussi, Giovanni; Quesenberry, Peter

2016-01-01

Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells. PMID:27150009

DKK1 rescues osteogenic differentiation of mesenchymal stem cells isolated from periodontal ligaments of patients with diabetes mellitus induced periodontitis

PubMed Central

Liu, Qi; Hu, Cheng-Hu; Zhou, Cui-Hong; Cui, Xiao-Xia; Yang, Kun; Deng, Chao; Xia, Jia-Jia; Wu, Yan; Liu, Lu-Chuan; Jin, Yan

2015-01-01

Multiple studies have shown that diabetes mellitus is an established risk factor for periodontitis. Recently mesenchymal stem cells derived from periodontal ligament (PDLSCs) have been utilized to reconstruct tissues destroyed by chronic inflammation. However, impact of periodontitis with diabetes mellitus on PDLSCs and mechanisms mediating effects of complex microenvironments remain poorly understood. In this study, we found multiple differentiation potential of PDLSCs from chronic periodontitis with diabetes mellitus donors (D-PDLSCs) was damaged significantly. Inhibition of NF-κB signaling could rescue osteogenic potential of PDLSCs from simple chronic periodontitis patients (P-PDLSCs), whereas did not promote D-PDLSCs osteogenesis. In addition, we found expression of DKK1 in D-PDLSCs did not respond to osteogenic signal and decreased osteogenic potential of D-PDLSCs treated with DKK1 could be reversed. To further elucidate different character between P-PDLSCs and D-PDLSCs, we treated PDLSCs with TNF-α and advanced glycation end products (AGEs), and find out AGEs which enhance effect of TNF-α in PDLSCs might mediate special personality of D-PDLSCs. The adverse effect of AGEs in PDLSCs could be reversed when PDLSCs were treated with DKK1. These results suggested DKK1 mediating WNT signaling might be a therapy target to rescue potential of PDLSCs in periodontitis with diabetes mellitus. PMID:26278788

Administration of bone marrow derived mesenchymal stem cells into the liver: potential to rescue pseudoxanthoma elasticum in a mouse model (Abcc6-/-).

PubMed

Jiang, Qiujie; Takahagi, Shunsuke; Uitto, Jouni

2012-01-01

Pseudoxanthoma elasticum (PXE) is a heritable ectopic mineralization disorder caused by loss-of-function mutations in the ABCC6 gene which is primarily expressed in the liver. There is currently no effective treatment for PXE. In this study, we characterized bone marrow derived mesenchymal stem cells (MSCs) and evaluated their ability to contribute to liver regeneration, with the aim to rescue PXE phenotype. The MSCs, isolated from GFP-transgenic mice by magnetic cell sorting, were shown to have high potential for hepatic differentiation, with expression of Abcc6, in culture. These cells were transplanted into the livers of 4-week-old immunodeficient Abcc6⁻/⁻ mice by intrasplenic injection one day after partial hepatectomy, when peak expression of the stromal cell derived factor-1 (SDF-1) in the liver was observed. Fluorescent bioimaging analyses indicated that transplanted MSCs homed into liver between day 1 and 7, and significant numbers of GFP-positive cells were confirmed in the liver by immunofluorescence. Moreover, enhanced engraftment efficiency was observed with MSCs with high expression levels of the chemokine receptor Cxcr4, a receptor for SDF-1. These data suggest that purified MSCs have the capability of differentiating into hepatic lineages relevant to PXE pathogenesis and may contribute to partial correction of the PXE phenotype.

Whole-Body Radiation Therapy, Systemic Chemotherapy, and High-Dose Chemotherapy Followed By Stem Cell Rescue in Treating Patients With Poor-Risk Ewing Sarcoma

ClinicalTrials.gov

2015-01-07

Adult Supratentorial Primitive Neuroectodermal Tumor (PNET); Ewing Sarcoma of Bone; Extraosseous Ewing Sarcoma; Metastatic Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor; Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor; Recurrent Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumor; Untreated Childhood Supratentorial Primitive Neuroectodermal Tumor

Protein S Negatively Regulates Neural Stem Cell Self-Renewal through Bmi-1 Signaling

PubMed Central

Zelentsova-Levytskyi, Katya; Talmi, Ziv; Abboud-Jarrous, Ghada; Capucha, Tal; Sapir, Tamar; Burstyn-Cohen, Tal

2017-01-01

Revealing the molecular mechanisms underlying neural stem cell self-renewal is a major goal toward understanding adult brain homeostasis. The self-renewing potential of neural stem and progenitor cells (NSPCs) must be tightly regulated to maintain brain homeostasis. We recently reported the expression of Protein S (PROS1) in adult hippocampal NSPCs, and revealed its role in regulation of NSPC quiescence and neuronal differentiation. Here, we investigate the effect of PROS1 on NSPC self-renewal and show that genetic ablation of Pros1 in neural progenitors increased NSPC self-renewal by 50%. Mechanistically, we identified the upregulation of the polycomb complex protein Bmi-1 and repression of its downstream effectors p16Ink4a and p19Arf to promote NSPC self-renewal in Pros1-ablated cells. Rescuing Pros1 expression restores normal levels of Bmi-1 signaling, and reverts the proliferation and enhanced self-renewal phenotypes observed in Pros1-deleted cells. Our study identifies PROS1 as a novel negative regulator of NSPC self-renewal. We conclude PROS1 is instructive for NSPC differentiation by negatively regulating Bmi-1 signaling in adult and embryonic neural stem cells. PMID:28512399

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems

PubMed Central

Talts, Kristiina; Ilau, Birger; Ojangu, Eve-Ly; Tanner, Krista; Peremyslov, Valera V.; Dolja, Valerian V.; Truve, Erkki; Paves, Heiti

2016-01-01

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis. PMID:28066484

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

PubMed

van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

PubMed

Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

2018-01-01

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

Stem cell therapy emerging as the key player in treating type 1 diabetes mellitus.

PubMed

Vanikar, Aruna V; Trivedi, Hargovind L; Thakkar, Umang G

2016-09-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease causing progressive destruction of pancreatic β cells, ultimately resulting in loss of insulin secretion producing hyperglycemia usually affecting children. Replacement of damaged β cells by cell therapy can treat it. Currently available strategies are insulin replacement and islet/pancreas transplantation. Unfortunately these offer rescue for variable duration due to development of autoantibodies. For pancreas/islet transplantation a deceased donor is required and various shortfalls of treatment include quantum, cumbersome technique, immune rejection and limited availability of donors. Stem cell therapy with assistance of cellular reprogramming and β-cell regeneration can open up new therapeutic modalities. The present review describes the history and current knowledge of T1DM, evolution of cell therapies and different cellular therapies to cure this condition. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Modeling Fanconi Anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs

PubMed Central

Montserrat, Nuria; Tarantino, Carolina; Gu, Ying; Yi, Fei; Xu, Xiuling; Zhang, Weiqi; Ruiz, Sergio; Plongthongkum, Nongluk; Zhang, Kun; Masuda, Shigeo; Nivet, Emmanuel; Tsunekawa, Yuji; Soligalla, Rupa Devi; Goebl, April; Aizawa, Emi; Kim, Na Young; Kim, Jessica; Dubova, Ilir; Li, Ying; Ren, Ruotong; Benner, Chris; del Sol, Antonio; Bueren, Juan; Trujillo, Juan Pablo; Surralles, Jordi; Cappelli, Enrico; Dufour, Carlo; Esteban, Concepcion Rodriguez; Belmonte, Juan Carlos Izpisua

2014-01-01

Fanconi Anemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration-free induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA-patient bone marrow cells. PMID:24999918

ZFX controls propagation and prevents differentiation of acute T-lymphoblastic and myeloid leukemia

PubMed Central

Weisberg, Stuart P.; Smith-Raska, Matthew R.; Esquilin, Jose M.; Zhang, Ji; Arenzana, Teresita L.; Lau, Colleen M.; Churchill, Michael; Pan, Haiyan; Klinakis, Apostolos; Dixon, Jack E.; Mirny, Leonid A.; Mukherjee, Siddhartha; Reizis, Boris

2014-01-01

Summary Tumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem cell-related genetic regulator. PMID:24485662

Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs.

PubMed

Liu, Guang-Hui; Suzuki, Keiichiro; Li, Mo; Qu, Jing; Montserrat, Nuria; Tarantino, Carolina; Gu, Ying; Yi, Fei; Xu, Xiuling; Zhang, Weiqi; Ruiz, Sergio; Plongthongkum, Nongluk; Zhang, Kun; Masuda, Shigeo; Nivet, Emmanuel; Tsunekawa, Yuji; Soligalla, Rupa Devi; Goebl, April; Aizawa, Emi; Kim, Na Young; Kim, Jessica; Dubova, Ilir; Li, Ying; Ren, Ruotong; Benner, Chris; Del Sol, Antonio; Bueren, Juan; Trujillo, Juan Pablo; Surralles, Jordi; Cappelli, Enrico; Dufour, Carlo; Esteban, Concepcion Rodriguez; Belmonte, Juan Carlos Izpisua

2014-07-07

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells.

Rescue plan for Achilles: Therapeutics steering the fate and functions of stem cells in tendon wound healing.

PubMed

Schneider, Magdalena; Angele, Peter; Järvinen, Tero A H; Docheva, Denitsa

2017-12-24

Due to the increasing age of our society and a rise in engagement of young people in extreme and/or competitive sports, both tendinopathies and tendon ruptures present a clinical and financial challenge. Tendon has limited natural healing capacity and often responds poorly to treatments, hence it requires prolonged rehabilitation in most cases. Till today, none of the therapeutic options has provided successful long-term solutions, meaning that repaired tendons do not recover their complete strength and functionality. Our understanding of tendon biology and healing increases only slowly and the development of new treatment options is insufficient. In this review, following discussion on tendon structure, healing and the clinical relevance of tendon injury, we aim to elucidate the role of stem cells in tendon healing and discuss new possibilities to enhance stem cell treatment of injured tendon. To date, studies mainly apply stem cells, often in combination with scaffolds or growth factors, to surgically created tendon defects. Deeper understanding of how stem cells and vasculature in the healing tendon react to growth factors, common drugs used to treat injured tendons and promising cellular boosters could help to develop new and more efficient ways to manage tendon injuries. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6-deficient murine embryoid bodies.

PubMed

Pierre, Monique; Yoshimoto, Momoko; Huang, Lan; Richardson, Matthew; Yoder, Mervin C

2009-09-01

Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells. EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays. Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner. These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.

Sequence adaptations during growth of rescued classical swine fever viruses in cell culture and within infected pigs.

PubMed

Hadsbjerg, Johanne; Friis, Martin B; Fahnøe, Ulrik; Nielsen, Jens; Belsham, Graham J; Rasmussen, Thomas Bruun

2016-08-30

Classical swine fever virus (CSFV) causes an economically important disease of swine. Four different viruses were rescued from full-length cloned cDNAs derived from the Paderborn strain of CSFV. Three of these viruses had been modified by mutagenesis (with 7 or 8 nt changes) within stem 2 of the subdomain IIIf of the internal ribosome entry site (IRES) that directs the initiation of protein synthesis. Rescued viruses were inoculated into pigs. The rescued vPader10 virus, without modifications in the IRES, induced clinical disease in pigs that was very similar to that observed previously with the parental field strain and transmission to in-contact pigs occurred. Two sequence reversions, in the NS2 and NS5B coding regions, became dominant within the virus populations in these infected pigs. Rescued viruses, with mutant IRES elements, did not induce disease and only very limited circulation of viral RNA could be detected. However, the animals inoculated with these mutant viruses seroconverted against CSFV. Thus, these mutant viruses were highly attenuated in vivo. All 4 rescued viruses were also passaged up to 20 times in cell culture. Using full genome sequencing, the same two adaptations within each of four independent virus populations were observed that restored the coding sequence to that of the parental field strain. These adaptations occurred with different kinetics. The combination of reverse genetics and in depth, full genome sequencing provides a powerful approach to analyse virus adaptation and to identify key determinants of viral replication efficiency in cells and within host animals. Copyright © 2016 Elsevier B.V. All rights reserved.

How to grow a kidney: patient-specific kidney organoids come of age.

PubMed

Schmidt-Ott, Kai M

2017-01-01

The notion of regrowing a patient's kidney in a dish has fascinated researchers for decades and has spurred visions of revolutionary clinical applications. Recently, this option has come closer to reality. Key technologies have been developed to generate patient-specific pluripotent stem cells and to edit their genome. Several laboratories have devised protocols to differentiate patient-specific pluripotent stem cells into kidney cells or into in vitro organoids that resemble the kidney with respect to cell types, tissue architecture and disease pathology. This was possible because of rapidly expanding knowledge regarding the cellular and molecular basis of embryonic kidney development. Generating kidney cells or organoids from patient-specific stem cells may prove to be clinically useful in several ways. First, patient-specific kidney cells or organoids could be used to predict an individual's response to stressors, toxins or medications and thereby develop personalized treatment decisions. Second, patient-specific stem cells harbour the individual's genetic defects. This may potentially enable genetic rescue attempts to establish the significance of a genetic defect in a stem cell-derived organoid or it may allow testing of patient-specific targeted therapies for kidney disease in vitro. From a tissue engineering perspective, patient-specific kidney organoids might provide a key advance towards engineering immunocompatible transplantable kidneys. This review article summarizes recent developments in the field and discusses its current limitations and future perspectives. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Nutraceutical intervention reverses the negative effects of blood from aged rats on stem cells.

PubMed

Bickford, Paula C; Kaneko, Yuji; Grimmig, Bethany; Pappas, Colleen; Small, Brent; Sanberg, Cyndy D; Sanberg, Paul R; Tan, Jun; Douglas Shytle, R

2015-10-01

Aging is associated with a decline in function in many of the stem cell niches of the body. An emerging body of literature suggests that one of the reasons for this decline in function is due to cell non-autonomous influences on the niche from the body. For example, studies using the technique of parabiosis have demonstrated a negative influence of blood from aged mice on muscle satellite cells and neurogenesis in young mice. We examined if we could reverse this effect of aged serum on stem cell proliferation by treating aged rats with NT-020, a dietary supplement containing blueberry, green tea, vitamin D3, and carnosine that has been shown to increase neurogenesis in aged rats. Young and aged rats were administered either control NIH-31 diet or one supplemented with NT-020 for 28 days, and serum was collected upon euthanasia. The serum was used in cultures of both rat hippocampal neural progenitor cells (NPCs) and rat bone marrow-derived mesenchymal stem cells (MSCs). Serum from aged rats significantly reduced cell proliferation as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays in both NPCs and MSCs. Serum from aged rats treated with NT-020 was not different from serum from young rats. Therefore, NT-020 rescued the effect of serum from aged rats to reduce stem cell proliferation.

MiR-146b-5p overexpression attenuates stemness and radioresistance of glioma stem cells by targeting HuR/lincRNA-p21/β-catenin pathway

PubMed Central

Yang, Wei; Yu, Hongquan; Shen, Yueming; Liu, Yingying; Yang, Zhanshan; Sun, Ting

2016-01-01

A stem-like subpopulation existed in GBM cells, called glioma stem cells (GSCs), might contribute to cancer invasion, angiogenesis, immune evasion, and therapeutic resistance, providing a rationale to eliminate GSCs population and their supporting niche for successful GBM treatment. LincRNA-p21, a novel regulator of cell proliferation, apoptosis and DNA damage response, is found to be downregulated in several types of tumor. However, little is known about the role of lincRNA-p21 in stemness and radioresistance of GSCs and its regulating mechanisms. In this study, we found that lincRNA-p21 negatively regulated the expression and activity of β-catenin in GSCs. Downregulation of lincRNA-p21 in GSCs was resulted from upregulation of Hu antigen R (HuR) expression caused by miR-146b-5p downregulation. MiR-146b-5p overexpression increased apoptosis and radiosensitivity, decreased cell viability, neurosphere formation capacity and stem cell marker expression, and induced differentiation in GSCs. Moreover, knock-down lincRNA-p21 or HuR and β-catenin overexpression could rescue the phenotypic changes resulted from miR-146b-5p overexpression in GSCs. These findings suggest that targeting the miR-146b-5p/HuR/lincRNA-p21/β-catenin signaling pathway may be valuable therapeutic strategies against glioma. PMID:27166258

Satellite cells say NO to radiation.

PubMed

Cho-Lim, Jennie J; Caiozzo, Vincent J; Tseng, Bertrand P; Giedzinski, Erich; Baker, Mike J; Limoli, Charles L

2011-05-01

Skeletal muscles are commonly exposed to radiation for diagnostic procedures and the treatment of cancers and heterotopic bone formation. Few studies have considered the impact of clinical doses of radiation on the ability of satellite cells (myogenic stem cells) to proliferate, differentiate and contribute to recovering/maintaining muscle mass. The primary objective of this study was to determine whether the proliferation of irradiated satellite cells could be rescued by manipulating NO levels via pharmacological approaches and mechanical stretch (which is known to increase NO levels). We used both SNP (NO donor) and PTIO (NO scavenger) to manipulate NO levels in satellite cells. We observed that SNP was highly effective in rescuing the proliferation of irradiated satellite cells, especially at doses less than 5 Gy. The potential importance of NO was further illustrated by the effects of PTIO, which completely inhibited the rescue effect of SNP. Mechanical cyclic stretch was found to produce significant increases in NO levels of irradiated satellite cells, and this was associated with a robust increase in satellite cell proliferation. The effects of both radiation and NO on two key myogenic regulatory factors (MyoD and myogenin) were also explored. Irradiation of satellite cells produced a significant increase in both MyoD and myogenin, effects that were mitigated by manipulating NO levels via SNP. Given the central role of myogenic regulatory factors in the proliferation and differentiation of satellite cells, the findings of the current study underscore the need to more fully understand the relationship between radiation, NO and the functionality of satellite cells.

Phosphorylation of ULK1 by AMPK is essential for mouse embryonic stem cell self-renewal and pluripotency.

PubMed

Gong, Jiaqi; Gu, Haifeng; Zhao, Lin; Wang, Liang; Liu, Pinglei; Wang, Fuping; Xu, Haoyu; Zhao, Tongbiao

2018-01-18

Autophagy is a catabolic process to degrade both damaged organelles and aggregated proteins in somatic cells. We have recently identified that autophagy is an executor for mitochondrial homeostasis in embryonic stem cell (ESC), and thus contribute to stemness regulation. However, the regulatory and functional mechanisms of autophagy in ESC are still largely unknown. Here we have shown that activation of ULK1 by AMPK is essential for ESC self-renewal and pluripotency. Dysfunction of Ulk1 decreases the autophagic flux in ESC, leading to compromised self-renewal and pluripotency. These defects can be rescued by reacquisition of wild-type ULK1 and ULK1(S757A) mutant, but not ULK1(S317A, S555A and S777A) and kinase dead ULK1(K46I) mutant. These data indicate that phosphorylation of ULK1 by AMPK, but not mTOR, is essential for stemness regulation in ESC. The findings highlight a critical role for AMPK-dependent phosphorylation of ULK1 pathway to maintain ESC self-renewal and pluripotency.

OCIAD1 Controls Electron Transport Chain Complex I Activity to Regulate Energy Metabolism in Human Pluripotent Stem Cells.

PubMed

Shetty, Deeti K; Kalamkar, Kaustubh P; Inamdar, Maneesha S

2018-06-14

Pluripotent stem cells (PSCs) derive energy predominantly from glycolysis and not the energy-efficient oxidative phosphorylation (OXPHOS). Differentiation is initiated with energy metabolic shift from glycolysis to OXPHOS. We investigated the role of mitochondrial energy metabolism in human PSCs using molecular, biochemical, genetic, and pharmacological approaches. We show that the carcinoma protein OCIAD1 interacts with and regulates mitochondrial complex I activity. Energy metabolic assays on live pluripotent cells showed that OCIAD1-depleted cells have increased OXPHOS and may be poised for differentiation. OCIAD1 maintains human embryonic stem cells, and its depletion by CRISPR/Cas9-mediated knockout leads to rapid and increased differentiation upon induction, whereas OCIAD1 overexpression has the opposite effect. Pharmacological alteration of complex I activity was able to rescue the defects of OCIAD1 modulation. Thus, hPSCs can exist in energy metabolic substates. OCIAD1 provides a target to screen for additional modulators of mitochondrial activity to promote transient multipotent precursor expansion or enhance differentiation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Intraspinal Stem Cell Transplantation for Amyotrophic Lateral Sclerosis

PubMed Central

Chen, Kevin S.; Sakowski, Stacey A.; Feldman, Eva L.

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder in which the loss of upper and lower motor neurons produces progressive weakness and eventually death. In the decades since the approval of riluzole, the only FDA approved medication to moderately slow progression of ALS, no new therapeutics have arisen to alter the course of the disease. This is partly due to our incomplete understanding of the complex pathogenesis of motor neuron degeneration. Stem cells have emerged as an attractive option in treating ALS since they come armed with equally complex cellular machinery and may modulate the local microenvironment in many ways to rescue diseased motor neurons. While various stem cell types are being evaluated in preclinical and early clinical applications, here we review the preclinical strategies and advances supporting the recent clinical translation of neural progenitor cell therapy for ALS. Specifically, we focus on the use of spinal cord neural progenitor cells and the pipeline starting from preclinical studies to the designs of the Phase I and IIa clinical trials involving direct intraspinal transplantation in humans. PMID:26696091

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

PubMed

Schwörer, Simon; Becker, Friedrich; Feller, Christian; Baig, Ali H; Köber, Ute; Henze, Henriette; Kraus, Johann M; Xin, Beibei; Lechel, André; Lipka, Daniel B; Varghese, Christy S; Schmidt, Manuel; Rohs, Remo; Aebersold, Ruedi; Medina, Kay L; Kestler, Hans A; Neri, Francesco; von Maltzahn, Julia; Tümpel, Stefan; Rudolph, K Lenhard

2016-12-15

The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFβ, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

PubMed

Dietrich, Philipp A; Yang, Chen; Leung, Halina H L; Lynch, Jennifer R; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D; Wang, Jianlong; Wang, Jenny Yingzi

2014-11-20

β-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML. © 2014 by The American Society of Hematology.

FGFR1 promotes the stem cell-like phenotype of FGFR1-amplified non-small cell lung cancer cells through the Hedgehog pathway.

PubMed

Ji, Wenxiang; Yu, Yongfeng; Li, Ziming; Wang, Guan; Li, Fan; Xia, Weiliang; Lu, Shun

2016-03-22

Cancer stem cell-like phenotype is critical for tumor formation and treatment resistance. FGFR1 is found to be amplified in non-small cell lung cancer, particularly in the lung squamous cell cancer (LSCC). Whether FGFR1 contributes to the maintenance of stem cell-like phenotype of FGFR1-amplified lung cancer cells remains elusive. In this study, treatment with FGFR1 inhibitor AZD4547 suppressed the growth of tumor spheres and reduced ALDH positive proportion in FGFR1-amplified lung cancer cells in vitro, as well as inhibited the growth of oncospheres and parental cells in xenograft models. Knockdown of FGFR1 recaptured the similar effect as AZD4547 in vitro. Furthermore, activation of FGFR1 and subsequently its downstream ERK signaling enhanced the expression and transcriptional activity of GLI2, which could be blocked by FGFR1 inhibitor/silencing or ERK inhibitor. Knockdown of GLI2 directly inhibited the stem-like phenotype of FGFR1-amilified cells, whereas overexpression of GLI2 sufficiently rescued the phenotype caused by FGFR1 knockdown. Notably we also identified a correlation between FGFR1 and GLI2 expressions from clinical data, as well as an inverse relationship with progression free survival (PFS). Together our study suggests that the FGFR1/GLI2 axis promotes the lung cancer stem cell-like phenotype. These results support a rational strategy of combination of FGFR1 and GLI inhibitors for treatment of FGFR1-amplified lung cancers, especially LSCC.

Fibroblast Activation Protein (FAP) Is Essential for the Migration of Bone Marrow Mesenchymal Stem Cells through RhoA Activation

PubMed Central

Chung, Kuei-Min; Hsu, Shu-Ching; Chu, Yue-Ru; Lin, Mei-Yao; Jiaang, Weir-Tong; Chen, Ruey-Hwa; Chen, Xin

2014-01-01

Background The ability of human bone marrow mesenchymal stem cells (BM-MSCs) to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP) is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. Principal Findings We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1β) and transforming growth factor-beta (TGF-β) upregulated FAP expression, which coincided with better BM-MSC migration. Conclusions Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1β and TGF-β upregulate the expression level of FAP and thus enhance BM-MSC migration. PMID:24551161

Fibroblast activation protein (FAP) is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation.

PubMed

Chung, Kuei-Min; Hsu, Shu-Ching; Chu, Yue-Ru; Lin, Mei-Yao; Jiaang, Weir-Tong; Chen, Ruey-Hwa; Chen, Xin

2014-01-01

The ability of human bone marrow mesenchymal stem cells (BM-MSCs) to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP) is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1β) and transforming growth factor-beta (TGF-β) upregulated FAP expression, which coincided with better BM-MSC migration. Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1β and TGF-β upregulate the expression level of FAP and thus enhance BM-MSC migration.

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.

PubMed

Chen, B; Teng, Jiawen; Liu, Hongwei; Pan, X; Zhou, Y; Huang, Shu; Lai, Mowen; Bian, Guohui; Mao, Bin; Sun, Wencui; Zhou, Qiongxiu; Yang, Shengyong; Nakahata, Tatsutoshi; Ma, Feng

2017-08-01

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models. © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

A novel platform for minimally invasive delivery of cellular therapy as a thin layer across the subretina for treatment of retinal degeneration

NASA Astrophysics Data System (ADS)

Rotenstreich, Ygal; Tzameret, Adi; Kalish, Sapir E.; Belkin, Michael; Meir, Amilia; Treves, Avraham J.; Nagler, Arnon; Sher, Ifat

2015-03-01

Incurable retinal degenerations affect millions worldwide. Stem cell transplantation rescued visual functions in animal models of retinal degeneration. In those studies cells were transplanted in subretinal "blebs", limited number of cells could be injected and photoreceptor rescue was restricted to areas in proximity to the injection sites. We developed a minimally-invasive surgical platform for drug and cell delivery in a thin layer across the subretina and extravascular spaces of the choroid. The novel system is comprised of a syringe with a blunt-tipped needle and an adjustable separator. Human bone marrow mesenchymal stem cells (hBM-MSCs) were transplanted in eyes of RCS rats and NZW rabbits through a longitudinal triangular scleral incision. No immunosuppressants were used. Retinal function was determined by electroretinogram analysis and retinal structure was determined by histological analysis and OCT. Transplanted cells were identified as a thin layer across the subretina and extravascular spaces of the choroid. In RCS rats, cell transplantation delayed photoreceptor degeneration across the entire retina and significantly enhanced retinal functions. No retinal detachment or choroidal hemorrhages were observed in rabbits following transplantation. This novel platform opens a new avenue for drug and cell delivery, placing the transplanted cells in close proximity to the damaged RPE and retina as a thin layer, across the subretina and thereby slowing down cell death and photoreceptor degeneration, without retinal detachment or choroidal hemorrhage. This new transplantation system may increase the therapeutic effect of other cell-based therapies and therapeutic agents. This study is expected to directly lead to phase I/II clinical trials for autologous hBM-MSCs transplantation in retinal degeneration patients.

Poly(Lactide-Co-Glycolide)-Monomethoxy-Poly-(Polyethylene Glycol) Nanoparticles Loaded with Melatonin Protect Adipose-Derived Stem Cells Transplanted in Infarcted Heart Tissue.

PubMed

Ma, Qiang; Yang, Junjie; Huang, Xu; Guo, Weisheng; Li, Sulei; Zhou, Hao; Li, Jingwei; Cao, Feng; Chen, Yundai

2018-04-01

Stem cell transplantation is a promising therapeutic strategy for myocardial infarction. However, transplanted cells face low survival rates due to oxidative stress and the inflammatory microenvironment in ischemic heart tissue. Melatonin has been used as a powerful endogenous antioxidant to protect cells from oxidative injury. However, melatonin cannot play a long-lasting effect against the hostile microenvironment. Nano drug delivery carriers have the ability to protect the loaded drug from degradation in physiological environments in a controlled manner, which results in longer effects and decreased side effects. Therefore, we constructed poly(lactide-co-glycolide)-monomethoxy-poly-(polyethylene glycol) (PLGA-mPEG) nanoparticles to encapsulate melatonin. We tested whether the protective effect of melatonin encapsulated by PLGA-mPEG nanoparticles (melatonin nanoparticles [Mel-NPs]) on adipose-derived mesenchymal stem cells (ADSCs) was enhanced compared to that of free melatonin both in vitro and in vivo. In the in vitro study, we found that Mel-NPs reduced formation of the p53- cyclophilin D complex, prevented mitochondrial permeability transition pores from opening, and rescued ADSCs from hypoxia/reoxygenation injury. Moreover, Mel-NPs can achieve higher ADSC survival rates than free melatonin in rat myocardial infarction areas, and the therapeutic effects of ADSCs pretreated with Mel-NPs were more apparent. Hence, the combination of Mel-NPs and stem cell transplantation may be a promising strategy for myocardial infarction therapy. Stem Cells 2018;36:540-550. © AlphaMed Press 2018.

Randomized Trial of Interleukin-2 (IL-2) as Early Consolidation Following Marrow Ablative Therapy with Stem Cell Rescue for Matastatic Breast Cancer

DTIC Science & Technology

2007-10-01

survival, with minimal toxicity. Effectiveness of this approach may correlate with the effective induction of LAK precursor and effector cells, as...and safe after AC+T chemotherapy and is showing a promising effect on breast cancer relapses. This regimen should be further evaluated in high-risk...1337 Diarrhea, hypoglycemia, hypocalcemia , stomatitis, skin irritation (port), nasal congestion, chest pain fatigue, dyspnea S-W 40 T2 N1biv

Understanding the Role of TSC1/2 in Cerebellar Purkinje Neurons

DTIC Science & Technology

2017-09-01

patient-specific iPSC lines and rescue the disease phenotypes in patient specific neurons in vitro. We will employ CRISPR -Caspase 9 (Cas9) genome...development from human stem cells. For Aim 2. We at BCH have successfully generated CRISPR -cas9-mediated correction of TSC2- microdeletion in TSC...patient (TSC2+/-) derived hiPSC line (Figure 2). We used puromycin selection for isolation of the CRISPR -cas9 and ssODN transfected cells, and with

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

PubMed

Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.

Cigarette Smoking Impairs Adipose Stromal Cell Vasculogenic Activity and Abrogates Potency to Ameliorate Ischemia.

PubMed

Barwinska, Daria; Traktuev, Dmitry O; Merfeld-Clauss, Stephanie; Cook, Todd G; Lu, Hongyan; Petrache, Irina; March, Keith L

2018-06-01

Cigarette smoking (CS) adversely affects the physiologic function of endothelial progenitor, hematopoietic stem and progenitor cells. However, the effect of CS on the ability of adipose stem/stromal cells (ASC) to promote vasculogenesis and rescue perfusion in the context of ischemia is unknown. To evaluate this, ASC from nonsmokers (nCS-ASC) and smokers (CS-ASC), and their activity to promote perfusion in hindlimb ischemia models, as well as endothelial cell (EC) survival and vascular morphogenesis in vitro were assessed. While nCS-ASC improved perfusion in ischemic limbs, CS-ASC completely lost this therapeutic effect. In vitro vasculogenesis assays revealed that human CS-ASC and ASC from CS-exposed mice showed compromised support of EC morphogenesis into vascular tubes, and the CS-ASC secretome was less potent in supporting EC survival/proliferation. Comparative secretome analysis revealed that CS-ASC produced lower amounts of hepatocyte growth factor (HGF) and stromal cell-derived growth factor 1 (SDF-1). Conversely, CS-ASC secreted the angiostatic/pro-inflammatory factor Activin A, which was not detected in nCS-ASC conditioned media (CM). Furthermore, higher Activin A levels were measured in EC/CS-ASC cocultures than in EC/nCS-ASC cocultures. CS-ASC also responded to inflammatory cytokines with 5.2-fold increase in Activin A secretion, whereas nCS-ASC showed minimal Activin A induction. Supplementation of EC/CS-ASC cocultures with nCS-ASC CM or with recombinant vascular endothelial growth factor, HGF, or SDF-1 did not rescue vasculogenesis, whereas inhibition of Activin A expression or activity improved network formation up to the level found in EC/nCS-ASC cocultures. In conclusion, ASC of CS individuals manifest compromised in vitro vasculogenic activity as well as in vivo therapeutic activity. Stem Cells 2018;36:856-867. © 2018 AlphaMed Press.

Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications.

PubMed

Di Baldassarre, Angela; Cimetta, Elisa; Bollini, Sveva; Gaggi, Giulia; Ghinassi, Barbara

2018-05-25

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications.

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance.

PubMed

Yang, Wei; Wei, Jing; Guo, Tiantian; Shen, Yueming; Liu, Fenju

2014-08-01

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches. Copyright © 2014 Elsevier Inc. All rights reserved.

Just-in-time rescue plerixafor in combination with chemotherapy and granulocyte-colony stimulating factor for peripheral blood progenitor cell mobilization

PubMed Central

Smith, Veronica R.; Popat, Uday; Ciurea, Stefan; Nieto, Yago; Anderlini, Paolo; Rondon, Gabriela; Alousi, Amin; Qazilbash, Muzaffar; Kebriaei, Partow; Khouri, Issa; de Lima, Marcos; Champlin, Richard; Hosing, Chitra

2014-01-01

Plerixafor, a recently approved peripheral blood progenitor cell mobilizing agent, is often added to granulocyte-colony stimulating factor (G-CSF) to mobilize peripheral blood progenitor cells in patients with lymphoma or myeloma who cannot mobilize enough CD34+ cells with G-CSF alone to undergo autologous stem cell transplantation. However, data are lacking regarding the feasibility and efficacy of just-in-time plerixafor in combination with chemotherapy and G-CSF. We reviewed the peripheral blood stem cell collection data of 38 consecutive patients with lymphoma (Hodgkin’s and non-Hodgkin’s) and multiple myeloma who underwent chemomobilization and high-dose G-CSF and just-in-time plerixafor to evaluate the efficacy of this treatment combination. All patients with multiple myeloma and all but 1 patient with lymphoma collected the minimum required number of CD34+ cells to proceed with autologous stem cell transplantation (>2 × 106/kilogram of body weight). The median CD34+ cell dose collected in patients with non-Hodgkin lymphoma was 4.93 × 106/kilogram of body weight. The median CD34+ cell dose collected for patients with multiple myeloma was 8.81 × 106/kilogram of body weight. Plerixafor was well tolerated; no grade 2 or higher non- hematologic toxic effects were observed. PMID:23749720

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

PubMed

Ma, Hong; Folmes, Clifford D L; Wu, Jun; Morey, Robert; Mora-Castilla, Sergio; Ocampo, Alejandro; Ma, Li; Poulton, Joanna; Wang, Xinjian; Ahmed, Riffat; Kang, Eunju; Lee, Yeonmi; Hayama, Tomonari; Li, Ying; Van Dyken, Crystal; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Koski, Amy; Mitalipov, Nargiz; Amato, Paula; Wolf, Don P; Huang, Taosheng; Terzic, Andre; Laurent, Louise C; Izpisua Belmonte, Juan Carlos; Mitalipov, Shoukhrat

2015-08-13

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

The Potent Humanin Analogue (HNG) Protects Germ Cells and Leucocytes While Enhancing Chemotherapy-Induced Suppression of Cancer Metastases in Male Mice.

PubMed

Lue, YanHe; Swerdloff, Ronald; Wan, Junxiang; Xiao, Jialin; French, Samuel; Atienza, Vince; Canela, Victor; Bruhn, Kevin W; Stone, Brian; Jia, Yue; Cohen, Pinchas; Wang, Christina

2015-12-01

Humanin is a peptide that is cytoprotective against stresses in many cell types. We investigated whether a potent humanin analogue S14G-humanin (HNG) would protect against chemotherapy-induced damage to normal cells without interfering with the chemotherapy-induced suppression of cancer cells. Young adult male mice were inoculated iv with murine melanoma cells. After 1 week, cancer-bearing mice were randomized to receive either: no treatment, daily ip injection of HNG, a single ip injection of cyclophosphamide (CP), or CP+HNG and killed at the end of 3 weeks. HNG rescued the CP-induced suppression of leucocytes and protected germ cell from CP-induced apoptosis. Lung metastases were suppressed by HNG or CP alone, and further suppressed by CP+HNG treatment. Plasma IGF-1 levels were suppressed by HNG with or without CP treatment. To investigate whether HNG maintains its protective effects on spermatogonial stem cells, sperm output, and peripheral leucocytes after repeated doses of CP, normal adult male mice received: no treatment, daily sc injection of HNG, 6 ip injections of CP at 5-day intervals, and the same regimens of CP+HNG and killed at the end of 4 weeks of treatment. Cauda epididymal sperm counts were elevated by HNG and suppressed by CP. HNG rescued the CP-induced suppression of spermatogonial stem cells, sperm count and peripheral leucocytes. We conclude that HNG 1) protects CP-induced loss of male germ cells and leucocytes, 2) enhances CP-induced suppression of cancer metastases, and 3) acts as a caloric-restriction mimetic by suppressing IGF-1 levels. Our findings suggest that humanin analogues may be promising adjuvants to chemotherapy.

The Potent Humanin Analogue (HNG) Protects Germ Cells and Leucocytes While Enhancing Chemotherapy-Induced Suppression of Cancer Metastases in Male Mice

PubMed Central

Lue, YanHe; Swerdloff, Ronald; Wan, Junxiang; Xiao, Jialin; French, Samuel; Atienza, Vince; Canela, Victor; Bruhn, Kevin W.; Stone, Brian; Jia, Yue; Cohen, Pinchas

2015-01-01

Humanin is a peptide that is cytoprotective against stresses in many cell types. We investigated whether a potent humanin analogue S14G-humanin (HNG) would protect against chemotherapy-induced damage to normal cells without interfering with the chemotherapy-induced suppression of cancer cells. Young adult male mice were inoculated iv with murine melanoma cells. After 1 week, cancer-bearing mice were randomized to receive either: no treatment, daily ip injection of HNG, a single ip injection of cyclophosphamide (CP), or CP+HNG and killed at the end of 3 weeks. HNG rescued the CP-induced suppression of leucocytes and protected germ cell from CP-induced apoptosis. Lung metastases were suppressed by HNG or CP alone, and further suppressed by CP+HNG treatment. Plasma IGF-1 levels were suppressed by HNG with or without CP treatment. To investigate whether HNG maintains its protective effects on spermatogonial stem cells, sperm output, and peripheral leucocytes after repeated doses of CP, normal adult male mice received: no treatment, daily sc injection of HNG, 6 ip injections of CP at 5-day intervals, and the same regimens of CP+HNG and killed at the end of 4 weeks of treatment. Cauda epididymal sperm counts were elevated by HNG and suppressed by CP. HNG rescued the CP-induced suppression of spermatogonial stem cells, sperm count and peripheral leucocytes. We conclude that HNG 1) protects CP-induced loss of male germ cells and leucocytes, 2) enhances CP-induced suppression of cancer metastases, and 3) acts as a caloric-restriction mimetic by suppressing IGF-1 levels. Our findings suggest that humanin analogues may be promising adjuvants to chemotherapy. PMID:26384090

PDGFRα + pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo.

PubMed

Greicius, Gediminas; Kabiri, Zahra; Sigmundsson, Kristmundur; Liang, Chao; Bunte, Ralph; Singh, Manvendra K; Virshup, David M

2018-04-03

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5 -expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha ( PdgfRα ) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα + cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα + cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα . Copyright © 2018 the Author(s). Published by PNAS.

PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo

PubMed Central

Greicius, Gediminas; Kabiri, Zahra; Sigmundsson, Kristmundur; Liang, Chao; Bunte, Ralph; Singh, Manvendra K.

2018-01-01

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα. PMID:29559533

Characteristics and Outcome of Patients After Allogeneic Hematopoietic Stem Cell Transplantation Treated With Extracorporeal Membrane Oxygenation for Acute Respiratory Distress Syndrome.

PubMed

Wohlfarth, Philipp; Beutel, Gernot; Lebiedz, Pia; Stemmler, Hans-Joachim; Staudinger, Thomas; Schmidt, Matthieu; Kochanek, Matthias; Liebregts, Tobias; Taccone, Fabio Silvio; Azoulay, Elie; Demoule, Alexandre; Kluge, Stefan; Svalebjørg, Morten; Lueck, Catherina; Tischer, Johanna; Combes, Alain; Böll, Boris; Rabitsch, Werner; Schellongowski, Peter

2017-05-01

The acute respiratory distress syndrome is a frequent condition following allogeneic hematopoietic stem cell transplantation. Extracorporeal membrane oxygenation may serve as rescue therapy in refractory acute respiratory distress syndrome but has not been assessed in allogeneic hematopoietic stem cell transplantation recipients. Multicenter, retrospective, observational study. ICUs in 12 European tertiary care centers (Austria, Germany, France, and Belgium). All allogeneic hematopoietic stem cell transplantation recipients treated with venovenous extracorporeal membrane oxygenation for acute respiratory distress syndrome between 2010 and 2015. None. Thirty-seven patients, nine of whom underwent noninvasive ventilation at the time of extracorporeal membrane oxygenation initiation, were analyzed. ICU admission occurred at a median of 146 (interquartile range, 27-321) days after allogeneic hematopoietic stem cell transplantation. The main reason for acute respiratory distress syndrome was pneumonia in 81% of patients. All but one patient undergoing noninvasive ventilation at extracorporeal membrane oxygenation initiation had to be intubated thereafter. Overall, seven patients (19%) survived to hospital discharge and were alive and in remission of their hematologic disease after a follow-up of 18 (range, 5-30) months. Only one of 24 patients (4%) initiated on extracorporeal membrane oxygenation within 240 days after allogeneic hematopoietic stem cell transplantation survived compared to six of 13 (46%) of those treated thereafter (p < 0.01). Fourteen patients (38%) experienced bleeding events, of which six (16%) were associated with fatal outcomes. Discouraging survival rates in patients treated early after allogeneic hematopoietic stem cell transplantation do not support the use of extracorporeal membrane oxygenation for acute respiratory distress syndrome in this group. On the contrary, long-term allogeneic hematopoietic stem cell transplantation recipients otherwise eligible for full-code ICU management may be potential candidates for extracorporeal membrane oxygenation therapy in case of severe acute respiratory distress syndrome failing conventional measures.

The Coumarin Derivative Osthole Stimulates Adult Neural Stem Cells, Promotes Neurogenesis in the Hippocampus, and Ameliorates Cognitive Impairment in APP/PS1 Transgenic Mice.

PubMed

Kong, Liang; Hu, Yu; Yao, Yingjia; Jiao, Yanan; Li, Shaoheng; Yang, Jingxian

2015-01-01

It is believed that neuronal death caused by abnormal deposition of amyloid-beta peptide is the major cause of the cognitive decline in Alzheimer's disease. Adult neurogenesis plays a key role in the rescue of impaired neurons and amelioration of cognitive impairment. In the present study, we demonstrated that osthole, a natural coumarin derivative, was capable of promoting neuronal stem cell (NSC) survival and inducing NSC proliferation in vitro. In osthole-treated APP/PS1 transgenic mice, a significant improvement in learning and memory function was seen, which was associated with a significant increase in the number of new neurons (Ki67(+)/NF-M(+)) and a decrease in apoptotic cells in the hippocampal region of the brain. These observations suggested that osthole promoted NSC proliferation, supported neurogenesis, and thus efficiently rescued impaired neurons in the hippocampus and ameliorated cognitive impairment. We also found that osthole treatment activated the Notch pathway and upregulated the expression of self-renewal genes Notch 1 and Hes 1 mRNA in NSCs. However, when Notch activity was blocked by the γ-secretase inhibitor DAPT, the augmentation of Notch 1 and Hes 1 protein was ameliorated, and the proliferation-inducing effect of osthole was abolished, suggesting that the effects of osthole are at least in part mediated by activation of the Notch pathway.

Naringin rescued the TNF-α-induced inhibition of osteogenesis of bone marrow-derived mesenchymal stem cells by depressing the activation of NF-кB signaling pathway.

PubMed

Cao, Xvhai; Lin, Weilong; Liang, Chengwei; Zhang, Dong; Yang, Fengjian; Zhang, Yan; Zhang, Xuelin; Feng, Jianyong; Chen, Cong

2015-07-01

Naringin exhibits antiinflammatory activity and is shown to induce bone formation. Yet the impact of naringin on inflammation-affected bone marrow-derived mesenchymal stem cell (BM-MSC), a promising tool for the regenerative treatment of bone injury, remained to be investigated. We first cultured and characterized the BM-MSCs in vitro and observe the effects of treatments of TNF-α, naringin, or the combination of both on osteogenic differentiation. TNF-α administered at the concentration of 20 ng/ml results in significant reductions in MSC's cell survival, alkaline phosphatase activity and expressions of two osteogenic genes, Runx2 and Osx. Simultaneous treatment of both TNF-α and naringin is able to rescue such reductions. Further mechanistic studies indicate that TNF-α treatment activates the NF-кB signaling pathway, evidenced by elevated p-IкBα level as well as the increased nuclear fraction of NF-кB subunit, p65. Finally, treatment with both TNF-α and naringin decreases expressions of p-IкBα and nuclear p65, and thus represses NF-кB pathway activated by sole TNF-α treatment. Our findings provide a molecular basis by which naringin restores the TNF-α-induced damage in MSCs and provide novel insights into the application of naringin in the MSC-based treatments for inflammation-induced bone injury.

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

PubMed Central

Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

2014-01-01

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

Reduced Erg Dosage Impairs Survival of Hematopoietic Stem and Progenitor Cells.

PubMed

Xie, Ying; Koch, Mia Lee; Zhang, Xin; Hamblen, Melanie J; Godinho, Frank J; Fujiwara, Yuko; Xie, Huafeng; Klusmann, Jan-Henning; Orkin, Stuart H; Li, Zhe

2017-07-01

ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Erg kd ) in which Erg expression can be conditionally restored by Cre recombinase. Erg kd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Erg kd/+ mice, ∼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin - Sca-1 + c-Kit + (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Erg kd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785. © 2017 AlphaMed Press.

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells

PubMed Central

Lafaille, Fabien G; Pessach, Itai M.; Zhang, Shen-Ying; Ciancanelli, Michael J.; Herman, Melina; Abhyankar, Avinash; Ying, Shui-Wang; Keros, Sotirios; Goldstein, Peter A.; Mostoslavsky, Gustavo; Ordovas-Montanes, Jose; Jouanguy, Emmanuelle; Plancoulaine, Sabine; Tu, Edmund; Elkabetz, Yechiel; Al-Muhsen, Saleh; Tardieu, Marc; Schlaeger, Thorsten M.; Daley, George Q.; Abel, Laurent; Casanova, Jean-Laurent; Studer, Lorenz; Notarangelo, Luigi D.

2012-01-01

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of TLR3 immunity are prone to HSV-1 encephalitis (HSE) 1–3. We tested the hypothesis that the pathogenesis of HSE involves non hematopoietic central nervous system (CNS)-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of IFN-β and/or IFN-γ1 in response to poly(I:C) stimulation was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-β and IFN-γ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele demonstrated that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was further rescued by treatment with exogenous IFN-α/β, but not IFN-γ1.Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3 pathway deficiencies. PMID:23103873

Evidence for organ-specific stem cell microenvironments.

PubMed

Ghinassi, Barbara; Martelli, Fabrizio; Verrucci, Maria; D'Amore, Emanuela; Migliaccio, Giovanni; Vannucchi, Alessandro Maria; Hoffman, Ronald; Migliaccio, Anna Rita

2010-05-01

The X-linked Gata1(low) mutation in mice induces strain-restricted myeloproliferative disorders characterized by extramedullary hematopoiesis in spleen (CD1 and DBA/2) and liver (CD1 only). To assess the role of the microenvironment in establishing this myeloproliferative trait, progenitor cell compartments of spleen and marrow from wild-type and Gata1(low) mice were compared. Phenotype and clonal assay of non-fractionated cells indicated that Gata1(low) mice contain progenitor cell numbers 4-fold lower and 10-fold higher than normal in marrow and spleen, respectively. However, progenitor cells prospectively isolated from spleen, but not from marrow, of Gata1(low) mice expressed colony-forming function in vitro. Therefore, calculation of cloning activity of purified cells demonstrated that the total number of Gata1(low) progenitor cells was 10- to 100-fold lower than normal in marrow and >1,000 times higher than normal in spleen. This observation indicates that Gata1(low) hematopoiesis is favored by the spleen and is in agreement with our previous report that removal of this organ induces wild-type hematopoiesis in heterozygous Gata1(low/+) females (Migliaccio et al., 2009, Blood 114:2107). To clarify if rescue of wild-type hematopoiesis by splenectomy prevented extramedullary hematopoiesis in liver, marrow cytokine expression profile and liver histopathology of splenectomized Gata1(low/+) females were investigated. After splenectomy, the marrow expression levels of TGF-beta, VEGF, osteocalcin, PDGF-alpha, and SDF-1 remained abnormally high while Gata1(low) hematopoiesis was detectable in liver of both CD1 and DBA/2 mutants. Therefore, in the absence of the spleen, Gata1(low) hematopoiesis is supported by the liver suggesting that treatment of myelofibrosis in these animals requires the rescue of both stem cell and microenvironmental functions.

Effects of Epigenetic Modulation on Reporter Gene Expression: Implications for Stem Cell Imaging

PubMed Central

Krishnan, Manickam; Park, Jinha M.; Cao, Feng; Wang, Dongxu; Paulmurugan, Ramasay; Tseng, Jeffrey R.; Gonzalgo, Mark L.; Gambhir, Sanjiv S.; Wu, Joseph C.

2013-01-01

Tracking stem cell localization, survival, differentiation, and proliferation following transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage-1 (843±28) to passage-20 (250±10) to passage-40 (44±3) to passage-60 (3±1 RLU/µg) (P<0.05 vs. passage-1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared to trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator) (P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 weeks compared to 1 week for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation. PMID:16246867

Lysophosphatidic acid rescues bone mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

PubMed

Wang, Xian-Yun; Fan, Xue-Song; Cai, Lin; Liu, Si; Cong, Xiang-Feng; Chen, Xi

2015-03-01

The increase of reactive oxygen species in infracted heart significantly reduces the survival of donor mesenchymal stem cells, thereby attenuating the therapeutic efficacy for myocardial infarction. In our previous study, we demonstrated that lysophosphatidic acid (LPA) protects bone marrow-derived mesenchymal stem cells (BMSCs) against hypoxia and serum deprivation-induced apoptosis. However, whether LPA protects BMSCs from H2O2-induced apoptosis was not examined. In this study, we report that H2O2 induces rat BMSC apoptosis whereas LPA pre-treatment effectively protects BMSCs from H2O2-induced apoptosis. LPA protection of BMSC from the induced apoptosis is mediated mostly through LPA3 receptor. Furthermore, we found that membrane G protein Gi2 and Gi3 are involved in LPA-elicited anti-apoptotic effects through activation of ERK1/2- and PI3 K-pathways. Additionally, H2O2 increases levels of type II of light chain 3B (LC3B II), an autophagy marker, and H2O2-induced autophagy thus protected BMSCs from apoptosis. LPA further increases the expression of LC3B II in the presence of H2O2. In contrast, autophagy flux inhibitor bafilomycin A1 has no effect on LPA's protection of BMSC from H2O2-induced apoptosis. Taken together, our data suggest that LPA rescues H2O2-induced apoptosis mainly by interacting with Gi-coupled LPA3, resulting activation of the ERK1/2- and PI3 K/AKT-pathways and inhibition caspase-3 cleavage, and LPA protection of BMSCs against the apoptosis is independent of it induced autophagy.

JMJD1C Ensures Mouse Embryonic Stem Cell Self-Renewal and Somatic Cell Reprogramming through Controlling MicroRNA Expression.

PubMed

Xiao, Feng; Liao, Bing; Hu, Jing; Li, Shuang; Zhao, Haixin; Sun, Ming; Gu, Junjie; Jin, Ying

2017-09-12

The roles of histone demethylases (HDMs) for the establishment and maintenance of pluripotency are incompletely characterized. Here, we show that JmjC-domain-containing protein 1c (JMJD1C), an H3K9 demethylase, is required for mouse embryonic stem cell (ESC) self-renewal. Depletion of Jmjd1c leads to the activation of ERK/MAPK signaling and epithelial-to-mesenchymal transition (EMT) to induce differentiation of ESCs. Inhibition of ERK/MAPK signaling rescues the differentiation phenotype caused by Jmjd1c depletion. Mechanistically, JMJD1C, with the help of pluripotency factor KLF4, maintains ESC identity at least in part by regulating the expression of the miR-200 family and miR-290/295 cluster to suppress the ERK/MAPK signaling and EMT. Additionally, we uncover that JMJD1C ensures efficient generation and maintenance of induced pluripotent stem cells, at least partially through controlling the expression of microRNAs. Collectively, we propose an integrated model of epigenetic and transcriptional control mediated by the H3K9 demethylase for ESC self-renewal and somatic cell reprogramming. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Klf5 controls bone marrow homing of stem cells and progenitors through Rab5-mediated β1/β2-integrin trafficking

PubMed Central

Taniguchi Ishikawa, E.; Chang, K.H.; Nayak, R.; Olsson, H.A; Ficker, A.; Dunn, S.K.; Madhu, M.; Sengupta, A.; Whitsett, J.A.; Grimes, H.L.; Cancelas, J.A.

2013-01-01

Kruppel-like factor 5 (Klf5) regulates pluripotent stem cell self-renewal but its role in somatic stem cells is unknown. Here we show that Klf5 deficient haematopoietic stem cells and progenitors (HSC/P) fail to engraft after transplantation. This HSC/P defect is associated with impaired bone marrow homing and lodging and decreased retention in bone marrow, and with decreased adhesion to fibronectin and expression of membrane-bound β1/β2-integrins. In vivo inducible gain-of-function of Klf5 in HSCs increases HSC/P adhesion. The expression of Rab5 family members, mediators of β1/β2-integrin recycling in the early endosome, is decreased in Klf5Δ/Δ HSC/Ps. Klf5 binds directly to the promoter of Rab5a/b and overexpression of Rab5b rescues the expression of activated β1/β2-integrins, adhesion and bone marrow homing of Klf5Δ/Δ HSC/Ps. Altogether, these data indicate that Klf5 is indispensable for adhesion, homing, lodging and retention of HSC/Ps in the bone marrow through Rab5-dependent post-translational regulation of β1/β2 integrins. PMID:23552075

YAP1 Regulates OCT4 Activity and SOX2 Expression to Facilitate Self-Renewal and Vascular Mimicry of Stem-Like Cells.

PubMed

Bora-Singhal, Namrata; Nguyen, Jonathan; Schaal, Courtney; Perumal, Deepak; Singh, Sandeep; Coppola, Domenico; Chellappan, Srikumar

2015-06-01

Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that stem-like cells contribute to the genesis and progression of NSCLC. Our results show that the transcriptional coactivator yes-associated protein 1 (YAP1), which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules. Inhibition of YAP1 by a small molecule or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor, Sox2. YAP1 could transcriptionally induce Sox2 through a physical interaction with Oct4; Sox2 induction occurred independent of TEAD2 transcription factor, which is the predominant mediator of YAP1 functions. The binding of Oct4 to YAP1 could be detected in cell lines as well as tumor tissues; the interaction was elevated in NSCLC samples compared to normal tissue as seen by proximity ligation assays. YAP1 bound to Oct4 through the WW domain, and a peptide corresponding to this region could disrupt the interaction. Delivery of the WW domain peptide to stem-like cells disrupted the interaction and abrogated Sox2 expression, self-renewal, and vascular mimicry. Depleting YAP1 reduced the expression of multiple epithelial-mesenchymal transition genes and prevented the growth and metastasis of tumor xenografts in mice; overexpression of Sox2 in YAP1 null cells rescued these functions. These results demonstrate a novel regulation of stem-like functions by YAP1, through the modulation of Sox2 expression. © 2015 AlphaMed Press.

Effect of Placenta-Derived Mesenchymal Stem Cells in a Dementia Rat Model via Microglial Mediation: a Comparison between Stem Cell Transplant Methods.

PubMed

Cho, Jae Sung; Lee, Jihyeon; Jeong, Da Un; Kim, Han Wool; Chang, Won Seok; Moon, Jisook; Chang, Jin Woo

2018-05-01

Loss of cholinergic neurons in the hippocampus is a hallmark of many dementias. Administration of stem cells as a therapeutic intervention for patients is under active investigation, but the optimal stem cell type and transplantation modality has not yet been established. In this study, we studied the therapeutic effects of human placenta-derived mesenchymal stem cells (pMSCs) in dementia rat model using either intracerebroventricular (ICV) or intravenous (IV) injections and analyzed their mechanisms of therapeutic action. Dementia modeling was established by intraventricular injection of 192 IgG-saporin, which causes lesion of cholinergic neurons. Sixty-five male Sprague-Dawley rats were divided into five groups: control, lesion, lesion+ICV injection of pMSCs, lesion+IV injection of pMSCs, and lesion+donepezil. Rats were subjected to the Morris water maze and subsequent immunostaining analyses. Both ICV and IV pMSC administrations allowed significant cognitive recovery compared to the lesioned rats. Acetylcholinesterase activity was significantly rescued in the hippocampus of rats injected with pMSCs post-lesion. Choline acetyltransferase did not co-localize with pMSCs, showing that pMSCs did not directly differentiate into cholinergic cells. Number of microglial cells increased in lesioned rats and significantly decreased back to normal levels with pMSC injection. Our results suggest that ICV and IV injections of pMSCs facilitate the recovery of cholinergic neuronal populations and cognitive behavior. This recovery likely occurs through paracrine effects that resemble microglia function rather than direct differentiation of injected pMSCs into cholinergic neurons. © Copyright: Yonsei University College of Medicine 2018.

Macrophages control vascular stem/progenitor cell plasticity through tumor necrosis factor-α-mediated nuclear factor-κB activation.

PubMed

Wong, Mei Mei; Chen, Yikuan; Margariti, Andriani; Winkler, Bernhard; Campagnolo, Paola; Potter, Claire; Hu, Yanhua; Xu, Qingbo

2014-03-01

Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Local application of IGFBP5 protein enhanced periodontal tissue regeneration via increasing the migration, cell proliferation and osteo/dentinogenic differentiation of mesenchymal stem cells in an inflammatory niche.

PubMed

Han, Nannan; Zhang, Fengqiu; Li, Guoqing; Zhang, Xiuli; Lin, Xiao; Yang, Haoqing; Wang, Lijun; Cao, Yangyang; Du, Juan; Fan, Zhipeng

2017-09-29

Periodontitis is a widespread infectious disease ultimately resulting in tooth loss. The number of mesenchymal stem cells (MSCs) in patients with periodontitis is decreased, and MSC functions are impaired. Rescuing the impaired function of MSCs in periodontitis is the key for treatment, especially in a manner independent of exogenous MSCs. Our previous study found that overexpressed insulin-like growth factor binding protein 5 (IGFBP5) could promote exogenous MSC-mediated periodontal tissue regeneration. Here, we investigate the role of IGFBP5 protein in MSCs and periodontal tissue regeneration independent of exogenous MSCs in an inflammatory niche. TNFα was used to mimic the inflammatory niche. Lentiviral IGFBP5 shRNA was used to silence IGFBP5 and recombinant human IGFBP5 protein (rhIGFBP5) was used to stimulate the periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). The effects of IGFBP5 on PDLSCs were evaluated using the scratch-simulated wound migration, Transwell chemotaxis, alkaline phosphatase (ALP) activity, Alizarin red staining, Cell Counting Kit-8, Western blot, Real-time PCR, Co-IP and ChIP assays. The swine model of periodontitis was used to investigate the functions of IGFBP5 for periodontal regeneration and its anti-inflammation effect. We discovered that 0.5 ng/ml rhIGFBP5 protein enhanced the migration, chemotaxis, osteo/dentinogenic differentiation and cell proliferation of MSCs under the inflammatory condition. Moreover, 0.5 ng/ml rhIGFBP5 application could rescue the impaired functions of IGFBP5-silenced-MSCs in the inflammatory niche. Furthermore, local injection of rhIGFBP5 could promote periodontal tissue regeneration and relieve the local inflammation in a minipig model of periodontitis. Mechanistically, we found that BCOR negatively regulated the expression of IGFBP5 in MSCs. BCOR formed a protein complex with histone demethylase KDM6B and raised histone K27 methylation in the IGFBP5 promoter. This study revealed that rhIGFBP5 could activate the functions of MSCs in an inflammatory niche, provided insight into the mechanism underlying the activated capacities of MSCs, and identified IGFBP5 as a potential cytokine for improving tissue regeneration and periodontitis treatment independent of exogenous MSCs and its potential application in dental clinic.

Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females.

PubMed

Ote, Manabu; Yamamoto, Daisuke

2018-04-27

The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). We isolated the TomO homologs from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomO w Pip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomO w Pip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. We propose that modular reshuffling is the basis for structural and functional diversification of TomO protein members. © 2018 Wiley Periodicals, Inc.

Genomic Instability in Human Pluripotent Stem Cells Arises from Replicative Stress and Chromosome Condensation Defects.

PubMed

Lamm, Noa; Ben-David, Uri; Golan-Lev, Tamar; Storchová, Zuzana; Benvenisty, Nissim; Kerem, Batsheva

2016-02-04

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells. Copyright © 2016 Elsevier Inc. All rights reserved.

The Immunomodulatory and Neuroprotective Effects of Mesenchymal Stem Cells (MSCs) in Experimental Autoimmune Encephalomyelitis (EAE): A Model of Multiple Sclerosis (MS)

PubMed Central

Al Jumah, Mohammed A.; Abumaree, Mohamed H.

2012-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into the mesenchymal lineages of adipocytes, osteocytes and chondrocytes. MSCs can also transdifferentiate and thereby cross lineage barriers, differentiating for example into neurons under certain experimental conditions. MSCs have anti-proliferative, anti-inflammatory and anti-apoptotic effects on neurons. Therefore, MSCs were tested in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), for their effectiveness in modulating the pathogenic process in EAE to develop effective therapies for MS. The data in the literature have shown that MSCs can inhibit the functions of autoreactive T cells in EAE and that this immunomodulation can be neuroprotective. In addition, MSCs can rescue neural cells via a mechanism that is mediated by soluble factors, which provide a suitable environment for neuron regeneration, remyelination and cerebral blood flow improvement. In this review, we discuss the effectiveness of MSCs in modulating the immunopathogenic process and in providing neuroprotection in EAE. PMID:22942767

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

PubMed

Sampaziotis, Fotios; de Brito, Miguel Cardoso; Madrigal, Pedro; Bertero, Alessandro; Saeb-Parsy, Kourosh; Soares, Filipa A C; Schrumpf, Elisabeth; Melum, Espen; Karlsen, Tom H; Bradley, J Andrew; Gelson, William Th; Davies, Susan; Baker, Alastair; Kaser, Arthur; Alexander, Graeme J; Hannan, Nicholas R F; Vallier, Ludovic

2015-08-01

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

Involvement of extracellular factors in maintaining self-renewal of neural stem cell by nestin.

PubMed

Di, Chun Guang; Xiang, Andy Peng; Jia, Lei; Liu, Jun Feng; Lahn, Bruce T; Ma, Bao Feng

2014-07-09

Nestin knockout leads to embryonic lethality and self-renewal deficiency in neural stem cells (NSCs). However, how nestin maintains self-renewal remains uncertain. Here, we used the dosage effect of nestin in heterozygous mice (Nes+/-) to study self-renewal of NSCs. With existing extracellular signaling in vivo or in vitro, nestin levels do not affect proliferation ability or apoptosis when compared between Nes+/- and Nes+/+ NSCs. However, self-renewal ability of Nes+/- NSCs is impaired when plated at a low cell density and completely lost at a clonal density. This deficiency in self-renewal at a clonal density is rescued using a medium conditioned by Nes+/+ NSCs. In addition, the Akt signaling pathway is altered at low density and reversed by conditioned medium. Our data show that secreted factors contribute toward maintaining self-renewal of NSCs by nestin, potentially through Akt signaling.

NCCN Guidelines Insights: Non-Hodgkin's Lymphomas, Version 3.2016.

PubMed

Horwitz, Steven M; Zelenetz, Andrew D; Gordon, Leo I; Wierda, William G; Abramson, Jeremy S; Advani, Ranjana H; Andreadis, C Babis; Bartlett, Nancy; Byrd, John C; Fayad, Luis E; Fisher, Richard I; Glenn, Martha J; Habermann, Thomas M; Lee Harris, Nancy; Hernandez-Ilizaliturri, Francisco; Hoppe, Richard T; Kaminski, Mark S; Kelsey, Christopher R; Kim, Youn H; Krivacic, Susan; LaCasce, Ann S; Lunning, Matthew; Nademanee, Auayporn; Press, Oliver; Rabinovitch, Rachel; Reddy, Nishitha; Reid, Erin; Roberts, Kenneth; Saad, Ayman A; Sokol, Lubomir; Swinnen, Lode J; Vose, Julie M; Yahalom, Joachim; Zafar, Nadeem; Dwyer, Mary; Sundar, Hema; Porcu, Pierluigi

2016-09-01

Peripheral T-cell lymphomas (PTCLs) represent a relatively uncommon heterogeneous group of non-Hodgkin's lymphomas (NHLs) with an aggressive clinical course and poor prognosis. Anthracycline-based multiagent chemotherapy with or without radiation therapy followed by first-line consolidation with high-dose therapy followed by autologous stem cell rescue (HDT/ASCR) is the standard approach to most of the patients with newly diagnosed PTCL. Relapsed or refractory disease is managed with second-line systemic therapy followed by HDT/ASCR or allogeneic stem cell transplant, based on the patient's eligibility for transplant. In recent years, several newer agents have shown significant activity in patients with relapsed or refractory disease across all 4 subtypes of PTCL. These NCCN Guideline Insights highlight the important updates to the NCCN Guidelines for NHL, specific to the management of patients with relapsed or refractory PTCL. Copyright © 2016 by the National Comprehensive Cancer Network.

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

PubMed

Shalom-Feuerstein, Ruby; Serror, Laura; Aberdam, Edith; Müller, Franz-Josef; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing; Aberdam, Daniel; Petit, Isabelle

2013-02-05

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation

PubMed Central

McGill, Trevor J.; Bohana-Kashtan, Osnat; Stoddard, Jonathan W.; Andrews, Michael D.; Pandit, Neelay; Rosenberg-Belmaker, Lior R.; Wiser, Ofer; Matzrafi, Limor; Banin, Eyal; Reubinoff, Benjamin; Netzer, Nir; Irving, Charles

2017-01-01

Purpose Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. Methods Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. Results OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. Conclusions OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. Translational Relevance Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina. PMID:28626601

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme

PubMed Central

Tejada-Romero, Belen; Carter, Jean-Michel; Mihaylova, Yuliana; Neumann, Bjoern; Aboobaker, A. Aziz

2015-01-01

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration. PMID:26062938

Chronic High Dose Alcohol Induces Osteopenia via Activation of mTOR Signaling in Bone Marrow Mesenchymal Stem Cells.

PubMed

Liu, Yao; Kou, Xiaoxing; Chen, Chider; Yu, Wenjing; Su, Yingying; Kim, Yong; Shi, Songtao; Liu, Yi

2016-08-01

Chronic consumption of excessive alcohol results in reduced bone mass, impaired bone structure, and increased risk of bone fracture. However, the mechanisms underlying alcohol-induced osteoporosis are not fully understood. Here, we show that high dose chronic alcohol consumption reduces osteogenic differentiation and enhances adipogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), leading to osteopenia in a mouse model. Mechanistically, impaired osteo/adipogenic lineage differentiation of BMMSCs is due to activation of a phosphatidylinositide 3-kinase/AKT/mammalian target of rapamycin (mTOR) signaling cascade, resulting in downregulation of runt-related transcription factor 2 and upregulation of peroxisome proliferator-activated receptor gamma via activation of p70 ribosomal protein S6 kinase. Blockage of the mTOR pathway by rapamycin treatment ameliorates alcohol-induced osteopenia by rescuing impaired osteo/adipogenic lineage differentiation of BMMSCs. In this study, we identify a previously unknown mechanism by which alcohol impairs BMMSC lineage differentiation and reveal a potential rapamycin-based drug therapy for alcohol-induced osteoporosis. Stem Cells 2016;34:2157-2168. © 2016 AlphaMed Press.

Randomized Trial of Interleukin-2 (IL-2) as Early Consolidation Following Marrow Ablative Therapy with Stem Cell Rescue for Matastatic Breast Cancer

DTIC Science & Technology

2006-10-01

that a single course of IL-2 will result in a significant improvement in disease-free survival, with minimal toxicity. Effectiveness of this approach...may correlate with the effective induction of LAK precursor and effector cells, as well as evidence for reduction in the burden of minimal residual...following completion of the IL-2 infusion. Low dose IL-2 infusion is feasible and safe after AC+T chemotherapy and is showing a promising effect on

Sevoflurane represses the self-renewal ability by regulating miR-7a,7b/Klf4 signalling pathway in mouse embryonic stem cells.

PubMed

Wang, Qimin; Li, Guifeng; Li, Baolin; Chen, Qiu; Lv, Dongdong; Liu, Jiaying; Ma, Jieyu; Sun, Nai; Yang, Longqiu; Fei, Xuejie; Song, Qiong

2016-10-01

Sevoflurane is a frequently-used clinical inhalational anaesthetic and can cause toxicity to embryos during foetal development. Embryonic stem cells (ESCs) are derived from the inner cell mass of blastospheres and can be used as a useful model of early development. Here, we found that sevoflurane significantly influenced self-renewal ability of mESCs on stemness maintenance and cell proliferation. The cell cycle was arrested via G1 phase delay. We further found that sevoflurane upregulated expression of miR-7a,7b to repress self-renewal. Next we performed rescue experiments and found that after adding miR-7a,7b inhibitor into mESCs treated with sevoflurane, its influence on self-renewal could be blocked. Further we identified stemness factor Klf4 as the direct target of miR-7a,7b. Overexpression of Klf4 restored self-renewal ability repressed by miR-7a,7b or sevoflurane. In this work, we determined that sevoflurane repressed self-renewal ability by regulating the miR-7a,7b/Klf4 signalling pathway in mESCs. Our study demonstrated molecular mechanism underlying the side effects of sevoflurane during early development, laying the foundation for studies on safe usage of inhalational anaesthetic during non-obstetric surgery. © 2016 John Wiley & Sons Ltd.

Just-in-time rescue plerixafor in combination with chemotherapy and granulocyte-colony stimulating factor for peripheral blood progenitor cell mobilization.

PubMed

Smith, Veronica R; Popat, Uday; Ciurea, Stefan; Nieto, Yago; Anderlini, Paolo; Rondon, Gabriela; Alousi, Amin; Qazilbash, Muzaffar; Kebriaei, Partow; Khouri, Issa; de Lima, Marcos; Champlin, Richard; Hosing, Chitra

2013-09-01

Plerixafor, a recently approved peripheral blood progenitor cell mobilizing agent, is often added to granulocyte-colony stimulating factor (G-CSF) to mobilize peripheral blood progenitor cells in patients with lymphoma or myeloma who cannot mobilize enough CD34+ cells with G-CSF alone to undergo autologous stem cell transplantation. However, data are lacking regarding the feasibility and efficacy of just-in-time plerixafor in combination with chemotherapy and G-CSF. We reviewed the peripheral blood stem cell collection data of 38 consecutive patients with lymphoma (Hodgkin's and non-Hodgkin's) and multiple myeloma who underwent chemomobilization and high-dose G-CSF and just-in-time plerixafor to evaluate the efficacy of this treatment combination. All patients with multiple myeloma and all but one patient with lymphoma collected the minimum required number of CD34+ cells to proceed with autologous stem cell transplantation (>2 × 10(6) /kg of body weight). The median CD34+ cell dose collected in patients with non-Hodgkin lymphoma was 4.93 × 10(6) /kg of body weight. The median CD34+ cell dose collected for patients with multiple myeloma was 8.81 × 10(6) /kg of body weight. Plerixafor was well tolerated; no grade 2 or higher non-hematologic toxic effects were observed. Copyright © 2013 Wiley Periodicals, Inc.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

PubMed

Gu, Yue; Jones, Amanda E; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M; Townes, Tim M; Klug, Christopher A; Chen, Dongquan; Wang, Hengbin

2016-01-05

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection.

PubMed

De Feo, Donatella; Merlini, Arianna; Laterza, Cecilia; Martino, Gianvito

2012-06-01

Transplantation of neural stem/precursor cells (NPCs) has been proposed as a promising therapeutic strategy in almost all neurological disorders characterized by the failure of central nervous system (CNS) endogenous repair mechanisms in restoring the tissue damage and rescuing the lost function. Nevertheless, recent evidence consistently challenges the limited view that transplantation of these cells is solely aimed at protecting the CNS from inflammatory and neurodegenerative damage through cell replacement. Recent preclinical data confirmed that transplanted NPCs may also exert a 'bystander' neuroprotective effect and identified a series of molecules - for example, immunomodulatory substances, neurotrophic growth factors, stem cell regulators as well as guidance molecules - whose in-situ secretion by NPCs is temporally and spatially orchestrated by environmental needs. A better understanding of the molecular and cellular mechanisms sustaining this 'therapeutic plasticity' is of pivotal importance for defining crucial aspects of the bench-to-beside translation of neural stem cell therapy, that is route and timing of administration as well as the best cellular source. Further insight into those latter issues is eagerly expected from the ongoing phase I/II clinical trials, while, on the other hand, new cellular sources are being developed, mainly by exploiting the new possibilities offered by cellular reprogramming. Nowadays, the research on NPC transplantation in neurological disorders is advancing on two different fronts: on one hand, recent preclinical data are uncovering the molecular basis of NPC therapeutic plasticity, offering a more solid rational framework for the design of clinical studies. On the other hand, pilot trials are highlighting the safety and feasibility issues of neural stem cell transplantation that need to be addressed before efficacy could be properly evaluated.

Disease-in-a-dish: the contribution of patient-specific induced pluripotent stem cell technology to regenerative rehabilitation.

PubMed

Mack, David L; Guan, Xuan; Wagoner, Ashley; Walker, Stephen J; Childers, Martin K

2014-11-01

Advances in regenerative medicine technologies will lead to dramatic changes in how patients in rehabilitation medicine clinics are treated in the upcoming decades. The multidisciplinary field of regenerative medicine is developing new tools for disease modeling and drug discovery based on induced pluripotent stem cells. This approach capitalizes on the idea of personalized medicine by using the patient's own cells to discover new drugs, increasing the likelihood of a favorable outcome. The search for compounds that can correct disease defects in the culture dish is a conceptual departure from how drug screens were done in the past. This system proposes a closed loop from sample collection from the diseased patient, to in vitro disease model, to drug discovery and Food and Drug Administration approval, to delivering that drug back to the same patient. Here, recent progress in patient-specific induced pluripotent stem cell derivation, directed differentiation toward diseased cell types, and how those cells can be used for high-throughput drug screens are reviewed. Given that restoration of normal function is a driving force in rehabilitation medicine, the authors believe that this drug discovery platform focusing on phenotypic rescue will become a key contributor to therapeutic compounds in regenerative rehabilitation.

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

USDA-ARS?s Scientific Manuscript database

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic st...

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

PubMed Central

Rittelmeyer, Ina; Sharma, Amar Deep; Sgodda, Malte; Zaehres, Holm; Bleidißel, Martina; Greber, Boris; Gentile, Luca; Han, Dong Wook; Rudolph, Cornelia; Steinemann, Doris; Schambach, Axel; Ott, Michael; Schöler, Hans R.; Cantz, Tobias

2011-01-01

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. PMID:21765802

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

PubMed Central

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-01-01

ABSTRACT Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance. PMID:27575019

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance.

PubMed

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-11-01

Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production. 1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors

PubMed Central

Doulatov, Sergei; Vo, Linda T.; Macari, Elizabeth R.; Wahlster, Lara; Kinney, Melissa A.; Taylor, Alison M.; Barragan, Jessica; Gupta, Manav; McGrath, Katherine; Lee, Hsiang-Ying; Humphries, Jessica M.; DeVine, Alex; Narla, Anupama; Alter, Blanche P.; Beggs, Alan H.; Agarwal, Suneet; Ebert, Benjamin L.; Gazda, Hanna T.; Lodish, Harvey F.; Sieff, Colin A.; Schlaeger, Thorsten M.; Zon, Leonard I.; Daley, George Q.

2017-01-01

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA. PMID:28179501

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

PubMed

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-03-11

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish

PubMed Central

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-01-01

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis. PMID:25757417

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells.

PubMed

Xu, Zhuojin; Robitaille, Aaron M; Berndt, Jason D; Davidson, Kathryn C; Fischer, Karin A; Mathieu, Julie; Potter, Jennifer C; Ruohola-Baker, Hannele; Moon, Randall T

2016-10-18

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis

PubMed Central

Nakamura, Takahiro; Hamuro, Junji; Takaishi, Mikiro; Simmons, Szandor; Maruyama, Kazuichi; Zaffalon, Andrea; Bentley, Adam J.; Kawasaki, Satoshi; Nagata-Takaoka, Maho; Fullwood, Nigel J.; Itami, Satoshi; Sano, Shigetoshi; Ishii, Masaru; Barrandon, Yann; Kinoshita, Shigeru

2013-01-01

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1–/– mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1–/– mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow–derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis. PMID:24316976

Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium

PubMed Central

2013-01-01

Introduction Early pregnancy has a strong protective effect against breast cancer in humans and rodents, but the underlying mechanism is unknown. Because breast cancers are thought to arise from specific cell subpopulations of mammary epithelia, we studied the effect of parity on the transcriptome and the differentiation/proliferation potential of specific luminal and basal mammary cells in mice. Methods Mammary epithelial cell subpopulations (luminal Sca1-, luminal Sca1+, basal stem/progenitor, and basal myoepithelial cells) were isolated by flow cytometry from parous and age-matched virgin mice and examined by using a combination of unbiased genomics, bioinformatics, in vitro colony formation, and in vivo limiting dilution transplantation assays. Specific findings were further investigated with immunohistochemistry in entire glands of parous and age-matched virgin mice. Results Transcriptome analysis revealed an upregulation of differentiation genes and a marked decrease in the Wnt/Notch signaling ratio in basal stem/progenitor cells of parous mice. Separate bioinformatics analyses showed reduced activity for the canonical Wnt transcription factor LEF1/TCF7 and increased activity for the Wnt repressor TCF3. This finding was specific for basal stem/progenitor cells and was associated with downregulation of potentially carcinogenic pathways and a reduction in the proliferation potential of this cell subpopulation in vitro and in vivo. As a possible mechanism for decreased Wnt signaling in basal stem/progenitor cells, we found a more than threefold reduction in the expression of the secreted Wnt ligand Wnt4 in total mammary cells from parous mice, which corresponded to a similar decrease in the proportion of Wnt4-secreting and estrogen/progesterone receptor-positive cells. Because recombinant Wnt4 rescued the proliferation defect of basal stem/progenitor cells in vitro, reduced Wnt4 secretion appears to be causally related to parity-induced alterations of basal stem/progenitor cell properties in mice. Conclusions By revealing that parity induces differentiation and downregulates the Wnt/Notch signaling ratio and the in vitro and in vivo proliferation potential of basal stem/progenitor cells in mice, our study sheds light on the long-term consequences of an early pregnancy. Furthermore, it opens the door to future studies assessing whether inhibitors of the Wnt pathway may be used to mimic the parity-induced protective effect against breast cancer. PMID:23621987

REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence.

PubMed

Maioli, Margherita; Rinaldi, Salvatore; Pigliaru, Gianfranco; Santaniello, Sara; Basoli, Valentina; Castagna, Alessandro; Fontani, Vania; Ventura, Carlo

2016-06-24

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30(th) passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

PHABULOSA Controls the Quiescent Center-Independent Root Meristem Activities in Arabidopsis thaliana

PubMed Central

Sebastian, Jose; Ryu, Kook Hui; Zhou, Jing; Tarkowská, Danuše; Tarkowski, Petr; Cho, Young-Hee; Yoo, Sang-Dong; Kim, Eun-Sol; Lee, Ji-Young

2015-01-01

Plant growth depends on stem cell niches in meristems. In the root apical meristem, the quiescent center (QC) cells form a niche together with the surrounding stem cells. Stem cells produce daughter cells that are displaced into a transit-amplifying (TA) domain of the root meristem. TA cells divide several times to provide cells for growth. SHORTROOT (SHR) and SCARECROW (SCR) are key regulators of the stem cell niche. Cytokinin controls TA cell activities in a dose-dependent manner. Although the regulatory programs in each compartment of the root meristem have been identified, it is still unclear how they coordinate one another. Here, we investigate how PHABULOSA (PHB), under the posttranscriptional control of SHR and SCR, regulates TA cell activities. The root meristem and growth defects in shr or scr mutants were significantly recovered in the shr phb or scr phb double mutant, respectively. This rescue in root growth occurs in the absence of a QC. Conversely, when the modified PHB, which is highly resistant to microRNA, was expressed throughout the stele of the wild-type root meristem, root growth became very similar to that observed in the shr; however, the identity of the QC was unaffected. Interestingly, a moderate increase in PHB resulted in a root meristem phenotype similar to that observed following the application of high levels of cytokinin. Our protoplast assay and transgenic approach using ARR10 suggest that the depletion of TA cells by high PHB in the stele occurs via the repression of B-ARR activities. This regulatory mechanism seems to help to maintain the cytokinin homeostasis in the meristem. Taken together, our study suggests that PHB can dynamically regulate TA cell activities in a QC-independent manner, and that the SHR-PHB pathway enables a robust root growth system by coordinating the stem cell niche and TA domain. PMID:25730098

p63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells

PubMed Central

Fatt, M P; Cancino, G I; Miller, F D; Kaplan, D R

2014-01-01

The p53 family members p73 and p63 have been implicated in various aspects of stem cell regulation. Here, we have asked whether they work together to regulate stem cell biology, focusing upon neural precursor cells (NPCs) in the adult murine brain. By studying mice that are haploinsufficient for p63 and/or p73, we show that these two proteins cooperate to ensure appropriate NPC self-renewal and long-term maintenance in the hippocampus and forebrain, and that when both are haploinsufficient, the NPC deficits are significantly greater than haploinsufficiency for either alone. We show that, in the case of p63+/− mice, this decrease in adult NPCs is caused by enhanced apoptosis. However, when p73 is coincidently haploinsufficient, this rescues the enhanced apoptosis of p63+/− NPCs under both basal conditions and following genotoxic stress, instead causing increased cellular senescence. This increase in cellular senescence is likely due, at least in part, to increased levels of basal DNA damage and p53 activation, as genetic ablation of p53 completely rescues the senescence phenotype observed in p63+/−; p73+/− mice. Thus, the presence of p73 determines whether p63+/− NPCs exhibit increased p53-dependent apoptosis or senescence. Together, these studies demonstrate that p63 and p73 cooperate to maintain adult NPC pools through regulation of p53 function; p63 antagonizes p53 to promote cellular survival, whereas p73 regulates self-renewal and p53-mediated apoptosis versus senescence. PMID:24809925

Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling

PubMed Central

Lin, Michelle I; Price, Emily N; Boatman, Sonja; Hagedorn, Elliott J; Trompouki, Eirini; Satishchandran, Sruthi; Carspecken, Charles W; Uong, Audrey; DiBiase, Anthony; Yang, Song; Canver, Matthew C; Dahlberg, Ann; Lu, Zhigang; Zhang, Cheng Cheng; Orkin, Stuart H; Bernstein, Irwin D; Aster, Jon C; White, Richard M; Zon, Leonard I

2015-01-01

Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34+ cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets. DOI: http://dx.doi.org/10.7554/eLife.05544.001 PMID:25714926

Niclosamide rescues microcephaly in a humanized in vivo model of Zika infection using human induced neural stem cells

PubMed Central

Boorgu, Devi Sai Sri Kavya; Levin, Michael; Kaplan, David L.

2018-01-01

ABSTRACT Zika virus (ZIKV) is a mosquito-transmitted flavivirus with a causative link to microcephaly, a condition resulting in reduced cranial size and brain abnormalities. Despite recent progress, there is a current lack of in vivo models that permit the study of systemic virus on human neurons in a developing organism that replicates the pathophysiology of human disease. Furthermore, no treatment to date has been reported to reduce ZIKV-induced microcephaly. We tested the effects of ZIKV on human induced neural stem cells (hiNSCs) in vitro and found that infected hiNSCs secrete inflammatory cytokines, display altered differentiation, and become apoptotic. We also utilized this in vitro system to assess the therapeutic effects of niclosamide, an FDA-approved anthelminthic, and found that it decreases ZIKV production, partially restores differentiation, and prevents apoptosis in hiNSCs. We intracranially injected hiNSCs into developing chicks, subjected them to systemic ZIKV infection via the chorioallantoic membrane (CAM), a tissue similar in structure and function to the mammalian placenta, and found that humanized ZIKV-infected embryos developed severe microcephaly including smaller crania, decreased forebrain volume and enlarged ventricles. Lastly, we utilized this humanized model to show that CAM-delivery of niclosamide can partially rescue ZIKV-induced microcephaly and attenuate infection of hiNSCs in vivo. This article has an associated First Person interview with the first author of the paper. PMID:29378701

Two-Step Functional Innovation of the Stem-Cell Factors WUS/WOX5 during Plant Evolution

PubMed Central

Zhang, Yuzhou; Jiao, Yue; Jiao, Hengwu

2017-01-01

WUS and WOX5, which are expressed, respectively, in the organizing center (OC) and the quiescent center (QC), are essential for shoot/root apical stem-cell maintenance in flowering plants. However, little is known about how these stem-cell factors evolved their functions in flowering plants. Here, we show that the WUS/WOX5 proteins acquired two distinct capabilities by a two-step functional innovation process in the course of plant evolution. The first-step is the apical stem-cell maintenance activity of WUS/WOX5, which originated in the common ancestor of ferns and seed plants, as evidenced by the interspecies complementation experiments, showing that ectopic expression of fern Ceratopteris richardii WUS-like (CrWUL) surrounding OC/QC, or exclusive OC-/QC-expressed gymnosperms/angiosperms WUS/WOX5 in Arabidopsis wus-1 and wox5-1 mutants, could rescue their phenotypes. The second-step is the intercellular mobility that emerged in the common ancestor of seed plants after divergence from the ferns. Evidence for this includes confocal imaging of GFP fusion proteins, showing that WUS/WOX5 from seed plants, rather than from the fern CrWUL, can migrate into cells adjacent to the OC/QC. Evolutionary analysis showed that the WUS-like gene was duplicated into two copies prior to the divergence of gymnosperms/angiosperms. Then the two gene copies (WUS and WOX5) have undergone similar levels of purifying selection, which is consistent with their conserved functions in angiosperm shoot/root stem-cell maintenance and floral organ formation. Our results highlight the critical roles and the essential prerequisites that the two-step functional innovation of these genes performs and represents in the origin of flowering plants. PMID:28053005

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21

PubMed Central

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21. PMID:28656059

Therapeutic Effect of Ligustilide-Stimulated Adipose-Derived Stem Cells in a Mouse Thromboembolic Stroke Model.

PubMed

Chi, Kang; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Huang, Pi-Chun; Lin, Po-Cheng; Chang, Fu-Kuei; Liu, Shih-Ping

2016-01-01

Stroke is a result of cerebral ischemia that triggers a cascade of both physiological and biochemical events. No effective treatment is available for stroke; however, stem cells have the potential to rescue tissue from the effects of stroke. Adipose-derived stem cells (ADSCs) are an abundant source of adult stem cells; therefore, ADSC therapy can be considered as a future strategy for regenerative medicine. However, more research is required to improve the effectiveness of transplanted ADSCs as a treatment for stroke in the mouse stroke model. Ligustilide, isolated from the herb Angelica sinensis, exhibits a protective effect on neurons and inhibits inflammation. We also demonstrated that ligustilide treatment increases the expression levels of homing factors such as SDF-1 and CXCR4. In the present study, we evaluated the therapeutic effects of ADSC transplantation and ligustilide treatment in a mouse thromboembolic stroke model by behavioral tests, including beam walking, locomotor activity, and rotarod analysis. ADSCs pretreated with ligustilide were transplanted into the brains of stroke mice. The results showed that the therapeutic effect of ADSCs pretreated with ligustilide was better than that of ADSCs without ligustilide pretreatment. There was no difference between the recovery of mice treated by ADSC transplantation combined with subcutaneous ligustilide injection and that of mice treated only with ADSCs. The TUNEL assay showed fewer apoptotic cells in the brains of mice transplanted with ADSCs pretreated with ligustilide as well as in those without pretreatment. In summary, pretreatment of ADSCs with ligustilide improves the therapeutic efficacy of ADSC transplantation. The results of this study will help improve stem cell therapies being developed for future clinical applications.

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21.

PubMed

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Arsenic inhibits hedgehog signaling during P19 cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Jui Tung; Bain, Lisa J., E-mail: lbain@clemson.edu; Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634

Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mousemore » embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH expression and GLI2 primary cilum accumulation. • Induction of the Shh pathway rescues arsenic's inhibitory effects on cell differentiation.« less

Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions

NASA Astrophysics Data System (ADS)

Lin, Shu-Hai; Liu, Tengfei; Ming, Xiaoyan; Tang, Zhi; Fu, Li; Schmitt-Kopplin, Philippe; Kanawati, Basem; Guan, Xin-Yuan; Cai, Zongwei

2016-02-01

Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

PubMed

Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

2016-11-14

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

PubMed

Stryjewska, Agata; Dries, Ruben; Pieters, Tim; Verstappen, Griet; Conidi, Andrea; Coddens, Kathleen; Francis, Annick; Umans, Lieve; van IJcken, Wilfred F J; Berx, Geert; van Grunsven, Leo A; Grosveld, Frank G; Goossens, Steven; Haigh, Jody J; Huylebroeck, Danny

2017-03-01

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Preparation, characterization, and banking of clinical-grade cells for neural transplantation: Scale up, fingerprinting, and genomic stability of stem cell lines.

PubMed

Natalwala, Ammar; Kunath, Tilo

2017-01-01

Parkinson's disease is a complex and progressive neurodegenerative condition that is characterized by the severe loss of midbrain dopaminergic (mDA) neurons, which innervate the striatum. Cell transplantation therapies to rebuild this dopaminergic network have been attempted for over 30 years. The most promising outcomes were observed when human fetal mesencephalic tissue was used as the source of cells for transplantation. However, reliance on terminations for a Parkinson's therapy presents significant logistical and ethical hurdles. An alternative source of transplantable mDA neurons is urgently needed, and the solution may come from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Protocols to differentiate hESCs/iPSCs toward mDA neurons are now robust and efficient, and upon grafting the cells rescue preclinical animal models of Parkinson's disease. The challenge now is to apply Good Manufacturing Practice (GMP) to the academic discoveries and protocols to produce clinical-grade transplantable mDA cells. Major technical and logistical considerations include (i) source of hESC or iPSC line, (ii) GMP compliance of the differentiation protocol and all reagents, (iii) characterization of the cell product in terms of identity, safety, and efficacy, (iv) characterization of genomic state and stability, and (v) banking of a transplantation-ready cell product. Approaches and solutions to these challenges are reviewed here. © 2017 Elsevier B.V. All rights reserved.

GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

PubMed Central

Gao, Xin; Wu, Tongyu; Johnson, Kirby D.; Lahvic, Jamie L.; Ranheim, Erik A.; Zon, Leonard I.; Bresnick, Emery H.

2016-01-01

Summary Hematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis. PMID:26905203

Platelet-derived growth factor receptors differentially inform intertumoral and intratumoral heterogeneity

PubMed Central

Kim, Youngmi; Kim, Eunhee; Wu, Qiulian; Guryanova, Olga; Hitomi, Masahiro; Lathia, Justin D.; Serwanski, David; Sloan, Andrew E.; Weil, Robert J.; Lee, Jeongwu; Nishiyama, Akiko; Bao, Shideng; Hjelmeland, Anita B.; Rich, Jeremy N.

2012-01-01

Growth factor-mediated proliferation and self-renewal maintain tissue-specific stem cells and are frequently dysregulated in cancers. Platelet-derived growth factor (PDGF) ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors, as proven in genetically engineered models. While PDGFRα alterations inform intertumoral heterogeneity toward a proneural glioblastoma (GBM) subtype, we interrogated the role of PDGFRs in intratumoral GBM heterogeneity. We found that PDGFRα is expressed only in a subset of GBMs, while PDGFRβ is more commonly expressed in tumors but is preferentially expressed by self-renewing tumorigenic GBM stem cells (GSCs). Genetic or pharmacological targeting of PDGFRβ (but not PDGFRα) attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ inhibition decreased activation of the cancer stem cell signaling node STAT3, while constitutively active STAT3 rescued the loss of GSC self-renewal caused by PDGFRβ targeting. In silico survival analysis demonstrated that PDGFRB informed poor prognosis, while PDGFRA was a positive prognostic factor. Our results may explain mixed clinical responses of anti-PDGFR-based approaches and suggest the need for integration of models of cancer as an organ system into development of cancer therapies. PMID:22661233

High-dose melphalan and autologous stem cell transplantation for AL amyloidosis: recent trends in treatment-related mortality and 1-year survival at a single institution

PubMed Central

Seldin, D. C.; Andrea, N.; Berenbaum, I.; Berk, J. L.; Connors, L.; Dember, L. M.; Doros, G.; Fennessey, S.; Finn, K.; Girnius, S.; Lerner, A.; Libbey, C.; Meier-Ewert, H. K.; O’Connell, R.; O’Hara, C.; Quillen, K.; Ruberg, F. L.; Sam, F.; Segal, A.; Shelton, A.; Skinner, M.; Sloan, J. M.; Wiesman, J. F.; Sanchorawala, V.

2017-01-01

Treatment with high-dose melphalan chemotherapy supported by hematopoietic rescue with autologous stem cells produces high rates of hematologic responses and improvement in survival and organ function for patients with AL amyloidosis. Ongoing clinical trials explore pre-transplant induction regimens, post-transplant consolidation or maintenance approaches, and compare transplant to non-transplant regimens. To put these studies into context, we reviewed our recent experience with transplant for AL amyloidosis in the Amyloid Treatment and Research Program at Boston Medical Center and Boston University School of Medicine. Over the past 10 years, there was a steady reduction in rates of treatment-related mortality and improvement in 1-year survival, now approximately 5% and 90%, respectively, based upon an intention-to-treat analysis. Median overall survival of patients treated with this approach at our center exceeds 7.5 years. PMID:21838459

Overexpression of HSPA1A enhances the osteogenic differentiation of bone marrow mesenchymal stem cells via activation of the Wnt/β-catenin signaling pathway

PubMed Central

Zhang, Wei; Xue, Deting; Yin, Houfa; Wang, Shengdong; Li, Chao; Chen, Erman; Hu, Dongcai; Tao, Yiqing; Yu, Jiawei; Zheng, Qiang; Gao, Xiang; Pan, Zhijun

2016-01-01

HSPA1A, which encodes cognate heat shock protein 70, plays important roles in various cellular metabolic pathways. To investigate its effects on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), its expression level was compared between undifferentiated and differentiated BMSCs. Rat HSPA1A overexpression in BMSCs increased osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Moreover, it upregulated β-catenin and downregulated DKK1 and SOST. The enhanced osteogenesis due to HSPA1A overexpression was partly rescued by a Wnt/β-catenin inhibitor. Additionally, using a rat tibial fracture model, a sheet of HSPA1A-overexpressing BMSCs improved bone fracture healing, as determined by imaging and histological analysis. Taken together, these findings suggest that HSPA1A overexpression enhances osteogenic differentiation of BMSCs, partly through Wnt/β-catenin. PMID:27279016

Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging

PubMed Central

Stein, Liana R; Imai, Shin-ichiro

2014-01-01

Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD+ and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD+ biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD+ levels. Nampt is the main source of NSPC NAD+ levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD+ biosynthesis is a mediator of age-associated functional declines in NSPCs. PMID:24811750

Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer

PubMed Central

Luo, Man-Li; Gong, Chang; Chen, Chun-Hau; Lee, Daniel Y.; Hu, Hai; Huang, Pengyu; Yao, Yandan; Guo, Wenjun; Reinhardt, Ferenc; Wulf, Gerburg; Lieberman, Judy; Zhou, Xiao Zhen; Song, Erwei; Lu, Kun Ping

2014-01-01

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness and tumorigenicity, defining it as a key target of miR-200c which is known to be a critical regluator in BSCS. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition (EMT). Conversely, genetic or pharmaacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR-200c-resistant Pin1 rescued the BCSC defect in miR-200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells isolated from clinical samples inhibited the expansion, self-renewal activity and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR-200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC. PMID:24786790

Chitosan derived co-spheroids of neural stem cells and mesenchymal stem cells for neural regeneration.

PubMed

Han, Hao-Wei; Hsu, Shan-Hui

2017-10-01

Chitosan has been considered as candidate biomaterials for neural applications. The effective treatment of neurodegeneration or injury to the central nervous system (CNS) is still in lack nowadays. Adult neural stem cells (NSCs) represents a promising cell source to treat the CNS diseases but they are limited in number. Here, we developed the core-shell spheroids of NSCs (shell) and mesenchymal stem cells (MSCs, core) by co-culturing cells on the chitosan surface. The NSCs in chitosan derived co-spheroids displayed a higher survival rate than those in NSC homo-spheroids. The direct interaction of NSCs with MSCs in the co-spheroids increased the Notch activity and differentiation tendency of NSCs. Meanwhile, the differentiation potential of MSCs in chitosan derived co-spheroids was significantly enhanced toward neural lineages. Furthermore, NSC homo-spheroids and NSC/MSC co-spheroids derived on chitosan were evaluated for their in vivo efficacy by the embryonic and adult zebrafish brain injury models. The locomotion activity of zebrafish receiving chitosan derived NSC homo-spheroids or NSC/MSC co-spheroids was partially rescued in both models. Meanwhile, the higher survival rate was observed in the group of adult zebrafish implanted with chitosan derived NSC/MSC co-spheroids as compared to NSC homo-spheroids. These evidences indicate that chitosan may provide an extracellular matrix-like environment to drive the interaction and the morphological assembly between NSCs and MSCs and promote their neural differentiation capacities, which can be used for neural regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

A Precision Medicine Approach to the Rescue of Function on Malignant Calmodulinopathic Long-QT Syndrome.

PubMed

Limpitikul, Worawan B; Dick, Ivy E; Tester, David J; Boczek, Nicole J; Limphong, Pattraranee; Yang, Wanjun; Choi, Myoung Hyun; Babich, Jennifer; DiSilvestre, Deborah; Kanter, Ronald J; Tomaselli, Gordon F; Ackerman, Michael J; Yue, David T

2017-01-06

Calmodulinopathies comprise a new category of potentially life-threatening genetic arrhythmia syndromes capable of producing severe long-QT syndrome (LQTS) with mutations involving CALM1, CALM2, or CALM3. The underlying basis of this form of LQTS is a disruption of Ca 2+ /calmodulin (CaM)-dependent inactivation of L-type Ca 2+ channels. To gain insight into the mechanistic underpinnings of calmodulinopathies and devise new therapeutic strategies for the treatment of this form of LQTS. We generated and characterized the functional properties of induced pluripotent stem cell-derived cardiomyocytes from a patient with D130G-CALM2-mediated LQTS, thus creating a platform with which to devise and test novel therapeutic strategies. The patient-derived induced pluripotent stem cell-derived cardiomyocytes display (1) significantly prolonged action potentials, (2) disrupted Ca 2+ cycling properties, and (3) diminished Ca 2+ /CaM-dependent inactivation of L-type Ca 2+ channels. Next, taking advantage of the fact that calmodulinopathy patients harbor a mutation in only 1 of 6 redundant CaM-encoding alleles, we devised a strategy using CRISPR interference to selectively suppress the mutant gene while sparing the wild-type counterparts. Indeed, suppression of CALM2 expression produced a functional rescue in induced pluripotent stem cell-derived cardiomyocytes with D130G-CALM2, as shown by the normalization of action potential duration and Ca 2+ /CaM-dependent inactivation after treatment. Moreover, CRISPR interference can be designed to achieve selective knockdown of any of the 3 CALM genes, making it a generalizable therapeutic strategy for any calmodulinopathy. Overall, this therapeutic strategy holds great promise for calmodulinopathy patients as it represents a generalizable intervention capable of specifically altering CaM expression and potentially attenuating LQTS-triggered cardiac events, thus initiating a path toward precision medicine. © 2016 American Heart Association, Inc.

Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

PubMed

Talbot, N C; Caperna, T J; Garrett, W M

2013-01-01

Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.

BAF180 regulates cellular senescence and hematopoietic stem cell homeostasis through p21

PubMed Central

Lee, Hyemin; Dai, Fangyan; Zhuang, Li; Xiao, Zhen-Dong; Kim, Jongchan; Zhang, Yilei; Ma, Li; You, M. James; Wang, Zhong; Gan, Boyi

2016-01-01

BAF180 (also called PBRM1), a subunit of the SWI/SNF complex, plays critical roles in the regulation of chromatin remodeling and gene transcription, and is frequently mutated in several human cancers. However, the role of mammalian BAF180 in tumor suppression and tissue maintenance in vivo remains largely unknown. Here, using a conditional somatic knockout approach, we explored the cellular and organismal functions of BAF180 in mouse. BAF180 deletion in primary mouse embryonic fibroblasts (MEFs) triggers profound cell cycle arrest, premature cellular senescence, without affecting DNA damage response or chromosomal integrity. While somatic deletion of BAF180 in adult mice does not provoke tumor development, BAF180 deficient mice exhibit defects in hematopoietic system characterized by progressive reduction of hematopoietic stem cells (HSCs), defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. BAF180 deletion results in elevated p21 expression in both MEFs and HSCs. Mechanistically, we showed that BAF180 binds to p21 promoter, and BAF180 deletion enhances the binding of modified histones associated with transcriptional activation on p21 promoter. Deletion of p21 rescues cell cycle arrest and premature senescence in BAF180 deficient MEFs, and partially rescues hematopoietic defects in BAF180 deficient mice. Together, our study identifies BAF180 as a critical regulator of cellular senescence and HSC homeostasis, which is at least partially regulated through BAF180-mediated suppression of p21 expression. Our results also suggest that senescence triggered by BAF180 inactivation may serve as a failsafe mechanism to restrain BAF180 deficiency-associated tumor development, providing a conceptual framework to further understand BAF180 function in tumor biology. PMID:26992241

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2

PubMed Central

Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM. PMID:26444992

FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.

PubMed

Lee, Yeri; Kim, Kang Ho; Kim, Dong Geon; Cho, Hee Jin; Kim, Yeonghwan; Rheey, Jinguen; Shin, Kayoung; Seo, Yun Jee; Choi, Yeon-Sook; Lee, Jung-Il; Lee, Jeongwu; Joo, Kyeung Min; Nam, Do-Hyun

2015-01-01

Glioblastoma (GBM) is the most aggressive and most lethal brain tumor. As current standard therapy consisting of surgery and chemo-irradiation provides limited benefit for GBM patients, novel therapeutic options are urgently required. Forkhead box M1 (FoxM1) transcription factor is an oncogenic regulator that promotes the proliferation, survival, and treatment resistance of various human cancers. The roles of FoxM1 in GBM remain incompletely understood, due in part to pleotropic nature of the FoxM1 pathway. Here, we show the roles of FoxM1 in GBM stem cell maintenance and radioresistance. ShRNA-mediated FoxM1 inhibition significantly impeded clonogenic growth and survival of patient-derived primary GBM cells with marked downregulation of Sox2, a master regulator of stem cell phenotype. Ectopic expression of Sox2 partially rescued FoxM1 inhibition-mediated effects. Conversely, FoxM1 overexpression upregulated Sox2 expression and promoted clonogenic growth of GBM cells. These data, with a direct binding of FoxM1 in the Sox2 promoter region in GBM cells, suggest that FoxM1 regulates stemness of primary GBM cells via Sox2. We also found significant increases in FoxM1 and Sox2 expression in GBM cells after irradiation both in vitro and in vivo orthotopic tumor models. Notably, genetic or a small-molecule FoxM1 inhibitor-mediated FoxM1 targeting significantly sensitized GBM cells to irradiation, accompanying with Sox2 downregulation. Finally, FoxM1 inhibition combined with irradiation in a patient GBM-derived orthotopic model significantly impeded tumor growth and prolonged the survival of tumor bearing mice. Taken together, these results indicate that the FoxM1-Sox2 signaling axis promotes clonogenic growth and radiation resistance of GBM, and suggest that FoxM1 targeting combined with irradiation is a potentially effective therapeutic approach for GBM.

Combination of Extracorporeal Life Support and Mesenchymal Stem Cell Therapy for Treatment of ARDS in Combat Casualties and Evacuation of Service Members with ARDS

DTIC Science & Technology

2016-10-01

devastating injuries. Aeromedical evacuation of patients with Acute Respiratory Distress Syndrome (ARDS) is sometimes beyond the possibilities because of...sheep induces lung injury equivalent to a moderated ARDS. In a second group of studies sheep in which respiratory support was providing by a low flow...low pressure ECMO (ALung) partially rescued the animals returned the parameters of respiratory function to normal values. It is our goal to now use

Recent progress and problems in animal cloning.

PubMed

Tsunoda, Y; Kato, Y

2002-01-01

It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.

Second allogeneic stem cell transplant for aplastic anaemia: a retrospective study by the Severe Aplastic Anaemia Working Party of the European Society for Blood and Marrow Transplantation.

PubMed

Cesaro, Simone; Peffault de Latour, Regis; Tridello, Gloria; Pillon, Marta; Carlson, Kristina; Fagioli, Franca; Jouet, Jean-Pierre; Koh, Mickey B C; Panizzolo, Irene Sara; Kyrcz-Krzemien, Slawomira; Maertens, Johan; Rambaldi, Alessandro; Strahm, Brigitte; Blaise, Didier; Maschan, Alexei; Marsh, Judith; Dufour, Carlo

2015-11-01

We analysed the outcome of a second allogeneic haematopoietic stem cell transplant (alloHSCT) in 162 patients reported to the European Society for Blood and Marrow Transplantation between 1998 and 2009. Donor origin was a sibling in 110 and an unrelated donor in 52 transplants, respectively. The stem cell source was bone marrow in 31% and peripheral blood in 69% of transplants. The same donor as for the first alloHSCT was used in 81% of transplants whereas a change in the choice of stem cell source was reported in 56% of patients, mainly from bone marrow to peripheral blood. Neutrophil and platelet engraftment occurred in 85% and 72% of patients, after a median time of 15 and 17 days, respectively. Grade II-IV acute graft-versus-host disease (GVHD) and chronic GVHD occurred in 21% and 37% of patients, respectively. Graft failure (GF) occurred in 42 patients (26%). After a median follow-up of 3·5 years, the 5-year overall survival (OS) was 60·7%. In multivariate analysis, the only factor significantly associated with a better outcome was a Karnofsky/Lansky score ≥80 (higher OS). We conclude that a second alloHSCT is feasible rescue option for GF in SAA, with a successful outcome in 60% of cases. © 2015 John Wiley & Sons Ltd.

RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells

PubMed Central

Ciaudo, Constance; Jay, Florence; Okamoto, Ikuhiro; Chen, Chong-Jian; Sarazin, Alexis; Servant, Nicolas; Barillot, Emmanuel; Heard, Edith; Voinnet, Olivier

2013-01-01

In most mouse tissues, long-interspersed elements-1 (L1s) are silenced via methylation of their 5′-untranslated regions (5′-UTR). A gradual loss-of-methylation in pre-implantation embryos coincides with L1 retrotransposition in blastocysts, generating potentially harmful mutations. Here, we show that Dicer- and Ago2-dependent RNAi restricts L1 accumulation and retrotransposition in undifferentiated mouse embryonic stem cells (mESCs), derived from blastocysts. RNAi correlates with production of Dicer-dependent 22-nt small RNAs mapping to overlapping sense/antisense transcripts produced from the L1 5′-UTR. However, RNA-surveillance pathways simultaneously degrade these transcripts and, consequently, confound the anti-L1 RNAi response. In Dicer−/− mESC complementation experiments involving ectopic Dicer expression, L1 silencing was rescued in cells in which microRNAs remained strongly depleted. Furthermore, these cells proliferated and differentiated normally, unlike their non-complemented counterparts. These results shed new light on L1 biology, uncover defensive, in addition to regulatory roles for RNAi, and raise questions on the differentiation defects of Dicer−/− mESCs. PMID:24244175

aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1.

PubMed

Chibly, Alejandro M; Wong, Wen Yu; Pier, Maricela; Cheng, Hongqiang; Mu, Yongxin; Chen, Ju; Ghosh, Sourav; Limesand, Kirsten H

2018-04-20

Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.

Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakano, T.; Waki, N.; Asai, H.

The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developedmore » the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5-fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3) were injected into 200-rad-irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.« less

Molecular and functional interactions between AKT and SOX2 in breast carcinoma

PubMed Central

Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

2015-01-01

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration.

PubMed

Deng, Zhili; Lei, Xiaohua; Zhang, Xudong; Zhang, Huishan; Liu, Shuang; Chen, Qi; Hu, Huimin; Wang, Xinyue; Ning, Lina; Cao, Yujing; Zhao, Tongbiao; Zhou, Jiaxi; Chen, Ting; Duan, Enkui

2015-02-01

Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration.

PubMed

Bellio, Michael A; Rodrigues, Claudia O; Landin, Ana Marie; Hatzistergos, Konstantinos E; Kuznetsov, Jeffim; Florea, Victoria; Valasaki, Krystalenia; Khan, Aisha; Hare, Joshua M; Schulman, Ivonne Hernandez

2016-12-01

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O 2 ). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O 2 concentrations. Physiological O 2 levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of β-galactosidase activity (-4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O 2 reduces senescence and promotes quiescence. Furthermore, physiological O 2 levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O 2 migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O 2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O 2 concentration on CSC biology and has important implications for refining stem cell therapies. Copyright © 2016 the American Physiological Society.

GLABROUS INFLORESCENCE STEMS regulates trichome branching by genetically interacting with SIM in Arabidopsis.

PubMed

Sun, Li-Li; Zhou, Zhong-Jing; An, Li-Jun; An, Yan; Zhao, Yong-Qin; Meng, Xiao-Fang; Steele-King, Clare; Gan, Yin-Bo

2013-07-01

Arabidopsis trichomes are large branched single cells that protrude from the epidermis. The first morphological indication of trichome development is an increase in nuclear content resulting from an initial cycle of endoreduplication. Our previous study has shown that the C2H2 zinc finger protein GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome initiation in the inflorescence organ and for trichome branching in response to gibberellic acid signaling, although GIS gene does not play a direct role in regulating trichome cell division. Here, we describe a novel role of GIS, controlling trichome cell division indirectly by interacting genetically with a key endoreduplication regulator SIAMESE (SIM). Our molecular and genetic studies have shown that GIS might indireclty control cell division and trichome branching by acting downstream of SIM. A loss of function mutation of SIM signficantly reduced the expression of GIS. Futhermore, the overexpression of GIS rescued the trichome cluster cell phenotypes of sim mutant. The gain or loss of function of GIS had no significant effect on the expression of SIM. These results suggest that GIS may play an indirect role in regulating trichome cell division by genetically interacting with SIM.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

PubMed

Rhee, Yong-Hee; Ko, Ji-Yun; Chang, Mi-Yoon; Yi, Sang-Hoon; Kim, Dohoon; Kim, Chun-Hyung; Shim, Jae-Won; Jo, A-Young; Kim, Byung-Woo; Lee, Hyunsu; Lee, Suk-Ho; Suh, Wonhee; Park, Chang-Hwan; Koh, Hyun-Chul; Lee, Yong-Sung; Lanza, Robert; Kim, Kwang-Soo; Lee, Sang-Hun

2011-06-01

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

[Construction and analysis of a forward and reverse subtractive cDNA library from leaves and stem of Polygonum sibiricum Laxm. under salt stress].

PubMed

Liu, Guan-Jun; Liu, Ming-Kun; Xu, Zhi-Ru; Yan, Xiu-Feng; Wei, Zhi-Gang; Yang, Chuan-Ping

2009-04-01

Using cDNAs prepared from the leaves and stems of Polygonum sibiricum Laxm. treated with NaHCO3 stress for 48 h as testers and cDNAs from unstressed P. sibiricum leaves and stems as drivers library, suppression subtractive hybridization (SSH) was employed to construct a cDNA subtracted library, which contained 2 282 valid sequences including 598 ESTs in the stems forward SSH library and 490 ESTs in the stem reverse SSH library, 627 ESTs in the leaf forward SSH library and 567 in the leaf reverse SSH library. According to the functional catalogue of MIPs and the comparison of the reverse and forward SSH libraries of the stem and leaf, the responses to NaHCO3 stress were different between leaf and stem, except for the same trend in cell rescue defense and transport facilitation. The trend in the metabolism, energy, photosynthesis, protein synthesis, transcription, and signal transduction was opposite. RT-PCR analysis demonstrated that the expression of 12 putative stress related genes in the NaHCO3-treated leaves and stems was different from that in the untreated leaves and stems. This indicated that different mechanisms might be responsible for reactions of leaf and stem in P. sibiricum. The results from this study are useful in understanding the molecular mechanism of saline-alkali tolerance in P. sibiricum.

Mesenchymal stem cells protective effect in adriamycin model of nephropathy.

PubMed

Magnasco, Alberto; Corselli, Mirko; Bertelli, Roberta; Ibatici, Adalberto; Peresi, Monica; Gaggero, Gabriele; Cappiello, Valentina; Chiavarina, Barbara; Mattioli, Girolamo; Gusmano, Rosanna; Ravetti, Jean Louis; Frassoni, Francesco; Ghiggeri, Gian Marco

2008-01-01

Mesenchymal stem cells (MSCs) may be of value in regeneration of renal tissue after damage; however, lack of biological knowledge and variability of results in animal models limit their utilization. We studied the effects of MSCs on podocytes in vitro and in vivo utilizing adriamycin (ADR) as a model of renal toxicity. The in vivo experimental approach was carried out in male Sprague-Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSCs were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSCs. MSCs rescued podocytes from apoptosis induced by ADR in vitro. The maximal effect (80% rescue) was obtained with MSCs/podocytes coculture ratio of 1:1 for 72 h. All rats treated with ADR developed nephrosis. MSCs did not modify the clinical parameters (i.e., proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSCs 60 days after ADR developed the same severe renal damage. Only a few MSCs were found in renal tubule-interstitial areas 1-24 h after injection and no MSCs were detected in glomeruli. MSCs reduced apoptosis of podocytes treated with ADR in vitro. Early and repeated MSCs infusion blunted glomerular damage in chronic ADR-induced nephropathy. MSCs did not modify proteinuria and progression to renal failure, which implies lack of regenerative potential in this model.

Mesenchymal stem cell transplantation for the infarcted heart: a role in minimizing abnormalities in cardiac-specific energy metabolism

PubMed Central

Johnsen, Virginia L.; Ma, Lianli; James, Freyja D.; Young, Pampee P.; Wasserman, David H.; Rottman, Jeffrey N.; Hittel, Dustin S.; Shearer, Jane

2012-01-01

Intense interest has been focused on cell-based therapy for the infarcted heart given that stem cells have exhibited the ability to reduce infarct size and mitigate cardiac dysfunction. Despite this, it is unknown whether mesenchymal stem cell (MSC) therapy can prevent metabolic remodeling following a myocardial infarction (MI). This study examines the ability of MSCs to rescue the infarcted heart from perturbed substrate uptake in vivo. C57BL/6 mice underwent chronic ligation of the left anterior descending coronary artery to induce a MI. Echocardiography was performed on conscious mice at baseline as well as 7 and 23 days post-MI. Twenty-eight days following the ligation procedure, hyperinsulinemic euglycemic clamps assessed in vivo insulin sensitivity. Isotopic tracer administration evaluated whole body, peripheral tissue, and cardiac-specific glucose and fatty acid utilization. To gain insight into the mechanisms by which MSCs modulate metabolism, mitochondrial function was assessed by high-resolution respirometry using permeabilized cardiac fibers. Data show that MSC transplantation preserves insulin-stimulated fatty acid uptake in the peri-infarct region (4.25 ± 0.64 vs. 2.57 ± 0.34 vs. 3.89 ± 0.54 μmol·100 g−1·min−1, SHAM vs. MI + PBS vs. MI + MSC; P < 0.05) and prevents increases in glucose uptake in the remote left ventricle (3.11 ± 0.43 vs. 3.81 ± 0.79 vs. 6.36 ± 1.08 μmol·100 g−1·min−1, SHAM vs. MI + PBS vs. MI + MSC; P < 0.05). This was associated with an enhanced efficiency of mitochondrial oxidative phosphorylation with a respiratory control ratio of 3.36 ± 0.18 in MSC-treated cardiac fibers vs. 2.57 ± 0.14 in the infarct-only fibers (P < 0.05). In conclusion, MSC therapy exhibits the potential to rescue the heart from metabolic aberrations following a MI. Restoration of metabolic flexibility is important given the metabolic demands of the heart and the role of energetics in the progression to heart failure. PMID:21971524

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

PubMed

Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

2016-04-01

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

PubMed Central

Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

2016-01-01

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts.

PubMed

Haramizu, Satoshi; Asano, Shinichi; Butler, David C; Stanton, David A; Hajira, Ameena; Mohamed, Junaith S; Alway, Stephen E

2017-12-01

High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 μM) or very high (1 mM) levels of H 2 O 2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H 2 O 2 . Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H 2 O 2 . Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 μM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H 2 O 2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts. Copyright © 2017 Elsevier Inc. All rights reserved.

ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells

PubMed Central

Valabrega, G; Fagioli, F; Corso, S; Madon, E; Brach del Prever, A; Biasin, E; Linari, A; Aglietta, M; Giordano, S

2003-01-01

Osteosarcoma is the most common malignant bone neoplasia occurring in young patients in the first two decades of life, and represents 20% of all primitive malignant bone tumours. At present, treatment of metastatic osteosarcoma is unsatisfactory. High-dose chemotherapy followed by CD34+ leukapheresis rescue may improve these poor results. Neoplastic cells contaminating the apheresis may, however, contribute to relapse. To identify markers suitable for detecting osteosarcoma cells in aphereses we analysed the expression of bone-specific genes (Bone Sialoprotein (BSP) and Osteocalcin) and oncogenes (Met and ErbB2) in 22 patients with metastatic osteosarcoma and six healthy stem cell donors. The expression of these genes in aphereses of patients affected by metastatic osteosarcoma was assessed by RT–PCR and Southern blot analysis. Met and Osteocalcin proved to be not useful markers since they are positive in aphereses of both patients with metastatic osteosarcoma and healthy stem cell donors. On the contrary, BSP was expressed at significant levels in 85% of patients. Moreover, 18% of patients showed a strong and significantly positive (seven to 16 times higher than healthy stem cell donors) ErbB2 expression. In all positive cases, neoplastic tissue also expressed ErbB2. Our data show that ErbB2 can be a useful marker for tumour contamination in aphereses of patients affected by ErbB2-expressing osteosarcomas and that analysis of Bone Sialoprotein expression can be an alternative useful marker. PMID:12569382

ATM kinase sustains breast cancer stem-like cells by promoting ATG4C expression and autophagy.

PubMed

Antonelli, Martina; Strappazzon, Flavie; Arisi, Ivan; Brandi, Rossella; D'Onofrio, Mara; Sambucci, Manolo; Manic, Gwenola; Vitale, Ilio; Barilà, Daniela; Stagni, Venturina

2017-03-28

The efficacy of Ataxia-Telangiectasia Mutated (ATM) kinase signalling inhibition in cancer therapy is tempered by the identification of new emerging functions of ATM, which suggests that the role of this protein in cancer progression is complex. We recently demonstrated that this tumor suppressor gene could act as tumor promoting factor in HER2 (Human Epidermal Growth Factor Receptor 2) positive breast cancer. Herein we put in evidence that ATM expression sustains the proportion of cells with a stem-like phenotype, measured as the capability to form mammospheres, independently of HER2 expression levels. Transcriptomic analyses revealed that, in mammospheres, ATM modulates the expression of cell cycle-, DNA repair- and autophagy-related genes. Among these, the silencing of the autophagic gene, autophagy related 4C cysteine peptidase (ATG4C), impairs mammosphere formation similarly to ATM depletion. Conversely, ATG4C ectopic expression in cells silenced for ATM expression, rescues mammospheres growth. Finally, tumor array analyses, performed using public data, identify a significant correlation between ATM and ATG4C expression levels in all human breast cancer subtypes, except for the basal-like one.Overall, we uncover a new connection between ATM kinase and autophagy regulation in breast cancer. We demonstrate that, in breast cancer cells, ATM and ATG4C are essential drivers of mammosphere formation, suggesting that their targeting may improve current approaches to eradicate breast cancer cells with a stem-like phenotype.

Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch.

PubMed

López-Arribillaga, Erika; Rodilla, Verónica; Pellegrinet, Luca; Guiu, Jordi; Iglesias, Mar; Roman, Angel Carlos; Gutarra, Susana; González, Susana; Muñoz-Cánoves, Pura; Fernández-Salguero, Pedro; Radtke, Freddy; Bigas, Anna; Espinosa, Lluís

2015-01-01

Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal. © 2015. Published by The Company of Biologists Ltd.

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

PubMed

Fütterer, Agnes; de Celis, Jésus; Navajas, Rosana; Almonacid, Luis; Gutiérrez, Julio; Talavera-Gutiérrez, Amaia; Pacios-Bras, Cristina; Bernascone, Ilenia; Martin-Belmonte, Fernando; Martinéz-A, Carlos

2017-04-11

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Adipose Tissue-Derived Mesenchymal Stem Cells Attenuate Staphylococcal Enterotoxin A-Induced Toxic Shock

PubMed Central

Asano, Krisana; Yoshimura, Sayuri

2015-01-01

Adipose tissue-derived stem cells (ASCs), which are mesenchymal stromal cells isolated from adipose tissues, exhibit immunomodulatory effects that are promising for several applications, including the therapeutics of inflammatory diseases. In the present study, the effect of ASCs on bacterial toxin-induced inflammation was investigated. Intraperitoneal administration of ASCs rescued mice from lethal shock induced by staphylococcal enterotoxin A (SEA) potentiated with lipopolysaccharide. In the sera and/or spleens of mice administered ASCs, the production of proinflammatory cytokines, including interferon gamma, tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-2 was reduced. By quantitative real-time PCR, the expression of Foxp3 in the mice administered ASCs was not altered. On the other hand, the expression of IL-12 receptor and STAT4 was decreased with ASC administration. These results imply that the effect of ASCs is not involved in the lineage of regulatory T cells but that these cells may modulate TH1 differentiation. This information provides evidence that ASCs have properties that are effective to attenuate SEA-induced toxic shock and should prompt further exploration on other inflammatory diseases caused by bacterial toxins or bacterial infections. PMID:26099581

Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown–Vialetto disease that is partially rescued by riboflavin

PubMed Central

Rizzo, Federica; Ramirez, Agnese; Compagnucci, Claudia; Salani, Sabrina; Melzi, Valentina; Bordoni, Andreina; Fortunato, Francesco; Niceforo, Alessia; Bresolin, Nereo; Comi, Giacomo P.; Bertini, Enrico; Nizzardo, Monica; Corti, Stefania

2017-01-01

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies. PMID:28382968

Activation of Postnatal Neural Stem Cells Requires Nuclear Receptor TLX

PubMed Central

Niu, Wenze; Zou, Yuhua; Shen, ChengCheng; Zhang, Chun-Li

2011-01-01

Neural stem cells (NSCs) continually produce new neurons in postnatal brains. However, the majority of these cells stay in a non-dividing, inactive state. The molecular mechanism that is required for these cells to enter proliferation still remains largely unknown. Here, we show that nuclear receptor TLX (NR2E1) controls the activation status of postnatal NSCs in mice. Lineage tracing indicates that TLX-expressing cells give rise to both activated and inactive postnatal NSCs. Surprisingly, loss of TLX function does not result in spontaneous glial differentiation, but rather leads to a precipitous age-dependent increase of inactive cells with marker expression and radial morphology for NSCs. These inactive cells are mis-positioned throughout the granular cell layer of the dentate gyrus during development and can proliferate again after reintroducing ectopic TLX. RNA-seq analysis of sorted NSCs revealed a TLX-dependent global expression signature, which includes the p53 signaling pathway. TLX regulates p21 expression in a p53-dependent manner and acute removal of p53 can rescue the proliferation defect of TLX-null NSCs in culture. Together, these findings suggest that TLX acts as an essential regulator that ensures the proliferative ability of postnatal NSCs by controlling their activation through genetic interaction with p53 and other signaling pathways. PMID:21957244

Activation of postnatal neural stem cells requires nuclear receptor TLX.

PubMed

Niu, Wenze; Zou, Yuhua; Shen, Chengcheng; Zhang, Chun-Li

2011-09-28

Neural stem cells (NSCs) continually produce new neurons in postnatal brains. However, the majority of these cells stay in a nondividing, inactive state. The molecular mechanism that is required for these cells to enter proliferation still remains largely unknown. Here, we show that nuclear receptor TLX (NR2E1) controls the activation status of postnatal NSCs in mice. Lineage tracing indicates that TLX-expressing cells give rise to both activated and inactive postnatal NSCs. Surprisingly, loss of TLX function does not result in spontaneous glial differentiation, but rather leads to a precipitous age-dependent increase of inactive cells with marker expression and radial morphology for NSCs. These inactive cells are mispositioned throughout the granular cell layer of the dentate gyrus during development and can proliferate again after reintroduction of ectopic TLX. RNA-seq analysis of sorted NSCs revealed a TLX-dependent global expression signature, which includes the p53 signaling pathway. TLX regulates p21 expression in a p53-dependent manner, and acute removal of p53 can rescue the proliferation defect of TLX-null NSCs in culture. Together, these findings suggest that TLX acts as an essential regulator that ensures the proliferative ability of postnatal NSCs by controlling their activation through genetic interaction with p53 and other signaling pathways.

The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

PubMed

Du, W; Amarachintha, S; Wilson, A; Pang, Q

2017-02-01

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca -/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca -/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca -/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca -/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

Reticular dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress

PubMed Central

Rissone, Alberto; Weinacht, Katja Gabriele; la Marca, Giancarlo; Bishop, Kevin; Giocaliere, Elisa; Jagadeesh, Jayashree; Felgentreff, Kerstin; Dobbs, Kerry; Al-Herz, Waleed; Jones, Marypat; Chandrasekharappa, Settara; Kirby, Martha; Wincovitch, Stephen; Simon, Karen Lyn; Itan, Yuval; DeVine, Alex; Schlaeger, Thorsten; Schambach, Axel; Sood, Raman

2015-01-01

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD. PMID:26150473

Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging.

PubMed

Hariharan, Nirmala; Quijada, Pearl; Mohsin, Sadia; Joyo, Anya; Samse, Kaitlen; Monsanto, Megan; De La Torre, Andrea; Avitabile, Daniele; Ormachea, Lucia; McGregor, Michael J; Tsai, Emily J; Sussman, Mark A

2015-01-20

Functional decline in stem cell-mediated regeneration contributes to aging associated with cellular senescence in c-kit+ cardiac progenitor cells (CPCs). Clinical implementation of CPC-based therapy in elderly patients would benefit tremendously from understanding molecular characteristics of senescence to antagonize aging. Nucleostemin (NS) is a nucleolar protein regulating stem cell proliferation and pluripotency. This study sought to demonstrate that NS preserves characteristics associated with "stemness" in CPCs and antagonizes myocardial senescence and aging. CPCs isolated from human fetal (fetal human cardiac progenitor cell [FhCPC]) and adult failing (adult human cardiac progenitor cell [AhCPC]) hearts, as well as young (young cardiac progenitor cell [YCPC]) and old mice (old cardiac progenitor cell [OCPC]), were studied for senescence characteristics and NS expression. Heterozygous knockout mice with 1 functional allele of NS (NS+/-) were used to demonstrate that NS preserves myocardial structure and function and slows characteristics of aging. NS expression is decreased in AhCPCs relative to FhCPCs, correlating with lowered proliferation potential and shortened telomere length. AhCPC characteristics resemble those of OCPCs, which have a phenotype induced by NS silencing, resulting in cell flattening, senescence, multinucleated cells, decreased S-phase progression, diminished expression of stemness markers, and up-regulation of p53 and p16. CPC senescence resulting from NS loss is partially p53 dependent and is rescued by concurrent silencing of p53. Mechanistically, NS induction correlates with Pim-1 kinase-mediated stabilization of c-Myc. Engineering OCPCs and AhCPCs to overexpress NS decreases senescent and multinucleated cells, restores morphology, and antagonizes senescence, thereby preserving phenotypic properties of "stemness." Early cardiac aging with a decline in cardiac function, an increase in senescence markers p53 and p16, telomere attrition, and accompanied CPC exhaustion is evident in NS+/- mice. Youthful properties and antagonism of senescence in CPCs and the myocardium are consistent with a role for NS downstream from Pim-1 signaling that enhances cardiac regeneration. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

A New, Dynamic Era for Somatic Cell Nuclear Transfer?

PubMed

Loi, Pasqualino; Iuso, Domenico; Czernik, Marta; Ogura, Atsuo

2016-10-01

Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs). Copyright © 2016 Elsevier Ltd. All rights reserved.

Dedifferentiation rescues senescence of progeria cells but only while pluripotent.

PubMed

Niedernhofer, Laura J; Glorioso, Joseph C; Robbins, Paul D

2011-06-01

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate filament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, affecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fibroblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in differentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence.

Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto.

PubMed

Pan, Hung-Chuan; Yang, Dar-Yu; Ho, Shu-Peng; Sheu, Meei-Ling; Chen, Chung-Jung; Hwang, Shiaw-Min; Chang, Ming-Hong; Cheng, Fu-Chou

2009-08-23

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

eIF4E Threshold Levels Differ in Governing Normal and Neoplastic Expansion of Mammary Stem and Luminal Progenitor cells

PubMed Central

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R.; Beadnell, Thomas C.; Schwertfeger, Kathryn L.; Benyumov, Alexey O.; Manivel, J. Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B.; Polunovsky, Vitaly A.

2015-01-01

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biological outputs remains unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased cell self-renewal, triggered DNA replication stress, and induced formation of pre-malignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biological output in lactating mammary glands, and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its pro-neoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. PMID:25524901

Two-Step Functional Innovation of the Stem-Cell Factors WUS/WOX5 during Plant Evolution.

PubMed

Zhang, Yuzhou; Jiao, Yue; Jiao, Hengwu; Zhao, Huabin; Zhu, Yu-Xian

2017-03-01

WUS and WOX5, which are expressed, respectively, in the organizing center (OC) and the quiescent center (QC), are essential for shoot/root apical stem-cell maintenance in flowering plants. However, little is known about how these stem-cell factors evolved their functions in flowering plants. Here, we show that the WUS/WOX5 proteins acquired two distinct capabilities by a two-step functional innovation process in the course of plant evolution. The first-step is the apical stem-cell maintenance activity of WUS/WOX5, which originated in the common ancestor of ferns and seed plants, as evidenced by the interspecies complementation experiments, showing that ectopic expression of fern Ceratopteris richardii WUS-like (CrWUL) surrounding OC/QC, or exclusive OC-/QC-expressed gymnosperms/angiosperms WUS/WOX5 in Arabidopsis wus-1 and wox5-1 mutants, could rescue their phenotypes. The second-step is the intercellular mobility that emerged in the common ancestor of seed plants after divergence from the ferns. Evidence for this includes confocal imaging of GFP fusion proteins, showing that WUS/WOX5 from seed plants, rather than from the fern CrWUL, can migrate into cells adjacent to the OC/QC. Evolutionary analysis showed that the WUS-like gene was duplicated into two copies prior to the divergence of gymnosperms/angiosperms. Then the two gene copies (WUS and WOX5) have undergone similar levels of purifying selection, which is consistent with their conserved functions in angiosperm shoot/root stem-cell maintenance and floral organ formation. Our results highlight the critical roles and the essential prerequisites that the two-step functional innovation of these genes performs and represents in the origin of flowering plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Impact of diet-induced obesity on intestinal stem cells: hyperproliferation but impaired intrinsic function that requires insulin/IGF1.

PubMed

Mah, Amanda T; Van Landeghem, Laurianne; Gavin, Hannah E; Magness, Scott T; Lund, P Kay

2014-09-01

Nutrient intake regulates intestinal epithelial mass and crypt proliferation. Recent findings in model organisms and rodents indicate nutrient restriction impacts intestinal stem cells (ISC). Little is known about the impact of diet-induced obesity (DIO), a model of excess nutrient intake on ISC. We used a Sox9-EGFP reporter mouse to test the hypothesis that an adaptive response to DIO or associated hyperinsulinemia involves expansion and hyperproliferation of ISC. The Sox9-EGFP reporter mouse allows study and isolation of ISC, progenitors, and differentiated lineages based on different Sox9-EGFP expression levels. Sox9-EGFP mice were fed a high-fat diet for 20 weeks to induce DIO and compared with littermates fed low-fat rodent chow. Histology, fluorescence activated cell sorting, and mRNA analyses measured impact of DIO on jejunal crypt-villus morphometry, numbers, and proliferation of different Sox9-EGFP cell populations and gene expression. An in vitro culture assay directly assessed functional capacity of isolated ISC. DIO mice exhibited significant increases in body weight, plasma glucose, insulin, and insulin-like growth factor 1 (IGF1) levels and intestinal Igf1 mRNA. DIO mice had increased villus height and crypt density but decreased intestinal length and decreased numbers of Paneth and goblet cells. In vivo, DIO resulted in a selective expansion of Sox9-EGFP(Low) ISC and percentage of ISC in S-phase. ISC expansion significantly correlated with plasma insulin levels. In vitro, isolated ISC from DIO mice formed fewer enteroids in standard 3D Matrigel culture compared to controls, indicating impaired ISC function. This decreased enteroid formation in isolated ISC from DIO mice was rescued by exogenous insulin, IGF1, or both. We conclude that DIO induces specific increases in ISC and ISC hyperproliferation in vivo. However, isolated ISC from DIO mice have impaired intrinsic survival and growth in vitro that can be rescued by exogenous insulin or IGF1.

Embryonal tumor with abundant neuropil and true rosettes: a systematic literature review and report of 2 new cases.

PubMed

Alexiou, George A; Stefanaki, Kalliopi; Vartholomatos, George; Sfakianos, George; Prodromou, Neofytos; Moschovi, Maria

2013-12-01

Embryonal tumor with abundant neuropil and true rosettes has been recently defined as a distinct central nervous system embryonal neoplasm, although it was initially regarded as a subtype of central nervous system primitive neuroectodermal tumor. To date 70 cases have been reported. We have performed a literature review and we present 2 new cases. Analysis of the reported data revealed that radiotherapy, tumor excision and high-dose adjuvant chemotherapy with sequential autologous hematopoietic stem cell rescue have a prognostic significance.

The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model

PubMed Central

Murai, Kiyohito; Sun, Guoqiang; Ye, Peng; Tian, E.; Yang, Su; Cui, Qi; Sun, Guihua; Trinh, Daniel; Sun, Olivia; Hong, Teresa; Wen, Zhexing; Kalkum, Markus; Riggs, Arthur D.; Song, Hongjun; Ming, Guo-li; Shi, Yanhong

2016-01-01

Dysregulated expression of miR-219, a brain-specific microRNA, has been observed in neurodevelopmental disorders, such as schizophrenia (SCZ). However, its role in normal mammalian neural stem cells (NSCs) and in SCZ pathogenesis remains unknown. We show here that the nuclear receptor TLX, an essential regulator of NSC proliferation and self-renewal, inhibits miR-219 processing. miR-219 suppresses mouse NSC proliferation downstream of TLX. Moreover, we demonstrate upregulation of miR-219 and downregulation of TLX expression in NSCs derived from SCZ patient iPSCs and DISC1-mutant isogenic iPSCs. SCZ NSCs exhibit reduced cell proliferation. Overexpression of TLX or inhibition of miR-219 action rescues the proliferative defect in SCZ NSCs. Therefore, this study uncovers an important role for TLX and miR-219 in both normal neurodevelopment and in SCZ patient iPSC-derived NSCs. Moreover, this study reveals an unexpected role for TLX in regulating microRNA processing, independent of its well-characterized role in transcriptional regulation. PMID:26965827

The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model.

PubMed

Murai, Kiyohito; Sun, Guoqiang; Ye, Peng; Tian, E; Yang, Su; Cui, Qi; Sun, Guihua; Trinh, Daniel; Sun, Olivia; Hong, Teresa; Wen, Zhexing; Kalkum, Markus; Riggs, Arthur D; Song, Hongjun; Ming, Guo-li; Shi, Yanhong

2016-03-11

Dysregulated expression of miR-219, a brain-specific microRNA, has been observed in neurodevelopmental disorders, such as schizophrenia (SCZ). However, its role in normal mammalian neural stem cells (NSCs) and in SCZ pathogenesis remains unknown. We show here that the nuclear receptor TLX, an essential regulator of NSC proliferation and self-renewal, inhibits miR-219 processing. miR-219 suppresses mouse NSC proliferation downstream of TLX. Moreover, we demonstrate upregulation of miR-219 and downregulation of TLX expression in NSCs derived from SCZ patient iPSCs and DISC1-mutant isogenic iPSCs. SCZ NSCs exhibit reduced cell proliferation. Overexpression of TLX or inhibition of miR-219 action rescues the proliferative defect in SCZ NSCs. Therefore, this study uncovers an important role for TLX and miR-219 in both normal neurodevelopment and in SCZ patient iPSC-derived NSCs. Moreover, this study reveals an unexpected role for TLX in regulating microRNA processing, independent of its well-characterized role in transcriptional regulation.

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation.

PubMed

Thomas, Charles A; Tejwani, Leon; Trujillo, Cleber A; Negraes, Priscilla D; Herai, Roberto H; Mesci, Pinar; Macia, Angela; Crow, Yanick J; Muotri, Alysson R

2017-09-07

Three-prime repair exonuclease 1 (TREX1) is an anti-viral enzyme that cleaves nucleic acids in the cytosol, preventing accumulation and a subsequent type I interferon-associated inflammatory response. Autoimmune diseases, including Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus, can arise when TREX1 function is compromised. AGS is a neuroinflammatory disorder with severe and persistent intellectual and physical problems. Here we generated a human AGS model that recapitulates disease-relevant phenotypes using pluripotent stem cells lacking TREX1. We observed abundant extrachromosomal DNA in TREX1-deficient neural cells, of which endogenous Long Interspersed Element-1 retrotransposons were a major source. TREX1-deficient neurons also exhibited increased apoptosis and formed three-dimensional cortical organoids of reduced size. TREX1-deficient astrocytes further contributed to the observed neurotoxicity through increased type I interferon secretion. In this model, reverse-transcriptase inhibitors rescued the neurotoxicity of AGS neurons and organoids, highlighting their potential utility in therapeutic regimens for AGS and related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain.

PubMed

Zhou, Ting; Tan, Lei; Cederquist, Gustav Y; Fan, Yujie; Hartley, Brigham J; Mukherjee, Suranjit; Tomishima, Mark; Brennand, Kristen J; Zhang, Qisheng; Schwartz, Robert E; Evans, Todd; Studer, Lorenz; Chen, Shuibing

2017-08-03

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells.

PubMed

Xu, Xiaohong; Tay, Yilin; Sim, Bernice; Yoon, Su-In; Huang, Yihui; Ooi, Jolene; Utami, Kagistia Hana; Ziaei, Amin; Ng, Bryan; Radulescu, Carola; Low, Donovan; Ng, Alvin Yu Jin; Loh, Marie; Venkatesh, Byrappa; Ginhoux, Florent; Augustine, George J; Pouladi, Mahmoud A

2017-03-14

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Induced Pluripotent Stem Cell Models of Progranulin-Deficient Frontotemporal Dementia Uncover Specific Reversible Neuronal Defects

PubMed Central

Almeida, Sandra; Zhang, Zhijun; Coppola, Giovanni; Mao, Wenjie; Futai, Kensuke; Karydas, Anna; Geschwind, Michael D.; Tartaglia, M. Carmela; Gao, Fuying; Gianni, Davide; Sena-Esteves, Miguel; Geschwind, Daniel H.; Miller, Bruce L.; Farese, Robert V.; Gao, Fen-Biao

2012-01-01

SUMMARY The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell (iPSC) lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel GRN mutation (PGRN S116X). In neurons and microglia differentiated from PGRN S116X iPSCs, the levels of intracellular and secreted progranulin were reduced, establishing patient-specific cellular models of progranulin haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the PI3K and MAPK pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by progranulin expression. Our findings identify cell-autonomous, reversible defects in patient neurons with progranulin deficiency and provide a new model for studying progranulin-dependent pathogenic mechanisms and testing potential therapies. PMID:23063362

Human adipose-derived stem cells ameliorate repetitive behavior, social deficit and anxiety in a VPA-induced autism mouse model.

PubMed

Ha, Sungji; Park, Hyunjun; Mahmood, Usman; Ra, Jeong Chan; Suh, Yoo-Hun; Chang, Keun-A

2017-01-15

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impairments in social interaction and communication, and patients often display co-occurring repetitive behaviors. Although the global prevalence of ASD has increased over time, the etiology and treatments for ASD are poorly understood. Recently, some researchers have suggested that stem cells have therapeutic potential for ASD. Thus, in the present study, we investigated the therapeutic effects of human adipose-derived stem cells (hASCs), a kind of autologous mesenchymal stem cells (MSCs) isolated from adipose tissue, on valproic acid (VPA)-induced autism model mice. Human ASCs were injected into the neonatal pups (P2 or P3) intraventricularly and then we evaluated major behavior symptoms of ASD. VPA-treated mice showed increased repetitive behaviors, decreased social interactions and increased anxiety but these autistic behaviors were ameliorated through transplantation of hASCs. In addition, hASCs transplantation restored the alteration of phosphatase and tensin homolog (PTEN) expression and p-AKT/AKT ratio in the brains of VPA-induced ASD model mice. The decreased level of vascular endothelial growth factor (VEGF) and interleukin 10 (IL-10) by VPA were rescued in the brains of the hASC-injected VPA mice. With these results, we experimentally found hASCs' therapeutic effects on autistic phenotypes in a ASD model mice for the first time. This animal model system can be used to elucidate further mechanisms of therapeutic effects of hASCs in ASD. Copyright © 2016 Elsevier B.V. All rights reserved.

Resveratrol activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against amyloid-beta-induced inflammation and oxidative stress.

PubMed

Chiang, Ming-Chang; Nicol, Christopher J; Cheng, Yi-Chuan

2018-05-01

Alzheimer's disease (AD) is a neurodegenerative disorder with progressive memory loss resulting in dementia. Amyloid-beta (Aβ) peptides play a critical role in the pathogenesis of this disease, and are thought to promote inflammation and oxidative stress leading to neurodegeneration in the neocortex and hippocampus of the AD brains. AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis, and cell survival in response to inflammation and oxidative stress. However, the neuroprotective mechanisms by which AMPK achieves these beneficial effects in human neural stem cells (hNSCs) exposed to Aβ is still not well understood. Resveratrol is a potent activator of AMPK suggesting it may have therapeutic potential against AD. Therefore, we will test the hypothesis that the AMPK activator resveratrol protects against Aβ mediated neuronal impairment (inflammation and oxidative stress) in hNSCs. Here, Aβ-treated hNSCs had significantly decreased cell viability that correlated with increased TNF-α and IL-1β inflammatory cytokine expression. Co-treatment with resveratrol significantly abrogated the Aβ-mediated effects in hNSCs, and was effectively blocked by the addition of the AMPK-specific antagonist (Compound C). These results suggest the neuroprotective effects of resveratrol are mediated by an AMPK-dependent pathway. In addition, resveratrol rescued the transcript expression levels of inhibitory kappa B kinase (IKK) in Aβ-treated hNSCs. NF-κB is a transcription factor with a key role in the expression of a variety of genes involved in inflammatory responses. Resveratrol prevented the Aβ-mediated increases in NF-κB mRNA and protein levels, and its nuclear translocation in hNSCs. Co-treatment with resveratrol also significantly restored iNOS and COX-2 levels in Aβ-treated hNSCs. Furthermore, hNSCs co-treated with resveratrol were significantly rescued from Aβ-induced oxidative stress, which correlated with reversal of the Aβ-induced mRNA decrease in oxidative defense genes (SOD-1, NRF2, Gpx1, Catalase, GSH and HO-1). Taken together, these novel findings show that activation of AMPK-dependent signaling by resveratrol rescues Aβ-mediated neurotoxicity in hNSCs, and provides evidence supporting a neuroprotective role for AMPK activating drugs in Aβ-related diseases such as AD. Copyright © 2017 Elsevier Ltd. All rights reserved.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

PubMed Central

Cho, Lily Ting-yin; Andrews, Robert; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G.; Fisher, Amanda G.; Skarnes, William C.

2017-01-01

Abstract Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the ‘knockout-first’ allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. PMID:28981838

Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

PubMed

Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

2010-04-29

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway.

PubMed

Wang, Yanping; Yan, Ming; Yu, Yan; Wu, Jintao; Yu, Jinhua; Fan, Zhipeng

2013-06-01

Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells.

PubMed

Kawamura, Norihiko; Nimura, Keisuke; Nagano, Hiromichi; Yamaguchi, Sohei; Nonomura, Norio; Kaneda, Yasufumi

2015-09-08

NANOG expression in prostate cancer is highly correlated with cancer stem cell characteristics and resistance to androgen deprivation. However, it is not clear whether NANOG or its pseudogenes contribute to the malignant potential of cancer. We established NANOG- and NANOGP8-knockout DU145 prostate cancer cell lines using the CRISPR/Cas9 system. Knockouts of NANOG and NANOGP8 significantly attenuated malignant potential, including sphere formation, anchorage-independent growth, migration capability, and drug resistance, compared to parental DU145 cells. NANOG and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG- and NANOGP8-rescued cell lines. These results indicate that NANOG and NANOGP8 proteins are expressed in prostate cancer cell lines, and NANOG and NANOGP8 equally contribute to the high malignant potential of prostate cancer.

eIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

PubMed

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R; Beadnell, Thomas C; Schwertfeger, Kathryn L; Benyumov, Alexey O; Manivel, J Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2015-02-15

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. ©2014 American Association for Cancer Research.

Human Umbilical Cord Matrix Stem Cells Efficiently Rescue Acute Liver Failure Through Paracrine Effects Rather than Hepatic Differentiation

PubMed Central

Chen, Li; Liu, Tao; Zhang, Bo; Xiang, Dedong; Wang, Zhengguo

2012-01-01

There is increasing evidence that mesenchymal stem cells (MSCs) derived from different tissues could act as an alternative source of mature hepatocytes for treatment of acute liver failure (ALF). Human umbilical cord matrix stem cells (hUCMSCs) represent a novel source of MSCs. We examined the therapeutic potential and the different mechanisms of hUCMSCs by their transplantation into nonobese diabetic severe combined-immunodeficient (NOD-SCID) mice with carbon tetrachloride (CCl4)-induced ALF in comparison to adult human hepatocytes (AHHs). The characteristics of isolated hUCMSCs were determined from MSCs and hepatocyte marker expression, hepatic function, and differentiation. Native hUCMSCs constitutively expressed some hepatic markers, though weaker hepatocyte-specific functions were observed when compared to AHHs. When native hUCMSCs or AHHs were transplanted into livers of NOD-SCID mice with ALF induced by CCl4, both hUCMSCs and AHHs provided a significant survival benefit and prevented the release of liver injury biomarkers. hUCMSCs were found to engraft within the recipient liver and differentiated into functional hepatocytes, whereas the HepPar1-/albumin (ALB)-positive cells of the hUCMSC group were less than the AHH group in the recipient liver. Higher values of human ALB in the serum of mice-transplanted AHHs were determined in comparison with levels in mice-transplanted hUCMSCs. The analysis of mouse serum cytokine levels showed that hUCMSC transplantation was even more effective than treatment with AHHs and successfully downregulated the systemic inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, and IL-1 receptor antagonist (IL-1RA). Furthermore, paracrine effects produced by hUCMSCs were identified by indirect coculture with damaged mouse hepatocytes (MHs) induced by CCl4. Coculture with hUCMSCs significantly increased the viability, ALB secretion of damaged MHs, and greatly enhanced the regeneration of MHs in vitro when compared with AHHs. These data suggest that direct transplantation of native hUCMSCs can rescue ALF and repopulate livers of mice through paracrine effects to stimulate endogenous liver regeneration rather than hepatic differentiation for compensated liver function, which is the primary effect of AHHs. Thus, hUCMSCs can be a potential alternative source of AHHs for cell therapy of ALF and eliminate the shortage of hepatocytes. PMID:22519429

A Reinterpretation of Cell Transplantation: GFP Transfer From Donor to Host Photoreceptors.

PubMed

Ortin-Martinez, Arturo; Tsai, En Leh Samuel; Nickerson, Philip E; Bergeret, Miriam; Lu, Yao; Smiley, Sheila; Comanita, Lacrimioara; Wallace, Valerie A

2017-04-01

The utilization of fluorescent reporter transgenes to discriminate donor versus host cells has been a mainstay of photoreceptor transplantation research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of donor photoreceptors. We previously reported that GFP + cells in the ONL of cone-GFP transplanted retinas exhibited rod-like characteristics, raising the possibility that GFP signal in recipient tissue may not be a consequence of donor cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and recipient models, and assessed cell identity using nuclear architecture, immunocytochemistry, and DNA prelabeling. Our results indicate that GFP + cells in the ONL fail to exhibit hallmark elements of donor cells, including nuclear hetero/euchromatin architecture. Furthermore, GFP signal does not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between donor and host photoreceptors. This transfer can be mediated by rods and cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in second-order retinal neurons and Müller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing fluorescent reporters in transplantation studies involving the retina and other CNS tissues. Furthermore, the reinterpretation of previous functional rescue data, based on material exchange, rather than cell integration, may offer a novel approach to vision rescue. Stem Cells 2017;35:932-939. © 2016 AlphaMed Press.

Intensified chemotherapy with stem-cell rescue in germ-cell tumors.

PubMed

Simonelli, M; Rosti, G; Banna, G L; Pedrazzoli, P

2012-04-01

Based on the high chemosensitivity of germ-cell tumors (GCTs), the concept of high-dose chemotherapy (HDCT) has been developed worldwide and investigated through many clinical trials. It has been carried out in different clinical settings, ranging from resistant or absolute refractory disease to chemosensitive relapse. HDCT with stem-cell support has been also explored as a part of first-line strategy for poor-prognosis patients. Our review summarized results from clinical trials evaluating the role of HDCT in patients with advanced GCTs. So far available data were obtained through a Medline search of English-language literature. Several phase II trials and retrospective series have shown a possible benefit for GCT patients with recurrent disease as well as in first-line setting. Despite these results, data derived from randomized phase III studies failed to demonstrate any survival advantage for HDCT over conventional chemotherapy. The role of HDCT in GCTs remains controversial. We need new prospective studies based on prognostic factors with multiple transplants of carboplatin and etoposide as the preferred high dose regimen. At present, based mainly on retrospective and phase II studies, HDCT may represent a therapeutic option for patients with primary refractory disease or for those with a second or further relapse.

Impaired redox environment modulates cardiogenic and ion-channel gene expression in cardiac-resident and non-resident mesenchymal stem cells.

PubMed

Subramani, Baskar; Subbannagounder, Sellamuthu; Ramanathanpullai, Chithra; Palanivel, Sekar; Ramasamy, Rajesh

2017-03-01

Redox homeostasis plays a crucial role in the regulation of self-renewal and differentiation of stem cells. However, the behavioral actions of mesenchymal stem cells in redox imbalance state remain elusive. In the present study, the effect of redox imbalance that was induced by either hydrogen peroxide (H 2 O 2 ) or ascorbic acid on human cardiac-resident (hC-MSCs) and non-resident (umbilical cord) mesenchymal stem cells (hUC-MSCs) was evaluated. Both cells were sensitive and responsive when exposed to either H 2 O 2 or ascorbic acid at a concentration of 400 µmol/L. Ascorbic acid pre-treated cells remarkably ameliorated the reactive oxygen species level when treated with H 2 O 2 . The endogenous antioxidative enzyme gene (Sod1, Sod2, TRXR1 and Gpx1) expressions were escalated in both MSCs in response to reactive oxygen species elevation. In contrast, ascorbic acid pre-treated hUC-MSCs attenuated considerable anti-oxidative gene (TRXR1 and Gpx1) expressions, but not the hC-MSCs. Similarly, the cardiogenic gene (Nkx 2.5, Gata4, Mlc2a and β-MHC) and ion-channel gene ( I KDR , I KCa , I to and I Na.TTX ) expressions were significantly increased in both MSCs on the oxidative state. On the contrary, reduced environment could not alter the ion-channel gene expression and negatively regulated the cardiogenic gene expressions except for troponin-1 in both cells. In conclusion, redox imbalance potently alters the cardiac-resident and non-resident MSCs stemness, cardiogenic, and ion-channel gene expressions. In comparison with cardiac-resident MSC, non-resident umbilical cord-MSC has great potential to tolerate the redox imbalance and positively respond to cardiac regeneration. Impact statement Human mesenchymal stem cells (h-MSCs) are highly promising candidates for tissue repair in cardiovascular diseases. However, the retention of cells in the infarcted area has been a major challenge due to its poor viability and/or low survival rate after transplantation. The regenerative potential of mesenchymal stem cells (MSCs) repudiate and enter into premature senescence via oxidative stress. Thus, various strategies have been attempted to improve the MSC survival in 'toxic' conditions. Similarly, we investigated the response of cardiac resident MSC (hC-MSCs) and non-resident MSCs against the oxidative stress induced by H 2 O 2 . Supplementation of ascorbic acid (AA) into MSCs culture profoundly rescued the stem cells from oxidative stress induced by H 2 O 2 . Our data showed that the pre-treatment of AA is able to inhibit the cell death and thus preserving the viability and differentiation potential of MSCs.

3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair.

PubMed

Hsieh, Fu-Yu; Lin, Hsin-Hua; Hsu, Shan-Hui

2015-12-01

The 3D bioprinting technology serves as a powerful tool for building tissue in the field of tissue engineering. Traditional 3D printing methods involve the use of heat, toxic organic solvents, or toxic photoinitiators for fabrication of synthetic scaffolds. In this study, two thermoresponsive water-based biodegradable polyurethane dispersions (PU1 and PU2) were synthesized which may form gel near 37 °C without any crosslinker. The stiffness of the hydrogel could be easily fine-tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation. The dispersions containing NSCs were subsequently printed and maintained at 37 °C. The NSCs in 25-30% PU2 hydrogels (∼680-2400 Pa) had excellent proliferation and differentiation but not in 25-30% PU1 hydrogels. Moreover, NSC-laden 25-30% PU2 hydrogels injected into the zebrafish embryo neural injury model could rescue the function of impaired nervous system. However, NSC-laden 25-30% PU1 hydrogels only showed a minor repair effect in the zebrafish model. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden 25% PU2 constructs. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermoresponsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of single and combined low frequency electromagnetic fields and simulated microgravity on gene expression of human mesenchymal stem cells during chondrogenesis

PubMed Central

Hammerschmid, Florian; Blum, Helmut; Krebs, Stefan; Redeker, Julia I.; Holzapfel, Boris M.; Jansson, Volkmar; Müller, Peter E.

2016-01-01

Introduction Low frequency electromagnetic fields (LF-EMF) and simulated microgravity (SMG) have been observed to affect chondrogenesis. A controlled bioreactor system was developed to apply LF-EMF and SMG singly or combined during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in 3D culture. Material and methods An external motor gear SMG bioreactor was combined with magnetic Helmholtz coils for EMF (5 mT; 15 Hz). Pellets of hMSCs (±TGF-β3) were cultured (P5) under SMG, LF-EMF, LF-EMF/SMG and control (1 g) conditions for 3 weeks. Sections were stained with safranin-O and collagen type II. Gene expression was evaluated by microarray and real-time polymerase chain reaction analysis. Results Simulated microgravity application significantly changed gene expression; specifically, COLXA1 but also COL2A1, which represents the chondrogenic potential, were reduced (p < 0.05). Low frequency electromagnetic fields application showed no gene expression changes on a microarray basis. LF-EMF/SMG application obtained significant different expression values from cultures obtained under SMG conditions with a re-increase of COL2A1, therefore rescuing the chondrogenic potential, which had been lowered by SMG. Conclusions Simulated microgravity lowered hypertrophy but also the chondrogenic potential of hMSCs. Combined LF-EMF/SMG provided a rescue effect of the chondrogenic potential of hMSCs although no LF-EMF effect was observed under optimal conditions. The study provides new insights into how LF-EMF and SMG affect chondrogenesis of hMSCs and how they generate interdependent effects. PMID:29765449

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

PubMed

Mosqueira, Diogo; Mannhardt, Ingra; Bhagwan, Jamie R; Lis-Slimak, Katarzyna; Katili, Puspita; Scott, Elizabeth; Hassan, Mustafa; Prondzynski, Maksymilian; Harmer, Stephen C; Tinker, Andrew; Smith, James G W; Carrier, Lucie; Williams, Philip M; Gaffney, Daniel; Eschenhagen, Thomas; Hansen, Arne; Denning, Chris

2018-05-08

Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Amniotic fluid stem cells rescue both in vitro and in vivo growth, innervation, and motility in nitrofen-exposed hypoplastic rat lungs through paracrine effects.

PubMed

Pederiva, F; Ghionzoli, M; Pierro, A; De Coppi, P; Tovar, J A

2013-01-01

Lung hypoplasia can be prevented in vitro by retinoic acid (RA). Recent evidence suggests that amniotic fluid stem (AFS) cells may integrate injured lungs and influence their recovery. We tested the hypothesis that AFS cells might improve lung growth and motility by paracrine mechanisms. Pregnant rats received either nitrofen or vehicle on E9.5. In vitro E13 embryonic lungs were cultured in the presence of culture medium alone or with RA, basophils, or AFS cells. In vivo green fluorescent protein-expressing (GFP(+)) rat AFS cells were transplanted in nitrofen-exposed rats on E10.5. E13 lung explants were cultured before analysis. The surface, the number of terminal buds, and the frequency of bronchial contractions were assessed. Protein gene product 9.5 (PGP 9.5) and α-actin protein levels were measured. The lung explants transplanted with AFS cells were stained for α-actin, PGP 9.5, and TTF-1. The levels of FGF-10, VEGFα, and TGF-β1 secreted by the AFS cells in the culture medium were measured. Comparison between groups was made by ANOVA. In vitro, the surface, the number of terminal buds, and the bronchial peristalsis were increased in nitrofen+AFS cell explants in comparison with nitrofen-exposed lungs. While nitrofen+RA lungs were similar to nitrofen+AFS ones, basophils did not normalize these measurements. PGP 9.5 protein was decreased in nitrofen lungs, but after adding AFS cells, the value was similar to controls. No differences were found in the expression of α-actin. In vivo, the surface, number of terminal buds, and peristalsis were similar to control after injection of AFS cells in nitrofen-exposed rats. Colocalization with TTF-1-positive cells was found. The levels of FGF-10 and VEGFα were increased in nitrofen+AFS cell explants, while the levels of TGF-β1 were similar to controls. Lung growth, bronchial motility, and innervation were decreased in nitrofen explants and rescued by AFS cells both in vitro and in vivo, similarly to that observed before with RA. The AFS cell beneficial effect was probably related to paracrine action of growth factor secretion.

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells

PubMed Central

Stryjewska, Agata; Dries, Ruben; Pieters, Tim; Verstappen, Griet; Conidi, Andrea; Coddens, Kathleen; Francis, Annick; Umans, Lieve; van IJcken, Wilfred F. J.; Berx, Geert; van Grunsven, Leo A.; Grosveld, Frank G.; Goossens, Steven; Haigh, Jody J.

2016-01-01

Abstract In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell‐fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA‐sequencing (RNA‐seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast‐like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial‐to‐mesenchymal transition (EMT), and DNA‐(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1‐binding sites correlate with loss‐of‐methylation in neural‐stimulating conditions, however, after the cells initially acquired the correct DNA‐methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re‐adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA‐methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611–625 PMID:27739137

Nucleostemin Rejuvenates Cardiac Progenitor Cells and Antagonizes Myocardial Aging

PubMed Central

Hariharan, Nirmala; Quijada, Pearl; Mohsin, Sadia; Joyo, Anya; Samse, Kaitlen; Monsanto, Megan; De La Torre, Andrea; Avitabile, Daniele; Ormachea, Lucia; McGregor, Michael J.; Tsai, Emily J; Sussman, Mark A.

2015-01-01

BACKGROUND Functional decline in stem cell-mediated regeneration contributes to aging associated with cellular senescence in c-kit+ cardiac progenitor cells (CPCs). Clinical implementation of CPC-based therapy with elderly patients would benefit tremendously from understanding molecular characteristics of senescence to antagonize aging. Nucleostemin (NS) is a nucleolar protein regulating stem cell proliferation and pluripotency. OBJECTIVES The goal is to demonstrate that NS preserves characteristics associated with “stemness” in CPCs and antagonizes myocardial senescence and aging. METHODS CPCs isolated from human fetal (FhCPC) and adult failing (AhCPC) hearts, as well as young (YCPC) and old mice (OCPC), were studied for senescence characteristics and NS expression. Heterozygous knockout mice with one functional allele of NS (NS+/−) were used to demonstrate that NS preserves myocardial structure and function and slows characteristics of aging. RESULTS NS expression is decreased in AhCPCs relative to FhCPC, correlating with lowered proliferation potential and shortened telomere length. AhCPC characteristics resemble OCPCs, which have a phenotype induced by NS silencing, resulting in cell flattening, senescence, multinucleated cells, decreased S phase progression, diminished expression of stemness markers and up-regulation of p53 and p16. CPC senescence resulting from NS loss is partially p53 dependent and is rescued by concurrent silencing of p53. Mechanistically, NS induction correlates with Pim-1 kinase-mediated stabilization of c-Myc. Engineering OCPCs and AhCPCs to overexpress NS decreases senescent and multinucleated cells, restores morphology, and antagonizes senescence, thereby preserving phenotypic properties of “stemness.” Early cardiac aging with decline in cardiac function, increase in senescence markers p53 and p16, telomere attrition, and accompanied CPC exhaustion is evident in NS+/− mice. CONCLUSIONS Youthful properties and antagonism of senescence in CPCs and the myocardium is consistent with a role for NS downstream from Pim-1 signaling that enhances cardiac regeneration. PMID:25593054

Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections.

PubMed

Hetzel, Miriam; Mucci, Adele; Blank, Patrick; Nguyen, Ariane Hai Ha; Schiller, Jan; Halle, Olga; Kühnel, Mark-Philipp; Billig, Sandra; Meineke, Robert; Brand, Daniel; Herder, Vanessa; Baumgärtner, Wolfgang; Bange, Franz-Christoph; Goethe, Ralph; Jonigk, Danny; Förster, Reinhold; Gentner, Bernhard; Casanova, Jean-Laurent; Bustamante, Jacinta; Schambach, Axel; Kalinke, Ulrich; Lachmann, Nico

2018-02-01

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 ( IFNGR1 or IFNGR2 ) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express Ifnγr1 either constitutively or myeloid specifically. Transduction of mouse Ifnγr1 -/- HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against Mycobacterium avium and Mycobacterium bovis Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into Ifnγr1 -/- mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients. © 2018 by The American Society of Hematology.

Melatonin protects bone marrow mesenchymal stem cells against iron overload-induced aberrant differentiation and senescence.

PubMed

Yang, Fan; Yang, Lei; Li, Yuan; Yan, Gege; Feng, Chao; Liu, Tianyi; Gong, Rui; Yuan, Ye; Wang, Ning; Idiiatullina, Elina; Bikkuzin, Timur; Pavlov, Valentin; Li, Yang; Dong, Chaorun; Wang, Dawei; Cao, Yang; Han, Zhenbo; Zhang, Lai; Huang, Qi; Ding, Fengzhi; Bi, Zhengang; Cai, Benzhi

2017-10-01

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload-induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload-induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload-induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Recipient Glycemic Micro-environments Govern Therapeutic Effects of Mesenchymal Stem Cell Infusion on Osteopenia

PubMed Central

Sui, Bing-Dong; Hu, Cheng-Hu; Zheng, Chen-Xi; Shuai, Yi; He, Xiao-Ning; Gao, Ping-Ping; Zhao, Pan; Li, Meng; Zhang, Xin-Yi; He, Tao; Xuan, Kun; Jin, Yan

2017-01-01

Therapeutic effects of mesenchymal stem cell (MSC) infusion have been revealed in various human disorders, but impacts of diseased micro-environments are only beginning to be noticed. Donor diabetic hyperglycemia is reported to impair therapeutic efficacy of stem cells. However, whether recipient diabetic condition also affects MSC-mediated therapy is unknown. We and others have previously shown that MSC infusion could cure osteopenia, particularly in ovariectomized (OVX) mice. Here, we discovered impaired MSC therapeutic effects on osteopenia in recipient type 1 diabetes (T1D). Through intensive glycemic control by daily insulin treatments, therapeutic effects of MSCs on osteopenia were maintained. Interestingly, by only transiently restoration of recipient euglycemia using single insulin injection, MSC infusion could also rescue T1D-induced osteopenia. Conversely, under recipient hyperglycemia induced by glucose injection in OVX mice, MSC-mediated therapeutic effects on osteopenia were diminished. Mechanistically, recipient hyperglycemic micro-environments reduce anti-inflammatory capacity of MSCs in osteoporotic therapy through suppressing MSC interaction with T cells via the Adenosine monophosphate-activated protein kinase (AMPK) pathway. We further revealed in diabetic micro-environments, double infusion of MSCs ameliorated osteopenia by anti-inflammation, attributed to the first transplanted MSCs which normalized the recipient glucose homeostasis. Collectively, our findings uncover a previously unrecognized role of recipient glycemic conditions controlling MSC-mediated therapy, and unravel that fulfillment of potent therapeutic effects of MSCs requires tight control of recipient micro-environments. PMID:28435461

Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning

PubMed Central

Bhattacharya, Deepta; Rossi, Derrick J.; Bryder, David; Weissman, Irving L.

2006-01-01

In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that ∼0.1–1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation. PMID:16380511

The reprogramming factor nuclear receptor subfamily 5, group A, member 2 cannot replace octamer-binding transcription factor 4 function in the self-renewal of embryonic stem cells.

PubMed

Choi, Kyeng-Won; Oh, Hye-Rim; Lee, Jaeyoung; Lim, Bobae; Han, Yong-Mahn; Oh, Junseo; Kim, Jungho

2014-02-01

Although octamer-binding transcription factor 4 (Oct-4) is one of the most intensively studied factors in mammalian development, no cellular genes capable of replacing Oct-4 function in embryonic stem (ES) cells have been found. Recent data show that nuclear receptor subfamily 5, group A, member 2 (Nr5a2) is able to replace Oct-4 function in the reprogramming process; however, it is unclear whether Nr5a2 can replace Oct-4 function in ES cells. In this study, the ability of Nr5a2 to maintain self-renewal and pluripotency in ES cells was investigated. Nr5a2 localized to the nucleus in ES cells, similarly to Oct-4. However, expression of Nr5a2 failed to rescue the stem cell phenotype or to maintain the self-renewal ability of ES cells. Furthermore, as compared with Oct-4-expressing ES cells, Nr5a2-expressing ES cells showed a reduced number of cells in S-phase, did not expand normally, and did not remain in an undifferentiated state. Ectopic expression of Nr5a2 in ES cells was not able to activate transcription of ES cell-specific genes, and gene expression profiling demonstrated differences between Nr5a2-expressing and Oct-4-expressing ES cells. In addition, Nr5a2-expressing ES cells were not able to form teratomas in nude mice. Taken together, these results strongly suggest that the gene regulation properties of Nr5a2 and Oct-4 and their abilities to confer self-renewal and pluripotency of ES cells differ. The present study provides strong evidence that Nr5a2 cannot replace Oct-4 function in ES cells. © 2013 FEBS.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

PubMed

Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

2017-12-01

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

PubMed

Cuenca, Nicolás; Fernández-Sánchez, Laura; McGill, Trevor J; Lu, Bin; Wang, Shaomei; Lund, Raymond; Huhn, Stephen; Capela, Alexandra

2013-10-15

Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

PubMed

Qi, Y; Liu, J; Saadat, S; Tian, X; Han, Y; Fong, G-H; Pandolfi, P P; Lee, L Y; Li, S

2015-05-01

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

Efficacy of vincristine and etoposide with escalating cyclophosphamide in poor-prognosis pediatric brain tumors1

PubMed Central

Ziegler, David S.; Cohn, Richard J.; McCowage, Geoffrey; Alvaro, Frank; Oswald, Cecilia; Mrongovius, Robert; White, Les

2006-01-01

The objective of this study was to assess the efficacy of the VETOPEC regimen, a regimen of vincristine and etoposide with escalating doses of cyclophosphamide (CPA), in pediatric patients with high-risk brain tumors. Three consecutive studies by the Australia and New Zealand Children’s Cancer Study Group—VETOPEC I, Baby Brain 91, and VETOPEC II—have used a specific chemotherapy regimen of vincristine (VCR), etoposide (VP-16) and escalating CPA in patients with relapsed, refractory, or high-risk solid tumors. Patients in the VETOPEC II cohort were treated with very high dose CPA with peripheral blood stem cell (PBSC) rescue. We analyzed the subset of patients with high-risk brain tumors treated with these intensive VETOPEC-based protocols to assess the response, toxicity, and survival. We also assessed whether the use of very high dose chemotherapy with stem cell rescue improved the response rate or affected toxicity. Seventy-one brain tumor patients were treated with VETOPEC-based protocols. Of the 54 patients evaluable for tumor response, 17 had a complete response (CR) and 20 a partial response (PR) to treatment, which yielded an overall response rate of 69%. The CR + PR was 83% (19/23) for medulloblastomas, 56% (5/9) for primitive neuroectodermal tumors, 55% (6/11) for grade 3 and 4 astrocytomas, and 80% (6/8) for ependymomas. At a median follow-up of 36 months, overall survival for the entire cohort of 71 patients was 32%, with event-free survival of 13%. There were no toxic deaths within the PBSC-supported VETOPEC II cohort, despite higher CPA doses, compared with 7% among the non-PBSC patients. This regimen produces high response rates in a variety of very poor prognosis pediatric brain tumors. The maximum tolerated dose of CPA was not reached. Higher escalation in doses of CPA did not deliver a further improvement in response. With PBSC rescue in the VETOPEC II study, hematologic toxicity was no longer a limiting factor. The response rates observed support further development of this chemotherapy regimen. PMID:16443948

Maternal aldehyde elimination during pregnancy preserves the fetal genome.

PubMed

Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P; Patel, Ketan J

2014-09-18

Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Maternal Aldehyde Elimination during Pregnancy Preserves the Fetal Genome

PubMed Central

Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P.; Patel, Ketan J.

2014-01-01

Summary Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2−/−Fanca−/− embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. PMID:25155611

Successful Combination of Sequential Gene Therapy and Rescue Allo-HSCT in Two Children with X-CGD - Importance of Timing.

PubMed

Siler, Ulrich; Paruzynski, Anna; Holtgreve-Grez, Heidi; Kuzmenko, Elena; Koehl, Ulrike; Renner, Eleonore D; Alhan, Canan; de Loosdrecht, Arjan A van; Schwäble, Joachim; Pfluger, Thomas; Tchinda, Joelle; Schmugge, Markus; Jauch, Anna; Naundorf, Sonja; Kühlcke, Klaus; Notheis, Gundula; Güngor, Tayfun; Kalle, Christof V; Schmidt, Manfred; Grez, Manuel; Seger, Reinhard; Reichenbach, Janine

2015-01-01

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.

Genetic Correction of Human Induced Pluripotent Stem Cells from Patients with Spinal Muscular Atrophy

PubMed Central

Corti, Stefania; Nizzardo, Monica; Simone, Chiara; Falcone, Marianna; Nardini, Martina; Ronchi, Dario; Donadoni, Chiara; Salani, Sabrina; Riboldi, Giulietta; Magri, Francesca; Menozzi, Giorgia; Bonaglia, Clara; Rizzo, Federica; Bresolin, Nereo; Comi, Giacomo P.

2016-01-01

Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA. PMID:23253609

Transplantation of CX3CL1-expressing mesenchymal stem cells provides neuroprotective and immunomodulatory effects in a rat model of retinal degeneration.

PubMed

Huang, Libin; Xu, Wei; Xu, Guoxing

2013-08-01

To investigate the neuroprotective and immunomodulatory effects of mesenchymal stem cells (MSCs) engineered to secrete CX3CL1 on the light-injured retinal structure and function. Normal MSCs and CX3CL1-expressing MSCs (CX3CL1-MSCs) were transplanted into the subretinal space of light-injured rats. By ERG and TUNEL methods, their rescue effect of the host retina was compared with untreated light-injured and vehicle-injected rats. Activated microglia in the retina were stained by ED-1 antibody, and Western blot was performed to quantify cytokines secreted by the retina post-transplantation. ERG analysis showed better function in CX3CL1-MSC-injected group than other groups at 21 days after transplantation (p < 0.05). CX3CL1-MSCs inhibited apoptosis of the retinal cells and microglial activation. Neurotrophic factors expression in host retina that received CX3CL1-MSCs was stronger than in the retina that received normal MSCs. Conversely, the expression of proinflammatory factors was downregulated. CX3CL1-MSCs subretinal transplantation may enhance protective effect against light-induced retinal degeneration.

Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

PubMed

Young, Kira; Borikar, Sneha; Bell, Rebecca; Kuffler, Lauren; Philip, Vivek; Trowbridge, Jennifer J

2016-10-17

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging. © 2016 Young et al.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) recruits bone marrow-derived cells to the murine pulmonary vasculature.

PubMed

Angelini, Daniel J; Su, Qingning; Kolosova, Irina A; Fan, Chunling; Skinner, John T; Yamaji-Kegan, Kazuyo; Collector, Michael; Sharkis, Saul J; Johns, Roger A

2010-06-22

Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)(+) transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+) BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+) cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+) and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.

Modulation of the Innate Immune Response by Human Neural Precursors Prevails over Oligodendrocyte Progenitor Remyelination to Rescue a Severe Model of Pelizaeus-Merzbacher Disease.

PubMed

Marteyn, Antoine; Sarrazin, Nadège; Yan, Jun; Bachelin, Corinne; Deboux, Cyrille; Santin, Mathieu D; Gressens, Pierre; Zujovic, Violetta; Baron-Van Evercooren, Anne

2016-04-01

Pelizaeus-Merzbacher disease (PMD) results from an X-linked misexpression of proteolipid protein 1 (PLP1). This leukodystrophy causes severe hypomyelination with progressive inflammation, leading to neurological dysfunctions and shortened life expectancy. While no cure exists for PMD, experimental cell-based therapy in the dysmyelinated shiverer model suggested that human oligodendrocyte progenitor cells (hOPCs) or human neural precursor cells (hNPCs) are promising candidates to treat myelinopathies. However, the fate and restorative advantages of human NPCs/OPCs in a relevant model of PMD has not yet been addressed. Using a model of Plp1 overexpression, resulting in demyelination with progressive inflammation, we compared side-by-side the therapeutic benefits of intracerebrally grafted hNPCs and hOPCs. Our findings reveal equal integration of the donor cells within presumptive white matter tracks. While the onset of exogenous remyelination was earlier in hOPCs-grafted mice than in hNPC-grafted mice, extended lifespan occurred only in hNPCs-grafted animals. This improved survival was correlated with reduced neuroinflammation (microglial and astrocytosis loads) and microglia polarization toward M2-like phenotype followed by remyelination. Thus modulation of neuroinflammation combined with myelin restoration is crucial to prevent PMD pathology progression and ensure successful rescue of PMD mice. These findings should help to design novel therapeutic strategies combining immunomodulation and stem/progenitor cell-based therapy for disorders associating hypomyelination with inflammation as observed in PMD. © 2015 AlphaMed Press.

A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development.

PubMed

Xiao, Ying; Thoresen, Daniel T; Miao, Lingling; Williams, Jonathan S; Wang, Chaochen; Atit, Radhika P; Wong, Sunny Y; Brownell, Isaac

2016-07-01

The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.

Mechanism of As2O3-Induced Action Potential Prolongation and Using hiPS-CMs to Evaluate the Rescue Efficacy of Drugs With Different Rescue Mechanism.

PubMed

Yan, Meng; Feng, Lifang; Shi, Yanhui; Wang, Junnan; Liu, Yan; Li, Fengmei; Li, Baoxin

2017-08-01

Arsenic trioxide (As2O3) has been verified as a breakthrough in the management of acute promyelocytic leukemia in recent decades. However, cardiotoxicity, especially long QT syndrome (LQTS) has become the most important issue during As2O3 treatment. The characterized mechanisms behind this adverse effect are inhibition of cardiac hERG channel trafficking and increase of cardiac calcium currents. In our study, we found a new pathway underlying As2O3-induced cardiotoxicity that As2O3 accelerates lysosomal degradation of hERG on plasma membrane after using brefeldin A (BFA) to block protein trafficking. Then we explored pharmacological rescue strategies on As2O3-induced LQTS, and found that 4 therapeutic agents exert rescue efficacy via 3 different pathways: fexofenadine and astemizole facilitate hERG trafficking via promotion of channel-chaperone formation after As2O3 incubation; ranolazine slows hERG degradation in the presence of As2O3; and resveratrol shows significant attenuation on calcium current increase triggered by As2O3. Moreover, we used human-induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) to evaluate the rescue effects of the above agents on As2O3-induced prolongation of action potential duration (APD) and demonstrated that fexofenadine and resveratrol significantly ameliorate the prolonged APD. These observations suggested that pharmacological chaperone like fexofenadine and resveratrol might have the potential to protect against the cardiotoxicity of As2O3. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Non-Hodgkin’s Lymphomas, Version 4.2014

PubMed Central

Zelenetz, Andrew D.; Gordon, Leo I.; Wierda, William G.; Abramson, Jeremy S.; Advani, Ranjana H.; Andreadis, C. Babis; Bartlett, Nancy; Byrd, John C.; Czuczman, Myron S.; Fayad, Luis E.; Fisher, Richard I.; Glenn, Martha J.; Harris, Nancy Lee; Hoppe, Richard T.; Horwitz, Steven M.; Kelsey, Christopher R.; Kim, Youn H.; Krivacic, Susan; LaCasce, Ann S.; Nademanee, Auayporn; Porcu, Pierluigi; Press, Oliver; Rabinovitch, Rachel; Reddy, Nishitha; Reid, Erin; Saad, Ayman A.; Sokol, Lubomir; Swinnen, Lode J.; Tsien, Christina; Vose, Julie M.; Yahalom, Joachim; Zafar, Nadeem; Dwyer, Mary; Sundar, Hema

2016-01-01

Non-Hodgkin’s lymphomas (NHL) are a heterogeneous group of lymphoproliferative disorders originating in B lymphocytes, T lymphocytes, or natural killer cells. Mantle cell lymphoma (MCL) accounts for approximately 6% of all newly diagnosed NHL cases. Radiation therapy with or without systemic therapy is a reasonable approach for the few patients who present with early-stage disease. Rituximab-based chemoimmunotherapy followed by high-dose therapy and autologous stem cell rescue (HDT/ASCR) is recommended for patients presenting with advanced-stage disease. Induction therapy followed by rituximab maintenance may provide extended disease control for those who are not candidates for HDT/ASCR. Ibrutinib, a Bruton tyrosine kinase inhibitor, was recently approved for the treatment of relapsed or refractory disease. This manuscript discusses the recommendations outlined in the NCCN Guidelines for NHL regarding the diagnosis and management of patients with MCL. PMID:25190696

Drosophila Activin- and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain.

PubMed

Zhu, Changqi C; Boone, Jason Q; Jensen, Philip A; Hanna, Scott; Podemski, Lynn; Locke, John; Doe, Chris Q; O'Connor, Michael B

2008-02-01

The Drosophila Activin-like ligands Activin-beta and Dawdle control several aspects of neuronal morphogenesis, including mushroom body remodeling, dorsal neuron morphogenesis and motoneuron axon guidance. Here we show that the same two ligands act redundantly through the Activin receptor Babo and its transcriptional mediator Smad2 (Smox), to regulate neuroblast numbers and proliferation rates in the developing larval brain. Blocking this pathway results in the development of larvae with small brains and aberrant photoreceptor axon targeting, and restoring babo function in neuroblasts rescued these mutant phenotypes. These results suggest that the Activin signaling pathway is required for producing the proper number of neurons to enable normal connection of incoming photoreceptor axons to their targets. Furthermore, as the Activin pathway plays a key role in regulating propagation of mouse and human embryonic stem cells, our observation that it also regulates neuroblast numbers and proliferation in Drosophila suggests that involvement of Activins in controlling stem cell propagation may be a common regulatory feature of this family of TGF-beta-type ligands.

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells.

PubMed

Takebe, Takanori; Sekine, Keisuke; Kimura, Masaki; Yoshizawa, Emi; Ayano, Satoru; Koido, Masaru; Funayama, Shizuka; Nakanishi, Noriko; Hisai, Tomoko; Kobayashi, Tatsuya; Kasai, Toshiharu; Kitada, Rina; Mori, Akira; Ayabe, Hiroaki; Ejiri, Yoko; Amimoto, Naoki; Yamazaki, Yosuke; Ogawa, Shimpei; Ishikawa, Momotaro; Kiyota, Yasujiro; Sato, Yasuhiko; Nozawa, Kohei; Okamoto, Satoshi; Ueno, Yasuharu; Taniguchi, Hideki

2017-12-05

Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive "reverse" screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>10 8 ). Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Gadd45a deletion aggravates hematopoietic stem cell dysfunction in ATM-deficient mice.

PubMed

Chen, Yulin; Yang, Runan; Guo, Peng; Ju, Zhenyu

2014-01-01

Ataxia telangiectasia mutated (ATM) kinase plays an essential role in the maintenance of genomic stability. ATM-deficient (ATM(-/-)) mice exhibit hematopoietic stem cell (HSC) dysfunction and a high incidence of lymphoma. Gadd45a controls cell cycle arrest, apoptosis and DNA repair, and is involved in the ATM-p53 mediated DNA damage response. However, the role of Gadd45a in regulating the functionality of ATM(-/-) HSCs is unknown. Here we report that Gadd45a deletion did not rescue the defects of T-cells and B-cells development in ATM(-/-) mice. Instead, ATM and Gadd45a double knockout (ATM(-/-) Gadd45a(-/-)) HSCs exhibited an aggravated defect in long-term self-renewal capacity compared to ATM(-/-) HSCs in HSC transplantation experiments. Further experiments revealed that the aggravated defect of ATM(-/-) Gadd45a(-/-) HSCs was due to a reduction of cell proliferation, associated with an accumulation of DNA damage and subsequent activation of DNA damage response including an up-regulation of p53-p21 signaling pathway. Additionally, ATM(-/-) Gadd45a(-/-) mice showed an increased incidence of hematopoietic malignancies, as well as an increased rate of metastasis than ATM(-/-) mice. In conclusion, Gadd45a deletion aggravated the DNA damage accumulation, which subsequently resulted in a further impaired self-renewal capacity and an increased malignant transformation in ATM(-/-) HSCs.

Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity.

PubMed

Varshney, Rohan; Ali, Quaisar; Wu, Chengxiang; Sun, Zhongjie

2016-11-01

The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity. © 2016 American Heart Association, Inc.

ALS Patient Stem Cells for Unveiling Disease Signatures of Motoneuron Susceptibility: Perspectives on the Deadly Mitochondria, ER Stress and Calcium Triad

PubMed Central

Kaus, Anjoscha; Sareen, Dhruv

2015-01-01

Amyotrophic lateral sclerosis (ALS) is a largely sporadic progressive neurodegenerative disease affecting upper and lower motoneurons (MNs) whose specific etiology is incompletely understood. Mutations in superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TARDBP/TDP-43) and C9orf72, have been identified in subsets of familial and sporadic patients. Key associated molecular and neuropathological features include ubiquitinated TDP-43 inclusions, stress granules, aggregated dipeptide proteins from mutant C9orf72 transcripts, altered mitochondrial ultrastructure, dysregulated calcium homeostasis, oxidative and endoplasmic reticulum (ER) stress, and an unfolded protein response (UPR). Such impairments have been documented in ALS animal models; however, whether these mechanisms are initiating factors or later consequential events leading to MN vulnerability in ALS patients is debatable. Human induced pluripotent stem cells (iPSCs) are a valuable tool that could resolve this “chicken or egg” causality dilemma. Relevant systems for probing pathophysiologically affected cells from large numbers of ALS patients and discovering phenotypic disease signatures of early MN susceptibility are described. Performing unbiased ‘OMICS and high-throughput screening in relevant neural cells from a cohort of ALS patient iPSCs, and rescuing mitochondrial and ER stress impairments, can identify targeted therapeutics for increasing MN longevity in ALS. PMID:26635528

Delayed rhabdomyolysis with paclitaxel, ifosfamide, carboplatin, and etoposide regimen: a case report.

PubMed

Sokolova, Alexandra; Chan, Onyee; Ullah, Waqas; Hamdani, Auon Abbas; Anwer, Faiz

2017-04-11

High-dose chemotherapy with autologous stem cell rescue is commonly used for the treatment of relapsed germ cell tumors. We report the first case of delayed rhabdomyolysis with paclitaxel, ifosfamide, carboplatin, and etoposide regimen. We report a case of a 21-year-old African-American man diagnosed with relapsed non-seminomatous germ cell tumor who received high-dose chemotherapy with carboplatin and etoposide following TIGER trial arm B off-protocol. His course was complicated by muscle pain and rhabdomyolysis after cycle 4 on day +12 after infusion of autologous stem cells. To the best of our knowledge, this complication has not been reported with this regimen. A differential diagnosis of sepsis and neutropenic fever along with side effects of high-dose chemotherapy were considered, but based on the timing of events, it was concluded that the etiology of rhabdomyolysis is high-dose chemotherapy. Rhabdomyolysis was successfully treated with hydration and did not recur during subsequent cycle 5. Delayed rhabdomyolysis after high-dose chemotherapy with paclitaxel, ifosfamide, carboplatin, and etoposide regimen has not been previously reported and needs to be considered for preventive strategy and prompt diagnosis and treatment to avoid renal complications. Physicians should have a low threshold to check creatine kinase enzymes in patients with unexplained muscle pain or renal insufficiency after high-dose chemotherapy.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

PubMed

Zhou, Fangfang; Xie, Feng; Jin, Ke; Zhang, Zhengkui; Clerici, Marcello; Gao, Rui; van Dinther, Maarten; Sixma, Titia K; Huang, Huizhe; Zhang, Long; Ten Dijke, Peter

2017-06-01

SMAD4 is a common intracellular effector for TGF-β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-β family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity. © 2017 The Authors.

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

PubMed Central

El Haddad, Najib; Heathcote, Dean; Moore, Robert; Yang, Sunmi; Azzi, Jamil; Mfarrej, Bechara; Atkinson, Mark; Sayegh, Mohamed H.; Lee, Jeng-Shin; Ashton-Rickardt, Philip G.

2011-01-01

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases. PMID:21076046

A single center’s experience using four different front line mobilization strategies in lymphoma patients planned to undergo autologous hematopoietic cell transplantation

PubMed Central

Haverkos, Bradley M.; Huang, Ying; Elder, Patrick; O’Donnell, Lynn; Scholl, Diane; Whittaker, Becky; Vasu, Sumi; Penza, Sam; Andritsos, Leslie A.; Devine, Steven M.; Jaglowski, Samantha M.

2016-01-01

In an otherwise eligible patient with relapsed lymphoma, inadequate mobilization of peripheral blood stem cells is a limiting factor to proceeding with an autologous hematopoietic cell transplantation (auto-HCT). Multiple strategies have been used to mobilize an adequate number of hematopoietic stem cells (HSCs) with no obvious front-line strategy. We report a single institutional experience mobilizing HSCs using four different approaches in lymphoma patients. We prospectively collected mobilization outcomes on patients planning to undergo auto-HCT at Ohio State University. We report results of first mobilization attempt for all relapsed or refractory lymphoma patients between 2008–2014. We identified 255 lymphoma patients who underwent mobilization for planned auto-HCT. The 255 lymphoma patients underwent the following front line mobilization strategies: 95 (37%) GCSF alone, 38 (15%) chemomobilization (GCSF+chemotherapy), 97 (38%) preemptive day 4 plerixafor, and 25 (10%) rescue day 5 plerixafor. As expected, there were significant differences between cohorts including age, comorbid indices, histology, and amount of prior chemotherapy. After controlling for differences between groups, the odds of collecting 2×106/kg HSCs on the first day of collection and 5×106/kg HSCs in total was highest in the cohort undergoing chemomobilization. In conclusion, our experience highlights the effectiveness of chemomobilization. PMID:28067870

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

PubMed

Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

2017-09-06

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Conductive polymers for controlled release and treatment of central nervous system injury

NASA Astrophysics Data System (ADS)

Saigal, Rajiv

As one of the most devastating forms of neurotrauma, spinal cord injury remains a challenging clinical problem. The difficulties in treatment could potentially be resolved by better technologies for therapeutic delivery. In order to develop new approaches to treating central nervous system injury, this dissertation focused on using electrically-conductive polymers, controlled drug release, and stem cell transplantation. We first sought to enhance the therapeutic potential of neural stem cells by electrically increasing their production of neurotrophic factors (NTFs), important molecules for neuronal cell survival, differentiation, synaptic development, plasticity, and growth. We fabricated a new cell culture device for growing neural stem cells on a biocompatible, conductive polymer. Electrical stimulation via the polymer led to upregulation of NTF production by neural stem cells. This approach has the potential to enhance stem cell function while avoiding the pitfalls of genetic manipulation, possibly making stem cells more viable as a clinical therapy. Seeing the therapeutic potential of conductive polymers, we extended our studies to an in vivo model of spinal cord injury (SCI). Using a novel fabrication and extraction technique, a conductive polymer was fabricated to fit to the characteristic pathology that follows contusive SCI. Assessed via quantitative analysis of MR images, the conductive polymer significantly reduced compression of the injured spinal cord. Further characterizing astroglial and neuronal response of injured host tissue, we found significant neuronal sparing as a result of this treatment. The in vivo studies also demonstrated improved locomotor recovery mediated by a conductive polymer scaffold over a non-conductive control. We next sought to take advantage of conductive polymers for local, electronically-controlled release of drugs. Seeking to overcome reported limitations in drug delivery via polypyrrole, we first embedded drugs in poly[(D,L-lactide-co-glycolide)-co-polyethylene glycol] (PLGA-PEG) nanoparticles and then demonstrated scalable incorporation and controlled release. In a functional application, electronically-controlled release of minocycline nanoparticles was used to rescue primary spinal cord neurons from an excitotoxic environment in vitro. This approach offers a wide range of therapeutic possibilities, especially for treating traumatic lesions of the central nervous system. Finally, we explored use of conductive polymers for directed differentiation of progenitor cells. Retinal progenitors were seeded on custom polypyrrole cell culture devices and subjected to a biomimetic pattern of electrical stimulation. Stimulated cells showed phenotypic changes, increased neurite outgrowth, increased immunocytochemical expression of cone rod homeobox (CRX) and protein kinase C (PK-C), and decreased expression of glial fibrillary acidic protein (GFAP). Biomimetic stimulation thus led cells towards early photoreceptor and bipolar cell fates, and away from an astrocytic cell fate. Electrical stimulation via a conductive polymer offers a novel approach for directing differentiation of progenitor cells.

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

PubMed Central

Chu, Pat P. Y.; Bari, Sudipto; Fan, Xiubo; Gay, Florence P. H.; Ang, Justina M. L.; Chiu, Gigi N. C.; Lim, Sai K.; Hwang, William Y. K.

2012-01-01

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture. PMID:22775077

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia

PubMed Central

Kenderian, Saad S.; Shen, Feng; Ruella, Marco; Shestova, Olga; Kozlowski, Miroslaw; Li, Yong; Schrank-Hacker, April; Morrissette, Jennifer J. D.; Carroll, Martin; June, Carl H.; Grupp, Stephan A.; Gill, Saar

2017-01-01

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA–electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti–CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML. PMID:28246194

Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation

PubMed Central

Kazemi, Zahra; Bergmayr, Christian; Prchal-Murphy, Michaela; Javaheri, Tahereh; Themanns, Madeleine; Pham, Ha T. T.; Strohmaier, Wolfgang; Sexl, Veronika; Zebedin-Brandl, Eva

2016-01-01

Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [3H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg−1 8 h−1) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application. PMID:26989084

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

PubMed

Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Pivoraitė, Ugnė; Venalis, Algirdas; Pivoriūnas, Augustas

2015-07-01

Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 μmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

PubMed Central

Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

2015-01-01

ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

Pyrimethamine as a Potent and Selective Inhibitor of Acute Myeloid Leukemia Identified by High-throughput Drug Screening.

PubMed

Sharma, Amit; Jyotsana, Nidhi; Lai, Courteney K; Chaturvedi, Anuhar; Gabdoulline, Razif; Görlich, Kerstin; Murphy, Cecilia; Blanchard, Jan E; Ganser, Arnold; Brown, Eric; Hassell, John A; Humphries, R Keith; Morgan, Michael; Heuser, Michael

2016-01-01

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.

Biotechnological approaches to the treatment of aspermatogenic men

PubMed Central

Aponte, Pedro Manuel; Schlatt, Stefan; de Franca, Luiz Renato

2013-01-01

Aspermatogenesis is a severe impairment of spermatogenesis in which germ cells are completely lacking or present in an immature form, which results in sterility in approximately 25% of patients. Because assisted reproduction techniques require mature germ cells, biotechnology is a valuable tool for rescuing fertility while maintaining biological fatherhood. However, this process involves, for instance, the differentiation of preexisting immature germ cells or the production/derivation of sperm from somatic cells. This review critically addresses four potential techniques: sperm derivation in vitro, germ stem cell transplantation, xenologous systems, and haploidization. Sperm derivation in vitro is already feasible in fish and mammals through organ culture or 3D systems, and it is very useful in conditions of germ cell arrest or in type II Sertoli-cell-only syndrome. Patients afflicted by type I Sertoli-cell-only syndrome could also benefit from gamete derivation from induced pluripotent stem cells of somatic origin, and human haploid-like cells have already been obtained by using this novel methodology. In the absence of alternative strategies to generate sperm in vitro, in germ cells transplantation fertility is restored by placing donor cells in the recipient germ-cell-free seminiferous epithelium, which has proven effective in conditions of spermatogonial arrest. Grafting also provides an approach for ex-vivo generation of mature sperm, particularly using prepubertal testis tissue. Although less feasible, haploidization is an option for creating gametes based on biological cloning technology. In conclusion, the aforementioned promising techniques remain largely experimental and still require extensive research, which should address, among other concerns, ethical and biosafety issues, such as gamete epigenetic status, ploidy, and chromatin integrity. PMID:23503966

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

PubMed

Wu, Xianfang; Dao Thi, Viet Loan; Liu, Peng; Takacs, Constantin N; Xiang, Kuanhui; Andrus, Linda; Gouttenoire, Jérôme; Moradpour, Darius; Rice, Charles M

2018-02-01

The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

A Novel Notch-YAP Circuit Drives Stemness and Tumorigenesis in Embryonal Rhabdomyosarcoma.

PubMed

Slemmons, Katherine K; Crose, Lisa E S; Riedel, Stefan; Sushnitha, Manuela; Belyea, Brian; Linardic, Corinne M

2017-12-01

Rhabdomyosarcoma (RMS), a cancer characterized by skeletal muscle features, is the most common soft-tissue sarcoma of childhood. While low- and intermediate-risk groups have seen improved outcomes, high-risk patients still face a 5-year survival rate of <30%, a statistic that has not changed in over 40 years. Understanding the biologic underpinnings of RMS is critical. The developmental pathways of Notch and YAP have been identified as potent but independent oncogenic signals that support the embryonal variant of RMS (eRMS). Here, the cross-talk between these pathways and the impact on eRMS tumorigenesis is reported. Using human eRMS cells grown as three-dimensional (3D) rhabdospheres, which enriches in stem cells, it was found that Notch signaling transcriptionally upregulates YAP1 gene expression and YAP activity. Reciprocally, YAP transcriptionally upregulates the Notch ligand genes JAG1 and DLL1 and the core Notch transcription factor RBPJ This bidirectional circuit boosts expression of key stem cell genes, including SOX2 , which is functionally required for eRMS spheres. Silencing this circuit for therapeutic purposes may be challenging, because the inhibition of one node (e.g., pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Finally, supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases Notch signaling and SOX2 expression. Implications: This study identifies a novel oncogenic signaling circuit driving eRMS stemness and tumorigenesis, and provides evidence and rationale for combination therapies co-targeting Notch and YAP. Mol Cancer Res; 15(12); 1777-91. ©2017 AACR . ©2017 American Association for Cancer Research.

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation.

PubMed

Dikov, Mikhail M; Ohm, Joyce E; Ray, Neelanjan; Tchekneva, Elena E; Burlison, Jared; Moghanaki, Drew; Nadaf, Sorena; Carbone, David P

2005-01-01

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.

Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2

PubMed Central

Micheli, Laura; Ceccarelli, Manuela; Gioia, Roberta; D’Andrea, Giorgio; Farioli-Vecchioli, Stefano; Costanzi, Marco; Saraulli, Daniele; Cestari, Vincenzo; Tirone, Felice

2017-01-01

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate. PMID:28740463

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

PubMed Central

Zhang, Xiaoling; Shang, Xun; Guo, Fukun; Murphy, Kim; Kirby, Michelle; Kelly, Patrick; Reeves, Lilith; Smith, Franklin O.; Williams, David A.

2008-01-01

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway. PMID:18565850

Genetic Information, the Principle of Rescue, and Special Obligations.

PubMed

Liao, S Matthew; Mackenzie, Jordan

2018-05-01

In "Genetic Privacy, Disease Prevention, and the Principle of Rescue," Madison Kilbride argues that patients have a duty to warn biological family members about clinically actionable adverse genetic findings. The duty does not stem from the special obligations that we may have to family members, she argues, but rather follows from the principle of rescue, which she understands as the idea that one ought to prevent, reduce, or mitigate the risk of harm to another person when the expected harm is serious and the cost or risk to oneself is sufficiently moderate. We doubt, however, whether the principle of rescue can ground a duty to warn in the cases Kilbride envisages, and we suggest that Kilbride may have underappreciated the role that special obligations could play in generating a duty to warn family members. © 2018 The Hastings Center.

A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages.

PubMed

Radzisheuskaya, Aliaksandra; Chia, Gloryn Le Bin; dos Santos, Rodrigo L; Theunissen, Thorold W; Castro, L Filipe C; Nichols, Jennifer; Silva, José C R

2013-06-01

Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

Altered proliferation and networks in neural cells derived from idiopathic autistic individuals.

PubMed

Marchetto, Maria C; Belinson, Haim; Tian, Yuan; Freitas, Beatriz C; Fu, Chen; Vadodaria, Krishna; Beltrao-Braga, Patricia; Trujillo, Cleber A; Mendes, Ana P D; Padmanabhan, Krishnan; Nunez, Yanelli; Ou, Jing; Ghosh, Himanish; Wright, Rebecca; Brennand, Kristen; Pierce, Karen; Eichenfield, Lawrence; Pramparo, Tiziano; Eyler, Lisa; Barnes, Cynthia C; Courchesne, Eric; Geschwind, Daniel H; Gage, Fred H; Wynshaw-Boris, Anthony; Muotri, Alysson R

2017-06-01

Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells, neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1), a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle.

PubMed

Morosetti, Roberta; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; De Angelis, Luciana; Tagliafico, Enrico; Sampaolesi, Maurilio; Gidaro, Teresa; Papacci, Manuela; Roncaglia, Enrica; Rutella, Sergio; Ferrari, Stefano; Tonali, Pietro Attilio; Ricci, Enzo; Cossu, Giulio

2006-11-07

Inflammatory myopathies (IM) are acquired diseases of skeletal muscle comprising dermatomyositis (DM), polymyositis (PM), and inclusion-body myositis (IBM). Immunosuppressive therapies, usually beneficial for DM and PM, are poorly effective in IBM. We report the isolation and characterization of mesoangioblasts, vessel-associated stem cells, from diagnostic muscle biopsies of IM. The number of cells isolated, proliferation rate and lifespan, markers expression, and ability to differentiate into smooth muscle do not differ among normal and IM mesoangioblasts. At variance with normal, DM and PM mesoangioblasts, cells isolated from IBM, fail to differentiate into skeletal myotubes. These data correlate with lack in connective tissue of IBM muscle of alkaline phosphatase (ALP)-positive cells, conversely dramatically increased in PM and DM. A myogenic inhibitory basic helix-loop-helix factor B3 is highly expressed in IBM mesoangioblasts. Indeed, silencing this gene or overexpressing MyoD rescues the myogenic defect of IBM mesoangioblasts, opening novel cell-based therapeutic strategies for this crippling disorder.

MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle

PubMed Central

Morosetti, Roberta; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; De Angelis, Luciana; Tagliafico, Enrico; Sampaolesi, Maurilio; Gidaro, Teresa; Papacci, Manuela; Roncaglia, Enrica; Rutella, Sergio; Ferrari, Stefano; Tonali, Pietro Attilio; Ricci, Enzo; Cossu, Giulio

2006-01-01

Inflammatory myopathies (IM) are acquired diseases of skeletal muscle comprising dermatomyositis (DM), polymyositis (PM), and inclusion-body myositis (IBM). Immunosuppressive therapies, usually beneficial for DM and PM, are poorly effective in IBM. We report the isolation and characterization of mesoangioblasts, vessel-associated stem cells, from diagnostic muscle biopsies of IM. The number of cells isolated, proliferation rate and lifespan, markers expression, and ability to differentiate into smooth muscle do not differ among normal and IM mesoangioblasts. At variance with normal, DM and PM mesoangioblasts, cells isolated from IBM, fail to differentiate into skeletal myotubes. These data correlate with lack in connective tissue of IBM muscle of alkaline phosphatase (ALP)-positive cells, conversely dramatically increased in PM and DM. A myogenic inhibitory basic helix–loop–helix factor B3 is highly expressed in IBM mesoangioblasts. Indeed, silencing this gene or overexpressing MyoD rescues the myogenic defect of IBM mesoangioblasts, opening novel cell-based therapeutic strategies for this crippling disorder. PMID:17077152

Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

PubMed

Poleshko, Andrey; Shah, Parisha P; Gupta, Mudit; Babu, Apoorva; Morley, Michael P; Manderfield, Lauren J; Ifkovits, Jamie L; Calderon, Damelys; Aghajanian, Haig; Sierra-Pagán, Javier E; Sun, Zheng; Wang, Qiaohong; Li, Li; Dubois, Nicole C; Morrisey, Edward E; Lazar, Mitchell A; Smith, Cheryl L; Epstein, Jonathan A; Jain, Rajan

2017-10-19

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery. Copyright © 2017 Elsevier Inc. All rights reserved.

Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells.

PubMed

Di Felice, Valentina; Serradifalco, Claudia; Rizzuto, Luigi; De Luca, Angela; Rappa, Francesca; Barone, Rosario; Di Marco, Patrizia; Cassata, Giovanni; Puleio, Roberto; Verin, Lucia; Motta, Antonella; Migliaresi, Claudio; Guercio, Annalisa; Zummo, Giovanni

2015-11-01

The use of three-dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patient's life. Silk fibroin-based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly. Copyright © 2013 John Wiley & Sons, Ltd.

Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.

PubMed

Wang, Nan-Kai; Tosi, Joaquin; Kasanuki, Jennifer Mie; Chou, Chai Lin; Kong, Jian; Parmalee, Nancy; Wert, Katherine J; Allikmets, Rando; Lai, Chi-Chun; Chien, Chung-Liang; Nagasaki, Takayuki; Lin, Chyuan-Sheng; Tsang, Stephen H

2010-04-27

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.

PubMed

Jiang, Wei-Cheng; Cheng, Yu-Hao; Yen, Meng-Hua; Chang, Yin; Yang, Vincent W; Lee, Oscar K

2014-04-01

Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

PubMed

Pittermann, Erik; Lachmann, Nico; MacLean, Glenn; Emmrich, Stephan; Ackermann, Mania; Göhring, Gudrun; Schlegelberger, Brigitte; Welte, Karl; Schambach, Axel; Heckl, Dirk; Orkin, Stuart H; Cantz, Tobias; Klusmann, Jan-Henning

2017-06-13

Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 ( HAX1 ) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1 W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1 -centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1 W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue.

PubMed

Tzeng, Wen-Pin; Matthews, Jason D; Frey, Teryl K

2006-04-01

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.

Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Shin-Hae; Park, Joung-Sun; Kim, Young-Shin

Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. In the present study, we utilize the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed inmore » the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or Timp-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. Our data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis. -- Highlights: Black-Right-Pointing-Pointer Mmp1 is expressed in the adult midgut. Black-Right-Pointing-Pointer Mmp1 is involved in the regulation of ISC proliferation activity. Black-Right-Pointing-Pointer Mmp1-related ISC proliferation is associated with EGFR signaling. Black-Right-Pointing-Pointer Mmp1 in the gut is required for the intestinal homeostasis and longevity.« less

Zinc finger protein 274 regulates imprinted expression of transcripts in Prader-Willi syndrome neurons.

PubMed

Langouët, Maéva; Glatt-Deeley, Heather R; Chung, Michael S; Dupont-Thibert, Clémence M; Mathieux, Elodie; Banda, Erin C; Stoddard, Christopher E; Crandall, Leann; Lalande, Marc

2018-02-01

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity and is caused by the absence of paternal contribution to chromosome 15q11-q13. Using induced pluripotent stem cell (iPSC) models of PWS, we previously discovered an epigenetic complex that is comprised of the zinc-finger protein ZNF274 and the SET domain bifurcated 1 (SETDB1) histone H3 lysine 9 (H3K9) methyltransferase and that silences the maternal alleles at the PWS locus. Here, we have knocked out ZNF274 and rescued the expression of silent maternal alleles in neurons derived from PWS iPSC lines, without affecting DNA methylation at the PWS-Imprinting Center (PWS-IC). This suggests that the ZNF274 complex is a separate imprinting mark that represses maternal PWS gene expression in neurons and is a potential target for future therapeutic applications to rescue the PWS phenotype. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment.

PubMed

Ponnath, Abhilash; Depreux, Frederic F; Jodelka, Francine M; Rigo, Frank; Farris, Hamilton E; Hastings, Michelle L; Lentz, Jennifer J

2018-02-01

The absence of functional outer hair cells is a component of several forms of hereditary hearing impairment, including Usher syndrome, the most common cause of concurrent hearing and vision loss. Antisense oligonucleotide (ASO) treatment of mice with the human Usher mutation, Ush1c c.216G>A, corrects gene expression and significantly improves hearing, as measured by auditory-evoked brainstem responses (ABRs), as well as inner and outer hair cell (IHC and OHC) bundle morphology. However, it is not clear whether the improvement in hearing achieved by ASO treatment involves the functional rescue of outer hair cells. Here, we show that Ush1c c.216AA mice lack OHC function as evidenced by the absence of distortion product otoacoustic emissions (DPOAEs) in response to low-, mid-, and high-frequency tone pairs. This OHC deficit is rescued by treatment with an ASO that corrects expression of Ush1c c.216G>A. Interestingly, although rescue of inner hairs cells, as measured by ABR, is achieved by ASO treatment as late as 7 days after birth, rescue of outer hair cells, measured by DPOAE, requires treatment before post-natal day 5. These results suggest that ASO-mediated rescue of both IHC and OHC function is age dependent and that the treatment window is different for the different cell types. The timing of treatment for congenital hearing disorders is of critical importance for the development of drugs such ASO-29 for hearing rescue.

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

PubMed

Wu, Yong; Hu, Zhiqing; Li, Zhuo; Pang, Jialun; Feng, Mai; Hu, Xuyun; Wang, Xiaolin; Lin-Peng, Siyuan; Liu, Bo; Chen, Fangping; Wu, Lingqian; Liang, Desheng

2016-01-08

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5' part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23-26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs

PubMed Central

Wu, Yong; Hu, Zhiqing; Li, Zhuo; Pang, Jialun; Feng, Mai; Hu, Xuyun; Wang, Xiaolin; Lin-Peng, Siyuan; Liu, Bo; Chen, Fangping; Wu, Lingqian; Liang, Desheng

2016-01-01

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition. PMID:26743572

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

PubMed

Srivastava, A; Singh, S; Rajpurohit, C S; Srivastava, P; Pandey, A; Kumar, D; Khanna, V K; Pant, A B

2018-06-01

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Dose Dependent Side Effect of Superparamagnetic Iron Oxide Nanoparticle Labeling on Cell Motility in Two Fetal Stem Cell Populations

PubMed Central

Diana, Valentina; Bossolasco, Patrizia; Moscatelli, Davide; Silani, Vincenzo; Cova, Lidia

2013-01-01

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies. PMID:24244310

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway.

PubMed

Zhang, Liqiang; Liu, Wenjia; Zhao, Jiangdong; Ma, Xiaojie; Shen, Lin; Zhang, Yongjie; Jin, Fang; Jin, Yan

2016-10-01

The balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood. Here, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100kpa static pressure for 1h or 12h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells. In vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1h relative to 12h. In contrast, the expression ratio of RANKL/OPG was reduced at 1h and significantly increased at 12h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1h. Our findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity. We describe a novel potential mechanism related to tooth movement. Copyright © 2016 Elsevier B.V. All rights reserved.

Neural Stem Cells Expressing bFGF Reduce Brain Damage and Restore Sensorimotor Function after Neonatal Hypoxia-Ischemia.

PubMed

Ye, Qingsong; Wu, Yanqing; Wu, Jiamin; Zou, Shuang; Al-Zaazaai, Ali Ahmed; Zhang, Hongyu; Shi, Hongxue; Xie, Ling; Liu, Yanlong; Xu, Ke; He, Huacheng; Zhang, Fabiao; Ji, Yiming; He, Yan; Xiao, Jian

2018-01-01

Neonatal hypoxia-ischemia (HI) causes severe brain damage and significantly increases neonatal morbidity and mortality. Increasing evidences have verified that stem cell-based therapy has the potential to rescue the ischemic tissue and restore function via secreting growth factors after HI. Here, we had investigated whether intranasal neural stem cells (NSCs) treatment improves the recovery of neonatal HI, and NSCs overexpressing basic fibroblast growth factor (bFGF) has a better therapeutic effect for recovery than NSCs treatment only. We performed permanent occlusion of the right common carotid artery in 9-day old ICR mice as animal model of neonatal hypoxia-ischemia. At 3 days post-HI, NSC, NSC-GFP, NSC-bFGF and vehicle were delivered intranasally. To determine the effect of intranasal NSC, NSC-GFP and NSC-bFGF treatment on recovery after HI, we analyzed brain damage, sensor-motor function and cell differentiation. It was observed that intranasal NSC, NSC-GFP and NSC-bFGF treatment decreased gray and white matter loss area in comparison with vehicle-treated mouse. NSC, NSC-GFP and NSC-bFGF treatment also significantly improved sensor motor function in cylinder rearing test and adhesive removal test, however, NSC-bFGF-treatment was more effective than NSC-treatment in the improvement of somatosensory function. Furthermore, compared with NSC and NSC-GFP, NSC-bFGF treatment group appeared to differentiate into more neurons. Taken together, intranasal administration of NSCs is a promising therapy for treatment of neonatal HI, but NSCs overexpressing bFGF promotes the survival and differentiation of NSCs, and consequently achieves a better therapeutic effect in improving recovery after neonatal HI. © 2018 The Author(s). Published by S. Karger AG, Basel.

Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways.

PubMed

Lalli, Matthew A; Jang, Jiwon; Park, Joo-Hye C; Wang, Yidi; Guzman, Elmer; Zhou, Hongjun; Audouard, Morgane; Bridges, Daniel; Tovar, Kenneth R; Papuc, Sorina M; Tutulan-Cunita, Andreea C; Huang, Yadong; Budisteanu, Magdalena; Arghir, Aurora; Kosik, Kenneth S

2016-04-01

Williams syndrome (WS) is a neurodevelopmental disorder caused by a genomic deletion of ∼28 genes that results in a cognitive and behavioral profile marked by overall intellectual impairment with relative strength in expressive language and hypersocial behavior. Advancements in protocols for neuron differentiation from induced pluripotent stem cells allowed us to elucidate the molecular circuitry underpinning the ontogeny of WS. In patient-derived stem cells and neurons, we determined the expression profile of the Williams-Beuren syndrome critical region-deleted genes and the genome-wide transcriptional consequences of the hemizygous genomic microdeletion at chromosome 7q11.23. Derived neurons displayed disease-relevant hallmarks and indicated novel aberrant pathways in WS neurons including over-activated Wnt signaling accompanying an incomplete neurogenic commitment. We show that haploinsufficiency of the ATP-dependent chromatin remodeler, BAZ1B, which is deleted in WS, significantly contributes to this differentiation defect. Chromatin-immunoprecipitation (ChIP-seq) revealed BAZ1B target gene functions are enriched for neurogenesis, neuron differentiation and disease-relevant phenotypes. BAZ1B haploinsufficiency caused widespread gene expression changes in neural progenitor cells, and together with BAZ1B ChIP-seq target genes, explained 42% of the transcriptional dysregulation in WS neurons. BAZ1B contributes to regulating the balance between neural precursor self-renewal and differentiation and the differentiation defect caused by BAZ1B haploinsufficiency can be rescued by mitigating over-active Wnt signaling in neural stem cells. Altogether, these results reveal a pivotal role for BAZ1B in neurodevelopment and implicate its haploinsufficiency as a likely contributor to the neurological phenotypes in WS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

VEGF preconditioning leads to stem cell remodeling and attenuates age-related decay of adult hippocampal neurogenesis

PubMed Central

Licht, Tamar; Rothe, Gadiel; Kreisel, Tirzah; Wolf, Brachi; Benny, Ofra; Rooney, Alasdair G.; ffrench-Constant, Charles; Enikolopov, Grigori; Keshet, Eli

2016-01-01

Several factors are known to enhance adult hippocampal neurogenesis but a factor capable of inducing a long-lasting neurogenic enhancement that attenuates age-related neurogenic decay has not been described. Here, we studied hippocampal neurogenesis following conditional VEGF induction in the adult brain and showed that a short episode of VEGF exposure withdrawn shortly after the generation of durable new vessels (but not under conditions where newly made vessels failed to persist) is sufficient for neurogenesis to proceed at a markedly elevated level for many months later. Continual neurogenic increase over several months was not accompanied by accelerated exhaustion of the neuronal stem cell (NSC) reserve, thereby allowing neurogenesis to proceed at a markedly elevated rate also in old mice. Neurogenic enhancement by VEGF preconditioning was, in part, attributed to rescue of age-related NSC quiescence. Remarkably, VEGF caused extensive NSC remodelling manifested in transition of the enigmatic NSC terminal arbor onto long cytoplasmic processes engaging with and spreading over even remote blood vessels, a configuration reminiscent of early postnatal “juvenile” NSCs. Together, these findings suggest that VEGF preconditioning might be harnessed for long-term neurogenic enhancement despite continued exposure to an “aged” systemic milieu. PMID:27849577

Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich’s ataxia

PubMed Central

Rocca, Celine J.; Goodman, Spencer M.; Dulin, Jennifer N.; Haquang, Joseph H.; Gertsman, Ilya; Blondelle, Jordan; Smith, Janell L. M.; Heyser, Charles J.; Cherqui, Stephanie

2017-01-01

Friedreich’s ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the FXN gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart. Transplanted HSPCs engrafted and then differentiated into microglia in the brain and spinal cord and into macrophages in the DRGs, heart, and muscle of YG8R FRDA mice. We observed the transfer of wild-type frataxin and Cox8 mitochondrial proteins from HSPC-derived microglia/macrophages to FRDA mouse neurons and muscle myocytes in vivo. Our results show the HSPC-mediated phenotypic rescue of FRDA in YG8R mice and suggest that this approach should be investigated further as a strategy for treating FRDA. PMID:29070698

Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2.

PubMed

Liu, Ke; Jing, Ying; Zhang, Wen; Fu, Xuejie; Zhao, Huan; Zhou, Xichao; Tao, Yunxia; Yang, Huilin; Zhang, Yan; Zen, Ke; Zhang, Chenyu; Li, Donghai; Shi, Qin

2017-04-01

Osteoporosis is a serious health problem worldwide. MicroRNA is a post-transcriptional regulator of gene expression by either promoting mRNA degradation or interfering with mRNA translation of specific target genes. It plays a significant role in the pathogenesis of osteoporosis. Here, we first demonstrated that miR-106b (miR-106b-5p) negatively regulated osteogenic differentiation of mesenchymal stem cells in vitro. Then, we found that miR-106b expression increased in C57BL/6 mice with glucocorticoid-induced osteoporosis (GIOP), and that silencing of miR-106b signaling protected mice against GIOP through promoting bone formation and inhibiting bone resorption. At last, we showed that miR-106b inhibited osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) both in vitro and in the GIOP model. Together, our findings have identified the role and mechanism of miR-106b in negatively regulating osteogenesis. Inhibition of miR-106b might be a potential new strategy for treating osteoporosis and bone defects. Copyright © 2017. Published by Elsevier Inc.

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence.

PubMed

Yoshihara, Takeshi; Spalding, Edgar P; Iino, Moritoshi

2013-04-01

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Plasma cell leukaemia and other aggressive plasma cell malignancies

PubMed Central

Sher, Taimur; Miller, Kena C.; Deeb, George; Lee, Kelvin; Chanan-Khan, Asher

2014-01-01

Summary Extramedullary plasma cell cancers, such as plasma cell leukemia (PCL) and multiple extramedullary plasmacytomas (MEP) are very aggressive malignancies. These can be primary (de-novo) or secondary due to progressive prior multiple myeloma (MM). Recent reports suggest an increase in incidence of these disorders. Compared to MM, organ invasion is common in PCL, while soft tissue tumors involving the head, neck or paraspinal area are common sites for MEP. Markers of poor prognosis are frequently observed in these extramedullary forms of plasma cell cancers, and survival is significantly inferior compared to patients with MM. Conventional chemotherapeutic and radiotherapy approaches have been employed with variable results. Even high dose chemotherapy with autologous stem cell rescue has not been able to demonstrate consistent improvement in survival outcome. Although not specifically evaluated, novel anti-plasma cell agents, such as the proteasome inhibitor bortezomib, and immunomodulatory drugs, such as lenalidomide, appear to be active against these aggressive cancers. Clinical and translational research directed at improved understanding of disease biology and development of novel therapeutics is urgently needed. PMID:20701603

p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

PubMed Central

Deng, Wen; Zhou, Yuan; Tiwari, Agnes FY; Su, Hang; Yang, Jie; Zhu, Dandan; Lau, Victoria Ming Yi; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie LM; Guan, Xin-Yuan; Tsao, Sai Wah

2015-01-01

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21. PMID:25131797

Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

PubMed Central

2017-01-01

Neural cultures derived from Huntington’s disease (HD) patient-derived induced pluripotent stem cells were used for ‘omics’ analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. PMID:28319609

A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

PubMed Central

Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

2015-01-01

SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

Rescue of CD8+ T cell vaccine memory following sublethal γ irradiation.

PubMed

McFarland, Hugh I; Berkson, Julia D; Lee, Jay P; Elkahloun, Abdel G; Mason, Karen P; Rosenberg, Amy S

2015-07-31

Sublethal γ irradiation eliminates CD8+ T cell mediated memory responses. In this work, we explored how these memory responses could be rescued in the aftermath of such exposure. We utilized two models of CD8+ T cell mediated immunity: a mouse model of Listeria monocytogenes (LM) infection in which CD8+ T cells specific for LM expressed antigens (Listeriolysin O, LLO) can be tracked, and a murine skin graft model in which CD8+ T cells mediate rejection across a MHC class I (D(d)) disparity. In the LM immunized mice, LL0 specific CD8+ T memory cells were lost on irradiation, preserved with rapid revaccination with an attenuated strain 1-3 days post-irradiation (PI), and these mice survived a subsequent wild type LM challenge. A genetic "signature of rescue" identified a group of immune-associated mRNA maintained or upregulated following irradiation and rescue. A number of these factors, including IL-36γ, dectin-2 (Clec4n), and mir101c are upregulated rapidly after exposure of mice to sublethal γ radiation alone and are sustained by early, but not later rescue. Such factors will be evaluated as potential therapeutics to replace individual vaccines for global rescue of CD8+ T memory cell responses following sublethal γ irradiation. The skin allograft model mirrored that of the LM model in that the accelerated D(d) skin allograft rejection response was lost in mice exposed to sublethal γ radiation, but infusion of allogeneic D(d) expressing bone marrow cells 1-4 days PI preserved the CD8+ T memory mediated accelerated rejection response, further suggesting that innate immune responses may not always be essential to rescue of CD8+ memory T cells following γ irradiation. Published by Elsevier Ltd.

Nicotine Deteriorates the Osteogenic Differentiation of Periodontal Ligament Stem Cells through α7 Nicotinic Acetylcholine Receptor Regulating wnt Pathway

PubMed Central

Dong, Zhiwei; Liu, Fen; Zhang, Yu; Yu, Yang; Shang, Fengqing; Wu, Lizheng; Wang, Xiaojing; Jin, Yan

2013-01-01

Aims Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through activating α7 nicotinic acetylcholine receptor (α7 nAChR). Methods hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels. Results Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency. Conclusions These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis. PMID:24376645

MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression.

PubMed

Huang, Yanxia; Liu, Xiaoguai; Wang, Yaping

2015-10-16

Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3'-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

In Vivo Identification and Induction of Articular Cartilage Stem Cells by Inhibiting NF-κB Signaling in Osteoarthritis.

PubMed

Tong, Wenxue; Geng, Yiyun; Huang, Yan; Shi, Yu; Xiang, Shengnan; Zhang, Ning; Qin, Ling; Shi, Qin; Chen, Qian; Dai, Kerong; Zhang, Xiaoling

2015-10-01

Osteoarthritis (OA) is a highly prevalent and debilitating joint disorder characterized by the degeneration of articular cartilage. However, no effective medical therapy has been found yet for such condition. In this study, we directly confirmed the existence of articular cartilage stem cells (ACSCs) in vivo and in situ for the first time both in normal and OA articular cartilage, and explored their chondrogenesis in Interleukin-1β (IL-1β) induced inflammation environment and disclose whether the inhibition of NF-κB signaling can induce ACSCs activation thus improve the progression of experimental OA. We found an interesting phenomenon that ACSCs were activated and exhibited a transient proliferative response in early OA as an initial attempt for self-repair. During the in vitro mechanism study, we discovered IL-1β can efficiently activate the NF-κB pathway and potently impair the responsiveness of ACSCs, whereas the NF-κB pathway inhibitor rescued the ACSCs chondrogenesis. The final in vivo experiments further confirmed ACSCs' activation were maintained by NF-κB pathway inhibitor, which induced cartilage regeneration, and protected articular cartilage from injury in an OA animal model. Our results provided in vivo evidence of the presence of ACSCs, and disclosed their action in the early OA stage and gradual quiet as OA process, presented a potential mechanism for both cartilage intrinsic repair and its final degradation, and demonstrated the feasibility of inducing endogenous adult tissue-specific mesenchymal stem cells for articular cartilage repair and OA therapy. © 2015 AlphaMed Press.

A Dual Role for SOX10 in the Maintenance of the Postnatal Melanocyte Lineage and the Differentiation of Melanocyte Stem Cell Progenitors

PubMed Central

Harris, Melissa L.; Buac, Kristina; Shakhova, Olga; Hakami, Ramin M.; Wegner, Michael; Sommer, Lukas; Pavan, William J.

2013-01-01

During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10fl; Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitfvga9) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitfvga9 does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage. PMID:23935512

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

PubMed Central

Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

2013-01-01

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

Signaling hierarchy regulating human endothelial cell development.

PubMed

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

Anti-aging Effect of Transplanted Amniotic Membrane Mesenchymal Stem Cells in a Premature Aging Model of Bmi-1 Deficiency

PubMed Central

Xie, Chunfeng; Jin, Jianliang; Lv, Xianhui; Tao, Jianguo; Wang, Rong; Miao, Dengshun

2015-01-01

To determine whether transplanted amniotic membrane mesenchymal stem cells (AMSCs) ameliorated the premature senescent phenotype of Bmi-1-deficient mice, postnatal 2-day-old Bmi-1−/− mice were injected intraperitoneally with the second-passage AMSCs from amniotic membranes of β-galactosidase (β-gal) transgenic mice or wild-type (WT) mice labeled with DiI. Three reinjections were given, once every seven days. Phenotypes of 5-week-old β-gal+ AMSC-transplanted or 6-week-old DiI+ AMSC-transplanted Bmi-1−/− mice were compared with vehicle-transplanted Bmi-1−/− and WT mice. Vehicle-transplanted Bmi-1−/− mice displayed growth retardation and premature aging with decreased cell proliferation and increased cell apoptosis; a decreased ratio and dysmaturity of lymphocytic series; premature osteoporosis with reduced osteogenesis and increased adipogenesis; redox imbalance and DNA damage in multiple organs. Transplanted AMSCs carried Bmi-1 migrated into multiple organs, proliferated and differentiated into multiple tissue cells, promoted growth and delayed senescence in Bmi-1−/− transplant recipients. The dysmaturity of lymphocytic series were ameliorated, premature osteoporosis were rescued by promoting osteogenesis and inhibiting adipogenesis, the oxidative stress and DNA damage in multiple organs were inhibited by the AMSC transplantation in Bmi-1−/− mice. These findings indicate that AMSC transplantation ameliorated the premature senescent phenotype of Bmi-1-deficient mice and could be a novel therapy to delay aging and prevent aging-associated degenerative diseases. PMID:26370922

Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage.

PubMed

Jiang, Dan; Gao, Fei; Zhang, Yuelin; Wong, David Sai Hung; Li, Qing; Tse, Hung-Fat; Xu, Goufeng; Yu, Zhendong; Lian, Qizhou

2016-11-10

Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Improvement of Carbon Tetrachloride-Induced Acute Hepatic Failure by Transplantation of Induced Pluripotent Stem Cells without Reprogramming Factor c-Myc

PubMed Central

Chang, Hua-Ming; Liao, Yi-Wen; Chiang, Chih-Hung; Chen, Yi-Jen; Lai, Ying-Hsiu; Chang, Yuh-Lih; Chen, Hen-Li; Jeng, Shaw-Yeu; Hsieh, Jung-Hung; Peng, Chi-Hsien; Li, Hsin-Yang; Chien, Yueh; Chen, Szu-Yu; Chen, Liang-Kung; Huo, Teh-Ia

2012-01-01

The only curative treatment for hepatic failure is liver transplantation. Unfortunately, this treatment has several major limitations, as for example donor organ shortage. A previous report demonstrated that transplantation of induced pluripotent stem cells without reprogramming factor c-Myc (3-genes iPSCs) attenuates thioacetamide-induced hepatic failure with minimal incidence of tumorigenicity. In this study, we investigated whether 3-genes iPSC transplantation is capable of rescuing carbon tetrachloride (CCl4)-induced fulminant hepatic failure and hepatic encephalopathy in mice. Firstly, we demonstrated that 3-genes iPSCs possess the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that exhibit biological functions and express various hepatic specific markers. 3-genes iPSCs also exhibited several antioxidant enzymes that prevented CCl4-induced reactive oxygen species production and cell death. Intraperitoneal transplantation of either 3-genes iPSCs or 3-genes iPSC-Heps significantly reduced hepatic necrotic areas, improved hepatic functions, and survival rate in CCl4-treated mice. CCl4-induced hepatic encephalopathy was also improved by 3-genes iPSC transplantation. Hoechst staining confirmed the successful engraftment of both 3-genes iPSCs and 3-genes iPSC-Heps, indicating the homing properties of these cells. The most pronounced hepatoprotective effect of iPSCs appeared to originate from the highest antioxidant activity of 3-gene iPSCs among all transplanted cells. In summary, our findings demonstrated that 3-genes iPSCs serve as an available cell source for the treatment of an experimental model of acute liver diseases. PMID:22489170

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.

PubMed

Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

2015-03-15

Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. © 2015. Published by The Company of Biologists Ltd.

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.

PubMed

Wu, Ting-Hsiang; Sagullo, Enrico; Case, Dana; Zheng, Xin; Li, Yanjing; Hong, Jason S; TeSlaa, Tara; Patananan, Alexander N; McCaffery, J Michael; Niazi, Kayvan; Braas, Daniel; Koehler, Carla M; Graeber, Thomas G; Chiou, Pei-Yu; Teitell, Michael A

2016-05-10

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.

PubMed

Rochette-Egly, Cécile

2015-01-01

Retinoic acid (RA), the active derivative of vitamin A, a fat-soluble vitamin, plays key roles in cell growth and differentiation by activating nuclear receptors, RARs (α, β and γ), which are ligand dependent regulators of transcription. The past years highlighted several novelties in the field that increased the complexity of RA effects. Indeed, in addition to its classical genomic effects, RA also has extranuclear and non-transcriptional effects. RA induces the rapid and transient activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of RARs at a conserved serine residue located in the N-terminal domain and their coregulators. In order to investigate the relevance of RARs' phosphorylation in cell differentiation, mouse embryonic stem (mES) cells were used as a model. When treated with RA, these pluripotent cells give rise to neuronal cells. Cells invalidated for each RAR were generated as well as stable rescue lines expressing RARs mutated in phosphor acceptor sites. Such a strategy revealed that RA-induced neuronal differentiation involves the RARγ2 subtype and requires RARγ2 phosphorylation. Moreover, in gene expression profiling experiments, the phosphorylated form of RARγ2 was found to regulate a small subset of genes through binding a novel RA response element consisting of two direct repeats with a 7 base pair spacer. These new findings suggest an important role for RAR phosphorylation during cell differentiation, and pave the way for further investigations with other cell types and during embryonic development. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina

PubMed Central

2012-01-01

Background Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina’s stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. Results The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. Conclusions These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins. PMID:23111152

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina.

PubMed

Luo, Jing; Uribe, Rosa A; Hayton, Sarah; Calinescu, Anda-Alexandra; Gross, Jeffrey M; Hitchcock, Peter F

2012-10-30

Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina's stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.

SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

PubMed Central

Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.

2011-01-01

SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168

Evaluation of the spectra Optia® mononuclear cell collection procedure in multiple myeloma patients.

PubMed

Long, Gwynn; Waller, Edmund K; Gregurek, Steven; Tricot, Guido; Marschner, Susanne; Bill, Jerry

2015-02-01

Peripheral blood stem cell (PBSC) rescue following myeloablative therapy is a mainstay of cancer therapy. To evaluate the ability of the Spectra Optia Apheresis System (SO), a newly developed apheresis device, the device was studied in multiple myeloma patients undergoing a first autologous PBSC transplant. To demonstrate that neutrophil recovery was not inferior to historical controls when SO harvested PBSCs were reinfused following myeloablative therapy. Multiple myeloma patients were mobilized according to the standard practice at four clinical sites. Following mobilization, MNC collections were performed on the SO. The collected cells were cryopreserved and reinfused following myeloablative chemotherapy. Neutrophil recovery defined by an absolute neutrophil count exceeding 500/μL (ANC500) was compared to historical data for patients transplanted following apheresis using the COBE Spectra (CS) device. The median day to neutrophil recovery was 12 days (range 10-14 days), with no significant difference in engraftment comparing patients transplanted with stem cells collected using the SO versus historical cohort of patients collected with the CS. CD34+ cell and MNC collection efficiency (CE) were 69.3% and 65.0% for the SO and CS, respectively. Platelet CE, product hematocrit and product granulocytes (as % of WBCs) using the SO were 21%, 2.3% and 28%, respectively. There were no device-related severe adverse events. The study's results confirm that the Spectra Optia Apheresis System's MNC Collection Protocol is safe and effective for its intended use and that engraftment kinetics of cells collected by SO is not inferior to the CS System. © 2014 Wiley Periodicals, Inc.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells

PubMed Central

Hyatt, Sam; Cheung, Kat; Skelton, Andrew J.; Xu, Yaobo; Clark, Ian M.

2017-01-01

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA. PMID:29084806

Energy utilization of induced pluripotent stem cell-derived cardiomyocyte in Fabry disease.

PubMed

Chou, Shih-Jie; Yu, Wen-Chung; Chang, Yuh-Lih; Chen, Wen-Yeh; Chang, Wei-Chao; Chien, Yueh; Yen, Jiin-Cherng; Liu, Yung-Yang; Chen, Shih-Jen; Wang, Chien-Ying; Chen, Yu-Han; Niu, Dau-Ming; Lin, Shing-Jong; Chen, Jaw-Wen; Chiou, Shih-Hwa; Leu, Hsin-Bang

2017-04-01

Fabry disease (FD) is a lysosomal storage disease in which glycosphingolipids (GB3) accumulate in organs of the human body, leading to idiopathic hypertrophic cardiomyopathy and target organ damage. Its pathophysiology is still poorly understood. We aimed to generate patient-specific induced pluripotent stem cells (iPSC) from FD patients presenting cardiomyopathy to determine whether the model could recapitulate key features of the disease phenotype and to investigate the energy metabolism in Fabry disease. Peripheral blood mononuclear cells from a 30-year-old Chinese man with a diagnosis of Fabry disease, GLA gene (IVS4+919G>A) mutation were reprogrammed into iPSCs and differentiated into iPSC-CMs and energy metabolism was analyzed in iPSC-CMs. The FD-iPSC-CMs recapitulated numerous aspects of the FD phenotype including reduced GLA activity, cellular hypertrophy, GB3 accumulation and impaired contractility. Decreased energy metabolism with energy utilization shift to glycolysis was observed, but the decreased energy metabolism was not modified by enzyme rescue replacement (ERT) in FD-iPSCs-CMs. This model provided a promising in vitro model for the investigation of the underlying disease mechanism and development of novel therapeutic strategies for FD. This potential remedy for enhancing the energetic network and utility efficiency warrants further study to identify novel therapies for the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

PubMed Central

Mills, Richard J.; Titmarsh, Drew M.; Koenig, Xaver; Parker, Benjamin L.; Ryall, James G.; Quaife-Ryan, Gregory A.; Voges, Holly K.; Hodson, Mark P.; Ferguson, Charles; Drowley, Lauren; Plowright, Alleyn T.; Needham, Elise J.; Wang, Qing-Dong; Gregorevic, Paul; Xin, Mei; Thomas, Walter G.; Parton, Robert G.; Nielsen, Lars K.; Elliott, David A.; Porrello, Enzo R.

2017-01-01

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies. PMID:28916735

Embryonal tumors with abundant neuropil and true rosettes: 2 illustrative cases and a review of the literature.

PubMed

Manjila, Sunil; Ray, Abhishek; Hu, Yin; Cai, Dan X; Cohen, Mark L; Cohen, Alan R

2011-01-01

Embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a recently identified variant of primitive neuroectodermal tumor, with fewer than 50 cases reported in the literature to date. Histologically, this tumor has features of ependymoblastoma and neuroblastoma, demonstrating areas of fine fibrillary neuropil intermingled with ependymoblastic rosettes and zones of undifferentiated neuroepithelial cells. However, ETANTR is distinguished pathologically from other embryonal tumors by the striking abundance of neuropil. Clinically, ETANTRs have shown high malignant potential and poor clinical outcome despite aggressive treatment. The authors describe 2 illustrative surgical cases of ETANTR, one involving the longest reported survival in the literature to date. The other had a poor outcome despite high-dose adjuvant chemotherapy with sequential autologous hematopoietic stem cell rescue. The authors review the natural history and treatment strategies available for this unusual malignant pediatric brain tumor.

Outcome of aplastic anaemia in children. A study by the severe aplastic anaemia and paediatric disease working parties of the European group blood and bone marrow transplant.

PubMed

Dufour, Carlo; Pillon, Marta; Sociè, Gerard; Rovò, Alicia; Carraro, Elisa; Bacigalupo, Andrea; Oneto, Rosi; Passweg, Jakob; Risitano, Antonio; Tichelli, Andrè; Peffault de Latour, Regis; Schrezenmeier, Hubert; Hocshmann, Britta; Peters, Christina; Kulasekararaj, Austin; Van Biezen, Anja; Samarasinghe, Sujith; Hussein, Ayad Ahmed; Ayas, Mouhab; Aljurf, Mahmoud; Marsh, Judith

2015-05-01

This study analysed the outcome of 563 Aplastic Anaemia (AA) children aged 0-12 years reported to the Severe Aplastic Anaemia Working Party database of the European Society for Blood and Marrow Transplantation, according to treatment received. Overall survival (OS) after upfront human leucocyte antigen-matched family donor (MFD) haematopoietic stem cell transplantation (HSCT) or immunosuppressive treatment (IST) was 91% vs. 87% (P 0·18). Event-free survival (EFS) after upfront MFD HSCT or IST was 87% vs. 33% (P 0·001). Ninety-one of 167 patients (55%) failed front-line IST and underwent rescue HSCT. The OS of this rescue group was 83% compared with 91% for upfront MFD HSCT patients and 97% for those who did not fail IST up-front (P 0·017). Rejection was 2% for MFD HSCT and HSCT post-IST failure (P 0·73). Acute graft-versus-host disease (GVHD) grade II-IV was 8% in MFD graft vs. 25% for HSCT post-IST failure (P < 0·0001). Chronic GVHD was 6% in MFD HSCT vs. 20% in HSCT post-IST failure (P < 0·0001). MFD HSCT is an excellent therapy for children with AA. IST has a high failure rate, but remains a reasonable first-line choice if MFD HSCT is not available because high OS enables access to HSCT, which is a very good rescue option. © 2015 John Wiley & Sons Ltd.

Selective T cell-depleted haploidentical hematopoietic stem cell transplantation for relapsed/refractory neuroblastoma.

PubMed

Liu, Anthony P Y; Lee, Pamela P W; Kwok, Janette S Y; Leung, Rock Y Y; Chiang, Alan K S; Ha, Shau-Yin; Cheuk, Daniel K L; Chan, Godfrey C F

2018-06-19

Relapsed/refractory NB carries a bleak outcome, warranting novel treatment options. HaploHSCT induces a graft-versus-NB effect via natural killer cell alloreactivity. Review of patients with relapsed/refractory NB who underwent haploHSCT with ex vivo T-cell depletion in our unit from 2013 through 2018. Ten patients were identified (male=5; median age at haploHSCT=6.45 y, range: 3.49-11.02 y). Indications were relapsed in 7 and refractoriness in 3; disease status at haploHSCT was CR in 2, PR in 6, and PD in 2. All patients received peripheral blood stem cell grafts after ex vivo T-cell depletion (CD3/CD19-depletion=1; TCR-αβ/CD19-depletion=4; CD3/CD45RA-depletion=4; and TCR-αβ/CD45RA-depletion=1). Conditioning regimens were fludarabine-based. Neutrophils engrafted on median D + 10 (range: D + 9 to +13), and platelets engrafted (≥20 × 10 9 /L) on median D + 8 (range: D + 5 to D + 14). Early T- and NK-cell recovery were evident. Of the 10 patients, acute rejection developed in 1 (who died of PD despite rescue HSCT), and 1 died of sepsis before engraftment; 8 experienced full donor-chimerism post-HSCT. Among the 8, 6 experienced CR, 1 died of PD, and 1 died of pulmonary hypertensive crisis before evaluation. At publication, 4 were in remission (2.8, 7.4, 28.5, and 58.9 months). No significant GvHD occurred. HaploHSCT with selective ex vivo T-cell depletion may be a safe and useful salvage strategy for relapsed/refractory NB. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Grafting of ARPE-19 and Schwann cells to the subretinal space in RCS rats.

PubMed

Wang, Shaomei; Lu, Bin; Wood, Patrick; Lund, Raymond D

2005-07-01

To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

High Glucose Concentrations Suppress the Proliferation of Human Periodontal Ligament Stem Cells and Their Differentiation Into Osteoblasts.

PubMed

Kato, Hirohito; Taguchi, Yoichiro; Tominaga, Kazuya; Kimura, Daisuke; Yamawaki, Isao; Noguchi, Masahiro; Yamauchi, Nobuhiro; Tamura, Isao; Tanaka, Akio; Umeda, Makoto

2016-04-01

Diabetes mellitus (DM) is a major risk factor for periodontal disease and affects various cellular functions. Periodontal ligament stem cells (PDLSCs) play an important role in periodontal tissue regeneration; however, the effect of hyperglycemia on PDLSCs is unclear. The aim of this study is to investigate whether hyperglycemia affects periodontal tissue regeneration, using human PDLSCs and high-glucose medium as a model of DM. PDLSCs were obtained from healthy adult human mandibular third molars. Cell proliferation, osteoblastic differentiation, and proinflammatory cytokine expression were investigated by culturing PDLSCs in media supplemented with four different glucose concentrations representative of control patients (5.5 mM), patients with postprandial or controlled DM (8.0 mM), and patients with uncontrolled DM (12.0 and 24.0 mM). The molecular effects of hyperglycemia on PDLSC physiology were examined with a focus on the nuclear factor (NF)-(κB signaling pathway. The involvement of NF-κB was investigated with a specific NF-κB inhibitor in PDLSCs under hyperglycemic conditions. High glucose levels inhibited PDLSC proliferation and differentiation into osteoblasts but induced NF-κB activation and subsequent interleukin (IL)-6 and IL-8 expression. Treatment with an NF-κB inhibitor rescued the defects in cell proliferation and osteoblastic differentiation and inhibited the IL-6 expression caused by the high-glucose environment. The results of this study demonstrate that hyperglycemia inhibits human PDLSC proliferation and osteoblastic differentiation.

Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells.

PubMed

Zhou, Miaojin; Hu, Zhiqing; Qiu, Liyan; Zhou, Tao; Feng, Mai; Hu, Qian; Zeng, Baitao; Li, Zhuo; Sun, Qianru; Wu, Yong; Liu, Xionghao; Wu, Lingqian; Liang, Desheng

2018-05-09

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

PubMed

Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

2018-05-01

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid Lymphocyte Reconstitution of Unconditioned Immunodeficient Mice with Non-Self-Renewing Multipotent Hematopoietic Progenitors

PubMed Central

Bhattacharya, Deepta; Bryder, David; Rossi, Derrick J.; Weissman, Irving L.

2015-01-01

The replacement of abnormal hematopoietic stem cells (HSCs) with normal transplanted HSCs can correct a wide range of hematologic disorders. Here, we provide evidence that transplantation of more differentiated progenitor cells can be used to more rapidly correct lymphoid deficiencies in unconditioned immunocompromised mice. Transplantation of flk2+ multipotent progenitors led to robust B and T cell reconstitution that was maintained for at least 16 weeks. Antigenic challenge at 16 weeks post-transplantation revealed that reconstituted lymphocytes maintained a functional repertoire. In contrast to the persistent lymphocytic engraftment, myeloid chimerism was lost by 12 weeks post-transplantation consistent with the fact that flk2+ progenitors are non-self-renewing. Thus, while more differentiated progenitors are capable of rescuing lymphoid deficiencies, transplantation of HSCs must be used for the correction of non-lymphoid disorders, and, we propose, very long-term immune reconstitution. Based on recent evidence, we discuss novel strategies to achieve the replacement of abnormal HSCs without the use of cytotoxic conditioning regimens. PMID:16760650

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

PubMed

Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition with β-catenin signaling in inflammatory microenvironments

PubMed Central

Chen, X; Hu, C; Wang, G; Li, L; Kong, X; Ding, Y; Jin, Y

2013-01-01

Inflammation can influence multipotency and self-renewal of mesenchymal stem cells (MSCs), resulting in their awakened bone-regeneration ability. Human periodontal ligament tissue-derived MSCs (PDLSCs) have been isolated, and their differentiation potential was found to be defective due to β-catenin signaling indirectly regulated by inflammatory microenvironments. Nuclear factor-κB (NF-κB) is well studied in inflammation by many different groups. The role of NF-κB needs to be studied in PDLSCs, although genetic evidences have recently shown that NF-κB inhibits osteoblastic bone formation in mice. However, the mechanism as to how inflammation leads to the modulation of β-catenin and NF-κB signaling remains unclear. In this study, we investigated β-catenin and NF-κB signaling through regulation of glycogen synthase kinase 3β activity (GSK-3β, which modulates β-catenin and NF-κB signaling) using a specific inhibitor LiCl and a phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002. We identified that NF-κB signaling might be more important for the regulation of osteogenesis in PDLSCs from periodontitis compared with β-catenin. BAY 11-7082 (an inhibitor of NF-κB) could inhibit phosphorylation of p65 and partly rescue the differentiation potential of PDLSCs in inflammation. Our data indicate that NF-κB has a central role in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. Given the molecular mechanisms of NF-κB in osteogenic differentiation governed by inflammation, it can be said that NF-κB helps in improving stem cell-mediated inflammatory bone disease therapy. PMID:23449446

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

PubMed

Valanejad, Leila; Lewis, Kyle; Wright, Mary; Jiang, Yanjun; D'Souza, Amber; Karns, Rebekah; Sheridan, Rachel; Gupta, Anita; Bove, Kevin; Witte, David; Geller, James; Tiao, Gregory; Nelson, David L; Timchenko, Lubov; Timchenko, Nikolai

2017-07-01

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Yanxia; Department of Rehabilitation, Xi'an Children's Hospital, Xi'an 710003; Liu, Xiaoguai

Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis alsomore » showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.« less

Feasibility, toxicity and response of upfront metaiodobenzylguanidine therapy therapy followed by German Pediatric Oncology Group Neuroblastoma 2004 protocol in newly diagnosed stage 4 neuroblastoma patients.

PubMed

Kraal, K C J M; Bleeker, G M; van Eck-Smit, B L F; van Eijkelenburg, N K A; Berthold, F; van Noesel, M M; Caron, H N; Tytgat, G A M

2017-05-01

Radiolabelled meta-iodobenzylguanidine (MIBG) is an effective option in treatment of neuroblastoma (NBL) tumours. We studied feasibility, toxicity and efficacy of upfront 131 I-MIBG and induction treatment in stage 4 NBL patients. Retrospective, multi-centre (AMC and EMC) pilot regimen (1/1/2005-2011). Newly diagnosed stage 4 NBL patients, were treated with 2 courses of 131 I-MIBG, GPOH 2004 NBL protocol, myeloablative therapy (MAT) and autologous stem cell rescue (ASCT). 131 I-MIBG was administered in a fixed dose. Response rate (RR) was defined as complete remission, very good partial response and partial response. Thirty-two patients, (median age [range] 2.9 [0-11.4] years), 21 received 131 I-MIBG therapy, 11 did not because of: MIBG non-avid (N = 5) and poor clinical condition (N = 6). In 95% of eligible patients 131 I-MIBG treatment was feasible within 2 weeks from diagnosis. Interval between chemotherapy courses was 25 days ( 131 I-MIBG group) versus 22 days (chemotherapy group). No stem cell support was needed after 131 I-MIBG therapy. Stem cell harvest in both groups was feasible, neutrophil recovery was comparable, but platelet recovery post MAT, ASCT was slower for 131 I-MIBG-treated patients. RR post 131 I-MIBG was 38%, post MAT + ASCT was 71% ( 131 I-MIBG group), 36% (chemotherapy group) and overall 59%. Induction therapy with 131 I-MIBG before the HR GPOH NB 2004 protocol is feasible, tolerable and effective in newly diagnosed stage 4 NBL patients. 131 I-MIBG upfront therapy induces early responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

PubMed

Romero-Moya, Damià; Santos-Ocaña, Carlos; Castaño, Julio; Garrabou, Gloria; Rodríguez-Gómez, José A; Ruiz-Bonilla, Vanesa; Bueno, Clara; González-Rodríguez, Patricia; Giorgetti, Alessandra; Perdiguero, Eusebio; Prieto, Cristina; Moren-Nuñez, Constanza; Fernández-Ayala, Daniel J; Victoria Cascajo, Maria; Velasco, Iván; Canals, Josep Maria; Montero, Raquel; Yubero, Delia; Jou, Cristina; López-Barneo, José; Cardellach, Francesc; Muñoz-Cánoves, Pura; Artuch, Rafael; Navas, Plácido; Menendez, Pablo

2017-07-01

Coenzyme Q 10 (CoQ 10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ 10 biosynthesis (COQ genes) cause primary CoQ 10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ 10 ], CoQ 10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4 ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4 ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ 10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ 10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703. © 2017 AlphaMed Press.

Genetic analysis of the role of amyloplasts in shoot gravisensing

NASA Astrophysics Data System (ADS)

Tasaka, M.; Morita, M.

Plant can change the growth direction after sensing the gravity orientation This response calls gravitropism and the initial step is the gravisensing We have isolated many Arabidopsis mutants shoot gravitropism sgr with reduced or no gravitropic response in inflorescence stems The analysis of sgr1 and sgr7 revealed that endoderm cells in the inflorescence stems were gravisensing sites zig zigzag sgr4 and sgr3 showed no or reduced gravitropism in shoot respectively and their amyloplasts thought to be statoliths did not sedimented to the orientation of gravity in the endoderm cells ZIG encoded a SNARE AtVTI11 and SGR3 encoded other SNARE AtVAM3 These two SNAREs made a complex in the shoot endoderm cells suggesting that the vesicle transport from trans-Golgi network TGN to prevacuolar compartment PVC and or vacuole was involved in the amyloplasts localization and movement The analysis to visualize amyloplasts and vacuolar membrane in living endoderm cells supported that the vacuole function was important for the amyloplasts movement Recently we have isolated many suppressor mutants of zig One of them named zig suppressor zip 1 had a point mutation in the gene encoded other SNARE of AtVTI12 This protein is a homologous to ZIG AtVTI11 and these two proteins have partially redundant functions Although wild type At VTI 12 could not rescued zig mutated AtVTI12 protein ZIP1 could almost completely play the part of ZIG In zigzip1 amyloplasts in endoderm cells sedimented normally and the shoots showed normal gravitropic response The other

Umbilical cord blood as an alternative source of reduced-intensity hematopoietic stem cell transplantation for chronic Epstein-Barr virus-associated T or natural killer cell lymphoproliferative diseases.

PubMed

Sawada, Akihisa; Inoue, Masami; Koyama-Sato, Maho; Kondo, Osamu; Yamada, Kayo; Shimizu, Mariko; Isaka, Kanako; Kimoto, Tomiko; Kikuchi, Hiroaki; Tokimasa, Sadao; Yasui, Masahiro; Kawa, Keisei

2014-02-01

Chronic Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases represented by chronic active Epstein-Barr virus infection are lethal but are curable with several courses of chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). Recently, we reported that reduced-intensity conditioning (RIC) provided better outcomes than myeloablative conditioning because RIC was less toxic. However, it was unclear whether cord blood transplantation (CBT) works in the context of RIC. We retrospectively analyzed 17 patients who underwent RIC followed by bone marrow transplantation (RIC-BMT) and 15 patients who underwent RIC followed by CBT (RIC-CBT). The representative regimen was fludarabine and melphalan based. The overall survival rates with RIC-BMT and RIC-CBT were 92.9% ± 6.9% and 93.3% ± 6.4%, respectively (P = .87). One patient died of lung graft-versus-host disease after RIC-BMT, and 1 patient died of multiple viral infections after RIC-CBT. Although cytotoxic chemotherapy was also immunosuppressive and might contribute to better donor cell engraftment after RIC-HSCT, the rate of engraftment failure after RIC-CBT was still higher than that after RIC-BMT (not significant); however, patients who had experienced graft failure were successfully rescued with a second HSCT. Unrelated cord blood can be an alternative source for RIC-HSCT if a patient has no family donor. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin.

PubMed

Zhang, Shiqiang; Chen, Shen; Li, Wen; Guo, Xiangpeng; Zhao, Ping; Xu, Jianyong; Chen, Yan; Pan, Qiong; Liu, Xiaorong; Zychlinski, Daniela; Lu, Hai; Tortorella, Micky D; Schambach, Axel; Wang, Yan; Pei, Duanqing; Esteban, Miguel A

2011-08-15

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging

PubMed Central

Tang, Duozhuang; Tao, Si; Chen, Zhiyang; Koliesnik, Ievgen Oleksandrovich; Calmes, Philip Gerald; Hoerr, Verena; Han, Bing; Gebert, Nadja; Zörnig, Martin; Löffler, Bettina

2016-01-01

Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways. PMID:26951333

Neuroprotective effect of mesenchymal stem cell through complement component 3 downregulation after transient focal cerebral ischemia in mice.

PubMed

Jung, Hye-Seon; Jeong, Si-Yeon; Yang, Jiwon; Kim, So-Dam; Zhang, Baojin; Yoo, Hyun Seung; Song, Sun U; Jeon, Myung-Shin; Song, Yun Seon

2016-10-28

Bone marrow-derived mesenchymal stem cells (MSCs) are used in stroke treatment despite the poor understanding of its mode of action. The immune suppressive and anti-inflammatory properties of MSCs possibly play important roles in regulating neuroinflammation after stroke. We investigated whether MSCs reduce the inflammatory complement component 3 (C3) levels, thus, providing neuroprotection during stroke. Mice were subjected to transient focal cerebral ischemia (tFCI), after which MSCs were intravenously injected. The infarct volume of the brain was reduced in MSC-injected tFCI mice, and C3 expression was significantly reduced in both the brain and the blood. Additionally, the profiles of other inflammatory mediators demonstrated neuroprotective changes in the MSCs-treated group. In order to analyze the effect of MSCs on neurons during cerebral ischemia, primary cortical neurons were co-cultured with MSCs under oxygen-glucose deprivation (OGD). Primary neurons co-cultured with MSCs exhibited reduced levels of C3 expression and increased protection against OGD, indicating that treatment with MSCs reduces excessive C3 expression and rescues ischemia-induced neuronal damage. Our finding suggests that reduction of C3 expression by MSCs can help to ameliorate ischemic brain damage, offering a new neuroprotective strategy in stroke therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

Positive feedback between RNA-binding protein HuD and transcription factor SATB1 promotes neurogenesis.

PubMed

Wang, Feifei; Tidei, Joseph J; Polich, Eric D; Gao, Yu; Zhao, Huashan; Perrone-Bizzozero, Nora I; Guo, Weixiang; Zhao, Xinyu

2015-09-08

The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.

Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation.

PubMed

Huang, Xinghua; Chen, Mo; Ding, Yan; Wang, Qin

2017-03-01

Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC-induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC-differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo. © 2017 Wiley Periodicals, Inc.

Delayed Administration of Bone Marrow Mesenchymal Stem Cell Conditioned Medium Significantly Improves Outcome After Retinal Ischemia in Rats

PubMed Central

Dreixler, John C.; Poston, Jacqueline N.; Balyasnikova, Irina; Shaikh, Afzhal R.; Tupper, Kelsey Y.; Conway, Sineadh; Boddapati, Venkat; Marcet, Marcus M.; Lesniak, Maciej S.; Roth, Steven

2014-01-01

Purpose. Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. Methods. Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-μm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. Results. Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. Conclusions. By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect. PMID:24699381

Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

PubMed

Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

2016-09-15

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

PubMed

Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

2007-05-30

Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Xiao; Sun, Yong; Han, Siqi

2015-01-02

Highlights: • First reported deregulation of miR-203 and up-regulation of BMI1 in metastatic melanoma. • miR-203 decreased BMI1 expression by directly binding to 3′UTR. • Further found miR-203 overexpression suppressed cell invasion and stemness. • Re-expression of BMI1 rescued miR-203-mediated suppression. • miR-203-BMI1 axis may be potential therapeutic targets of melanoma metastasis. - Abstract: Metastasis is the major problem in malignant melanoma, posing a therapeutic challenge to clinicians. The investigation of the underlying mechanism driving this progress remains a large unmet need. In this study, we revealed a miR-203-BMI1 axis that regulated melanoma metastasis. We found significantly deregulation of miR-203more » and up-regulation of BMI1 in melanoma, particularly in metastatic melanoma. An inverse correlation between the levels of miR-203 and BMI1 was further observed in melanoma tissues and cell lines. We also identified BMI1 as a downstream target gene of miR-203, which bound to the 3′UTR of BMI1. Overexpression of miR-203 was associated with decreased BMI1 expression and impaired cell invasion and tumor sphere formation activities. Re-expression of BMI1 markedly rescued miR-203-mediated suppression of these events. Taken together, our results demonstrated that miR-203 regulated melanoma invasive and proliferative abilities in part by targeting BMI1, providing new insights into potential mechanisms of melanoma metastasis.« less

MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells

PubMed Central

Collins, Carol M.; Ellis, Joseph A.

2017-01-01

ABSTRACT Mutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo. PMID:28188262

Arenavirus reverse genetics for vaccine development

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; Carlos de la Torre, Juan

2013-01-01

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1. PMID:23364194

Cell fusion in the brain: two cells forward, one cell back.

PubMed

Kemp, Kevin; Wilkins, Alastair; Scolding, Neil

2014-11-01

Adult stem cell populations, notably those which reside in the bone marrow, have been shown to contribute to several neuronal cell types in the rodent and human brain. The observation that circulating bone marrow cells can migrate into the central nervous system and fuse with, in particular, cerebellar Purkinje cells has suggested, at least in part, a potential mechanism behind this process. Experimentally, the incidence of cell fusion in the brain is enhanced with age, radiation exposure, inflammation, chemotherapeutic drugs and even selective damage to the neurons themselves. The presence of cell fusion, shown by detection of increased bi-nucleated neurons, has also been described in a variety of human central nervous system diseases, including both multiple sclerosis and Alzheimer's disease. Accumulating evidence is therefore raising new questions into the biological significance of cell fusion, with the possibility that it represents an important means of cell-mediated neuroprotection or rescue of highly complex neurons that cannot be replaced in adult life. Here, we discuss the evidence behind this phenomenon in the rodent and human brain, with a focus on the subsequent research investigating the physiological mechanisms of cell fusion underlying this process. We also highlight how these studies offer new insights into endogenous neuronal repair, opening new exciting avenues for potential therapeutic interventions against neurodegeneration and brain injury.

Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat.

PubMed

Michalkiewicz, Mieczyslaw; Michalkiewicz, Teresa; Ettinger, Ruth A; Rutledge, Elizabeth A; Fuller, Jessica M; Moralejo, Daniel H; Van Yserloo, Brian; MacMurray, Armand J; Kwitek, Anne E; Jacob, Howard J; Lander, Eric S; Lernmark, Ake

2004-10-04

A single point mutation in a novel immune-associated nucleotide gene 5 (Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344.lyp/lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344.lyp/lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.

Advanced glycation end product 3 (AGE3) suppresses the mineralization of mouse stromal ST2 cells and human mesenchymal stem cells by increasing TGF-β expression and secretion.

PubMed

Notsu, Masakazu; Yamaguchi, Toru; Okazaki, Kyoko; Tanaka, Ken-ichiro; Ogawa, Noriko; Kanazawa, Ippei; Sugimoto, Toshitsugu

2014-07-01

In diabetic patients, advanced glycation end products (AGEs) cause bone fragility because of deterioration of bone quality. We previously showed that AGEs suppressed the mineralization of mouse stromal ST2 cells. TGF-β is abundant in bone, and enhancement of its signal causes bone quality deterioration. However, whether TGF-β signaling is involved in the AGE-induced suppression of mineralization during the osteoblast lineage remains unknown. We therefore examined the roles of TGF-β in the AGE-induced suppression of mineralization of ST2 cells and human mesenchymal stem cells. AGE3 significantly (P < .001) inhibited mineralization in both cell types, whereas transfection with small interfering RNA for the receptor for AGEs (RAGEs) significantly (P < .05) recovered this process in ST2 cells. AGE3 increased (P < .001) the expression of TGF-β mRNA and protein, which was partially antagonized by transfection with RAGE small interfering RNA. Treatment with a TGF-β type I receptor kinase inhibitor, SD208, recovered AGE3-induced decreases in osterix (P < .001) and osteocalcin (P < .05) and antagonized the AGE3-induced increase in Runx2 mRNA expression in ST2 cells (P < .001). Moreover, SD208 completely and dose dependently rescued AGE3-induced suppression of mineralization in both cell types. In contrast, SD208 intensified AGE3-induced suppression of cell proliferation as well as AGE3-induced apoptosis in proliferating ST2 cells. These findings indicate that, after cells become confluent, AGE3 partially inhibits the differentiation and mineralization of osteoblastic cells by binding to RAGE and increasing TGF-β expression and secretion. They also suggest that TGF-β adversely affects bone quality not only in primary osteoporosis but also in diabetes-related bone disorder.

Melatonin rescued interleukin 1β-impaired chondrogenesis of human mesenchymal stem cells.

PubMed

Gao, Bo; Gao, Wenjie; Wu, Zizhao; Zhou, Taifeng; Qiu, Xianjian; Wang, Xudong; Lian, Chengjie; Peng, Yan; Liang, Anjing; Qiu, Jincheng; Zhu, Yuanxin; Xu, Caixia; Li, Yibing; Su, Peiqiang; Huang, Dongsheng

2018-06-14

Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted. Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs. The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling. The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

Mutual inhibition between HDAC9 and miR-17 regulates osteogenesis of human periodontal ligament stem cells in inflammatory conditions.

PubMed

Li, Liya; Liu, Wenjia; Wang, Hong; Yang, Qianjuan; Zhang, Liqiang; Jin, Fang; Jin, Yan

2018-04-24

Histone deacetylases (HDAC) plays important roles in the post-translational modifications of histone cores as well as non-histone targets. Many of them are involved in key inflammatory processes. Despite their importance, whether and how HDAC9 is regulated under inflammatory conditions remains unclear. The aim of this study was to evaluate the effects of HDAC9 under chronic inflammation condition in human periodontal ligament stromal cell (PDLSCs) and to explore the underlying regulatory mechanism. PDLSCs from healthy or periodontitis human tissue was compared. The therapeutic effects of HDAC inhibitors was determined in PDLSC pellet transplanted nude mice and LPS-induced rat periodontitis. We report that HDAC9 was the most affected HDAC family member under inflammatory conditions in PDLSCs. HDAC9 impaired osteogenic differentiation capacity of PDLSCs under inflammatory conditions. Downregulation of HDAC9 by HDAC inhibitors or si-HDAC9 rescued the osteogenic differentiation capacity of inflammatory PDLSC to a similar level with the healthy PDLSC. In this context, HDAC9 and miR-17 formed an inhibitory loop. The inhibition of miR-17 aggravated loss of calcified nodules in inflamed PDLSCs and interrupted the effect of HDAC inhibitor in rescuing osteogenesis. In vivo experiments using nude mice and LPS-induced periodontitis model confirmed that HDAC inhibitors could improve new bone formation. We conclude that HDAC inhibitors improved osteogenesis of PDLSCs in vitro and periodontitis in vivo.

Nell-1, a key Functional Mediator of Runx2, Partially Rescues Calvarial Defects in Runx2+/− Mice

PubMed Central

Zhang, Xinli; Ting, Kang; Bessette, Catherine M; Culiat, Cymbeline T; Sung, Sang Jin; Lee, Haofu; Chen, Feng; Shen, Jia; Wang, James J; Kuroda, Shun'ichi; Soo, Chia

2011-01-01

Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)–associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2+/−) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)–like phenotype of Runx2+/− mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2+/− but not Runx2−/− calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2+/+ newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)–induced Nell-1−/− NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2−/−) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent. © 2011 American Society for Bone and Mineral Research. PMID:20939017

Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

PubMed Central

Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

2014-01-01

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing

PubMed Central

Perriman, Rhonda J.; Ares, Manuel

2007-01-01

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6–U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing. PMID:17403781

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

PubMed Central

Chatterjee, Paroma; Padmanarayana, Murugesh; Abdullah, Nazish; Holman, Chelsea L.; LaDu, Jane; Tanguay, Robert L.

2015-01-01

Sensory hair cells convert mechanical motion into chemical signals. Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance. PMID:25582200

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

PubMed

Fonoudi, Hananeh; Ansari, Hassan; Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Kiani, Sahar; Hashemizadeh, Shiva; Zarchi, Ali Sharifi; Bosman, Alexis; Blue, Gillian M; Pahlavan, Sara; Perry, Matthew; Orr, Yishay; Mayorchak, Yaroslav; Vandenberg, Jamie; Talkhabi, Mahmood; Winlaw, David S; Harvey, Richard P; Aghdami, Nasser; Baharvand, Hossein

2015-12-01

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease. ©AlphaMed Press.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

PubMed Central

Fonoudi, Hananeh; Ansari, Hassan; Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Kiani, Sahar; Hashemizadeh, Shiva; Zarchi, Ali Sharifi; Bosman, Alexis; Blue, Gillian M.; Pahlavan, Sara; Perry, Matthew; Orr, Yishay; Mayorchak, Yaroslav; Vandenberg, Jamie; Talkhabi, Mahmood; Winlaw, David S.; Harvey, Richard P.; Aghdami, Nasser

2015-01-01

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Significance Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease. PMID:26511653

Efficient and Robust Paramyxoviridae Reverse Genetics Systems

PubMed Central

Beaty, Shannon M.; Won, Sohui T.; Hong, Patrick; Lyons, Michael; Vigant, Frederic; Freiberg, Alexander N.; tenOever, Benjamin R.; Duprex, W. Paul

2017-01-01

ABSTRACT The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors. PMID:28405630

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

PubMed Central

Tong, Kevin; Skibbens, Robert V.

2015-01-01

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures—processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated. PMID:25986377

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

PubMed

Tong, Kevin; Skibbens, Robert V

2015-06-02

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Upfront plerixafor plus G-CSF versus cyclophosphamide plus G-CSF for stem cell mobilization in multiple myeloma: efficacy and cost analysis study.

PubMed

Afifi, S; Adel, N G; Devlin, S; Duck, E; Vanak, J; Landau, H; Chung, D J; Lendvai, N; Lesokhin, A; Korde, N; Reich, L; Landgren, O; Giralt, S; Hassoun, H

2016-04-01

Cyclophosphamide plus G-CSF (C+G-CSF) is one of the most widely used stem cell (SC) mobilization regimens for patients with multiple myeloma (MM). Plerixafor plus G-CSF (P+G-CSF) has demonstrated superior SC mobilization efficacy when compared with G-CSF alone and has been shown to rescue patients who fail mobilization with G-CSF or C+G-CSF. Despite the proven efficacy of P+G-CSF in upfront SC mobilization, its use has been limited, mostly due to concerns of high price of the drug. However, a comprehensive comparison of the efficacy and cost effectiveness of SC mobilization using C+G-CSF versus P+G-CSF is not available. In this study, we compared 111 patients receiving C+G-CSF to 112 patients receiving P+G-CSF. The use of P+G-CSF was associated with a higher success rate of SC collection defined as ⩾5 × 10(6) CD34+ cells/kg (94 versus 83%, P=0.013) and less toxicities. Thirteen patients in the C+G-CSF arm were hospitalized owing to complications while none in the P+G-CSF group. C+G-CSF was associated with higher financial burden as assessed using institutional-specific costs and charges (P<0.001) as well as using Medicare reimbursement rates (P=0.27). Higher rate of hospitalization, increased need for salvage mobilization, and increased G-CSF use account for these differences.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

PubMed

Turan, Soeren; Farruggio, Alfonso P; Srifa, Waracharee; Day, John W; Calos, Michele P

2016-04-01

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

PubMed

Kobayashi, Isao; Kobayashi-Sun, Jingjing; Kim, Albert D; Pouget, Claire; Fujita, Naonobu; Suda, Toshio; Traver, David

2014-08-21

Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

Analysing the bioactive makeup of demineralised dentine matrix on bone marrow mesenchymal stem cells for enhanced bone repair.

PubMed

Avery, S J; Sadaghiani, L; Sloan, A J; Waddington, R J

2017-07-10

Dentine matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, the range of bioactive components in dentine and specific biological effects on bone-derived mesenchymal stem cells (MSCs) in humans are less well understood. The aims of this study were to further elucidate the biological response of MSCs to demineralised dentine matrix (DDM) in enhancing wound repair responses and ascertain key contributing components. Dentine was obtained from human teeth and DDM proteins solubilised with ethylenediaminetetraacetic acid (EDTA). Bone marrow derived MSCs were commercially obtained. Cells with a more immature phenotype were then selected by preferential fibronectin adhesion (FN-BMMSCs) for use in subsequent in vitro assays. DDM at 10 µg/mL reduced cell expansion, attenuated apoptosis and was the minimal concentration capable of inducing osteoblastic differentiation. Enzyme-linked immunosorbent assay (ELISA) quantification of growth factors indicated physiological levels produced the above responses; transforming growth factor β (TGF-β1) was predominant (15.6 ng/mg DDM), with relatively lower concentrations of BMP-2, FGF, VEGF and PDGF (6.2-4.7 ng/mg DDM). Fractionation of growth factors from other DDM components by heparin affinity chromatography diminished osteogenic responses. Depletion of biglycan from DDM also attenuated osteogenic potency, which was partially rescued by the isolated biglycan. Decorin depletion from DDM had no influence on osteogenic potency. Collectively, these results demonstrate the potential of DDM for the delivery of physiological levels of growth factors for bone repair processes, and substantiate a role for biglycan as an additional adjuvant for driving osteogenic pathways.

Deletion of TAK1 in the Myeloid Lineage Results in the Spontaneous Development of Myelomonocytic Leukemia in Mice

PubMed Central

Lamothe, Betty; Lai, YunJu; Hur, Lana; Orozco, Natalia Martin; Wang, Jing; Campos, Alejandro D.; Xie, Min; Schneider, Michael D.; Lockworth, Cynthia R.; Jakacky, Jared; Tran, Diep; Ho, Michael; Dawud, Sity; Dong, Chen; Lin, Hui-Kuan; Hu, Peter; Estrov, Zeev; Bueso-Ramos, Carlos E.; Darnay, Bryant G.

2012-01-01

Previous studies of the conditional ablation of TGF-β activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells’ increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1’s role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1’s role in leukemogenesis. PMID:23251462

OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

PubMed Central

Kaur, Ravinder; Aiken, Christopher; Morrison, Ludivine Coudière; Rao, Radhika; Del Bigio, Marc R.; Rampalli, Shravanti; Werbowetski-Ogilvie, Tamra

2015-01-01

ABSTRACT Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. PMID:26398939

Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue.

PubMed

Zhang, Miao; D'Aniello, Cristina; Verkerk, Arie O; Wrobel, Eva; Frank, Stefan; Ward-van Oostwaard, Dorien; Piccini, Ilaria; Freund, Christian; Rao, Jyoti; Seebohm, Guiscard; Atsma, Douwe E; Schulze-Bahr, Eric; Mummery, Christine L; Greber, Boris; Bellin, Milena

2014-12-16

Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and β subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K(+) current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.

Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution.

PubMed

Mosquna, Assaf; Katz, Aviva; Decker, Eva L; Rensing, Stefan A; Reski, Ralf; Ohad, Nir

2009-07-01

The Polycomb group (PcG) complex is involved in the epigenetic control of gene expression profiles. In flowering plants, PcG proteins regulate vegetative and reproductive programs. Epigenetically inherited states established in the gametophyte generation are maintained after fertilization in the sporophyte generation, having a profound influence on seed development. The gametophyte size and phase dominance were dramatically reduced during angiosperm evolution, and have specialized in flowering plants to support the reproductive process. The moss Physcomitrella patens is an ideal organism in which to study epigenetic processes during the gametophyte stage, as it possesses a dominant photosynthetic gametophytic haploid phase and efficient homologous recombination, allowing targeted gene replacement. We show that P. patens PcG protein FIE (PpFIE) accumulates in haploid meristematic cells and in cells that undergo fate transition during dedifferentiation programs in the gametophyte. In the absence of PpFIE, meristems overproliferate and are unable to develop leafy gametophytes or reach the reproductive phase. This aberrant phenotype might result from failure of the PcG complex to repress proliferation and differentiation of three-faced apical stem cells, which are designated to become lateral shoots. The PpFIE phenotype can be partially rescued by FIE of Arabidopsis thaliana, a flowering plant that diverged >450 million years ago from bryophytes. PpFIE can partially complement the A. thaliana fie mutant, illustrating functional conservation of the protein during evolution in regulating the differentiation of meristematic cells in gametophyte development, both in bryophytes and angiosperms. This mechanism was harnessed at the onset of the evolution of alternating generations, facilitating the establishment of sporophytic developmental programs.

Human amniotic fluid mesenchymal stem cells in combination with hyperbaric oxygen augment peripheral nerve regeneration.

PubMed

Pan, Hung-Chuan; Chin, Chun-Shih; Yang, Dar-Yu; Ho, Shu-Peng; Chen, Chung-Jung; Hwang, Shiaw-Min; Chang, Ming-Hong; Cheng, Fu-Chou

2009-07-01

Attenuation of pro-inflammatory cytokines and associated inflammatory cell deposits rescues human amniotic fluid mesenchymal stem cells (AFS) from apoptosis. Hyperbaric oxygen (HBO) suppressed stimulus-induced pro-inflammatory cytokine production in blood-derived monocyte-macrophages. Herein, we evaluate the beneficial effect of hyperbaric oxygen on transplanted AFS in a sciatic nerve injury model. Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. Hyperbaric oxygen (100% oxygen, 2 ATA, 60 min/day) was administered 12 h after operation for seven consecutive days. Transplanted cell apoptosis, oxidative stress, inflammatory cell deposits and associated chemokines, pro-inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 and 28 days after injury. Crush injury induced an inflammatory response, disrupted nerve integrity, and impaired nerve function in the sciatic nerve. However, crush injury-provoked inflammatory cytokines, deposits of inflammatory cytokines, and associated macrophage migration chemokines were attenuated in groups receiving hyperbaric oxygen but not in the AFS-only group. No significant increase in oxidative stress was observed after administration of HBO. In transplanted AFS, marked apoptosis was detected and this event was reduced by HBO treatment. Increased nerve myelination and improved motor function were observed in AFS-transplant, HBO-administrated, and AFS/HBO-combined treatment groups. Significantly, the AFS/HBO combined treatment showed the most beneficial effect. AFS in combination with HBO augment peripheral nerve regeneration, which may involve the suppression of apoptotic death in implanted AFS and the attenuation of an inflammatory response detrimental to peripheral nerve regeneration.

Post-treatment effects of local GDNF administration to the inner ears of deafened guinea pigs.

PubMed

Fransson, Anette; Maruyama, Jun; Miller, Josef M; Ulfendahl, Mats

2010-09-01

For patients with profound hearing loss, a cochlear implant is the only treatment available today. The function of a cochlear implant depends in part on the function and survival of spiral ganglion neurons. Following deafferentation, glial cell-derived neurotrophic factor (GDNF) is known to affect spiral ganglion neuron survival. The purpose of this study was to assess delayed GDNF treatment after deafening, the effects of cessation of GDNF treatment, and the effects of subsequent antioxidants on responsiveness and survival of the spiral ganglion neurons. Three-week deafened (by local neomycin administration) guinea pigs were implanted in the scala tympani with a combined cochlear implant electrode and cannula. GDNF (1 μg/mL) or artificial perilymph was then delivered for 4 weeks, following which the animals received systemic ascorbic acid  +  Trolox or saline for an additional 4 weeks. Thresholds for electrically-evoked auditory brain stem responses (eABRs) were significantly elevated at 3 weeks with deafness, stabilized with GDNF, and showed no change with GDNF cessation and treatment with antioxidants or saline. The populations of spiral ganglion neurons were reduced with deafness (by 40% at 3 weeks and 70% at 11 weeks), and rescued from cell death by GDNF with no further reduction at 8 weeks following 4 weeks of cessation of GDNF treatment equally in both the antioxidant- and saline-treated groups. Local growth factor treatment of the deaf ear may prevent deterioration in electrical responsiveness and rescue auditory nerve cells from death; these effects outlast the period of treatment, and may enhance the benefits of cochlear implant therapy for the deaf.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse.

PubMed

Engert, Silvia; Burtscher, Ingo; Liao, W Perry; Dulev, Stanimir; Schotta, Gunnar; Lickert, Heiko

2013-08-01

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Preservation of photoreceptors in dystrophic RCS rats following allo- and xenotransplantation of IPE cells.

PubMed

Thumann, Gabriele; Salz, Anna Katharina; Walter, Peter; Johnen, Sandra

2009-03-01

To examine whether iris pigment epithelial (IPE) cells transplanted into the subretinal space of Royal College of Surgeons (RCS) rats have the ability to rescue photoreceptors. Rat IPE (rIPE) or human IPE (hIPE) cells were transplanted subretinally in 23-day-old RCS rats. Sham injection and transplantation of ARPE-19 cells served as controls. After 12 weeks, eyes were evaluated for photoreceptor survival by morphometric analysis and electron microscopy. Morphometric analysis showed photoreceptor rescue in all transplanted and sham-injected animals (number of photoreceptors/300 microm retina+/-sd: rIPE 41.67 +/- 28; hIPE 29.50 +/- 16; ARPE-19 36.12 +/- 21; sham 16.56 +/- 6) compared to age-matched, control rats (number of photoreceptors/300 microm retina+/-sd: 9.71 +/- 4). Photoreceptor rescue was prominent in IPE cell-transplanted rats and was significantly greater than sham-injected eyes (p = 0.02 for rIPE and p = 0.04 for hIPE). Since IPE cells transplanted into the subretinal space have the ability to rescue photoreceptors from degeneration in the RCS rat without any harmful effects, IPE cells may represent an ideal cell to genetically modify and thus carry essential genetic information for the repair of defects in the subretinal space.

Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2 mediated regeneration of sinusoidal endothelial cells

PubMed Central

Hooper, Andrea T.; Butler, Jason M.; Nolan, Daniel J; Kranz, Andrea; Iida, Kaoruko; Kobayashi, Mariko; Kopp, Hans-Georg; Shido, Koji; Petit, Isabelle; Yanger, Kilangsungla; James, Daylon; Witte, Larry; Zhu, Zhenping; Wu, Yan; Pytowski, Bronislaw; Rosenwaks, Zev; Mittal, Vivek; Sato, Thomas N.; Rafii, Shahin

2011-01-01

SUMMARY The phenotypic attributes and molecular determinants for the regeneration of bone marrow (BM) sinusoidal endothelial cells (SECs) and their contribution to hematopoiesis are unknown. We show that after myelosuppression VEGFR2 activation promotes reassembly of regressed SECs, reconstituting hematopoietic stem and progenitor cells (HSPCs). VEGFR2 and VEGFR3 expression are restricted to BM vasculature, demarcating a continuous network of VEGFR2+VEGFR3+Sca1− SECs and VEGFR2+VEGFR3−Sca1+ arterioles. While chemotherapy (5FU) and sublethal irradiation (650 rad) induce minor SEC regression, lethal irradiation (950 rad) induces severe regression of SECs requiring BM transplantation (BMT) for regeneration. Conditional deletion of VEGFR2 in adult mice blocks regeneration of SECs in sublethally irradiated animals, preventing hematopoietic reconstitution. Inhibition of VEGFR2 signaling in lethally irradiated wild type mice rescued with BMT severely impairs SEC reconstruction, preventing engraftment and reconstitution of HSPCs. Therefore, activation of VEGFR2 is critical for regeneration of VEGFR3+Sca1− SECs that are essential for engraftment and restoration of HSPCs and hematopoiesis. PMID:19265665

Cyclic stretch induced miR-146a upregulation delays C2C12 myogenic differentiation through inhibition of Numb

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuang Wei; Department of Stomatology, Guangzhou General Hospital, Guangzhou Military Command, Guangzhou 510010; Tan Jiali

2009-01-09

Proliferation and differentiation of muscle stem cells must be tightly regulated by intrinsic and extrinsic signals for effective regeneration and adaptive response. MicroRNAs have been implicated as potent regulators in diverse biological processes at the level of posttranscriptional repression. In this study, we found that miR-146a was significantly upregulated upon a 48-h cyclic stretch of 5% elongation/10cycles/min. Importantly, miR-146 was predicted to base-pair with sequences in the 3' UTR of Numb, which promotes satellite cell differentiation towards muscle cells by inhibiting Notch signaling. Through reporter assay and exogenous expression experiment, we confirmed Numb was inhibited by miR-146a. Inhibition of miR-146amore » by antago-miR-146a rescued the expression of Numb and facilitated the differentiation of C2C12 at a cost of compromised proliferation. Thus, for the first time, we propose a role of miR-146a in skewing the balance of muscle differentiation and proliferation through inhibiting the expression of Numb.« less

FGFR4 signaling couples to Bim and not Bmf to discriminate subsets of alveolar rhabdomyosarcoma cells.

PubMed

Wachtel, Marco; Rakic, Jelena; Okoniewski, Michal; Bode, Peter; Niggli, Felix; Schäfer, Beat W

2014-10-01

Biological heterogeneity represents a major obstacle for cancer treatment. Therefore, characterization of treatment-relevant tumor heterogeneity is necessary to develop more effective therapies in the future. Here, we uncovered population heterogeneity among PAX/FOXO1-positive alveolar rhabdomyosarcoma by characterizing prosurvival networks initiated by FGFR4 signaling. We found that FGFR4 signaling rescues only subgroups of alveolar rhabdomyosarcoma cells from apoptosis induced by compounds targeting the IGF1R-PI3K-mTOR pathway. Differences in both proapoptotic machinery and FGFR4-activated signaling are involved in the different behavior of the phenotypes. Proapoptotic stress induced by the kinase inhibitors is sensed by Bim/Bad in rescue cells and by Bmf in nonrescue cells. Anti-apoptotic ERK1/2 signaling downstream of FGFR4 is long-lasting in rescue and short-termed in most non-rescue cells. Gene expression analysis detected signatures specific for these two groups also in biopsy samples. The different cell phenotypes are present in different ratios in alveolar rhabdomyosarcoma tumors and can be identified by AP2β expression levels. Hence, inhibiting FGFR signaling might represent an important strategy to enhance efficacy of current RMS treatments. © 2014 UICC.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

PubMed

Lorenz, Carmen; Lesimple, Pierre; Bukowiecki, Raul; Zink, Annika; Inak, Gizem; Mlody, Barbara; Singh, Manvendra; Semtner, Marcus; Mah, Nancy; Auré, Karine; Leong, Megan; Zabiegalov, Oleksandr; Lyras, Ekaterini-Maria; Pfiffer, Vanessa; Fauler, Beatrix; Eichhorst, Jenny; Wiesner, Burkhard; Huebner, Norbert; Priller, Josef; Mielke, Thorsten; Meierhofer, David; Izsvák, Zsuzsanna; Meier, Jochen C; Bouillaud, Frédéric; Adjaye, James; Schuelke, Markus; Wanker, Erich E; Lombès, Anne; Prigione, Alessandro

2017-05-04

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system. Copyright © 2016 Elsevier Inc. All rights reserved.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

PubMed

Swee, Lee Kim; Tardivel, Aubry; Schneider, Pascal; Rolink, Antonius

2010-06-15

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Adenovirus infection in pediatric transplant recipients: are effective antiviral agents coming our way?

PubMed

Lopez, Santiago M C; Michaels, Marian G; Green, Michael

2018-05-24

Adenoviruses (AdVs) infection is a self-limited disease in the majority of immunocompetent children and adults, but can cause disseminated and life-threatening illness in immunocompromised hosts. This article will discuss therapeutic strategies for AdV infection in the pediatrics transplant recipient. Currently, there is no FDA approved antiviral therapy for AdV infection. Accordingly, the primary initial therapy would be decreasing immunosuppression, whenever possible. Cidofovir (CDV) is an antiviral drug whose use has been associated with significant reductions of AdV viral load and, in some series improved survival in recipients of solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT). However, its use is also associated with significant toxicity. Brincidofovir (BCV) is a lipid formulation of CDV, which has an improved oral bioavailability and favorable toxicity profile compared with CDV. However, studies have only shown modest benefit from BCV for AdV disease or viremia. Immunotherapy is a growing field in the management of this virus infection on HSCT patients with promising results. Current evidence support the use of CDV and BCV, as rescue therapy, on SOT and HSCT transplant patients. Immunotherapy had only been proven successful in HSCT patients, as an option for refractory cases or rescue therapy for AdV infection.

Transfection of embryonated Muscovy duck eggs with a recombinant plasmid is suitable for rescue of infectious Muscovy duck parvovirus.

PubMed

Wang, Jianye; Huang, Yu; Ling, Jueyi; Wang, Zhixiang; Zhu, Guoqiang

2017-12-01

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

PubMed

Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

2017-05-30

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

PubMed Central

Nayak, Ramesh C.; Trump, Lisa R.; Aronow, Bruce J.; Myers, Kasiani; Mehta, Parinda; Kalfa, Theodosia; Wellendorf, Ashley M.; Valencia, C. Alexander; Paddison, Patrick J.; Horwitz, Marshall S.; Grimes, H. Leighton; Lutzko, Carolyn; Cancelas, Jose A.

2015-01-01

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient–derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient–derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization. PMID:26193632

Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter.

PubMed

Yang, Ruili; Yu, Tingting; Kou, Xiaoxing; Gao, Xiang; Chen, Chider; Liu, Dawei; Zhou, Yanheng; Shi, Songtao

2018-06-01

Ten-eleven translocation (Tet) family-mediated DNA oxidation represents an epigenetic modification capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), which regulates various biological processes. However, it is unknown whether Tet family affects mesenchymal stem cells (MSCs) or the skeletal system. Here we show that depletion of Tet1 and Tet2 results in impaired self-renewal and differentiation of bone marrow MSCs (BMMSCs) and a significant osteopenia phenotype. Tet1 and Tet2 deficiency reduces demethylation of the P2rX7 promoter and downregulates exosome release, leading to intracellular accumulation of miR-297a-5p, miR-297b-5p, and miR-297c-5p. These miRNAs inhibit Runx2 signaling to impair BMMSC function. We show that overexpression of P2rX7 rescues the impaired BMMSCs and osteoporotic phenotype in Tet1 and Tet2 double knockout mice. These results indicate that Tet1 and Tet2 play a critical role in maintaining BMMSC and bone homeostasis through demethylation of P2rX7 to control exosome and miRNA release. This Tet/P2rX7/Runx2 cascade may serve as a target for the development of novel therapies for osteopenia disorders.

An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ran-yi, E-mail: liuranyi@mail.sysu.edu.cn; Zhou, Ling; Zhang, Yan-ling

2013-12-13

Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endomore » via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.« less

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling.

PubMed

Alvarado, Alvaro G; Turaga, Soumya M; Sathyan, Pratheesh; Mulkearns-Hubert, Erin E; Otvos, Balint; Silver, Daniel J; Hale, James S; Flavahan, William A; Zinn, Pascal O; Sinyuk, Maksim; Li, Meizhang; Guda, Maheedhara R; Velpula, Kiran K; Tsung, Andrew J; Nakano, Ichiro; Vogelbaum, Michael A; Majumder, Sadhan; Rich, Jeremy N; Lathia, Justin D

2016-05-01

Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Artificial cloning of domestic animals

PubMed Central

Keefer, Carol L.

2015-01-01

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

Artificial cloning of domestic animals.

PubMed

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway.

PubMed

Pini, Jonathan; Giuliano, Serena; Matonti, Julia; Simkin, Dina; Rouleau, Matthieu; Bendahhou, Saïd

2018-05-29

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone, that is associated with mutations leading to a loss of function of the inwardly rectifying K + channel Kir2.1. While the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome induced Pluripotent Stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the down regulation master gene expression. This down regulation is due to an impairment of the bone morphogenetic proteins signaling pathway through de-phosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression, and restored normal osteoblasts and chondrocytes behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Parkin absence accelerates microtubule aging in dopaminergic neurons.

PubMed

Cartelli, Daniele; Amadeo, Alida; Calogero, Alessandra Maria; Casagrande, Francesca Vittoria Marialuisa; De Gregorio, Carmelita; Gioria, Mariarosa; Kuzumaki, Naoko; Costa, Ilaria; Sassone, Jenny; Ciammola, Andrea; Hattori, Nobutaka; Okano, Hideyuki; Goldwurm, Stefano; Roybon, Laurent; Pezzoli, Gianni; Cappelletti, Graziella

2018-01-01

Loss-of-function caused by mutations in the parkin gene (PARK2) lead to early-onset familial Parkinson's disease. Recently, mechanistic studies proved the ability of parkin in regulating mitochondria homeostasis and microtubule (MT) stability. Looking at these systems during aging of PARK2 knockout mice, we found that loss of parkin induced an accelerated (over)acetylation of MT system both in dopaminergic neuron cell bodies and fibers, localized in the substantia nigra and corpus striatum, respectively. Interestingly, in PARK2 knockout mice, changes of MT stability preceded the alteration of mitochondria transport. Moreover, in-cell experiments confirmed that loss of parkin affects mitochondria mobility and showed that this defect depends on MT system as it is rescued by paclitaxel, a well-known MT-targeted agent. Furthermore, both in PC12 neuronal cells and in patients' induced pluripotent stem cell-derived midbrain neurons, we observed that parkin deficiencies cause the fragmentation of stable MTs. Therefore, we suggest that parkin acts as a regulator of MT system during neuronal aging, and we endorse the hypothesis that MT dysfunction may be crucial in the pathogenesis of Parkinson's disease. Copyright © 2017 Elsevier Inc. All rights reserved.

A role for Lin28 in primordial germ cell development and germ cell malignancy

PubMed Central

West, Jason A.; Viswanathan, Srinivas R.; Yabuuchi, Akiko; Cunniff, Kerianne; Takeuchi, Ayumu; Park, In-Hyun; Sero, Julia E.; Zhu, Hao; Perez-Atayde, Antonio; Frazier, A. Lindsay; Surani, M. Azim; Daley, George Q.

2009-01-01

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwarts efforts to investigate molecular mechanisms of germ cell specification. Stella marks the minute founder population of the germ lineage1,2. Here we differentiate mouse embryonic stem cells (ESCs) carrying a Stella transgenic reporter into putative PGCs in vitro. The Stella+ cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella+ cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing3-6, is essential for proper PGC development. We further show that Blimp1, a let-7 target and a master regulator of PGC specification7-9, can rescue the effect of Lin28-deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Over-expression of Lin28 promotes formation of Stella+ cells in vitro and PGCs in chimeric embryos, and is associated with human germ cell tumours. The differentiation of putative PGCs from ESCs in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ cell development and malignancy. PMID:19578360

Macrophages sustain HIV replication in vivo independently of T cells.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Madden, Victoria; Sharpe, Garrett; Haase, Ashley T; Eron, Joseph J; Garcia, J Victor

2016-04-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

PubMed

Gao, Yuan; Xiao, Fei; Wang, Chenglong; Wang, Chuandong; Cui, Penglei; Zhang, Xiaoling; Chen, Xiaodong

2018-05-09

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation. © 2018 Wiley Periodicals, Inc.

Types of Stem Cells

MedlinePlus

... Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice

PubMed Central

Lv, Ya-Jie; Yang, Yi; Sui, Bing-Dong; Hu, Cheng-Hu; Zhao, Pan; Liao, Li; Chen, Ji; Zhang, Li-Qiang; Yang, Tong-Tao; Zhang, Shao-Feng; Jin, Yan

2018-01-01

Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging. Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents. However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown. Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism. Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models. In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM. Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated. Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined. Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments. Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs. Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription. Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS). Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss. More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence. Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence. PMID:29721087

Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice.

PubMed

Lv, Ya-Jie; Yang, Yi; Sui, Bing-Dong; Hu, Cheng-Hu; Zhao, Pan; Liao, Li; Chen, Ji; Zhang, Li-Qiang; Yang, Tong-Tao; Zhang, Shao-Feng; Jin, Yan

2018-01-01

Rational: Senescence of mesenchymal stem cells (MSCs) and the related functional decline of osteogenesis have emerged as the critical pathogenesis of osteoporosis in aging. Resveratrol (RESV), a small molecular compound that safely mimics the effects of dietary restriction, has been well documented to extend lifespan in lower organisms and improve health in aging rodents. However, whether RESV promotes function of senescent stem cells in alleviating age-related phenotypes remains largely unknown. Here, we intend to investigate whether RESV counteracts senescence-associated bone loss via osteogenic improvement of MSCs and the underlying mechanism. Methods: MSCs derived from bone marrow (BMMSCs) and the bone-specific, senescence-accelerated, osteoblastogenesis/osteogenesis-defective mice (the SAMP6 strain) were used as experimental models. In vivo application of RESV was performed at 100 mg/kg intraperitoneally once every other day for 2 months, and in vitro application of RESV was performed at 10 μM. Bone mass, bone formation rates and osteogenic differentiation of BMMSCs were primarily evaluated. Metabolic statuses of BMMSCs and the mitochondrial activity, transcription and morphology were also examined. Mitofilin expression was assessed at both mRNA and protein levels, and short hairpin RNA (shRNA)-based gene knockdown was applied for mechanistic experiments. Results: Chronic intermittent application of RESV enhances bone formation and counteracts accelerated bone loss, with RESV improving osteogenic differentiation of senescent BMMSCs. Furthermore, in rescuing osteogenic decline under BMMSC senescence, RESV restores cellular metabolism through mitochondrial functional recovery via facilitating mitochondrial autonomous gene transcription. Molecularly, in alleviating senescence-associated mitochondrial disorders of BMMSCs, particularly the mitochondrial morphological alterations, RESV upregulates Mitofilin, also known as inner membrane protein of mitochondria (Immt) or Mic60, which is the core component of the mitochondrial contact site and cristae organizing system (MICOS). Moreover, Mitofilin is revealed to be indispensable for mitochondrial homeostasis and osteogenesis of BMMSCs, and that insufficiency of Mitofilin leads to BMMSC senescence and bone loss. More importantly, Mitofilin mediates resveratrol-induced mitochondrial and osteogenic improvements of BMMSCs in senescence. Conclusion: Our findings uncover osteogenic functional improvements of senescent MSCs as critical impacts in anti-osteoporotic practice of RESV, and unravel Mitofilin as a novel mechanism mediating RESV promotion on mitochondrial function in stem cell senescence.

miRNA-regulated cancer stem cells: understanding the property and the role of miRNA in carcinogenesis.

PubMed

Chakraborty, Chiranjib; Chin, Kok-Yong; Das, Srijit

2016-10-01

Over the last few years, microRNAs (miRNA)-controlled cancer stem cells have drawn enormous attention. Cancer stem cells are a small population of tumor cells that possess the stem cell property of self-renewal. Recent data shows that miRNA regulates this small population of stem cells. In the present review, we explained different characteristics of cancer stem cells as well as miRNA regulation of self-renewal and differentiation in cancer stem cells. We also described the migration and tumor formation. Finally, we described the different miRNAs that regulate various types of cancer stem cells, such as prostate cancer stem cells, head and neck cancer stem cells, breast cancer stem cells, colorectal cancer stem cells, lung cancer stem cells, gastric cancer stem cells, pancreatic cancer stem cells, etc. Extensive research is needed in order to employ miRNA-based therapeutics to control cancer stem cell population in various cancers in the future.

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

PubMed

Schiroli, Giulia; Ferrari, Samuele; Conway, Anthony; Jacob, Aurelien; Capo, Valentina; Albano, Luisa; Plati, Tiziana; Castiello, Maria C; Sanvito, Francesca; Gennery, Andrew R; Bovolenta, Chiara; Palchaudhuri, Rahul; Scadden, David T; Holmes, Michael C; Villa, Anna; Sitia, Giovanni; Lombardo, Angelo; Genovese, Pietro; Naldini, Luigi

2017-10-11

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Targeting p38 Mitogen-Activated Protein Kinase Signaling Restores Subventricular Zone Neural Stem Cells and Corrects Neuromotor Deficits in Atm Knockout Mouse

PubMed Central

Kim, Jeesun

2012-01-01

Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm−/− mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm−/− mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm−/− mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm−/− mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm−/− mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm−/− mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders. PMID:23197859

What is a stem cell?

PubMed

Slack, Jonathan M W

2018-05-15

The historical roots of the stem cell concept are traced with respect to its usage in embryology and in hematology. The modern consensus definition of stem cells, comprising both pluripotent stem cells in culture and tissue-specific stem cells in vivo, is explained and explored. Methods for identifying stem cells are discussed with respect to cell surface markers, telomerase, label retention and transplantability, and properties of the stem cell niche are explored. The CreER method for identifying stem cells in vivo is explained, as is evidence in favor of a stochastic rather than an obligate asymmetric form of cell division. In conclusion, it is found that stem cells do not possess any unique and specific molecular markers; and stem cell behavior depends on the environment of the cell as well as the stem cell's intrinsic qualities. Furthermore, the stochastic mode of division implies that stem cell behavior is a property of a cell population not of an individual cell. In this sense, stem cells do not exist in isolation but only as a part of multicellular system. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells. © 2018 Wiley Periodicals, Inc.

A combination of cytokines EGF and CNTF protects the functional beta cell mass in mice with short-term hyperglycaemia.

PubMed

Lemper, Marie; De Groef, Sofie; Stangé, Geert; Baeyens, Luc; Heimberg, Harry

2016-09-01

When the beta cell mass or function declines beyond a critical point, hyperglycaemia arises. Little is known about the potential pathways involved in beta cell rescue. As two cytokines, epidermal growth factor (EGF) and ciliary neurotrophic factor (CNTF), restored a functional beta cell mass in mice with long-term hyperglycaemia by reprogramming acinar cells that transiently expressed neurogenin 3 (NGN3), the current study assesses the effect of these cytokines on the functional beta cell mass after an acute chemical toxic insult. Glycaemia and insulin levels, pro-endocrine gene expression and beta cell origin, as well as the role of signal transducer and activator of transcription 3 (STAT3) signalling, were assessed in EGF+CNTF-treated mice following acute hyperglycaemia. The mice were hyperglycaemic 1 day following i.v. injection of the beta cell toxin alloxan, when the two cytokines were applied. One week later, 68.6 ± 4.6% of the mice had responded to the cytokine treatment and increased their insulin(+) cell number to 30% that of normoglycaemic control mice, resulting in restoration of euglycaemia. Although insulin(-) NGN3(+) cells appeared following acute EGF+CNTF treatment, genetic lineage tracing showed that the majority of the insulin(+) cells originated from pre-existing beta cells. Beta cell rescue by EGF+CNTF depends on glycaemia rather than on STAT3-induced NGN3 expression in acinar cells. In adult mice, EGF+CNTF allows the rescue of beta cells in distress when treatment is given shortly after the diabetogenic insult. The rescued beta cells restore a functional beta cell mass able to control normal blood glucose levels. These findings may provide new insights into compensatory pathways activated early after beta cell loss.

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.

PubMed

Yang, Zhong-Fa; Zhang, Haojian; Ma, Leyuan; Peng, Cong; Chen, Yaoyu; Wang, Junling; Green, Michael R; Li, Shaoguang; Rosmarin, Alan G

2013-02-05

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility*

PubMed Central

Mitzelfelt, Katie A.; Limphong, Pattraranee; Choi, Melinda J.; Kondrat, Frances D. L.; Lai, Shuping; Kolander, Kurt D.; Kwok, Wai-Meng; Dai, Qiang; Grzybowski, Michael N.; Zhang, Huali; Taylor, Graydon M.; Lui, Qiang; Thao, Mai T.; Hudson, Judith A.; Barresi, Rita; Bushby, Kate; Jungbluth, Heinz; Wraige, Elizabeth; Geurts, Aron M.; Benesch, Justin L. P.; Riedel, Michael; Christians, Elisabeth S.; Minella, Alex C.; Benjamin, Ivor J.

2016-01-01

Mutations of HSPB5 (also known as CRYAB or αB-crystallin), a bona fide heat shock protein and molecular chaperone encoded by the HSPB5 (crystallin, alpha B) gene, are linked to multisystem disorders featuring variable combinations of cataracts, cardiomyopathy, and skeletal myopathy. This study aimed to investigate the pathological mechanisms involved in an early-onset myofibrillar myopathy manifesting in a child harboring a homozygous recessive mutation in HSPB5, 343delT. To study HSPB5 343delT protein dynamics, we utilize model cell culture systems including induced pluripotent stem cells derived from the 343delT patient (343delT/343delT) along with isogenic, heterozygous, gene-corrected control cells (WT KI/343delT) and BHK21 cells, a cell line lacking endogenous HSPB5 expression. 343delT/343delT and WT KI/343delT-induced pluripotent stem cell-derived skeletal myotubes and cardiomyocytes did not express detectable levels of 343delT protein, contributable to the extreme insolubility of the mutant protein. Overexpression of HSPB5 343delT resulted in insoluble mutant protein aggregates and induction of a cellular stress response. Co-expression of 343delT with WT prevented visible aggregation of 343delT and improved its solubility. Additionally, in vitro refolding of 343delT in the presence of WT rescued its solubility. We demonstrate an interaction between WT and 343delT both in vitro and within cells. These data support a loss-of-function model for the myopathy observed in the patient because the insoluble mutant would be unavailable to perform normal functions of HSPB5, although additional gain-of-function effects of the mutant protein cannot be excluded. Additionally, our data highlight the solubilization of 343delT by WT, concordant with the recessive inheritance of the disease and absence of symptoms in carrier individuals. PMID:27226619

Human Umbilical Cord Mesenchymal Stem Cells: Subpopulations and Their Difference in Cell Biology and Effects on Retinal Degeneration in RCS Rats.

PubMed

Wang, L; Li, P; Tian, Y; Li, Z; Lian, C; Ou, Q; Jin, C; Gao, F; Xu, J-Y; Wang, J; Wang, F; Zhang, J; Zhang, J; Li, W; Tian, H; Lu, L; Xu, G-T

2017-01-01

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential candidates for treating retinal degeneration (RD). To further study the biology and therapeutic effects of the hUC-MSCs on retinal degeneration. Two hUC-MSC subpopulations, termed hUC-MSC1 and hUC-MSC2, were isolated by single-cell cloning method and their therapeutic functions were compared in RCS rat, a RD model. Although both subsets satisfied the basic requirements for hUC-MSCs, they were significantly different in morphology, proliferation rate, differentiation capacity, phenotype and gene expression. Furthermore, only the smaller, fibroblast-like, faster growing subset hUC-MSC1 displayed stronger colony forming potential as well as adipogenic and osteogenic differentiation capacities. When the two subsets were respectively transplanted into the subretinal spaces of RCS rats, both subsets survived, but only hUC-MSC1 expressed RPE cell markers Bestrophin and RPE65. More importantly, hUC-MSC1 showed stronger rescue effect on the retinal function as indicated by the higher b-wave amplitude on ERG examination, thicker retinal nuclear layer, and decreased apoptotic photoreceptors. When both subsets were treated with interleukin-6, mimicking the inflammatory environment when the cells were transplanted into the eyes with degenerated retina, hUC-MSC1 expressed much higher levels of trophic factors in comparison with hUC-MSC2. The data here, in addition to prove the heterogeneity of hUC-MSCs, confirmed that the stronger therapeutic effects of hUC-MSC1 were attributed to its stronger anti-apoptotic effect, paracrine of trophic factors and potential RPE cell differentiation capacity. Thus, the subset hUC-MSC1, not the other subset or the ungrouped hUC-MSCs should be used for effective treatment of RD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Compass in Need of Alignment: Does United States Air Force Fighter Doctrine Adequately Cover Contingency Operations?

DTIC Science & Technology

1989-12-13

New York: Harcourt Brace Jovancv’.ch, 1983. Ben-Porat, Yeshayahu, Haber , Eitan and Schiff, Zeev. Entebbe Rescue. Trans. Louis Williams. New York...Times 25 Sept.e:ner 1989: pp. 16-17, 22-24, 84. Barth, Fritz J., Captain, USMC. "Stemming Nuclear Terrorism.- U.S. Naval Institute Proceedings, December

SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells.

PubMed

Kato, Toshiki; Khanh, Vuong Cat; Sato, Kazutoshi; Takeuchi, Kosuke; Carolina, Erica; Yamashita, Toshiharu; Sugaya, Hisashi; Yoshioka, Tomokazu; Mishima, Hajime; Ohneda, Osamu

2017-11-18

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons

PubMed Central

Mesngon, Mariano; McNutt, Patrick

2011-01-01

The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K+ had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca2+ influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery. PMID:22069721

Designer Self-Assemble Peptides Maximize the Therapeutic Benefits of Neural Stem Cell Transplantation for Alzheimer's Disease via Enhancing Neuron Differentiation and Paracrine Action.

PubMed

Cui, Guo-hong; Shao, Shui-jin; Yang, Jia-jun; Liu, Jian-ren; Guo, Hai-dong

2016-03-01

The neuropathological hallmarks of Alzheimer's disease (AD) include the presence of extracellular amyloid-β peptide (Aβ) in the form of amyloid plaques and neuronal loss. Neural stem cell (NSC) is being scrutinized as a promising cell replacement therapy for various neurodegenerative diseases. However, the unfavorable niche at the site of degenerative disease is hostile to the survival and differentiation of transplanted cells. Here, we undertook in vitro and in vivo works to examine whether a designer self-assemble peptide (DSP), which contains one functional domain Tyr-Ile-Gly-Ser-Arg (YIGSR) derived from laminin, promotes the survival and neuronal differentiation of NSC and behavioral improvement. We found that DSP could undergo spontaneous assembly into well-ordered nanofibers, and it not only facilitated the cell viability in normal culture condition, but also decreased the number of apoptotic cells induced by Aβ in vitro. NSC seeded in DSP showed much more neuronal differentiation than that seeded in self-assemble peptide (SP) or alone. In the AD model, NSC transplantation in DSP-treated AD rats demonstrated much more obvious cognitive rescue with restoration of learning/memory function compared with NSC transplantation in SP, NSC alone, or DSP alone treated ones. Interestingly, DSP enhanced the survival and neuronal differentiation of transplanted NSC. Apoptosis levels in the CA1 region and Aβ level in the hippocampus were significantly decreased in the group of NSC transplantation in DSP. Moreover, synaptic function, indicated by the expression of pre-synaptic protein synapsin-1, was restored and the secretion of anti-inflammatory and neurotrophic factors were increased, such as IL-10, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and insulin-like growth factor 1 (IGF-1), while the expression of pro-inflammatory factors were decreased, such as TNF-α and IL-1β. These data firstly unveiled that the biomaterial DSP can maximize the therapeutic benefits of NSC transplantation for AD through improving the survival and differentiation of transplanted stem cells and promoting the effects of neuroprotection, anti-neuroinflammatory and paracrine action. Our results may have important clinical implications for the design of future NSC-based strategies using the biomaterials for various neurodegenerative diseases including AD.

Defective Endochondral Ossification-Derived Matrix and Bone Cells Alter the Lymphopoietic Niche in Collagen X Mouse Models

PubMed Central

Sweeney, Elizabeth; Roberts, Douglas; Lin, Angela; Guldberg, Robert

2013-01-01

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders. PMID:23656481

Defective endochondral ossification-derived matrix and bone cells alter the lymphopoietic niche in collagen X mouse models.

PubMed

Sweeney, Elizabeth; Roberts, Douglas; Lin, Angela; Guldberg, Robert; Jacenko, Olena

2013-10-01

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders.

Differential marker expression by cultures rich in mesenchymal stem cells

PubMed Central

2013-01-01

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

Macrophages sustain HIV replication in vivo independently of T cells

PubMed Central

Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Sharpe, Garrett; Haase, Ashley T.; Eron, Joseph J.; Garcia, J. Victor

2016-01-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection. PMID:26950420

Learn About Stem Cells

MedlinePlus

... Handbook Stem Cell Glossary Search Toggle Nav Stem Cell Basics Stem cells are the foundation from which ... Home > Learn About Stem Cells > Stem Cell Basics Cells in the human body The human body comprises ...

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing.

PubMed

Chung, Woo Young; Song, Myungjae; Park, Jinhong; Namkung, Wan; Lee, Jinu; Kim, Hyongbum; Lee, Min Goo; Kim, Joo Young

2016-12-01

To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

PubMed

Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

2015-01-01

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. Copyright© Ferrata Storti Foundation.

[Progress in stem cells and regenerative medicine].

PubMed

Wang, Libin; Zhu, He; Hao, Jie; Zhou, Qi

2015-06-01

Stem cells have the ability to differentiate into all types of cells in the body and therefore have great application potential in regenerative medicine, in vitro disease modelling and drug screening. In recent years, stem cell technology has made great progress, and induced pluripotent stem cell technology revolutionizes the whole stem cell field. At the same time, stem cell research in our country has also achieved great progress and becomes an indispensable power in the worldwide stem cell research field. This review mainly focuses on the research progress in stem cells and regenerative medicine in our country since the advent of induced pluripotent stem cell technology, including induced pluripotent stem cells, transdifferentiation, haploid stem cells, and new gene editing tools.

Application of Graphene Based Nanotechnology in Stem Cells Research.

PubMed

Hu, Shanshan; Zeng, Yongxiang; Yang, Shuying; Qin, Han; Cai, He; Wang, Jian

2015-09-01

The past several years have witnessed significant advances in stem cell therapy, tissue engineering and regenerative medicine. Graphene, with its unique properties such as high electrical conductivity, elasticity and good molecule absorption, have potential for creating the next generation of biomaterials. This review summarizes the interrelationship between graphene and stem cells. The analysis of graphene when applied on mesenchymal stem cells, neural stem cells, induced pluripotent stem cells, embryonic stem cells, periodontal ligament stem cells, human adipose-derived stem cells and cancer stem cells, and how graphene influences cell behavior and differentiation are discussed in details.

A revisionist history of adult marrow stem cell biology or 'they forgot about the discard'.

PubMed

Quesenberry, P; Goldberg, L

2017-08-01

The adult marrow hematopoietic stem cell biology has largely been based on studies of highly purified stem cells. This is unfortunate because during the stem cell purification the great bulk of stem cells are discarded. These cells are actively proliferating. The final purified stem cell is dormant and not representative of the whole stem cell compartment. Thus, a large number of studies on the cellular characteristics, regulators and molecular details of stem cells have been carried on out of non-represented cells. Niche studies have largely pursued using these purified stem cells and these are largely un-interpretable. Other considerations include the distinction between baseline and transplant stem cells and the modulation of stem cell phenotype by extracellular vesicles, to cite a non-inclusive list. Work needs to proceed on characterizing the true stem cell population.

Perspectives on stem cell therapy for cardiac regeneration. Advances and challenges.

PubMed

Choi, Sung Hyun; Jung, Seok Yun; Kwon, Sang-Mo; Baek, Sang Hong

2012-01-01

Ischemic heart disease (IHD) accelerates cardiomyocyte loss, but the developing stem cell research could be useful for regenerating a variety of tissue cells, including cardiomyocytes. Diverse sources of stem cells for IHD have been reported, including embryonic stem cells, induced pluripotent stem cells, skeletal myoblasts, bone marrow-derived stem cells, mesenchymal stem cells, and cardiac stem cells. However, stem cells have unique advantages and disadvantages for cardiac tissue regeneration, which are important considerations in determining the specific cells for improving cell survival and long-term engraftment after transplantation. Additionally, the dosage and administration method of stem cells need to be standardized to increase stability and efficacy for clinical applications. Accordingly, this review presents a summary of the stem cell therapies that have been studied for cardiac regeneration thus far, and discusses the direction of future cardiac regeneration research for stem cells.

Stem Cells

MedlinePlus

Stem cells are cells with the potential to develop into many different types of cells in the body. They serve as a repair ... body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats.

PubMed

Arnhold, Stefan; Heiduschka, Peter; Klein, Helmut; Absenger, Yvonne; Basnaoglu, Serkan; Kreppel, Florian; Henke-Fahle, Sylvia; Kochanek, Stefan; Bartz-Schmidt, Karl-Ulrich; Addicks, Klaus; Schraermeyer, Ulrich

2006-09-01

To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats. Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected. In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments. Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.

The Role of Integrin α6 (CD49f) in Stem Cells: More than a Conserved Biomarker.

PubMed

Krebsbach, Paul H; Villa-Diaz, Luis G

2017-08-01

Stem cells have the capacity for self-renewal and differentiation into specialized cells that form and repopulated all tissues and organs, from conception to adult life. Depending on their capacity for differentiation, stem cells are classified as totipotent (ie, zygote), pluripotent (ie, embryonic stem cells), multipotent (ie, neuronal stem cells, hematopoietic stem cells, epithelial stem cells, etc.), and unipotent (ie, spermatogonial stem cells). Adult or tissue-specific stem cells reside in specific niches located in, or nearby, their organ or tissue of origin. There, they have microenvironmental support to remain quiescent, to proliferate as undifferentiated cells (self-renewal), and to differentiate into progenitors or terminally differentiated cells that migrate from the niche to perform specialized functions. The presence of proteins at the cell surface is often used to identify, classify, and isolate stem cells. Among the diverse groups of cell surface proteins used for these purposes, integrin α6, also known as CD49f, may be the only biomarker commonly found in more than 30 different populations of stem cells, including some cancer stem cells. This broad expression among stem cell populations indicates that integrin α6 may play an important and conserved role in stem cell biology, which is reaffirmed by recent demonstrations of its role maintaining self-renewal of pluripotent stem cells and breast and glioblastoma cancer stem cells. Therefore, this review intends to highlight and synthesize new findings on the importance of integrin α6 in stem cell biology.

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

DOE PAGES

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; ...

2016-02-04

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

3-O-sulfated heparan sulfate recognized by the antibody HS4C3 contributes [corrected] to the differentiation of mouse embryonic stem cells via fas signaling.

PubMed

Hirano, Kazumi; Sasaki, Norihiko; Ichimiya, Tomomi; Miura, Taichi; Van Kuppevelt, Toin H; Nishihara, Shoko

2012-01-01

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.

3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

PubMed Central

Hirano, Kazumi; Sasaki, Norihiko; Ichimiya, Tomomi; Miura, Taichi; Van Kuppevelt, Toin H.; Nishihara, Shoko

2012-01-01

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs. PMID:22916262

Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells.

PubMed

Cremers, Niels A J; Lundvig, Ditte M S; van Dalen, Stephanie C M; Schelbergen, Rik F; van Lent, Peter L E M; Szarek, Walter A; Regan, Raymond F; Carels, Carine E; Wagener, Frank A D T G

2014-10-08

Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

PubMed

Jones, Melissa K; Lu, Bin; Saghizadeh, Mehrnoosh; Wang, Shaomei

2016-01-01

Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell-based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. RNA-seq data of retinas from RCS rats injected with hNPCs (RCS(hNPCs)) were compared to sham surgery in RCS (RCS(sham)) and wild-type Long Evans (LE(sham)) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCS(sham) and LE(sham) samples. Additionally, 283 genes were differentially expressed between the RCS(hNPCs) and RCS(sham) samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCS(sham). Pathway analysis of the differential expression gene sets identified three affected pathways in RCS(hNPCs), which all play roles in phagocytosis signaling. Immunofluorescent staining detected the increased presence of macrophages and microglia in RCS(sham) retinas, which decreased in RCS(hNPCs) retinas similar to the patterns detected in LE(sham). The results from this study provide evidence of the gene expression changes that occur following treatment with hNPCs in the degenerating retina. This information can be used in future studies to potentially enhance or predict responses to hNPC and other stem cell therapies for retinal degenerative diseases.

Drosophila's contribution to stem cell research.

PubMed

Singh, Gyanesh

2015-01-01

The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. Recent developments in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs) are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub). Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd) proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila.

Drosophila's contribution to stem cell research

PubMed Central

Singh, Gyanesh

2016-01-01

The discovery of Drosophila stem cells with striking similarities to mammalian stem cells has brought new hope for stem cell research. Recent developments in Drosophila stem cell research is bringing wider opportunities for contemporary stem cell biologists. In this regard, Drosophila germ cells are becoming a popular model of stem cell research. In several cases, genes that controlled Drosophila stem cells were later discovered to have functional homologs in mammalian stem cells. Like mammals, Drosophila germline stem cells (GSCs) are controlled by both intrinsic as well as external signals. Inside the Drosophila testes, germline and somatic stem cells form a cluster of cells (the hub). Hub cells depend on JAK-STAT signaling, and, in absence of this signal, they do not self-renew. In Drosophila, significant changes occur within the stem cell niche that contributes to a decline in stem cell number over time. In case of aging Drosophila, somatic niche cells show reduced DE-cadherin and unpaired (Upd) proteins. Unpaired proteins are known to directly decrease stem cell number within the niches, and, overexpression of upd within niche cells restored GSCs in older males also . Stem cells in the midgut of Drosophila are also very promising. Reduced Notch signaling was found to increase the number of midgut progenitor cells. On the other hand, activation of the Notch pathway decreased proliferation of these cells. Further research in this area should lead to the discovery of additional factors that regulate stem and progenitor cells in Drosophila. PMID:26180635

Current overview on dental stem cells applications in regenerative dentistry.

PubMed

Bansal, Ramta; Jain, Aditya

2015-01-01

Teeth are the most natural, noninvasive source of stem cells. Dental stem cells, which are easy, convenient, and affordable to collect, hold promise for a range of very potential therapeutic applications. We have reviewed the ever-growing literature on dental stem cells archived in Medline using the following key words: Regenerative dentistry, dental stem cells, dental stem cells banking, and stem cells from human exfoliated deciduous teeth. Relevant articles covering topics related to dental stem cells were shortlisted and the facts are compiled. The objective of this review article is to discuss the history of stem cells, different stem cells relevant for dentistry, their isolation approaches, collection, and preservation of dental stem cells along with the current status of dental and medical applications.

Hip Osteoarthritis in Dogs: A Randomized Study Using Mesenchymal Stem Cells from Adipose Tissue and Plasma Rich in Growth Factors

PubMed Central

Cuervo, Belen; Rubio, Monica; Sopena, Joaquin; Dominguez, Juan Manuel; Vilar, Jose; Morales, Manuel; Cugat, Ramón; Carrillo, Jose Maria

2014-01-01

Purpose: The aim of this study was to compare the efficacy and safety of a single intra-articular injection of adipose mesenchymal stem cells (aMSCs) versus plasma rich in growth factors (PRGF) as a treatment for reducing symptoms in dogs with hip osteoarthritis (OA). Methods: This was a randomized, multicenter, blinded, parallel group. Thirty-nine dogs with symptomatic hip OA were assigned to one of the two groups, to receive aMSCs or PRGF. The primary outcome measures were pain and function subscales, including radiologic assessment, functional limitation and joint mobility. The secondary outcome measures were owners’ satisfaction questionnaire, rescue analgesic requirement and overall safety. Data was collected at baseline, then, 1, 3 and 6 months post-treatment. Results: OA degree did not vary within groups. Functional limitation, range of motion (ROM), owner’s and veterinary investigator visual analogue scale (VAS), and patient’s quality of life improved from the first month up to six months. The aMSCs group obtained better results at 6 months. There were no adverse effects during the study. Our findings show that aMSCs and PRGF are safe and effective in the functional analysis at 1, 3 and 6 months; provide a significant improvement, reducing dog’s pain, and improving physical function. With respect to basal levels for every parameter in patients with hip OA, aMSCs showed better results at 6 months. PMID:25089877

Enhanced susceptibility to stress and seizures in GAD65 deficient mice

PubMed Central

Qi, Jin; Kim, Minjung; Sanchez, Russell; Ziaee, Saba M; Kohtz, Jhumku D

2018-01-01

Reduced gamma-aminobutyric acid (GABA) inhibition has been implicated in both anxiety and epilepsy. GAD65-/- (NOD/LtJ) mice have significantly decreased basal GABA levels in the brain and a lowered threshold for seizure generation. One fifth of GAD65 -/- mice experienced stress-induced seizures upon exposure to an open field at 4 weeks of age. In each successive week until 8 weeks of age, the latency to seizures decreased with prior seizure experience. 100% of GAD65-/- mice exhibited stress-induced seizures by the end of 8 weeks. GAD65-/- mice also exhibited marked impairment in open field exploratory behavior and deficits in spatial learning acquisition on a Barnes maze. Anxiety-like behavior in an open field was observed prior to seizure onset and was predictive of subsequent seizures. Immunohistochemical characterization of interneuron subtypes in GAD65-/- mice showed a selective decrease in GABA and neuropeptide Y (NPY) levels and no change in calbindin (CLB) or calretinin (CLR) immunoreactivity in the hippocampus. Stem cells from the medial ganglionic eminence (MGE) were injected into the hippocampal hilus to restore GABAergic interneurons. One week after transplantation, MGE-transplanted mice demonstrated significant seizure resistance compared to sham surgical controls. The percent area of GFP+ MGE graft in the hippocampus correlated significantly with the increase in seizure latency. Our data indicate that impaired GABAergic neurotransmission can cause anxiety-like behavior and stress-induced seizures that can be rescued by MGE stem cell transplantation. PMID:29377906

Enhanced susceptibility to stress and seizures in GAD65 deficient mice.

PubMed

Qi, Jin; Kim, Minjung; Sanchez, Russell; Ziaee, Saba M; Kohtz, Jhumku D; Koh, Sookyong

2018-01-01

Reduced gamma-aminobutyric acid (GABA) inhibition has been implicated in both anxiety and epilepsy. GAD65-/- (NOD/LtJ) mice have significantly decreased basal GABA levels in the brain and a lowered threshold for seizure generation. One fifth of GAD65 -/- mice experienced stress-induced seizures upon exposure to an open field at 4 weeks of age. In each successive week until 8 weeks of age, the latency to seizures decreased with prior seizure experience. 100% of GAD65-/- mice exhibited stress-induced seizures by the end of 8 weeks. GAD65-/- mice also exhibited marked impairment in open field exploratory behavior and deficits in spatial learning acquisition on a Barnes maze. Anxiety-like behavior in an open field was observed prior to seizure onset and was predictive of subsequent seizures. Immunohistochemical characterization of interneuron subtypes in GAD65-/- mice showed a selective decrease in GABA and neuropeptide Y (NPY) levels and no change in calbindin (CLB) or calretinin (CLR) immunoreactivity in the hippocampus. Stem cells from the medial ganglionic eminence (MGE) were injected into the hippocampal hilus to restore GABAergic interneurons. One week after transplantation, MGE-transplanted mice demonstrated significant seizure resistance compared to sham surgical controls. The percent area of GFP+ MGE graft in the hippocampus correlated significantly with the increase in seizure latency. Our data indicate that impaired GABAergic neurotransmission can cause anxiety-like behavior and stress-induced seizures that can be rescued by MGE stem cell transplantation.

Microtubule catastrophe and rescue.

PubMed

Gardner, Melissa K; Zanic, Marija; Howard, Jonathon

2013-02-01

Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

The longest telomeres: a general signature of adult stem cell compartments

PubMed Central

Flores, Ignacio; Canela, Andres; Vera, Elsa; Tejera, Agueda; Cotsarelis, George; Blasco, María A.

2008-01-01

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age. PMID:18283121

Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

PubMed

Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

2015-11-01

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

Impaired Mitochondrial Dynamics Underlie Axonal Defects in Hereditary Spastic Paraplegias.

PubMed

Denton, Kyle; Mou, Yongchao; Xu, Chong-Chong; Shah, Dhruvi; Chang, Jaerak; Blackstone, Craig; Li, Xue-Jun

2018-05-02

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

Stanniocalcin-1 Rescued Photoreceptor Degeneration in Two Rat Models of Inherited Retinal Degeneration

PubMed Central

Roddy, Gavin W; Rosa Jr, Robert H; Youn Oh, Joo; Ylostalo, Joni H; Bartosh, Thomas J; Choi, Hosoon; Lee, Ryang Hwa; Yasumura, Douglas; Ahern, Kelly; Nielsen, Gregory; Matthes, Michael T; LaVail, Matthew M; Prockop, Darwin J

2012-01-01

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD). PMID:22294148

Context clues: the importance of stem cell-material interactions

PubMed Central

Murphy, William L.

2014-01-01

Understanding the processes by which stem cells give rise to de novo tissues is an active focus of stem cell biology and bioengineering disciplines. Instructive morphogenic cues surrounding the stem cell during morphogenesis create what is referred to as the stem cell microenvironment. An emerging paradigm in stem cell bioengineering involves “biologically driven assembly,” in which stem cells are encouraged to largely define their own morphogenesis processes. However, even in the case of biologically driven assembly, stem cells do not act alone. The properties of the surrounding microenvironment can be critical regulators of cell fate. Stem cell-material interactions are among the most well-characterized microenvironmental effectors of stem cell fate, and they establish a signaling “context” that can define the mode of influence for morphogenic cues. Here we describe illustrative examples of cell-material interactions that occur during in vitro stem cell studies, with an emphasis on how cell-material interactions create instructive contexts for stem cell differentiation and morphogenesis. PMID:24369691

Cancer stem cells and differentiation therapy.

PubMed

Jin, Xiong; Jin, Xun; Kim, Hyunggee

2017-10-01

Cancer stem cells can generate tumors from only a small number of cells, whereas differentiated cancer cells cannot. The prominent feature of cancer stem cells is its ability to self-renew and differentiate into multiple types of cancer cells. Cancer stem cells have several distinct tumorigenic abilities, including stem cell signal transduction, tumorigenicity, metastasis, and resistance to anticancer drugs, which are regulated by genetic or epigenetic changes. Like normal adult stem cells involved in various developmental processes and tissue homeostasis, cancer stem cells maintain their self-renewal capacity by activating multiple stem cell signaling pathways and inhibiting differentiation signaling pathways during cancer initiation and progression. Recently, many studies have focused on targeting cancer stem cells to eradicate malignancies by regulating stem cell signaling pathways, and products of some of these strategies are in preclinical and clinical trials. In this review, we describe the crucial features of cancer stem cells related to tumor relapse and drug resistance, as well as the new therapeutic strategy to target cancer stem cells named "differentiation therapy."

Gα13 Mediates a Signal That Is Essential for Proliferation and Survival of Thymocyte Progenitors

PubMed Central

McNeil Coffield, V.; Helms, Whitney S.; Jiang, Qi; Su, Lishan

2004-01-01

G protein signaling via the Gα12 family (Gα12 and Gα13) has not been well studied in T cells. To investigate whether Gα12 and Gα13 are involved in thymopoiesis, we expressed the regulator of G protein signaling domain of p115RhoGEF to inhibit Gα12 and Gα13 during thymopoiesis. Fetal thymus organ cultures seeded with p115ΔDH-expressing progenitor cells showed impaired thymopoiesis with a block at the CD4−CD8−CD44−CD25+ (DN3) stage. Using Gα13 or Gα12 minigenes, we demonstrated that Gα13, but not Gα12, is required for thymopoiesis. T progenitor cells expressing p115ΔDH showed reduced proliferation and increased cell death. T cell receptor stimulation of the fetal thymus organ cultures did not rescue the block. Overexpression of the antiapoptotic gene Bcl2 rescued the defect in DN3 cells and partially rescued T cell development. Therefore, Gα13-mediated signaling is necessary in early thymocyte proliferation and survival. PMID:15534370

Clinical trials for stem cell transplantation: when are they needed?

PubMed

Van Pham, Phuc

2016-04-27

In recent years, both stem cell research and the clinical application of these promising cells have increased rapidly. About 1000 clinical trials using stem cells have to date been performed globally. More importantly, more than 10 stem cell-based products have been approved in some countries. With the rapid growth of stem cell applications, some countries have used clinical trials as a tool to diminish the rate of clinical stem cell applications. However, the point at which stem cell clinical trials are essential remains unclear. This commentary discusses when stem cell clinical trials are essential for stem cell transplantation therapies.

Stem cells - biological update and cell therapy progress

PubMed Central

GIRLOVANU, MIHAI; SUSMAN, SERGIU; SORITAU, OLGA; RUS-CIUCA, DAN; MELINCOVICI, CARMEN; CONSTANTIN, ANNE-MARIE; MIHU, CARMEN MIHAELA

2015-01-01

In recent years, the advances in stem cell research have suggested that the human body may have a higher plasticity than it was originally expected. Until now, four categories of stem cells were isolated and cultured in vivo: embryonic stem cells, fetal stem cells, adult stem cells and induced pluripotent stem cells (hiPSCs). Although multiple studies were published, several issues concerning the stem cells are still debated, such as: the molecular mechanisms of differentiation, the methods to prevent teratoma formation or the ethical and religious issues regarding especially the embryonic stem cell research. The direct differentiation of stem cells into specialized cells: cardiac myocytes, neural cells, pancreatic islets cells, may represent an option in treating incurable diseases such as: neurodegenerative diseases, type I diabetes, hematologic or cardiac diseases. Nevertheless, stem cell-based therapies, based on stem cell transplantation, remain mainly at the experimental stages and their major limitation is the development of teratoma and cancer after transplantation. The induced pluripotent stem cells (hiPSCs) represent a prime candidate for future cell therapy research because of their significant self-renewal and differentiation potential and the lack of ethical issues. This article presents an overview of the biological advances in the study of stem cells and the current progress made in the field of regenerative medicine. PMID:26609255

Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome.

PubMed

Chin, Eunice W M; Marcy, Guillaume; Yoon, Su-In; Ma, Dongliang; Rosales, Francisco J; Augustine, George J; Goh, Eyleen L K

2016-09-01

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155Δ32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT.

Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy

PubMed Central

Xu, Huaigeng

2017-01-01

In the past decade, the development of two innovative technologies, namely, induced pluripotent stem cells (iPSCs) and the CRISPR Cas9 system, has enabled researchers to model diseases derived from patient cells and precisely edit DNA sequences of interest, respectively. In particular, Duchenne muscular dystrophy (DMD) has been an exemplary monogenic disease model for combining these technologies to demonstrate that genome editing can correct genetic mutations in DMD patient-derived iPSCs. DMD is an X-linked genetic disorder caused by mutations that disrupt the open reading frame of the dystrophin gene, which plays a critical role in stabilizing muscle cells during contraction and relaxation. The CRISPR Cas9 system has been shown to be capable of targeting the dystrophin gene and rescuing its expression in in vitro patient-derived iPSCs and in vivo DMD mouse models. In this review, we highlight recent advances made using the CRISPR Cas9 system to correct genetic mutations and discuss how emerging CRISPR technologies and iPSCs in a combined platform can play a role in bringing a therapy for DMD closer to the clinic. PMID:28607562

Embryonic stem cell trials for macular degeneration: a preliminary report.

PubMed

Schwartz, Steven D; Hubschman, Jean-Pierre; Heilwell, Gad; Franco-Cardenas, Valentina; Pan, Carolyn K; Ostrick, Rosaleen M; Mickunas, Edmund; Gay, Roger; Klimanskaya, Irina; Lanza, Robert

2012-02-25

It has been 13 years since the discovery of human embryonic stem cells (hESCs). Our report provides the first description of hESC-derived cells transplanted into human patients. We started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystrophy and dry age-related macular degeneration--the leading cause of blindness in the developed world. Preoperative and postoperative ophthalmic examinations included visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing. These studies are registered with ClinicalTrials.gov, numbers NCT01345006 and NCT01344993. Controlled hESC differentiation resulted in greater than 99% pure RPE. The cells displayed typical RPE behaviour and integrated into the host RPE layer forming mature quiescent monolayers after transplantation in animals. The stage of differentiation substantially affected attachment and survival of the cells in vitro after clinical formulation. Lightly pigmented cells attached and spread in a substantially greater proportion (>90%) than more darkly pigmented cells after culture. After surgery, structural evidence confirmed cells had attached and continued to persist during our study. We did not identify signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection in either patient during the first 4 months. Although there is little agreement between investigators on visual endpoints in patients with low vision, it is encouraging that during the observation period neither patient lost vision. Best corrected visual acuity improved from hand motions to 20/800 (and improved from 0 to 5 letters on the Early Treatment Diabetic Retinopathy Study [ETDRS] visual acuity chart) in the study eye of the patient with Stargardt's macular dystrophy, and vision also seemed to improve in the patient with dry age-related macular degeneration (from 21 ETDRS letters to 28). The hESC-derived RPE cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months. The future therapeutic goal will be to treat patients earlier in the disease processes, potentially increasing the likelihood of photoreceptor and central visual rescue. Advanced Cell Technology. Copyright © 2012 Elsevier Ltd. All rights reserved.

NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network.

PubMed

Anderson, David J; Kaplan, David I; Bell, Katrina M; Koutsis, Katerina; Haynes, John M; Mills, Richard J; Phelan, Dean G; Qian, Elizabeth L; Leitoguinho, Ana Rita; Arasaratnam, Deevina; Labonne, Tanya; Ng, Elizabeth S; Davis, Richard P; Casini, Simona; Passier, Robert; Hudson, James E; Porrello, Enzo R; Costa, Mauro W; Rafii, Arash; Curl, Clare L; Delbridge, Lea M; Harvey, Richard P; Oshlack, Alicia; Cheung, Michael M; Mummery, Christine L; Petrou, Stephen; Elefanty, Andrew G; Stanley, Edouard G; Elliott, David A

2018-04-10

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.

Establishment of mouse expanded potential stem cells

PubMed Central

Gao, Xuefei; Antunes, Liliana; Yu, Yong; Zhu, Zhexin; Wang, Juexuan; Kolodziejczyk, Aleksandra A.; Campos, Lia S.; Wang, Cui; Yang, Fengtang; Zhong, Zhen; Fu, Beiyuan; Eckersley-Maslin, Melanie A.; Woods, Michael; Tanaka, Yosuke; Chen, Xi; Wilkinson, Adam C.; Bussell, James; White, Jacqui; Ramirez-Solis, Ramiro; Reik, Wolf; Göttgens, Berthold; Teichmann, Sarah A.; Tam, Patrick P. L.; Nakauchi, Hiromitsu; Zou, Xiangang; Lu, Liming; Liu, Pentao

2018-01-01

Mouse embryonic stem cells derived from the epiblast1 contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm2 upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species. PMID:29019987

Donor CD8+ T Cells Prevent Toxoplasma gondii De-Encystation but Fail To Rescue the Exhausted Endogenous CD8+ T Cell Population

PubMed Central

Bhadra, Rajarshi; Cobb, Dustin A.

2013-01-01

Functional exhaustion of CD8+ T cells due to increased expression of inhibitory molecule PD-1 (Programmed Death-1) causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from a primarily encysted, quiescent bradyzoite to a fast-replicating, highly motile tachyzoite. Thus, reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting gamma interferon (IFN-γ), a cytokine known to control latent toxoplasmosis, in chronically infected prerecrudescent mice. Since CD8+ T cells (an important source of IFN-γ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8+ T cells from acutely infected donors to the chronically infected prerecrudescent hosts could impede parasite de-encystation and rescue exhausted CD8+ T cells. While the transfer of immune CD8+ T cells temporarily restricted the breakdown of cysts, the exhausted endogenous CD8+ T cell population was not rescued. Over time, the donor population got deleted, resulting in parasite de-encystation and host mortality. Considering that donor CD8+ T cells fail to become long-lived, one of the cardinal features of memory CD8+ T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover, our data strongly suggest that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in the chronic phase by itself is insufficient for rescue of exhausted CD8+ T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis. PMID:23817617

Adult Stem Cell Therapy for Stroke: Challenges and Progress

PubMed Central

Bang, Oh Young; Kim, Eun Hee; Cha, Jae Min; Moon, Gyeong Joon

2016-01-01

Stroke is one of the leading causes of death and physical disability among adults. It has been 15 years since clinical trials of stem cell therapy in patients with stroke have been conducted using adult stem cells like mesenchymal stem cells and bone marrow mononuclear cells. Results of randomized controlled trials showed that adult stem cell therapy was safe but its efficacy was modest, underscoring the need for new stem cell therapy strategies. The primary limitations of current stem cell therapies include (a) the limited source of engraftable stem cells, (b) the presence of optimal time window for stem cell therapies, (c) inherited limitation of stem cells in terms of growth, trophic support, and differentiation potential, and (d) possible transplanted cell-mediated adverse effects, such as tumor formation. Here, we discuss recent advances that overcome these hurdles in adult stem cell therapy for stroke. PMID:27733032

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

PubMed

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.