Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

PubMed

Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

2015-07-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

Identification of Multipotent Stem/Progenitor Cells in Murine Sclera

PubMed Central

Tsai, Chia-Ling; Wu, Pei-Chang; Fini, M. Elizabeth; Shi, Songtao

2011-01-01

Purpose. The sclera forms the fibrous outer coat of the eyeball and acts as a supportive framework. The purpose of this study was to examine whether the sclera contains mesenchymal stem/progenitor cells. Method. Scleral tissue from C57BL6/J mice was separated from the retina and choroid and subsequently enzyme digested to release single cells. Proliferation capacity, self-renewal capacity, and ability for multipotent differentiation were analyzed by BrdU labeling, flow cytometry, reverse transcriptase–polymerase chain reaction, immunocytochemistry, and in vivo transplantation. Results. The scleral stem/progenitor cells (SSPCs) possessed clonogenic and high doubling capacities. These cells were positive for the mesenchymal markers Sca-1, CD90.2, CD44, CD105, and CD73 and negative for the hematopoietic markers CD45, CD11b, Flk1, CD34, and CD117. In addition to expressing stem cell genes ABCG2, Six2, Notch1, and Pax6, SSPCs were able to differentiate to adipogenic, chondrogenic, and neurogenic lineages. Conclusions. This study indicates that the sclera contains multipotent mesenchymal stem cells. Further study of SSPCs may help elucidate the cellular and molecular mechanism of scleral diseases such as scleritis and myopia. PMID:21788434

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

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

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo.

PubMed

Cottle, Beverley J; Lewis, Fiona C; Shone, Victoria; Ellison-Hughes, Georgina M

2017-07-04

The development of cellular therapies to treat muscle wastage with disease or age is paramount. Resident muscle satellite cells are not currently regarded as a viable cell source due to their limited migration and growth capability ex vivo. This study investigated the potential of muscle-derived PW1 + /Pax7 - interstitial progenitor cells (PICs) as a source of tissue-specific stem/progenitor cells with stem cell properties and multipotency. Sca-1 + /PW1 + PICs were identified on tissue sections from hind limb muscle of 21-day-old mice, isolated by magnetic-activated cell sorting (MACS) technology and their phenotype and characteristics assessed over time in culture. Green fluorescent protein (GFP)-labelled PICs were used to determine multipotency in vivo in a tumour formation assay. Isolated PICs expressed markers of pluripotency (Oct3/4, Sox2, and Nanog), were clonogenic, and self-renewing with >60 population doublings, and a population doubling time of 15.8 ± 2.9 h. PICs demonstrated an ability to generate both striated and smooth muscle, whilst also displaying the potential to differentiate into cell types of the three germ layers both in vitro and in vivo. Moreover, PICs did not form tumours in vivo. These findings open new avenues for a variety of solid tissue engineering and regeneration approaches, utilising a single multipotent stem cell type isolated from an easily accessible source such as skeletal muscle.

Clonal analysis of lineage fate in native haematopoiesis.

PubMed

Rodriguez-Fraticelli, Alejo E; Wolock, Samuel L; Weinreb, Caleb S; Panero, Riccardo; Patel, Sachin H; Jankovic, Maja; Sun, Jianlong; Calogero, Raffaele A; Klein, Allon M; Camargo, Fernando D

2018-01-11

Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ.

Potential feasibility of dental stem cells for regenerative therapies: stem cell transplantation and whole-tooth engineering.

PubMed

Nakahara, Taka

2011-07-01

Multipotent mesenchymal stem cells from bone marrow are expected to be a somatic stem cell source for the development of new cell-based therapy in regenerative medicine. However, dental clinicians are unlikely to carry out autologous cell/tissue collection from patients (i.e., marrow aspiration) as a routine procedure in their clinics; hence, the utilization of bone marrow stem cells seems impractical in the dental field. Dental tissues harvested from extracted human teeth are well known to contain highly proliferative and multipotent stem cell compartments and are considered to be an alternative autologous cell source in cell-based medicine. This article provides a short overview of the ongoing studies for the potential application of dental stem cells and suggests the utilization of 2 concepts in future regenerative medicine: (1) dental stem cell-based therapy for hepatic and other systemic diseases and (2) tooth replacement therapy using the bioengineered human whole tooth, called the "test-tube dental implant." Regenerative therapies will bring new insights and benefits to the fields of clinical medicine and dentistry.

Combinatorial effect of substratum properties on mesenchymal stem cell sheet engineering and subsequent multi-lineage differentiation.

PubMed

Chuah, Yon Jin; Zhang, Ying; Wu, Yingnan; Menon, Nishanth V; Goh, Ghim Hian; Lee, Ann Charlene; Chan, Vincent; Zhang, Yilei; Kang, Yuejun

2015-09-01

Cell sheet engineering has been exploited as an alternative approach in tissue regeneration and the use of stem cells to generate cell sheets has further showed its potential in stem cell-mediated tissue regeneration. There exist vast interests in developing strategies to enhance the formation of stem cell sheets for downstream applications. It has been proved that stem cells are sensitive to the biophysical cues of the microenvironment. Therefore we hypothesized that the combinatorial substratum properties could be tailored to modulate the development of cell sheet formation and further influence its multipotency. For validation, polydimethylsiloxane (PDMS) of different combinatorial substratum properties (including stiffness, roughness and wettability) were created, on which the human bone marrow derived mesenchymal stem cells (BMSCs) were cultured to form cell sheets with their multipotency evaluated after induced differentiation. The results showed that different combinatorial effects of these substratum properties were able to influence BMSC behavior such as adhesion, spreading and proliferation during cell sheet development. Collagen formation within the cell sheet was enhanced on substrates with lower stiffness, higher hydrophobicity and roughness, which further assisted the induced chondrogenesis and osteogenesis, respectively. These findings suggested that combinatorial substratum properties had profound effects on BMSC cell sheet integrity and multipotency, which had significant implications for future biomaterials and scaffold designs in the field of BMSC-mediated tissue regeneration. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

PubMed Central

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-01-01

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

PubMed

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-06-06

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

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

Curtis, Brandon M.; Leix, Kyle Alexander; Ji, Yajing

Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to suchmore » injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.« less

Expansion of Multipotent Stem Cells from the Adult Human Brain

PubMed Central

Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

2013-01-01

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

Adherent culture conditions enrich the side population obtained from the cochlear modiolus-derived stem/progenitor cells.

PubMed

Chao, Ting-Ting; Wang, Chih-Hung; Chen, Hsin-Chien; Shih, Cheng-Ping; Sytwu, Huey-Kang; Huang, Kun-Lun; Chen, Shao-Yuan

2013-05-01

Previously, our group reported that sphere-forming cells derived from the organ of Corti represent the stem/progenitor cells (SPCs) of the cochlea due to their properties of self-renewal and multipotency. However, long-term propagation of sphere-forming cells under suspension culture conditions may fail to maintain the characteristic stemness of these cells. Therefore, this study investigated whether an adherent culture system would be beneficial in terms of preserving more stem-like cells for long-term manipulations in vitro. Isolated modiolus-derived SPCs were placed on poly-d-lysine-coated petri dishes to form the so-called "adherent" culture system. Modiolus SPCs cultured under adherent conditions exhibited a significantly increased percentage of cells with the side population (SP) phenotype (18.6%) compared with cells cultured under conventional suspension culture conditions (0.8%). Even after repeated passages, modiolus SPCs cultured under adherent culture conditions preserved more SP phenotype cells. In comparison with the non-SP phenotype cells, the sorted SP cells exhibited more stem-like but less differentiated properties, with an upregulated expression of the ATP-binding cassette subfamily G member 2 (ABCG2), Nestin, Sox2, and Nanog proteins. Furthermore, Retinoic acid (RA) treatment confirmed the expression of the multipotent differentiation markers in the SP cells, including TUJ1, pancytokeratin, glial fibrillary acidic protein (GFAP), and p27(Kip1). Employment of an adherent culture system, instead of a suspension culture system, resulted in the enrichment of the SP cells from SPCs while retaining their stemness and multipotency. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

PubMed Central

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-01-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes. PMID:25688859

Luminal progenitors restrict their lineage potential during mammary gland development.

PubMed

Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

2015-02-01

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy.

PubMed

Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

2010-06-26

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.

Labeling and Imaging Mesenchymal Stem Cells with Quantum Dots

EPA Science Inventory

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, adipose and muscle cells. Adult derived MSCs are being actively investigated because of their potential to be utilized for therapeutic cell-based transplantation. Methods...

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

PubMed

Ebert, Allison D; Shelley, Brandon C; Hurley, Amanda M; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N; Svendsen, Soshana P; Mattis, Virginia B; McGivern, Jered V; Schwab, Andrew J; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N

2013-05-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Prenatal Exposure to the Environmental Obesogen Tributyltin Predisposes Multipotent Stem Cells to Become Adipocytes

PubMed Central

Kirchner, Séverine; Kieu, Tiffany; Chow, Connie; Casey, Stephanie; Blumberg, Bruce

2010-01-01

The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor γ (PPARγ). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARγ activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARγ antagonists, suggesting that activation of PPARγ mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARγ target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time. PMID:20160124

Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes.

PubMed

Kirchner, Séverine; Kieu, Tiffany; Chow, Connie; Casey, Stephanie; Blumberg, Bruce

2010-03-01

The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARgamma). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARgamma activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARgamma antagonists, suggesting that activation of PPARgamma mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARgamma target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

PubMed

Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

2017-03-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy

PubMed Central

Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

2010-01-01

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivo studies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders. PMID:21607121

Yolk Sac Mesenchymal Progenitor Cells from New World Mice (Necromys lasiurus) with Multipotent Differential Potential

PubMed Central

Favaron, Phelipe Oliveira; Mess, Andrea; Will, Sônia Elisabete; Maiorka, Paulo César; de Oliveira, Moacir Franco; Miglino, Maria Angelica

2014-01-01

Fetal membranes are abundant, ethically acceptable and readily accessible sources of stem cells. In particular, the yolk sac is a source of cell lineages that do not express MHCs and are mainly free from immunological incompatibles when transferred to a recipient. Although data are available especially for hematopoietic stem cells in mice and human, whereas other cell types and species are dramatically underrepresented. Here we studied the nature and differentiation potential of yolk sac derived mesenchymal stem cells from a New World mouse, Necromys lasiurus. Explants from mid-gestation were cultured in DMEM-High glucose medium with 10% defined fetal bovine serum. The cells were characterized by standard methods including immunophenotyping by fluorescence and flow cytometry, growth and differentiation potential and tumorigenicity assays. The first adherent cells were observed after 7 days of cell culture and included small, elongated fibroblast-like cells (92.13%) and large, round epithelial-like cells with centrally located nuclei (6.5%). Only the fibroblast-like cells survived the first passages. They were positive to markers for mesenchymal stem cells (Stro-1, CD90, CD105, CD73) and pluripotency (Oct3/4, Nanog) as well as precursors of hematopoietic stem cells (CD117). In differentiation assays, they were classified as a multipotent lineage, because they differentiated into osteogenic, adipogenic, and chondrogenic lineages and, finally, they did not develop tumors. In conclusion, mesenchymal progenitor cells with multipotent differentiation potential and sufficient growth and proliferation abilities were able to be obtained from Necromys yolk sacs, therefore, we inferred that these cells may be promising for a wide range of applications in regenerative medicine. PMID:24918429

Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

PubMed Central

Matigian, Nicholas; Brooke, Gary; Zaibak, Faten; Rossetti, Tony; Kollar, Katarina; Pelekanos, Rebecca; Heazlewood, Celena; Mackay-Sim, Alan; Wells, Christine A.; Atkinson, Kerry

2014-01-01

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues. PMID:26484151

Hallmarks of pluripotency.

PubMed

De Los Angeles, Alejandro; Ferrari, Francesco; Xi, Ruibin; Fujiwara, Yuko; Benvenisty, Nissim; Deng, Hongkui; Hochedlinger, Konrad; Jaenisch, Rudolf; Lee, Soohyun; Leitch, Harry G; Lensch, M William; Lujan, Ernesto; Pei, Duanqing; Rossant, Janet; Wernig, Marius; Park, Peter J; Daley, George Q

2015-09-24

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

PubMed

Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

2016-08-01

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/β-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The development of human mast cells. An historical reappraisal

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

Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it

2016-03-15

The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34{sup +}/CD117{sup +}/CD13{sup +}multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, differentmore » MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.« less

Asymmetric Distribution of GFAP in Glioma Multipotent Cells

PubMed Central

Guichet, Pierre-Olivier; Guelfi, Sophie; Ripoll, Chantal; Teigell, Marisa; Sabourin, Jean-Charles; Bauchet, Luc; Rigau, Valérie; Rothhut, Bernard; Hugnot, Jean-Philippe

2016-01-01

Asymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate. PMID:26953813

Osteoarthritis-derived chondrocytes are a potential source of multipotent progenitor cells for cartilage tissue engineering

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

Oda, Tomoyuki; Sakai, Tadahiro; Hiraiwa, Hideki

The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growthmore » rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering. - Highlights: • Osteoarthritis chondrocytes (OACs) have multilineage differentiation capacity. • Articular chondrocytes (ACs) and OACs have similar gene expression profiles. • OACs have high chondrogenic potential. • OACs could be a cell resource for cartilage tissue engineering.« less

Novel Stem Cell Therapies for Applications to Wound Healing and Tissue Repair.

PubMed

Grada, Ayman; Falanga, Vincent

2016-10-26

The number of individuals with chronic cutaneous wounds has been increasing worldwide due to an aging population, diabetes, obesity, and cardiovascular disease. In the United States, almost seven million Americans have chronic skin ulcers. Many therapeutic approaches have been used. However, the treatment outcomes are not always ideal because of failure to achieve complete wound closure in around 60% of cases, scarring, and high rate of recurrence. Therefore, there is a need for more effective therapies. Stem cells offer promising possibilities. Pre-clinical studies have shown that bone- or adipose tissue-derived mesenchymal stem cells (MSCs) have a competitive advantage over other types of stem cells due to their better defined multipotent differentiating potential, paracrine effects, immunomodulatory properties, and safety. However, large controlled clinical trials are needed to examine the capabilities of MSCs in humans and to assess their safety profile. In this review, we highlight emerging treatments in tissue regeneration and repair and provide some perspectives on how to translate current knowledge about stem cells-both multipotent and pluripotent-into viable clinical approaches for treating patients with difficult to heal wounds.

Dissecting Stages of Human Kidney Development and Tumorigenesis with Surface Markers Affords Simple Prospective Purification of Nephron Stem Cells.

PubMed

Pode-Shakked, Naomi; Pleniceanu, Oren; Gershon, Rotem; Shukrun, Rachel; Kanter, Itamar; Bucris, Efrat; Pode-Shakked, Ben; Tam, Gal; Tam, Hadar; Caspi, Revital; Pri-Chen, Sara; Vax, Einav; Katz, Guy; Omer, Dorit; Harari-Steinberg, Orit; Kalisky, Tomer; Dekel, Benjamin

2016-03-29

When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1(+)CD133(-) marks SIX2(+) multipotent renal stem cells transiting to NCAM1(+)CD133(+) differentiating segment-specific SIX2(-) epithelial progenitors and NCAM1(-)CD133(+) differentiated nephron cells. In tumorigenesis, NCAM1(+)CD133(-) marks SIX2(+) blastema that includes the ALDH1(+) WT cancer stem/initiating cells, while NCAM1(+)CD133(+) and NCAM1(-)CD133(+) specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1(+) nephron stem cells in normal and malignant nephrogenesis.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

PubMed

He, Xiaoli; H'ng, Shiau-Chen; Leong, David T; Hutmacher, Dietmar W; Melendez, Alirio J

2010-08-01

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

PubMed Central

Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

2014-01-01

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672

Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years

PubMed Central

2014-01-01

Introduction Regenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years. Methods After thawing, hC-MSCs were isolated with a high efficiency (12 × 106) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches. Results In early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-β, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells. Conclusions The efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies. PMID:24429026

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Large-scale expansion of Wharton's jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing.

PubMed

Zhao, Guifang; Liu, Feilin; Lan, Shaowei; Li, Pengdong; Wang, Li; Kou, Junna; Qi, Xiaojuan; Fan, Ruirui; Hao, Deshun; Wu, Chunling; Bai, Tingting; Li, Yulin; Liu, Jin Yu

2015-03-19

Successful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing. hWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments. hWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1-1.30×10(7) cells on 0.5 g of microbeads by days 8-14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5×10(5) cells per well in a 24-well plate by days 6-10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P<0.05) and had a significantly higher proliferation index (P<0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing. Thus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.

Production Methods for a Mesenchymal Stem Cell Therapeutic as a Medical Defense Countermeasure

DTIC Science & Technology

2012-02-01

differentiation of murine embryonic stem cells into vascular progenitors. BMC Cell Biol. 2008;9:2. 56. Johnson EA, Dao TL, Kan RK. Status epilepticus ...their undifferentiated and multipotent status . BMC Cell Biol. 2011;12:12. 52. Sun Y, Chen L, Hou XG, Hou WK, Dong JJ, Sun L, et al. Differentiation of

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

PubMed Central

Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D.; Bullens, Dominique M.; Pinxteren, Jef; Verfaillie, Catherine M.; Van Gool, Stefaan W.

2016-01-01

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy. PMID:27465071

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

PubMed

Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

2016-12-01

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8 + T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy. ©AlphaMed Press.

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

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

Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook

2007-06-29

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types,more » including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.« less

Vascular Wall-Resident Multipotent Stem Cells of Mesenchymal Nature within the Process of Vascular Remodeling: Cellular Basis, Clinical Relevance, and Implications for Stem Cell Therapy.

PubMed

Klein, Diana

2016-01-01

Until some years ago, the bone marrow and the endothelial cell compartment lining the vessel lumen (subendothelial space) were thought to be the only sources providing vascular progenitor cells. Now, the vessel wall, in particular, the vascular adventitia, has been established as a niche for different types of stem and progenitor cells with the capacity to differentiate into both vascular and nonvascular cells. Herein, vascular wall-resident multipotent stem cells of mesenchymal nature (VW-MPSCs) have gained importance because of their large range of differentiation in combination with their distribution throughout the postnatal organism which is related to their existence in the adventitial niche, respectively. In general, mesenchymal stem cells, also designated as mesenchymal stromal cells (MSCs), contribute to the maintenance of organ integrity by their ability to replace defunct cells or secrete cytokines locally and thus support repair and healing processes of the affected tissues. This review will focus on the central role of VW-MPSCs within vascular reconstructing processes (vascular remodeling) which are absolute prerequisite to preserve the sensitive relationship between resilience and stability of the vessel wall. Further, a particular advantage for the therapeutic application of VW-MPSCs for improving vascular function or preventing vascular damage will be discussed.

Leptin differentially regulates STAT3 activation in the ob/ob mice adipose mesenchymal stem cells

USDA-ARS?s Scientific Manuscript database

Leptin-deficient genetically obese ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Studies have shown that multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute...

A Unique Opportunity to Test Whether Cell Fusion is a Mechanism of Breast Cancer Metastasis

DTIC Science & Technology

2015-06-01

conditions for T47D and human mesenchymal stem cell populations. As a result we have been able to conduct our first co-culture experiments to determine...spontaneously and reliably with mesenchymal stem cells . We found that fusion occurs more frequently with hypoxia and that one means by which...in hypoxic conditions, we decided to investigate whether the mechanism of breast cancer cell fusion with mesenchymal stem /multipotent stromal cells

Imperative role of dental pulp stem cells in regenerative therapies: a systematic review.

PubMed

Kabir, Ramchandra; Gupta, Manish; Aggarwal, Avanti; Sharma, Deepak; Sarin, Anurag; Kola, Mohammed Zaheer

2014-01-01

Stem cells are primitive cells that can differentiate and regenerate organs in different parts of the body such as heart, bones, muscles and nervous system. This has been a field of great clinical interest with immense possibilities of using the stem cells in regeneration of human organ those are damaged due to disease, developmental defects and accident. The knowledge of stem cell technology is increasing quickly in all medical specialties and in dental field too. Stem cells of dental origin appears to hold the key to various cell-based therapies in regenerative medicine, but most avenues are in experimental stages and many procedures are undergoing standardization and validation. Long-term preservation of SHED cells or DPSC is becoming a popular consideration, similar to the banking of umbilical cord blood. Dental pulp stem cells (DPSCs) are the adult multipotent cells that reside in the cell rich zone of the dental pulp. The multipotent nature of these DPSCs may be utilized in both dental and medical applications. A systematic review of the literature was performed using various internet based search engines (PubMed, Medline Plus, Cochrane, Medknow, Ebsco, Science Direct, Hinari, WebMD, IndMed, Embase) using keywords like "dental pulp stem cells", "regeneration", "medical applications", "tissue engineering". DPSCs appears to be a promising innovation for the re-growth of tissues however, long term clinical studies need to be carried out that could establish some authentic guidelines in this perspective.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Adipose-derived mesenchymal stem cells and regenerative medicine.

PubMed

Konno, Masamitsu; Hamabe, Atsushi; Hasegawa, Shinichiro; Ogawa, Hisataka; Fukusumi, Takahito; Nishikawa, Shimpei; Ohta, Katsuya; Kano, Yoshihiro; Ozaki, Miyuki; Noguchi, Yuko; Sakai, Daisuke; Kudoh, Toshihiro; Kawamoto, Koichi; Eguchi, Hidetoshi; Satoh, Taroh; Tanemura, Masahiro; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

2013-04-01

Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Imperative Role of Dental Pulp Stem Cells in Regenerative Therapies: A Systematic Review

PubMed Central

Kabir, Ramchandra; Gupta, Manish; Aggarwal, Avanti; Sharma, Deepak; Sarin, Anurag; Kola, Mohammed Zaheer

2014-01-01

Stem cells are primitive cells that can differentiate and regenerate organs in different parts of the body such as heart, bones, muscles and nervous system. This has been a field of great clinical interest with immense possibilities of using the stem cells in regeneration of human organ those are damaged due to disease, developmental defects and accident. The knowledge of stem cell technology is increasing quickly in all medical specialties and in dental field too. Stem cells of dental origin appears to hold the key to various cell-based therapies in regenerative medicine, but most avenues are in experimental stages and many procedures are undergoing standardization and validation. Long-term preservation of SHED cells or DPSC is becoming a popular consideration, similar to the banking of umbilical cord blood. Dental pulp stem cells (DPSCs) are the adult multipotent cells that reside in the cell rich zone of the dental pulp. The multipotent nature of these DPSCs may be utilized in both dental and medical applications. A systematic review of the literature was performed using various internet based search engines (PubMed, Medline Plus, Cochrane, Medknow, Ebsco, Science Direct, Hinari, WebMD, IndMed, Embase) using keywords like “dental pulp stem cells”, “regeneration”, “medical applications”, “tissue engineering”. DPSCs appears to be a promising innovation for the re-growth of tissues however, long term clinical studies need to be carried out that could establish some authentic guidelines in this perspective. PMID:24665194

Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle.

PubMed

Anastasia, Luigi; Sampaolesi, Maurilio; Papini, Nadia; Oleari, Diego; Lamorte, Giuseppe; Tringali, Cristina; Monti, Eugenio; Galli, Daniela; Tettamanti, Guido; Cossu, Giulio; Venerando, Bruno

2006-12-01

Stem cells hold a great potential for the regeneration of damaged tissues in cardiovascular or musculoskeletal diseases. Unfortunately, problems such as limited availability, control of cell fate, and allograft rejection need to be addressed before therapeutic applications may become feasible. Generation of multipotent progenitors from adult differentiated cells could be a very attractive alternative to the limited in vitro self-renewal of several types of stem cells. In this direction, a recently synthesized unnatural purine, named reversine, has been proposed to induce reversion of adult cells to a multipotent state, which could be then converted into other cell types under appropriate stimuli. Our study suggests that reversine treatment transforms primary murine and human dermal fibroblasts into myogenic-competent cells both in vitro and in vivo. Moreover, this is the first study to demonstrate that plasticity changes arise in primary mouse and human cells following reversine exposure.

The Emerging Role of PEDF in Stem Cell Biology

PubMed Central

Elahy, Mina; Baindur-Hudson, Swati; Dass, Crispin R.

2012-01-01

Encoded by a single gene, PEDF is a 50 kDa glycoprotein that is highly conserved and is widely expressed among many tissues. Most secreted PEDF deposits within the extracellular matrix, with cell-type-specific functions. While traditionally PEDF is known as a strong antiangiogenic factor, more recently, as this paper highlights, PEDF has been linked with stem cell biology, and there is now accumulating evidence demonstrating the effects of PEDF in a variety of stem cells, mainly in supporting stem cell survival and maintaining multipotency. PMID:22675247

Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease

PubMed Central

Ezquer, Fernando; Bruna, Flavia; Calligaris, Sebastián; Conget, Paulette; Ezquer, Marcelo

2016-01-01

Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression. PMID:26755858

Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad.

PubMed

Carroll, S F; Buckley, C T; Kelly, D J

2014-06-27

The objective of this study was to investigate how joint specific biomechanical loading influences the functional development and phenotypic stability of cartilage grafts engineered in vitro using stem/progenitor cells isolated from different source tissues. Porcine bone marrow derived multipotent stromal cells (BMSCs) and infrapatellar fat pad derived multipotent stromal cells (FPSCs) were seeded in agarose hydrogels and cultured in chondrogenic medium, while simultaneously subjected to 10MPa of cyclic hydrostatic pressure (HP). To mimic the endochondral phenotype observed in vivo with cartilaginous tissues engineered using BMSCs, the culture media was additionally supplemented with hypertrophic factors, while the loss of phenotype observed in vivo with FPSCs was induced by withdrawing transforming growth factor (TGF)-β3 from the media. The application of HP was found to enhance the functional development of cartilaginous tissues engineered using both BMSCs and FPSCs. In addition, HP was found to suppress calcification of tissues engineered using BMSCs cultured in chondrogenic conditions and acted to maintain a chondrogenic phenotype in cartilaginous grafts engineered using FPSCs. The results of this study point to the importance of in vivo specific mechanical cues for determining the terminal phenotype of chondrogenically primed multipotent stromal cells. Furthermore, demonstrating that stem or progenitor cells will appropriately differentiate in response to such biophysical cues might also be considered as an additional functional assay for evaluating their therapeutic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human reaming debris: a source of multipotent stem cells.

PubMed

Wenisch, Sabine; Trinkaus, Katja; Hild, Anne; Hose, Dirk; Herde, Katja; Heiss, Christian; Kilian, Olaf; Alt, Volker; Schnettler, Reinhard

2005-01-01

The biological characteristics of human reaming debris (HRD) generated in the course of surgical treatment of long bone diaphyseal fractures and nonunions are still a matter of dispute. Therefore, the objective of the present investigation has been to characterize the intrinsic properties of human reaming debris in vitro. Samples of reaming debris harvested from 12 patients with closed diaphyseal fractures were examined ultrastucturally and were cultured under standard conditions. After a lag phase of 4-7 days, cells started to grow out from small bone fragments and established a confluent monolayer within 20-22 days. The cells were characterized according to morphology, proliferation capacity, cell surface antigen profile, and differentiation repertoire. The results reveal that human reaming debris is a source of multipotent stem cells which are able to grow and proliferate in vitro. The cells differentiate along the osteogenic pathway after induction and can be directed toward a neuronal phenotype, as has been shown morphologically and by the expression of neuronal markers after DMSO induction. These findings have prompted interest in the use of reaming debris-derived stem cells in cell and bone replacement therapies.

Isolation, culture and biological characteristics of multipotent porcine tendon-derived stem cells.

PubMed

Yang, Jinjuan; Zhao, Qianjun; Wang, Kunfu; Ma, Caiyun; Liu, Hao; Liu, Yingjie; Guan, Weijun

2018-06-01

Tendon-derived stem cells (TDSCs), a postulated multi-potential stem cell population, play significant role in the postnatal replenishment of tendon injuries. However, the majority of experimental materials were obtained from horse, rat, human and rabbit, but rarely from pig. In this research, 1‑day‑old pig was chosen as experimental sample source to isolate and culture TDSCs in vitro. Specific markers of TDSCs were then characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT‑PCR) assays. The results showed that TDSCs could be expanded for 11 passages in vitro. The expression of specific markers, such as collagen Ⅰ, collagen Ⅲ, α‑smooth muscle actin (α‑SMA), CD105 and CD90 were observed by immunofluorescence and RT‑PCR. TDSCs were induced to differentiate into adipocytes, osteoblasts and chondrocytes, respectively. These results suggest that TDSCs isolated from porcine tendon exhibit the characteristics of multipotent stem cells. TDSCs, therefore, may be potential candidates for cellular transplantation therapy and tissue engineering in tendon injuries.

Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.

PubMed

Korytnikov, Roman; Nostro, Maria Cristina

2016-05-15

Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Inferring rules of lineage commitment in haematopoiesis.

PubMed

Pina, Cristina; Fugazza, Cristina; Tipping, Alex J; Brown, John; Soneji, Shamit; Teles, Jose; Peterson, Carsten; Enver, Tariq

2012-02-19

How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.

Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation

PubMed Central

Sundin, Mikael; Lindblom, Anna; Örvell, Claes; Barrett, A.John; Sundberg, Berit; Watz, Emma; Wikman, Agneta; Broliden, Kristina; Le Blanc, Katarina

2014-01-01

Multipotent mesenchymal stromal cells (MSC) are used to improve the outcome of hematopoietic stem cell transplantation and in regenerative medicine. However, MSC may harbor persistent viruses that may compromise their clinical benefit. Retrospectively screened, 1 of 20 MSC from healthy donors contained parvovirus B19 (B19) DNA. We found that MSC express the B19 receptor (the globoside P antigen) and a co-receptor (Ku 80), and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two stem cell transplant patients received the B19 positive MSC as treatment for graft-versus-host disease. Neither developed viremia nor symptomatic B19 infection. These results demonstrate for the first time that persistent B19 in MSC can infect hematopoietic cells and underscore the importance of monitoring B19 transmission by MSC products. PMID:18804048

Isolation and culture of human multipotent stromal cells from the pancreas.

PubMed

Seeberger, Karen L; Eshpeter, Alana; Korbutt, Gregory S

2011-01-01

Mesenchymal stem cells, also termed multipotent mesenchymal stromal cells (MSCs), can be isolated from most adult tissues. Although the exact origin of MSCs expanded from the human pancreas has not been resolved, we have developed protocols to isolate and expand MSCs from human pancreatic tissue that remains after islet procurement. Similar to techniques used to isolate MSCs from bone marrow, pancreatic MSCs are isolated based on their cell adherence, expression of several cell surface antigens, and multilineage differentiation. The protocols for isolating, characterizing, and differentiating MSCs from the pancreas are presented in this chapter.

Novel Regenerative Therapies Based on Regionally Induced Multipotent Stem Cells in Post-Stroke Brains: Their Origin, Characterization, and Perspective.

PubMed

Takagi, Toshinori; Yoshimura, Shinichi; Sakuma, Rika; Nakano-Doi, Akiko; Matsuyama, Tomohiro; Nakagomi, Takayuki

2017-12-01

Brain injuries such as ischemic stroke cause severe neural loss. Until recently, it was believed that post-ischemic areas mainly contain necrotic tissue and inflammatory cells. However, using a mouse model of cerebral infarction, we demonstrated that stem cells develop within ischemic areas. Ischemia-induced stem cells can function as neural progenitors; thus, we initially named them injury/ischemia-induced neural stem/progenitor cells (iNSPCs). However, because they differentiate into more than neural lineages, we now refer to them as ischemia-induced multipotent stem cells (iSCs). Very recently, we showed that putative iNSPCs/iSCs are present within post-stroke areas in human brains. Because iNSPCs/iSCs isolated from mouse and human ischemic tissues can differentiate into neuronal lineages in vitro, it is possible that a clearer understanding of iNSPC/iSC profiles and the molecules that regulate iNSPC/iSC fate (e.g., proliferation, differentiation, and survival) would make it possible to perform neural regeneration/repair in patients following stroke. In this article, we introduce the origin and traits of iNSPCs/iSCs based on our reports and recent viewpoints. We also discuss their possible contribution to neurogenesis through endogenous and exogenous iNSPC/iSC therapies following ischemic stroke.

Different effects of resveratrol on early and late passage mesenchymal stem cells through β-catenin regulation

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

Yoon, Dong Suk; Choi, Yoorim; Choi, Seong Mi

2015-11-27

Resveratrol is a sirtuin 1 (SIRT1) activator and can function as an anti-inflammatory and antioxidant factor. In mesenchymal stem cells (MSCs), resveratrol enhances the proliferation and differentiation potential and has an anti-aging effect. However, contradictory effects of resveratrol on MSC cultures have been reported. In this study, we found that resveratrol had different effects on MSC cultures according to their cell passage and SIRT1 expression. Resveratrol enhanced the self-renewal potential and multipotency of early passage MSCs, but accelerated cellular senescence of late passage MSCs. In early passage MSCs expressing SIRT1, resveratrol decreased ERK and GSK-3β phosphorylation, suppressing β-catenin activity. Inmore » contrast, in late passage MSCs, which did not express SIRT1, resveratrol increased ERK and GSK-3β phosphorylation, activating β-catenin. We confirmed that SIRT1-deficient early passage MSCs treated with resveratrol lost their self-renewal potential and multipotency, and became senescent due to increased β-catenin activity. Sustained treatment with resveratrol at early passages maintained the self-renewal potential and multipotency of MSCs up to passage 10. Our findings suggest that resveratrol can be effectively applied to early passage MSC cultures, whereas parameters such as cell passage and SIRT1 expression must be taken into consideration before applying resveratrol to late passage MSCs. - Highlights: • Resveratrol enhances self-renewal potential and multipotency of early passage MSCs. • Resveratrol accelerates the cellular senescence of late passage MSCs. • The effects of resveratrol on MSCs are dependent on the presence of SIRT1. • SIRT1 modulates ERK/GSK-3β/β-catenin signaling. • Sustained resveratrol treatment maintains MSC stemness up to P10.« less

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

PubMed Central

Nagao, Yusuke; Suzuki, Takao; Shimizu, Atsushi; Kimura, Tetsuaki; Seki, Ryoko; Adachi, Tomoko; Inoue, Chikako; Omae, Yoshihiro; Kamei, Yasuhiro; Hara, Ikuyo; Taniguchi, Yoshihito; Naruse, Kiyoshi; Wakamatsu, Yuko; Kelsh, Robert N.; Hibi, Masahiko; Hashimoto, Hisashi

2014-01-01

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor). PMID:24699463

Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

PubMed

Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

2017-04-11

In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

PubMed

Kehl, Debora; Generali, Melanie; Görtz, Sabrina; Geering, Diego; Slamecka, Jaroslav; Hoerstrup, Simon P; Bleul, Ulrich; Weber, Benedikt

2017-10-01

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Sustained release of stem cell factor in a double network hydrogel for ex vivo culture of cord blood-derived CD34+ cells.

PubMed

Zhang, Yuanhao; Pan, Xiuwei; Shi, Zhen; Cai, Haibo; Gao, Yun; Zhang, Weian

2018-04-01

Stem cell factor (SCF) is considered as a commonly indispensable cytokine for proliferation of haematopoietic stem cells (HSCs), which is used in large dosages during ex vivo culture. The work presented here aimed to reduce the consumption of SCF by sustained release but still support cells proliferation and maintain the multipotency of HSCs. Stem cell factor was physically encapsulated within a hyaluronic acid/gelatin double network (HGDN) hydrogel to achieve a slow release rate. CD34 + cells were cultured within the SCF-loaded HGDN hydrogel for 14 days. The cell number, phenotype and functional capacity were investigated after culture. The HGDN hydrogels had desirable properties and encapsulated SCF kept being released for more than 6 days. SCF remained the native bioactivity, and the proliferation of HSCs within the SCF-loaded HGDN hydrogel was not affected, although the consumption of SCF was only a quarter in comparison with the conventional culture. Moreover, CD34 + cells harvested from the SCF-loaded HGDN hydrogels generated more multipotent colony-forming units (CFU-GEMM). The data suggested that the SCF-loaded HGDN hydrogel could support ex vivo culture of HSCs, thus providing a cost-effective culture protocol for HSCs. © 2017 John Wiley & Sons Ltd.

Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

PubMed Central

Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

2015-01-01

Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

Production and characterization of immortal human neural stem cell line with multipotent differentiation property.

PubMed

Kim, Seung U; Nagai, Atsushi; Nakagawa, Eiji; Choi, Hyun B; Bang, Jung H; Lee, Hong J; Lee, Myung A; Lee, Yong B; Park, In H

2008-01-01

We document the protocols and methods for the production of immortalized cell lines of human neural stem cells from the human fetal central nervous system (CNS) cells by using a retroviral vector encoding v-myc oncogene. One of the human neural stem cell lines (HB1.F3) was found to express nestin and other specific markers for human neural stem cells, giving rise to three fundamental cell types of the CNS: neurons, astrocytes, and oligodendrocytes. After transplantation into the brain of mouse model of stroke, implanted human neural stem cells were observed to migrate extensively from the site of implantation into other anatomical sites and to differentiate into neurons and glial cells.

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

PubMed Central

Kirby, S L; Cook, D N; Walton, W; Smithies, O

1996-01-01

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells. Images Fig. 3 PMID:8790342

Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells

PubMed Central

Barberi, Tiziano; Willis, Lucy M; Socci, Nicholas D; Studer, Lorenz

2005-01-01

Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. PMID:15971941

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

PubMed

Lilja, Anna M; Rodilla, Veronica; Huyghe, Mathilde; Hannezo, Edouard; Landragin, Camille; Renaud, Olivier; Leroy, Olivier; Rulands, Steffen; Simons, Benjamin D; Fre, Silvia

2018-06-01

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.

The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

Cancer.gov

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue.

Multipotent progenitor cells are present in human peripheral blood.

PubMed

Cesselli, Daniela; Beltrami, Antonio Paolo; Rigo, Silvia; Bergamin, Natascha; D'Aurizio, Federica; Verardo, Roberto; Piazza, Silvano; Klaric, Enio; Fanin, Renato; Toffoletto, Barbara; Marzinotto, Stefania; Mariuzzi, Laura; Finato, Nicoletta; Pandolfi, Maura; Leri, Annarosa; Schneider, Claudio; Beltrami, Carlo Alberto; Anversa, Piero

2009-05-22

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of approximately 3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Bone morphogenetic protein 9 (BMP9) induces effective bone formation from reversibly immortalized multipotent adipose-derived (iMAD) mesenchymal stem cells.

PubMed

Lu, Shun; Wang, Jing; Ye, Jixing; Zou, Yulong; Zhu, Yunxiao; Wei, Qiang; Wang, Xin; Tang, Shengli; Liu, Hao; Fan, Jiaming; Zhang, Fugui; Farina, Evan M; Mohammed, Maryam M; Song, Dongzhe; Liao, Junyi; Huang, Jiayi; Guo, Dan; Lu, Minpeng; Liu, Feng; Liu, Jianxiang; Li, Li; Ma, Chao; Hu, Xue; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Zhou, Dongsheng; He, Tongchuan

2016-01-01

Regenerative medicine and bone tissue engineering using mesenchymal stem cells (MSCs) hold great promise as an effective approach to bone and skeletal reconstruction. While adipose tissue harbors MSC-like progenitors, or multipotent adipose-derived cells (MADs), it is important to identify and characterize potential biological factors that can effectively induce osteogenic differentiation of MADs. To overcome the time-consuming and technically challenging process of isolating and culturing primary MADs, here we establish and characterize the reversibly immortalized mouse multipotent adipose-derived cells (iMADs). The isolated mouse primary inguinal MAD cells are reversibly immortalized via the retrovirus-mediated expression of SV40 T antigen flanked with FRT sites. The iMADs are shown to express most common MSC markers. FLP-mediated removal of SV40 T antigen effectively reduces the proliferative activity and cell survival of iMADs, indicating the immortalization is reversible. Using the highly osteogenic BMP9, we find that the iMADs are highly responsive to BMP9 stimulation, express multiple lineage regulators, and undergo osteogenic differentiation in vitro upon BMP9 stimulation. Furthermore, we demonstrate that BMP9-stimulated iMADs form robust ectopic bone with a thermoresponsive biodegradable scaffold material. Collectively, our results demonstrate that the reversibly immortalized iMADs exhibit the characteristics of multipotent MSCs and are highly responsive to BMP9-induced osteogenic differentiation. Thus, the iMADs should provide a valuable resource for the study of MAD biology, which would ultimately enable us to develop novel and efficacious strategies for MAD-based bone tissue engineering.

FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro

PubMed Central

Shao, Meiying; Liu, Chao; Song, Yingnan; Ye, Wenduo; He, Wei; Yuan, Guohua; Gu, Shuping; Lin, Congxin; Ma, Liang; Zhang, Yanding; Tian, Weidong; Hu, Tao; Chen, YiPing

2015-01-01

The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration. PMID:26243590

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

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

Santamaria-Martinez, Albert; Universitat de Barcelona, Barcelona; Barquinero, Jordi

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture andmore » sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.« less

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

PubMed

Santamaria-Martínez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinós, Tomàs; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventós, Jaume; Munell, Francina

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

PubMed

Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

2016-09-13

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. Published by Elsevier Inc.

Long-term survival of donor bone marrow multipotent mesenchymal stromal cells implanted into the periosteum of patients with allogeneic graft failure.

PubMed

Kuzmina, L A; Petinati, N A; Sats, N V; Drize, N J; Risinskaya, N V; Sudarikov, A B; Vasilieva, V A; Drokov, M Y; Michalzova, E D; Parovichnikova, E N; Savchenko, V G

2016-09-01

The present study involved three patients with graft failure following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We obtained multipotent mesenchymal stromal cells (MSCs) from the original hematopoietic cell donors and implanted these cells in the periosteum to treat long-term bone marrow aplasia. The results showed that in all patients endogenous blood formation was recovered 2 weeks after MSC administration. Donor MSCs were found in recipient bone marrow three and 5 months following MSC implantation. Thus, our findings indicate that functional donor MSCs can persist in patient bone marrow.

Stem cells in dentistry--review of literature.

PubMed

Dziubińska, P; Jaskólska, M; Przyborowska, P; Adamiak, Z

2013-01-01

Stem cells have been successfully isolated from a variety of human and animal tissues, including dental pulp. This achievement marks progress in regenerative dentistry. This article reviews the latest improvements made in regenerative dental medicine with the involvement of stem cells. Although, various types of multipotent somatic cells can be applied in dentistry, two types of cells have been investigated in this review. Dental pulp cells are classified as: DPSCs, SCAPs and SHEDs.The third group includes two types of cell associated with the periodontium: PDL and DFPC. This review aims to systematize basic knowledge about cellular engineering in dentistry.

Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non-attached plates.

PubMed

Zheng, Yue-Mao; Dang, Yong-Hui; Qiu, Shuang; Qi, Ying-Pei; Xu, Yong-Ping; Sai, Wu-Jia-Fu

2011-08-01

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.

The mechanosensor of mesenchymal stem cells: mechanosensitive channel or cytoskeleton?

PubMed

Xiao, E; Chen, Chider; Zhang, Yi

2016-09-20

Mesenchymal stem cells (MSCs) are multipotent adult stem cells. MSCs and their potential for use in regenerative medicine have been investigated extensively. Recently, the mechanisms by which MSCs detect mechanical stimuli have been described in detail. As in other cell types, both mechanosensitive channels, such as transient receptor potential melastatin 7 (TRPM7), and the cytoskeleton, including actin and actomyosin, have been implicated in mechanosensation in MSCs. This review will focus on discussing the precise role of TRPM7 and the cytoskeleton in mechanosensation in MSCs.

Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death.

PubMed

Curtis, Brandon M; Leix, Kyle Alexander; Ji, Yajing; Glaves, Richard Samuel Elliot; Ash, David E; Mohanty, Dillip K

2014-07-18

Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well. Published by Elsevier Inc.

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

PubMed

Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

2017-03-01

Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Hyaluronic Acid (HA) Scaffolds and Multipotent Stromal Cells (MSCs) in Regenerative Medicine.

PubMed

Prè, Elena Dai; Conti, Giamaica; Sbarbati, Andrea

2016-12-01

Traditional methods for tissue regeneration commonly used synthetic scaffolds to regenerate human tissues. However, they had several limitations, such as foreign body reactions and short time duration. In order to overcome these problems, scaffolds made of natural polymers are preferred. One of the most suitable and widely used materials to fabricate these scaffolds is hyaluronic acid. Hyaluronic acid is the primary component of the extracellular matrix of the human connective tissue. It is an ideal material for scaffolds used in tissue regeneration, thanks to its properties of biocompatibility, ease of chemical functionalization and degradability. In the last few years, especially from 2010, scientists have seen that the cell-based engineering of these natural scaffolds allows obtaining even better results in terms of tissue regeneration and the research started to grow in this direction. Multipotent stromal cells, also known as mesenchymal stem cells, plastic-adherent cells isolated from bone marrow and other mesenchymal tissues, with self-renew and multi-potency properties are ideal candidates for this aim. Normally, they are pre-seeded onto these scaffolds before their implantation in vivo. This review discusses the use of hyaluronic acid-based scaffolds together with multipotent stromal cells, as a very promising tool in regenerative medicine.

Ceramide-1-phosphate regulates migration of multipotent stromal cells (MSCs) and endothelial progenitor cells (EPCs) – implications for tissue regeneration

PubMed Central

Kim, ChiHwa; Schneider, Gabriela; Abdel-Latif, Ahmed; Mierzejewska, Kasia; Sunkara, Manjula; Borkowska, Sylwia; Ratajczak, Janina; Morris, Andrew J.; Kucia, Magda; Ratajczak, Mariusz Z.

2012-01-01

Ceramide-1-phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an anti-apoptotic molecule released from cells that are damaged and “leaky”. As reported recently, C1P promotes migration of hematopoietic cells. In the current paper, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for a first time that C1P is upregulated in damaged tissues and chemoattracts BM-derived multipotent stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells (HUVECs) and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal derived factor-1 (SDF-1) from BM-derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization. PMID:23193025

Adult bone marrow-derived stem cells for organ regeneration and repair.

PubMed

Tögel, Florian; Westenfelder, Christof

2007-12-01

Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow-derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine. 2007 Wiley-Liss, Inc

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

PubMed

Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

2015-01-15

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

Stem cell ageing: does it happen and can we intervene?

PubMed

Bellantuono, Ilaria; Keith, W Nicol

2007-11-19

Adult stem cells have become the focus of intense research in recent years as a result of their role in the maintenance and repair of tissues. They exert this function through their extensive expansion (self-renewal) and multipotent differentiation capacity. Understanding whether adult stem cells retain this capacity throughout the lifespan of the individual, or undergo a process of ageing resulting in a decreased stem cell pool, is an important area of investigation. Progress in this area has been hampered by lack of suitable models and of appropriate markers and assays to identify stem cells. However, recent data suggest that an understanding of the mechanisms governing stem cell ageing can give insight into the mechanism of tissue ageing and, most importantly, advance our ability to use stem cells in cell and gene therapy strategies.

Neurodegeneration and adaptation in response to low-dose photon irradiation

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

Limoli, Charles L.

2014-10-27

Neural stem and precursor cells (i.e. multipotent neural cells) are concentrated in the neurogenic regions of the brain (hippocampal dentate gyrus, subventricular zones), and considerable evidence suggests that these cells are important in mediating the stress response of the CNS after damage from ionizing radiation. The capability of these cells to proliferate, migrate and differentiate (i.e. to undergo neurogenesis) suggests they can participate in the repair and maintenance of CNS functions by replacing brain cells damaged or depleted due to irradiation. Importantly, we have shown that multipotent neural cells are markedly sensitive to irradiation and oxidative stress, insults that compromisemore » neurogenesis and hasten the onset and progression of degenerative processes that are likely to have an adverse impact on cognition. Our past and current work has demonstrated that relatively low doses of radiation cause a persistent (weeks-months) oxidative stress in multipotent neural cells that can elicit a range of degenerative sequelae in the CNS. Therefore, our project is focused on determining the extent that endogenous and redox sensitive multipotent neural cells represent important radioresponsive targets for low dose radiation effects. We hypothesize that the activation of redox sensitive signaling can trigger radioadaptive changes in these cells that can be either harmful or beneficial to overall cognitive health.« less

Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture.

PubMed

Park, Y; Subramanian, K; Verfaillie, C M; Hu, W S

2010-10-01

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment. Copyright © 2010 Elsevier B.V. All rights reserved.

Intra-femoral injection of human mesenchymal stem cells.

PubMed

Mohanty, Sindhu T; Bellantuono, Ilaria

2013-01-01

In vivo transplantation of putative populations of hematopoietic stem cells (HSC) and assessment of their engraftment is considered the golden standard to assess their quality and degree of stemness. Transplantation is usually carried out by intravenous injection in murine models and assessment of engraftment is performed by monitoring the number and type of mature blood cells produced by the donor cells in time. In contrast intravenous injection of mesenchymal stem cells (MSC), the multipotent stem cells present in bone marrow and capable of differentiating to osteoblasts, chondrocytes and adipocytes, has not been successful. This is due to limited or absent engraftment levels. Here, we describe the use of intra-femoral injection as an improved method to assess MSC engraftment to bone and bone marrow and their quality.

Human multipotent adult progenitor cells are nonimmunogenic and exert potent immunomodulatory effects on alloreactive T-cell responses.

PubMed

Jacobs, Sandra A; Pinxteren, Jef; Roobrouck, Valerie D; Luyckx, Ariane; van't Hof, Wouter; Deans, Robert; Verfaillie, Catherine M; Waer, Mark; Billiau, An D; Van Gool, Stefaan W

2013-01-01

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived nonhematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and cannot only differentiate into typical mesenchymal cell types but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus-host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, and Crohn's disease. Therefore, we studied the immune phenotype, immunogenicity, and immunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are nonimmunogenic for T-cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T-cell alloreactivity and on T-cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro. In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

PubMed

Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

2015-07-15

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

Induced pluripotent stem cells for regenerative medicine.

PubMed

Hirschi, Karen K; Li, Song; Roy, Krishnendu

2014-07-11

With the discovery of induced pluripotent stem (iPS) cells, it is now possible to convert differentiated somatic cells into multipotent stem cells that have the capacity to generate all cell types of adult tissues. Thus, there is a wide variety of applications for this technology, including regenerative medicine, in vitro disease modeling, and drug screening/discovery. Although biological and biochemical techniques have been well established for cell reprogramming, bioengineering technologies offer novel tools for the reprogramming, expansion, isolation, and differentiation of iPS cells. In this article, we review these bioengineering approaches for the derivation and manipulation of iPS cells and focus on their relevance to regenerative medicine.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tumor-Like Stem Cells Derived from Human Keloid Are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis

PubMed Central

Zhang, Qunzhou; Yamaza, Takayoshi; Kelly, A. Paul; Shi, Shihong; Wang, Songlin; Brown, Jimmy; Wang, Lina; French, Samuel W.; Shi, Songtao; Le, Anh D.

2009-01-01

Background Alterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their “pathological” niche in the development of the benign tumor. Methods and Findings Subclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17. Conclusions/Significance These findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the “pathological” stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors. PMID:19907660

Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease.

PubMed

Kakinuma, Sei; Nakauchi, Hiromitsu; Watanabe, Mamoru

2009-01-01

Allogeneic liver transplantation is still the only effective treatment available to patients with liver failure. However, because there is a serious shortage of liver donors, an alternative therapeutic approach is needed. Transplantation of mature hepatocytes has been evaluated in clinical trials, but the long-term efficacy remains unclear and the paucity of donor cells limits this strategy. Stem-cell transplantation is a more promising alternative approach. Several studies have provided information about the mechanism underlying the proliferation and differentiation of hepatic stem/progenitor cells. Moreover, in experimental models of liver disease, transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells led to donor cell-mediated repopulation of the liver and improved survival rates. However, before stem-cell transplantation can be applied in the clinic to treat liver failure in humans, it will be necessary to overcome several difficulties associated with the technique.

Endothelial Cells Promote Expansion of Long‐Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Kunar, Balvir; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Norgaard, Zachary K.; Adair, Jennifer E.; Rafii, Shahin

2016-01-01

Abstract Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876 PMID:28297579

Pluripotent stem cells for cardiac regeneration: Overview of recent advances & emerging trends

PubMed Central

Pawani, Harsha; Bhartiya, Deepa

2013-01-01

Cell based regenerative therapy has emerged as one of the most promising options of treatment for patients suffering from heart failure. Various adult stem cells types have undergone extensive clinical trials with limited success which is believed to be more of a cytokine effect rather than cell therapy. Pluripotent human embryonic stem cells (hESCs) have emerged as an attractive candidate stem cell source for obtaining cardiomyocytes (CMs) because of their tremendous capacity for expansion and unquestioned potential to differentiate into CMs. Studies carried out in animal models indicate that ES-derived CMs can partially remuscularize infarcted hearts and improve contractile function; however, the effect was not sustained over long follow up periods due to their limited capacity of cell division in vivo. Thus, the concept of transplanting multipotent cardiovascular progenitors derived from ES cells has emerged since the progenitors retain robust proliferative ability and multipotent nature enabling repopulation of other myocardial elements also in addition to CMs. Transplantation of CMs (progenitors) seeded in biodegradable scaffold and gel based engineered constructs has met with modest success due to issues like cell penetration, nutrient and oxygen availability and inflammation triggered during scaffold degradation inversely affecting the seeded cells. Recently cell sheet based tissue engineering involving culturing cells on ‘intelligent’ polymers has been evolved. Generation of a 3-D pulsatile myocardial tissue has been achieved. However, these advances have to be looked at with cautious optimism as many challenges need to be overcome before using these in clinical practice. PMID:23563370

A novel two-step procedure to expand Sca-1+ cells clonally

PubMed Central

Tang, Yao Liang; Shen, Leping; Qian, Keping; Phillips, M. Ian

2007-01-01

Resident cardiac stem cells (CSCs) are characterized by their capacity to self-renew in culture, and are multi-potent for forming normal cell types in hearts. CSCs were originally isolated directly from enzymatically digested hearts using stem cell markers. However, long exposure to enzymatic digestion can affect the integrity of stem cell markers on the cell surface, and also compromise stem cell function. Alternatively resident CSCs can migrate from tissue explant and form cardiospheres in culture. However, fibroblast contamination can easily occur during CSC culture. To avoid these problems, we developed a two-step procedure by growing the cells before selecting the Sca1+ cells and culturing in cardiac fibroblast conditioned medium, they avoid fibroblast overgrowth. PMID:17577582

Genetic modification of hematopoietic stem cells: recent advances in the gene therapy of inherited diseases.

PubMed

Bueren, Juan A; Guenechea, Guillermo; Casado, José A; Lamana, María Luisa; Segovia, José C

2003-01-01

Hematopoietic stem cells constitute a rare population of precursor cells with remarkable properties for being used as targets in gene therapy protocols. The last years have been particularly productive both in the fields of gene therapy and stem cell biology. Results from ongoing clinical trials have shown the first unquestionable clinical benefits of immunodeficient patients transplanted with genetically modified autologous stem cells. On the other hand, severe side effects in a few patients treated with gene therapy have also been reported, indicating the usefulness of further improving the vectors currently used in gene therapy clinical trials. In the field of stem cell biology, evidence showing the plastic potential of adult hematopoietic stem cells and data indicating the multipotency of adult mesenchymal precursor cells have been presented. Also, the generation of embryonic stem cells by means of nuclear transfer techniques has appeared as a new methodology with direct implications in gene therapy.

Upper gastrointestinal carcinogenesis: H. pylori and stem cell cross-talk.

PubMed

Pilpilidis, Ioannis; Kountouras, Jannis; Zavos, Christos; Katsinelos, Panagiotis

2011-04-01

Chronic inflammation of the gastric epithelium has been associated with the pathogenesis of gastric cancer, as it was postulated by Corea's model of gastric carcinogenesis. Helicobacter pylori (Hp) regulates this inflammatory process and promotes gastric carcinogenesis through induction of gene mutations and protein modulation. Recent data raise the cancer stem cell hypothesis, which implies a central role of multipotent cancer cells in oncogenesis of various solid tumors. This review provides a synopsis of gastric cancer initiation and promotion through Hp and stem cell signaling pathways. The expanding research field of Hp-related cancer stem cell biology may offer novel implications for future treatment of upper gastrointestinal cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure.

PubMed

Yun, Hongmin; Zhou, Yi; Wills, Andrew; Du, Yiqin

2016-06-01

Intraocular pressure (IOP) is still the main treatment target for glaucoma. Outflow resistance mainly exists at the trabecular meshwork (TM) outflow pathway, which is responsible for IOP regulation. Changes of TM cellularity and TM extracellular matrix turnover may play important roles in IOP regulation. In this article, we review basic anatomy and physiology of the outflow pathway and TM stem cell characteristics regarding the location, isolation, identification and function. TM stem cells are localized at the insert region of the TM and are label-retaining in vivo. They can be isolated by side-population cell sorting, cloning culture, or sphere culture. TM stem cells are multipotent with the ability to home to the TM region and differentiate into TM cells in vivo. Other stem cell types, such as adipose-derived stem cells, mesenchymal stem cells and induced pluripotent stem cells have been discovered for TM cell differentiation and TM regeneration. We also review glaucomatous animal models, which are suitable to study stem cell-based therapies for TM regeneration.

Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure

PubMed Central

Yun, Hongmin; Zhou, Yi; Wills, Andrew

2016-01-01

Abstract Intraocular pressure (IOP) is still the main treatment target for glaucoma. Outflow resistance mainly exists at the trabecular meshwork (TM) outflow pathway, which is responsible for IOP regulation. Changes of TM cellularity and TM extracellular matrix turnover may play important roles in IOP regulation. In this article, we review basic anatomy and physiology of the outflow pathway and TM stem cell characteristics regarding the location, isolation, identification and function. TM stem cells are localized at the insert region of the TM and are label-retaining in vivo. They can be isolated by side-population cell sorting, cloning culture, or sphere culture. TM stem cells are multipotent with the ability to home to the TM region and differentiate into TM cells in vivo. Other stem cell types, such as adipose-derived stem cells, mesenchymal stem cells and induced pluripotent stem cells have been discovered for TM cell differentiation and TM regeneration. We also review glaucomatous animal models, which are suitable to study stem cell-based therapies for TM regeneration. PMID:27183473

MLL-ENL cooperates with SCF to transform primary avian multipotent cells.

PubMed

Schulte, Cathleen E; von Lindern, Marieke; Steinlein, Peter; Beug, Hartmut; Wiedemann, Leanne M

2002-08-15

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.

Human dental pulp stem cells: from biology to clinical applications.

PubMed

d'Aquino, Riccardo; De Rosa, Alfredo; Laino, Gregorio; Caruso, Filippo; Guida, Luigi; Rullo, Rosario; Checchi, Vittorio; Laino, Luigi; Tirino, Virginia; Papaccio, Gianpaolo

2009-07-15

Dental pulp stem cells (DPSCs) can be found within the "cell rich zone" of dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, two groups have studied these cells extensively, albeit with different results. One group claims that these cells produce a "dentin-like tissue", whereas the other research group has demonstrated that these cells are capable of producing bone, both in vitro and in vivo. In addition, it has been reported that these cells can be easily cryopreserved and stored for long periods of time and still retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells: several scaffolds have been used to promote 3-D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. These results enforce the notion that DPSCs can be used successfully for tissue engineering. (c) 2008 Wiley-Liss, Inc.

Gastric cancer stem cells: A novel therapeutic target

PubMed Central

Singh, Shree Ram

2013-01-01

Gastric cancer remains one of the leading causes of global cancer mortality. Multipotent gastric stem cells have been identified in both mouse and human stomachs, and they play an essential role in the self-renewal and homeostasis of gastric mucosa. There are several environmental and genetic factors known to promote gastric cancer. In recent years, numerous in vitro and in vivo studies suggest that gastric cancer may originate from normal stem cells or bone marrow–derived mesenchymal cells, and that gastric tumors contain cancer stem cells. Cancer stem cells are believed to share a common microenvironment with normal niche, which play an important role in gastric cancer and tumor growth. This mini-review presents a brief overview of the recent developments in gastric cancer stem cell research. The knowledge gained by studying cancer stem cells in gastric mucosa will support the development of novel therapeutic strategies for gastric cancer. PMID:23583679

Gelatinized Copper–Capillary Alginate Gel Functions as an Injectable Tissue Scaffolding System for Stem Cell Transplants

PubMed Central

Willenberg, Bradley Jay; Zheng, Tong; Meng, Fan-Wei; Meneses, Juan Carlos; Rossignol, Candace; Batich, Christopher D.; Terada, Naohiro; Steindler, Dennis A.; Weiss, Michael D.

2013-01-01

In severe hypoxic–ischemic brain injury, cellular components such as neurons and astrocytes are injured or destroyed along with the supporting extracellular matrix. This presents a challenge to the field of regenerative medicine since the lack of extracellular matrix and supporting structures makes the transplant milieu inhospitable to the transplanted cells. A potential solution to this problem is the use of a biomaterial to provide the extracellular components needed to keep cells localized in cystic brain regions, allowing the cells to form connections and repair lost brain tissue. Ideally, this biomaterial would be combined with stem cells, which have been proven to have therapeutic potentials, and could be delivered via an injection. To study this approach, we derived a hydrogel biomaterial tissue scaffold from oligomeric gelatin and copper–capillary alginate gel (GCCAG). We then demonstrated that our multipotent astrocytic stem cells (MASCs) could be maintained in GCCAG scaffolds for up to 2 weeks in vitro and that the cells retained their multipotency. We next performed a pilot transplant study in which GCCAG was mixed with MASCs and injected into the brain of a neonatal rat pup. After a week in vivo, our results showed that: the GCCAG biomaterial did not cause a significant reactive gliosis; viable cells were retained within the injected scaffolds; and some delivered cells migrated into the surrounding brain tissue. Therefore, GCCAG tissue scaffolds are a promising, novel injectable system for transplantation of stem cells to the brain. PMID:20699061

Haploidentical hematopoietic stem cell transplant with umbilical cord-derived multipotent mesenchymal cell infusion for the treatment of high-risk acute leukemia in children.

PubMed

Zhu, Ling; Wang, Zhidong; Zheng, Xiaoli; Ding, Li; Han, Dongmei; Yan, Hongmin; Guo, Zikuan; Wang, Hengxiang

2015-05-01

In this study, 25 children with high-risk acute leukemia received haploidentical hematopoietic stem cell transplant (haplo-HSCT) with co-transfusion of umbilical cord multipotent mesenchymal cells (UC-MSCs). Adverse effects, hematopoietic recovery, complications and outcome were observed during a median follow-up of 12.8 months (range: 3-25 months). Myeloid engraftment was rapid, and the median time to neutrophil and platelet recovery was 15.12 days and 20.08 days, respectively. Eight patients developed grade I skin acute graft-versus-host disease (aGVHD) that responded well to standard steroid therapy. Of note, cytomegalovirus viremia was observed in most patients (23/25 cases). Patients died mainly of leukemia relapse and pulmonary complication. Fourteen patients are currently alive and remain with full donor chimerism at the time of reporting. The present results suggest further clinical trials to testify the effectiveness of UC-MSCs to prevent aGVHD in haplo-HSCT for treating children with high-risk leukemia.

Stem cells in the Drosophila digestive system.

PubMed

Zeng, Xiankun; Chauhan, Chhavi; Hou, Steven X

2013-01-01

Adult stem cells maintain tissue homeostasis by continuously replenishing damaged, aged and dead cells in any organism. Five types of region and organ-specific multipotent adult stem cells have been identified in the Drosophila digestive system: intestinal stem cells (ISCs) in the posterior midgut; hindgut intestinal stem cells (HISCs) at the midgut/hindgut junction; renal and nephric stem cells (RNSCs) in the Malpighian Tubules; type I gastric stem cells (GaSCs) at foregut/midgut junction; and type II gastric stem cells (GSSCs) at the middle of the midgut. Despite the fact that each type of stem cell is unique to a particular organ, they share common molecular markers and some regulatory signaling pathways. Due to the simpler tissue structure, ease of performing genetic analysis, and availability of abundant mutants, Drosophila serves as an elegant and powerful model system to study complex stem cell biology. The recent discoveries, particularly in the Drosophila ISC system, have greatly advanced our understanding of stem cell self-renewal, differentiation, and the role of stem cells play in tissue homeostasis/regeneration and adaptive tissue growth.

Stem cells in gastroenterology and hepatology

PubMed Central

Quante, Michael; Wang, Timothy C.

2010-01-01

Cellular and tissue regeneration in the gastrointestinal tract and liver depends on stem cells with properties of longevity, self-renewal and multipotency. Progress in stem cell research and the identification of potential esophageal, gastric, intestinal, colonic, hepatic and pancreatic stem cells provides hope for the use of stem cells in regenerative medicine and treatments for disease. Embryonic stem cells and induced pluripotent stem cells have the potential to give rise to any cell type in the human body, but their therapeutic application remains challenging. The use of adult or tissue-restricted stem cells is emerging as another possible approach for the treatment of gastrointestinal diseases. The same self-renewal properties that allow stem cells to remain immortal and generate any tissue can occasionally make their proliferation difficult to control and make them susceptible to malignant transformation. This Review provides an overview of the different types of stem cell, focusing on tissue-restricted adult stem cells in the fields of gastroenterology and hepatology and summarizing the potential benefits and risks of using stems cells to treat gastroenterological and liver disorders. PMID:19884893

Dynamic niches in the origination and differentiation of haematopoietic stem cells

PubMed Central

Wang, Leo D.; Wagers, Amy J.

2014-01-01

Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic ‘niche’, or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche. PMID:21886187

Sheep, Wolf, or Werewolf: Cancer Stem Cells and the Epithelial-to-Mesenchymal Transition

PubMed Central

2013-01-01

Multiple cancers contain subpopulations that exhibit characteristics of cancer stem cells (CSCs), the ability to self-renew and seed heterogeneous tumors. Recent evidence suggests two potentially overlapping models for these phenotypes: one where stem cells arise from multipotent progenitor cells, and another where they are created via an epithelial to mesenchymal transition. Unraveling this issue is critical, as it underlies phenomena such as metastasis and therapeutic resistance. Therefore, there is intense interest in understanding these two types of CSSs, how they differ from differentiated cancer cells, the mechanisms that drive their phenotypes, and how that knowledge can be incorporated into therapeutics. PMID:23499890

Insights into neurogenesis and aging: potential therapy for degenerative disease?

PubMed Central

Marr, Robert A; Thomas, Rosanne M; Peterson, Daniel A

2010-01-01

Neurogenesis is the process by which new neural cells are generated from a small population of multipotent stem cells in the adult CNS. This natural generation of new cells is limited in its regenerative capabilities and also declines with age. The use of stem cells in the treatment of neurodegenerative disease may hold great potential; however, the age-related incidence of many CNS diseases coincides with reduced neurogenesis. This review concisely summarizes current knowledge related to adult neurogenesis and its alteration with aging and examines the feasibility of using stem cell and gene therapies to combat diseases of the CNS with advancing age. PMID:20806052

Adult multipotent stromal cell cryopreservation: Pluses and pitfalls

PubMed Central

Duan, Wei; Hicok, Kevin

2017-01-01

Abstract Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long‐term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post‐cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation. PMID:29023790

Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis1

PubMed Central

Zhang, Qunzhou; Shi, Shihong; Liu, Yi; Uyanne, Jettie; Shi, Yufang; Shi, Songtao; Le, Anh D.

2010-01-01

Aside from the well-established self-renewal and multipotent differentiation properties, mesenchymal stem cells exhibit both immunomodulatory and anti-inflammatory roles in several experimental autoimmune and inflammatory diseases. In this study, we isolated a new population of stem cells from human gingiva, a tissue source easily accessible from the oral cavity, namely, gingiva-derived mesenchymal stem cells (GMSCs), which exhibited clonogenicity, self-renewal, and multipotent differentiation capacities. Most importantly, GMSCs were capable of immunomodulatory functions, specifically suppressed peripheral blood lymphocyte proliferation, induced expression of a wide panel of immunosuppressive factors including IL-10, IDO, inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in response to the inflammatory cytokine, IFN-γ. Cell-based therapy using systemic infusion of GMSCs in experimental colitis significantly ameliorated both clinical and histopathological severity of the colonic inflammation, restored the injured gastrointestinal mucosal tissues, reversed diarrhea and weight loss, and suppressed the overall disease activity in mice. The therapeutic effect of GMSCs was mediated, in part, by the suppression of inflammatory infiltrates and inflammatory cytokines/mediators and the increased infiltration of regulatory T cells and the expression of anti-inflammatory cytokine IL-10 at the colonic sites. Taken together, GMSCs can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and is a promising cell source for cell-based treatment in experimental inflammatory diseases. PMID:19923445

Basal Cells Are a Multipotent Progenitor Capable of Renewing the Bronchial Epithelium

PubMed Central

Hong, Kyung U.; Reynolds, Susan D.; Watkins, Simon; Fuchs, Elaine; Stripp, Barry R.

2004-01-01

Commitment of the pulmonary epithelium to bronchial and bronchiolar airway lineages occurs during the transition from pseudoglandular to cannalicular phases of lung development, suggesting that regional differences exist with respect to the identity of stem and progenitor cells that contribute to epithelial maintenance in adulthood. We previously defined a critical role for Clara cell secretory protein-expressing (CE) cells in renewal of bronchiolar airway epithelium following injury. Even though CE cells are also the principal progenitor for maintenance of the bronchial airway epithelium, CE cell injury is resolved through a mechanism involving recruitment of a second progenitor cell population that we now identify as a GSI-B4 reactive, cytokeratin-14-expressing basal cell. These cells exhibit multipotent differentiation capacity as assessed by analysis of cellular phenotype within clones of LacZ-tagged cells. Clones were derived from K14-expressing cells tagged in a cell-type-specific fashion by ligand-regulable Cre recombinase-mediated genomic rearrangement of the ROSA26 recombination substrate allele. We conclude that basal cells represent an alternative multipotent progenitor cell population of bronchial airways and that progenitor cell selection is dictated by the type of airway injury. PMID:14742263

Stem cell-mediated accelerated bone healing observed with in vivo molecular and small animal imaging technologies in a model of skeletal injury.

PubMed

Lee, Sheen-Woo; Padmanabhan, Parasuraman; Ray, Pritha; Gambhir, Sanjiv Sam; Doyle, Timothy; Contag, Christopher; Goodman, Stuart B; Biswal, Sandip

2009-03-01

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route. (c) 2008 Orthopaedic Research Society.

21st Nantes Actualités Transplantation: "When Stem Cells Meet Immunology".

PubMed

Anegon, Ignacio; Nguyen, Tuan Huy

2017-01-01

"When Stem Cells Meet Immunology" has been the topic of the 21st annual "Nantes Actualités en Transplantation" meeting (June 9-10, 2016, Nantes, France). This meeting brought together pioneers and leading experts in the fields of stem cells, biomaterials and immunoregulation. Presentations covered multipotent (mesenchymal and hematopoietic) and pluripotent stem cells (embryonic and induced) for regenerative medicine of incurable diseases, immunotherapy and blood transfusions. An additional focus had been immune rejections and responses of allogeneic or autologous stem cells. Conversely, stem cells are also able to directly modulate the immune response through the production of immunoregulatory molecules. Moreover, stem cells may also provide an unlimited source of immune cells (DCs, NK cells, B cells, and T cells) that can operate as "super" immune cells, for example, through genetic engineering with chimeric antigen receptors.This meeting report puts presentations into an overall context highlighting new potential biomarkers for potency prediction of mesenchymal stem cell-derived and pluripotent stem cell-derived multicellular organoids. Finally, we propose future directions arising from the flourishing encounter of stem cell and immune biology.

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

PubMed

Pascucci, L; Curina, G; Mercati, F; Marini, C; Dall'Aglio, C; Paternesi, B; Ceccarelli, P

2011-12-15

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations. Copyright © 2011 Elsevier B.V. All rights reserved.

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.

PubMed

Li, Yi; Guo, Gang; Li, Li; Chen, Fei; Bao, Ji; Shi, Yu-Jun; Bu, Hong

2015-05-01

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Peptide modified nanofibrous scaffold promotes human mesenchymal stem cell proliferation and long-term passaging.

PubMed

Mobasseri, Rezvan; Tian, Lingling; Soleimani, Masoud; Ramakrishna, Seeram; Naderi-Manesh, Hossein

2018-03-01

Long-term culture, passage and proliferation of human mesenchymal stem cells (hMSCs) cause loss of their stemness properties including self-renewal and multipotency. By optimizing the MSCs environment in vitro, maintaining the stemness state and better controlling the cell fate might be possible. We have recently reported the significant effects of bioactive Tat protein-derived peptide named R-peptide on hMSC adhesion, morphology and proliferation, which has demonstrated R-peptide enhanced MSC early adhesion and proliferation in comparison to other bioactive molecules including RGD peptide, fibronectin and collagen. In this study, R-peptide was used to evaluate stemness properties of MSCs after long-term passaging. R-peptide conjugated poly caprolactone (PCL) nanofibrous scaffold and unmodified nanofibrous scaffold were used to study the impact of R-peptide modified PCL nanofibers and PCL nanofibers on cell behavior. The results showed early formation of focal adhesion (FA) complex on R-peptide modified scaffolds at 30min after cell seeding. The rate of cell proliferation was significantly increased due to presence of R-peptide, and the MSCs marker analyses using flow cytometry and immunocytochemistry staining proved the ability of R-peptide to maintain mesenchymal stem cell properties (high proliferation, expression of multipotent markers and differentiation capacity) even after long-term passage culturing. Accordingly, our (The) results concluded that bioactive R-peptide in combination with nanofibrous scaffold can mimic the native ECM comprising micro/nano architecture and biochemical molecules in a best way. The designed scaffold can link extracellular matrix (ECM) to nucleus via formation of FA and organization of cytoskeleton, causing fast and strong attachment of MSCs and allowing integrin-mediated signaling to start. Copyright © 2017 Elsevier B.V. All rights reserved.

Hedgehog and Resident Vascular Stem Cell Fate

PubMed Central

Mooney, Ciaran J.; Hakimjavadi, Roya; Fitzpatrick, Emma; Kennedy, Eimear; Walls, Dermot; Morrow, David; Redmond, Eileen M.; Cahill, Paul A.

2015-01-01

The Hedgehog pathway is a pivotal morphogenic driver during embryonic development and a key regulator of adult stem cell self-renewal. The discovery of resident multipotent vascular stem cells and adventitial progenitors within the vessel wall has transformed our understanding of the origin of medial and neointimal vascular smooth muscle cells (SMCs) during vessel repair in response to injury, lesion formation, and overall disease progression. This review highlights the importance of components of the Hh and Notch signalling pathways within the medial and adventitial regions of adult vessels, their recapitulation following vascular injury and disease progression, and their putative role in the maintenance and differentiation of resident vascular stem cells to vascular lineages from discrete niches within the vessel wall. PMID:26064136

Stem/progenitor cells in pituitary organ homeostasis and tumourigenesis

PubMed Central

Manshaei, Saba

2018-01-01

Evidence for the presence of pituitary gland stem cells has been provided over the last decade using a combination of approaches including in vitro clonogenicity assays, flow cytometric side population analysis, immunohistochemical analysis and genetic approaches. These cells have been demonstrated to be able to self-renew and undergo multipotent differentiation to give rise to all hormonal lineages of the anterior pituitary. Furthermore, evidence exists for their contribution to regeneration of the organ and plastic responses to changing physiological demand. Recently, stem-like cells have been isolated from pituitary neoplasms raising the possibility that a cytological hierarchy exists, in keeping with the cancer stem cell paradigm. In this manuscript, we review the evidence for the existence of pituitary stem cells, their role in maintaining organ homeostasis and the regulation of their differentiation. Furthermore, we explore the emerging concept of stem cells in pituitary tumours and their potential roles in these diseases. PMID:28855316

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

2015-01-01

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease.

PubMed

Ikeda, Etsuko; Yagi, Kiyohito; Kojima, Midori; Yagyuu, Takahiro; Ohshima, Akira; Sobajima, Satoshi; Tadokoro, Mika; Katsube, Yoshihiro; Isoda, Katsuhiro; Kondoh, Masuo; Kawase, Masaya; Go, Masahiro J; Adachi, Hisashi; Yokota, Yukiharu; Kirita, Tadaaki; Ohgushi, Hajime

2008-05-01

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells

PubMed Central

Wang, Limin; Ott, Lindsey; Seshareddy, Kiran; Weiss, Mark L; Detamore, Michael S

2011-01-01

Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton’s jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering. PMID:21175290

Computational prediction of strain-dependent diffusion of transcription factors through the cell nucleus.

PubMed

Nava, Michele M; Fedele, Roberto; Raimondi, Manuela T

2016-08-01

Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance for mesenchymal stromal cells cultured on three-dimensional engineered niche substrates, fabricated via two-photon laser polymerization. We correlated maintenance of multipotency to a more roundish morphology of these cells with respect to those cultured on conventional flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to passive diffusion across the cell nucleus. Fully three-dimensional reconstructions of cultured cells were developed on the basis of confocal images: in particular, the level of nuclear spreading resulted significantly dependent on the cell localization within the niche architecture. We assumed that the cell diffusivity varies as a function of the local volumetric strain. The model predictions indicate that the higher the level of spreading of the cell, the higher the flux across the nucleus of small solutes such as transcription factors. Our results point toward nuclear spreading as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e., by amplifying the diffusive flow of transcriptional activators into the nucleus.

[Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System].

PubMed

Krug, C; Beer, A; Saller, M M; Aszodi, A; Holzbach, T; Giunta, R E; Volkmer, E

2016-04-01

Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system. We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay. Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic activity. This study showed that sufficient multipotent ASPCs of appropriate quality can be isolated from the inguinal fat pad of Wistar rats using the ARC™-cell separation system. As shown in previous studies, cultivation of cells under hypoxic conditions increased their stemness. Our findings will enable future studies that focus on autologous transplantation of ASPCs in a rat model, which most closely resembles a possible clinical application. © Georg Thieme Verlag KG Stuttgart · New York.

Distinct adipogenic differentiation phenotypes of human umbilical cord mesenchymal cells dependent on adipogenic conditions

USDA-ARS?s Scientific Manuscript database

The umbilical cord (UC) matrix is a source of multipotent mesenchymal stem cells (MSCs) that have adipogenic potential and thus can be a model to study adipogenesis. However, existing variability in adipocytic differentiation outcomes may be due to discrepancies in methods utilized for adipogenic d...

RNAi as a Routine Route Toward Breast Cancer Therapy

DTIC Science & Technology

2014-05-01

hematopoietic stem/ progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with...Hematopoietic Cells (A and B) Genome browser tracks depict methylation profiles across a lymphoid (A) and myeloid (B) specific locus in blood cells ...multipotent populations, and two derived, mature cell types from the lymphoid and myeloid lineages, respectively. For comparison, we generated methylomes

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE PAGES

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; ...

2015-10-17

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

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

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

PubMed Central

Humphries, Adam; Cereser, Biancastella; Gay, Laura J.; Miller, Daniel S. J.; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R.; Rodriguez-Justo, Manuel; McDonald, Stuart A. C.; Wright, Nicholas A.; Graham, Trevor A.

2013-01-01

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis. PMID:23766371

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution.

PubMed

Humphries, Adam; Cereser, Biancastella; Gay, Laura J; Miller, Daniel S J; Das, Bibek; Gutteridge, Alice; Elia, George; Nye, Emma; Jeffery, Rosemary; Poulsom, Richard; Novelli, Marco R; Rodriguez-Justo, Manuel; McDonald, Stuart A C; Wright, Nicholas A; Graham, Trevor A

2013-07-02

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

Biophysical regulation of stem cell differentiation.

PubMed

Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

2013-06-01

Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

Parthenogenesis-derived Multipotent Stem Cells Adapted for Tissue Engineering Applications

PubMed Central

Koh, Chester J.; Delo, Dawn M.; Lee, Jang Won; Siddiqui, M. Minhaj; Lanza, Robert P.; Soker, Shay; Yoo, James J.; Atala, Anthony

2009-01-01

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications. PMID:18799133

Agonism of Wnt/β-catenin signaling promotes mesenchymal stem cell (MSC) expansion

PubMed Central

Hoffman, Michael D.; Benoit, Danielle S.W.

2014-01-01

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell-based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6-bromoindirubin-3’-oxime (BIO) increases MSC β-catenin activity 106-fold and stem cell-associated gene expression ~33-fold respectively over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8-fold in typical 2D culture conditions, as well as 1.3-fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency, where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes, and adipocytes using standard conditions. Taken together, our results demonstrate BIOs potential utility as a proliferative agent for cell transplantation and tissue regeneration. PMID:23554411

Ground-state transcriptional requirements for skin-derived precursors.

PubMed

Suflita, Michael T; Pfaltzgraff, Elise R; Mundell, Nathan A; Pevny, Larysa H; Labosky, Patricia A

2013-06-15

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell.

Ground-State Transcriptional Requirements for Skin-Derived Precursors

PubMed Central

Suflita, Michael T.; Pfaltzgraff, Elise R.; Mundell, Nathan A.; Pevny, Larysa H.

2013-01-01

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell. PMID:23316968

Sheep, wolf, or werewolf: cancer stem cells and the epithelial-to-mesenchymal transition.

PubMed

Chang, Jeffrey T; Mani, Sendurai A

2013-11-28

Multiple cancers contain subpopulations that exhibit characteristics of cancer stem cells (CSCs), the ability to self-renew and seed heterogeneous tumors. Recent evidence suggests two potentially overlapping models for these phenotypes: one where stem cells arise from multipotent progenitor cells, and another where they are created via an epithelial to mesenchymal transition. Unraveling this issue is critical, as it underlies phenomena such as metastasis and therapeutic resistance. Therefore, there is intense interest in understanding these two types of CSSs, how they differ from differentiated cancer cells, the mechanisms that drive their phenotypes, and how that knowledge can be incorporated into therapeutics. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Brain mesenchymal stem cells: The other stem cells of the brain?

PubMed

Appaix, Florence; Nissou, Marie-France; van der Sanden, Boudewijn; Dreyfus, Matthieu; Berger, François; Issartel, Jean-Paul; Wion, Didier

2014-04-26

Multipotent mesenchymal stromal cells (MSC), have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation. The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair. However, some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist. In brain, perivascular MSCs like pericytes and adventitial cells, could constitute another stem cell population distinct to the neural stem cell pool. The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes, the demonstration of neural biomarkers expression, electrophysiological recordings, and the absence of cell fusion. The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells. It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.

Brain mesenchymal stem cells: The other stem cells of the brain?

PubMed Central

Appaix, Florence; Nissou, Marie-France; van der Sanden, Boudewijn; Dreyfus, Matthieu; Berger, François; Issartel, Jean-Paul; Wion, Didier

2014-01-01

Multipotent mesenchymal stromal cells (MSC), have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation. The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair. However, some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist. In brain, perivascular MSCs like pericytes and adventitial cells, could constitute another stem cell population distinct to the neural stem cell pool. The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes, the demonstration of neural biomarkers expression, electrophysiological recordings, and the absence of cell fusion. The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells. It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression. PMID:24772240

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

PubMed Central

Emirandetti, Amanda; Lewicka, Michalina; Hermanson, Ola; Fisahn, André

2010-01-01

Background Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture. PMID:21079795

Pluripotency of Stem Cells from Human Exfoliated Deciduous Teeth for Tissue Engineering

PubMed Central

Rosa, Vinicius; Dubey, Nileshkumar; Islam, Intekhab; Min, Kyung-San; Nör, Jacques E.

2016-01-01

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative pluripotent cells that can be retrieved from primary teeth. Although SHED are isolated from the dental pulp, their differentiation potential is not limited to odontoblasts only. In fact, SHED can differentiate into several cell types including neurons, osteoblasts, adipocytes, and endothelial cells. The high plasticity makes SHED an interesting stem cell model for research in several biomedical areas. This review will discuss key findings about the characterization and differentiation of SHED into odontoblasts, neurons, and hormone secreting cells (e.g., hepatocytes and islet-like cell aggregates). The outcomes of the studies presented here support the multipotency of SHED and their potential to be used for tissue engineering-based therapies. PMID:27313627

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

PubMed

Cao, Kaiyue; Pan, Yunzhi; Yu, Long; Shu, Xiong; Yang, Jing; Sun, Linxin; Sun, Lichao; Yang, Zhihua; Ran, Yuliang

2017-02-01

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

Adult neural stem cells: The promise of the future

PubMed Central

Taupin, Philippe

2007-01-01

Stem cells are self-renewing undifferentiated cells that give rise to multiple types of specialized cells of the body. In the adult, stem cells are multipotents and contribute to homeostasis of the tissues and regeneration after injury. Until recently, it was believed that the adult brain was devoid of stem cells, hence unable to make new neurons and regenerate. With the recent evidences that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS), the adult brain has the potential to regenerate and may be amenable to repair. The function(s) of NSCs in the adult CNS remains the source of intense research and debates. The promise of the future of adult NSCs is to redefine the functioning and physiopathology of the CNS, as well as to treat a broad range of CNS diseases and injuries. PMID:19300610

Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

PubMed Central

Balic, Anamaria; Aguila, H. Leonardo; Caimano, Melissa J.; Francone, Victor P.; Mina, Mina

2010-01-01

In the past few years there have been significant advances in the identification of putative stem cells also referred to as “mesenchymal stem cells” (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our study showed that dental pulps from unerupted molars contain a significant number of cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45- and little if any CD45+ cells. Our in vitro functional studies showed that dental pulp cells from unerupted molars displayed extensive osteo-dentinogenic potential but were unable to differentiate into chondrocytes and adipocytes. Dental pulp from erupted molars displayed a reduced number of cells, contained higher percentage of CD45+ and lower percentage of cells expressing CD90+/CD45-, CD117+/CD45- as compared to unerupted molars. In vitro functional assays demonstrated the ability of a small fraction of cells to differentiate into odontoblasts, osteoblasts, adipocytes and chondrocytes. There was a significant reduction in the osteo-dentinogenic potential of the pulp cells derived from erupted molars compared to unerupted molars. Furthermore, the adipogenic and chondrogenic differentiation of pulp cells from erupted molars was dependent on a long induction period and infrequent. Based on these findings we propose that the dental pulp of the erupted molars contain a small population of multipotent cells, whereas the dental pulp of the unerupted molars does not contain multipotent cells but is enriched in osteo-dentinogenic progenitors engaged in the formation of coronal and radicular odontoblasts. PMID:20193787

Development and Differentiation of Mesenchymal Bone Marrow Cells in Porous Permeable Titanium Nickelide Implants In Vitro and In Vivo.

PubMed

Kokorev, O V; Khodorenko, V N; Radkevich, A A; Dambaev, G Ts; Gunter, V E

2016-08-01

We studied the structure of porous permeable titanium nickelide used as the scaffold. In vitro population of the porous scaffold with multipotent mesenchymal stem bone marrow cells on days 7, 14, 21, and 28 was analyzed by scanning electron microscopy. Stage-by-stage histogenesis of the tissues formed from the bone marrow cells in the titanium nickelide scaffold in vivo is described in detail. Using mesenchymal stem cells, we demonstrated that porous permeable titanium nickelide scaffolds are unique incubators for cell cultures applicable for tissue engineering.

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

PubMed

Yoon, Dong Suk; Choi, Yoorim; Jang, Yeonsue; Lee, Moses; Choi, Woo Jin; Kim, Sung-Hwan; Lee, Jin Woo

2014-12-01

SOX2 is crucial for the maintenance of the self-renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)-derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self-renewal and differentiation capacities of BM-MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM-MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM-MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA-mediated acetylation and ubiquitination of SOX2 in BM-MSCs. SIRT1 activation by resveratrol enhanced the colony-forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose-dependent manner. However, the enhancement of self-renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1-SOX2 axis plays an important role in maintaining the self-renewal capability and multipotency of BM-MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post-translational modification in BM-MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1-mediated deacetylation contributes to maintaining SOX2 protein in the nucleus. © 2014 AlphaMed Press.

Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency

PubMed Central

Chuah, Yon Jin; Koh, Yi Ting; Lim, Kaiyang; Menon, Nishanth V.; Wu, Yingnan; Kang, Yuejun

2015-01-01

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies. PMID:26647719

Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine

PubMed Central

Yamaza, Haruyoshi; Akiyama, Kentaro; Hoshino, Yoshihiro; Song, Guangtai; Kukita, Toshio; Nonaka, Kazuaki; Shi, Songtao; Yamaza, Takayoshi

2012-01-01

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine. PMID:23251621

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture

PubMed Central

Eildermann, K.; Gromoll, J.; Behr, R.

2012-01-01

BACKGROUND Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells. PMID:22442249

In vitro regeneration of kidney from pluripotent stem cells

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

Osafune, Kenji, E-mail: osafu@cira.kyoto-u.ac.jp; PRESTO, Japan Science and Technology Agency; JST Yamanaka iPS Cell Special Project, Japan Science and Technology Agency

2010-10-01

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitromore » is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.« less

A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

PubMed

Tsai, Yihuan; Cutts, Josh; Kimura, Azuma; Varun, Divya; Brafman, David A

2015-07-01

Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery. Copyright © 2015. Published by Elsevier B.V.

Human adipose-derived stem cells: definition, isolation, tissue-engineering applications.

PubMed

Nae, S; Bordeianu, I; Stăncioiu, A T; Antohi, N

2013-01-01

Recent researches have demonstrated that the most effective repair system of the body is represented by stem cells - unspecialized cells, capable of self-renewal through successive mitoses, which have also the ability to transform into different cell types through differentiation. The discovery of adult stem cells represented an important step in regenerative medicine because they no longer raises ethical or legal issues and are more accessible. Only in 2002, stem cells isolated from adipose tissue were described as multipotent stem cells. Adipose tissue stem cells benefits in tissue engineering and regenerative medicine are numerous. Development of adipose tissue engineering techniques offers a great potential in surpassing the existing limits faced by the classical approaches used in plastic and reconstructive surgery. Adipose tissue engineering clinical applications are wide and varied, including reconstructive, corrective and cosmetic procedures. Nowadays, adipose tissue engineering is a fast developing field, both in terms of fundamental researches and medical applications, addressing issues related to current clinical pathology or trauma management of soft tissue injuries in different body locations.

Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

PubMed Central

Panich, Uraiwan; Sittithumcharee, Gunya; Rathviboon, Natwarath

2016-01-01

Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been linked to impaired skin homeostasis (e.g., skin premature aging and skin cancers). Skin stem cells, with self-renewal capability and multipotency, are frequently affected by environment. Ultraviolet radiation (UVR), a major cause of stem cell DNA damage, can contribute to depletion of stem cells (ESCs and mesenchymal stem cells) and damage of stem cell niche, eventually leading to photoinduced skin aging. In this review, we discuss the role of UV-induced DNA damage and oxidative stress in the skin stem cell aging in order to gain insights into the pathogenesis and develop a way to reduce photoaging of skin cells. PMID:27148370

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

PubMed Central

Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

2009-01-01

Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. Reviewers This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter. PMID:19715575

Improving the Post-Stroke Therapeutic Potency of Mesenchymal Multipotent Stromal Cells by Cocultivation With Cortical Neurons: The Role of Crosstalk Between Cells.

PubMed

Babenko, Valentina A; Silachev, Denis N; Zorova, Ljubava D; Pevzner, Irina B; Khutornenko, Anastasia A; Plotnikov, Egor Y; Sukhikh, Gennady T; Zorov, Dmitry B

2015-09-01

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat cortical neurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain. The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to develop a detailed protocol for coculturing followed by cell separation. ©AlphaMed Press.

Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation.

PubMed

Abdallah, Basem M; Jafari, Abbas; Zaher, Walid; Qiu, Weimin; Kassem, Moustapha

2015-01-01

Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone. Copyright © 2014 Elsevier Inc. All rights reserved.

Dental pulp stem cells in regenerative dentistry.

PubMed

Casagrande, Luciano; Cordeiro, Mabel M; Nör, Silvia A; Nör, Jacques E

2011-01-01

Stem cells constitute the source of differentiated cells for the generation of tissues during development, and for regeneration of tissues that are diseased or injured postnatally. In recent years, stem cell research has grown exponentially owing to the recognition that stem cell-based therapies have the potential to improve the life of patients with conditions that span from Alzheimer's disease to cardiac ischemia to bone or tooth loss. Growing evidence demonstrates that stem cells are primarily found in niches and that certain tissues contain more stem cells than others. Among these tissues, the dental pulp is considered a rich source of mesenchymal stem cells that are suitable for tissue engineering applications. It is known that dental pulp stem cells have the potential to differentiate into several cell types, including odontoblasts, neural progenitors, osteoblasts, chondrocytes, and adipocytes. The dental pulp stem cells are highly proliferative. This characteristic facilitates ex vivo expansion and enhances the translational potential of these cells. Notably, the dental pulp is arguably the most accessible source of postnatal stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental pulp an attractive source of mesenchymal stem cells for tissue regeneration. This review discusses fundamental concepts of stem cell biology and tissue engineering within the context of regenerative dentistry.

Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

PubMed Central

Xu, Zijian; Wang, Wenjie; Jiang, Kaiju; Yu, Zhou; Huang, Huanwei; Wang, Fengchao; Zhou, Bin; Chen, Ting

2015-01-01

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. DOI: http://dx.doi.org/10.7554/eLife.10567.001 PMID:26653852

Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells.

PubMed

Gilbert, Jay; Reynolds, Nicholas P; Russell, Sarah M; Haylock, David; McArthur, Sally; Charnley, Mirren; Jones, Owen G

2017-10-01

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or β-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Can bone marrow differentiate into renal cells?

PubMed

Imai, Enyu; Ito, Takahito

2002-10-01

A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow-derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy.

Single-Factor SOX2 Mediates Direct Neural Reprogramming of Human Mesenchymal Stem Cells via Transfection of In Vitro Transcribed mRNA.

PubMed

Kim, Bo-Eun; Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Kang, Insung; Lee, Byung-Chul; Lee, Jin Young; Kook, Myoung Geun; Kang, Kyung-Sun

2018-01-01

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

PubMed

Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

2015-04-01

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

Cancer Stem Cells: Dynamic Entities in an Ever-Evolving Paradigm.

PubMed

Lopez-Bertoni, Hernando; Li, Yunqing; Laterra, John

2015-11-01

The cancer stem cell (CSC) hypothesis postulates that there is a hierarchy of cellular differentiation within cancers and that the bulk population of tumor cells is derived from a relatively small population of multi-potent neoplastic stem-like cells (CSCs). This tumor-initiating cell population plays an important role in maintaining tumor growth through their unlimited self-renewal, therapeutic resistance, and capacity to propagate tumors through asymmetric cell division. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to de-differentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of novel therapeutic strategies that focus on depleting tumors of their tumor-propagating cell population.

Regenerative Endodontics in light of the stem cell paradigm

PubMed Central

Rosa, Vinicius; Botero, Tatiana M.; Nör, Jacques E.

2013-01-01

Stem cells play a critical role in development and in tissue regeneration. The dental pulp contains a small sub-population of stem cells that are involved in the response of the pulp to caries progression. Specifically, stem cells replace odontoblasts that have undergone cell death as a consequence of the cariogenic challenge. Stem cells also secrete factors that have the potential to enhance pulp vascularization and provide the oxygen and nutrients required for the dentinogenic response that is typically observed in teeth with deep caries. However, the same angiogenic factors that are required for dentin regeneration may ultimately contribute to the demise of the pulp by enhancing vascular permeability and interstitial pressure. Recent studies focused on the biology of dental pulp stem cells revealed that the multipotency and angiogenic capacity of these cells could be exploited therapeutically in dental pulp tissue engineering. Collectively, these findings suggest new treatment paradigms in the field of Endodontics. The goal of this review is to discuss the potential impact of dental pulp stem cells to Regenerative Endodontics. PMID:21726222

In utero transplantation of human bone marrow-derived multipotent mesenchymal stem cells in mice.

PubMed

Chou, Shiu-Huey; Kuo, Tom K; Liu, Ming; Lee, Oscar K

2006-03-01

Mesenchymal stem cells (MSCs) are multipotent cells that can be isolated from human bone marrow and possess the potential to differentiate into progenies of embryonic mesoderm. However, current evidence is based predominantly on in vitro experiments. We used a murine model of in utero transplantation (IUT) to study the engraftment capabilities of human MSCs. MSCs were obtained from bone marrow by negative immunoselection and limiting dilution, and were characterized by flow cytometry and by in vitro differentiation into osteoblasts, chondrocytes, and adipocytes. MSCs were transplanted into fetal mice at a gestational age of 14 days. Engraftment of human MSCs was determined by flow cytometry, polymerase chain reaction, and fluorescence in situ hybridization (FISH). MSCs engrafted into tissues originating from all three germ layers and persisted for up to 4 months or more after delivery, as evidenced by the expression of the human-specific beta-2 microglobulin gene and by FISH for donor-derived cells. Donor-derived CD45+ cells were detectable in the peripheral blood of recipients, suggesting the participation of MSCs in hematopoiesis at the fetal stage. This model can further serve to evaluate possible applications of MSCs. Copyright 2006 Orthopaedic Research Society.

CRISPR/Cas9 in Stem Cell Research: Current Application and Future Perspective.

PubMed

Patmanathan, Sathya Narayanan; Gnanasegaran, Nareshwaran; Lim, Moon Nian; Husaini, Roslina; Fakiruddin, Kamal Shaik; Zakaria, Zubaidah

2018-06-12

The clustered regularly interspaced short palindromic repeats-associated protein 9 or CRISPR/Cas9 system is one of the hottest topics discussed lately due to its robustness and effectiveness in genome editing. The technology has been widely used in life science research including microbial, plant, animal, and human cell studies. Combined with the pluripotency of stem cells, the technology represents a powerful tool to generate various cell types for disease modeling, drug screening, toxicology, and targeted therapies. Generally, the CRISPR/Cas9 system has been applied in genetic modification of pluripotent or multipotent stem cells, after which the cells are differentiated into specific cell types and used for functional analysis or even clinical transplantation. Recent advancement in CRISPR/Cas9 technology has widened the scope of stem cell research and its therapeutic application. This review provides an overview of the current application and the prospect of CRISPR/Cas9 technology, particularly in stem cell research and therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art.

PubMed

Malgieri, Arianna; Kantzari, Eugenia; Patrizi, Maria Patrizia; Gambardella, Stefano

2010-09-07

Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in all tissues, as part of the perivascular population. As multipotent cells, MSCs can differentiate into different tissues originating from mesoderm ranging from bone and cartilage, to cardiac muscle. MSCs are an excellent candidate for cell therapy because they are easily accessible, their isolation is straightforward, they can be bio-preserved with minimal loss of potency, and they have shown no adverse reactions to allogeneic versus autologous MSCs transplants. Therefore, MSCs are being explored to regenerate damaged tissue and treat inflammation, resulting from cardiovascular disease and myo-cardial infarction (MI), brain and spinal cord injury, stroke, diabetes, cartilage and bone injury, Crohn's disease and graft versus host disease (GvHD). Most of the application and clinical trials involve MSCs from bone marrow (BMMSCs). Transplantation of MSCs from bone marrow is considered safe and has been widely tested in clinical trials of cardiovascular, neurological, and immunological disease with encouraging results. There are examples of MSCs utilization in the repair of kidney, muscle and lung. The cells were also found to promote angiogenesis, and were used in chronic skin wound treatment. Recent studies involve also mesenchymal stem cell transplant from umbilical cord (UCMSCt). One of these demonstrate that UCMSCt may improve symptoms and biochemical values in patients with severe refractory systemic lupus erythematosus (SLE), and therefore this source of MSCs need deeper studies and require more attention. However, also if there are 79 registered clinical trial sites for evaluating MSC therapy throughout the world, it is still a long way to go before using these cells as a routinely applied therapy in clinics.

Atherosclerosis is a vascular stem cell disease caused by insulin.

PubMed

Traunmüller, Friederike

2018-07-01

The present article proposes the hypothesis that when multipotent vascular stem cells are exposed to excessive insulin in a rhythmic pattern of sharply rising and falling concentrations, their differentiation is misdirected toward adipogenic and osteogenic cell lineages. This results in plaque-like accumulation of adipocytes with fat and cholesterol deposition from adipocyte debris, and osteogenic (progenitor) cells with a calcified matrix in advanced lesions. The ingrowth of capillaries and infiltration with macrophages, which upon uptake of lipids turn into foam cells, are unspecific pro-resolving reactions. Epidemiological, histopathological, pharmacological, and experimental evidence in favour of this hypothesis is summarised. Copyright © 2018. Published by Elsevier Ltd.

Nestin-expressing cells in the pancreatic islets of Langerhans.

PubMed

Hunziker, E; Stein, M

2000-04-29

The pancreatic islets of Langerhans produce several peptide hormones, predominantly the metabolically active hormones insulin and glucagon, which are critical for maintaining normal fuel homeostasis. Some evidence exists that pancreatic endocrine cells turn over at a slow rate and can regenerate in certain conditions. This could be due to the presence of pluripotent cells residing in the pancreas. Recently the intermediate filament protein nestin has been identified to be a marker for a multipotent stem cell in the central nervous system. Given the similarity between the pancreatic islets and neuronal cells, we hypothesized that stem cells expressing nestin might be present in the pancreas. Here we present evidence that a subset of cells in the pancreatic islets express the stem cell marker nestin. These cells might serve as precursors of differentiated pancreatic endocrine cells. Copyright 2000 Academic Press.

Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

PubMed

Beane, Olivia S; Darling, Eric M

2012-10-01

The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.

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.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

PubMed

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Mesenchymal Stem Cell Therapy for Nonhealing Cutaneous Wounds

PubMed Central

Hanson, Summer E.; Bentz, Michael L.; Hematti, Peiman

2014-01-01

Summary Chronic wounds remain a major challenge in modern medicine and represent a significant burden, affecting not only physical and mental health, but also productivity, health care expenditure, and long-term morbidity. Even under optimal conditions, the healing process leads to fibrosis or scar. One promising solution, cell therapy, involves the transplantation of progenitor/stem cells to patients through local or systemic delivery, and offers a novel approach to many chronic diseases, including nonhealing wounds. Mesenchymal stem cells are multipotent, adult progenitor cells of great interest because of their unique immunologic properties and regenerative potential. A variety of preclinical and clinical studies have shown that mesenchymal stem cells may have a useful role in wound-healing and tissue-engineering strategies and both aesthetic and reconstructive surgery. Recent advances in stem cell immunobiology can offer insight into the multiple mechanisms through which mesenchymal stem cells could affect underlying pathophysiologic processes associated with nonhealing mesenchymal stem cells. Critical evaluation of the current literature is necessary for understanding how mesenchymal stem cells could potentially revolutionize our approach to skin and soft-tissue defects and designing clinical trials to address their role in wound repair and regeneration. PMID:20124836

In vivo bone formation by human marrow stromal cells in biodegradable scaffolds that release dexamethasone and ascorbate-2-phosphate.

PubMed

Kim, Hyongbum; Suh, Hwal; Jo, Sangmee Ahn; Kim, Hyun Woo; Lee, Jung Min; Kim, Eun Hae; Reinwald, Yvonne; Park, Sang-Hyug; Min, Byoung-Hyun; Jo, Inho

2005-07-15

An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

Concise Review: Pluripotent Stem Cell-Derived Cardiac Cells, A Promising Cell Source for Therapy of Heart Failure: Where Do We Stand?

PubMed

Gouadon, Elodie; Moore-Morris, Thomas; Smit, Nicoline W; Chatenoud, Lucienne; Coronel, Ruben; Harding, Sian E; Jourdon, Philippe; Lambert, Virginie; Rucker-Martin, Catherine; Pucéat, Michel

2016-01-01

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials. © 2015 AlphaMed Press.

Ngn3+ endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

PubMed Central

Magenheim, Judith; Klein, Allon M.; Stanger, Ben Z.; Ashery-Padan, Ruth; Sosa-Pineda, Beatriz; Gu, Guoqiang; Dor, Yuval

2013-01-01

Summary During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta-cells from stem cells. PMID:21888903

Manipulation of immune system via immortal bone marrow stem cells.

PubMed

Ruedl, Christiane; Khameneh, Hanif Javanmard; Karjalainen, Klaus

2008-09-01

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

Commentary: "re-programming or selecting adult stem cells?".

PubMed

Trosko, James E

2008-01-01

The recent observations that embryonic stemness-associated genes could assist in the "de-differentiation" of adult skin fibroblast cells to "embryonic-like stem cells", using the "somatic cell nuclear transfer" techniques, have been interpreted as indicating a "re-programming" of genes. These reports have demonstrated a "proof of principle" approach to by-pass many, but not all, of the ethical, scientific and medical limitations of the "therapeutic cloning" of embryonic stem cells from embryos. However, while the interpretation that real "re-programming" of all those somatic fibroblastic differentiation genes might be correct, there does exists an alternative hypothesis of these exciting results. Based on the fact that multipotent adult stem cells exist in most, if not all, adult organs, the possibility exists that all these recent "re-programming" results, using the somatic nuclear transfer techniques, actually were the results of transferred rare nuclear material from the adult stem cells residing in the skin of the mouse, monkey and human samples. An examination of the rationale for this challenging hypothesis has been drawn from the hypothesis of the "stem cell theory of cancer", as well as from the field of human adult stem cells research.

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.

PubMed

Zhao, Yong; Jiang, Zhaoshun; Zhao, Tingbao; Ye, Mingliang; Hu, Chengjin; Zhou, Huimin; Yin, Zhaohui; Chen, Yana; Zhang, Ye; Wang, Shanfeng; Shen, Jie; Thaker, Hatim; Jain, Summit; Li, Yunxiang; Diao, Yalin; Chen, Yingjian; Sun, Xiaoming; Fisk, Mary Beth; Li, Heng

2013-07-09

The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. In an open-label, phase 1/phase 2 study, patients (N=36) with long-standing T2D were divided into three groups (Group A, oral medications, n=18; Group B, oral medications+insulin injections, n=11; Group C having impaired β-cell function with oral medications+insulin injections, n=7). All patients received one treatment with the Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient's circulation. Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61%±1.12 at baseline to 7.25%±0.58 at 12 weeks (P=2.62E-06), and 7.33%±1.02 at one year post-treatment (P=0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. ClinicalTrials.gov number, NCT01415726.

[Research Progress on Metabolic Regulatory Mechanisms of Hematopoietic Stem Cells -Review].

PubMed

Zhang, Ya-Wen; Cheng, Hui; Cheng, Tao

2018-06-01

Hematopoietic stem cells (HSC) are a class of stem cells with self-renewal and multipotent differentiation into a variety of blood cells and are most thoroughly studied, maturely applied in the clinic adult stem cell. Function of HSC is closely associated with metabolic regulation. The metabolic state mainly maintains HSC living in hypoxic bone marrow microenvironment depending on glycolysis for energy metabolism, and keeping low reactive oxygen species (ROS) level. Proteins like Hif-1, FoxO3, ATM, PTPMT1 protect HSC from ROS injury, maintaining HSC in hypoxic state. In addition, glucose metabolism-related enzymes, glutamine, fatty acid oxidation, purine and amino acid metabolism also play important roles in metabolic regulation of HSC. In this review the research progress on metabolism regnlation mechanisms of HSC is summurized, focusing on the mechanisms releted with oxydation metabolism regulation, carbohydrate metabolism level, purine metabolism and aminoacide metabolism.

Epidermal Growth Factor (EGF) Receptor Intron 1 CA Repeat Polymorphisms in African-American and Caucasian Males: Influence on Prostate Cancer Risk or Disease Progression and Interaction with Androgen Receptor CAG Repeat Polymorphisms

DTIC Science & Technology

2003-09-01

Adipose Stromal Cells from Tumescent Liposuction Procedures. American Society for Dermatologic Surgery, $15,000 direct, 11/01/01 -10/31/02. 1999...stromal cells from tumescent liposuction procedures" ASDS Annual Meeting, Chicago, IL, November 1,2002."Adult Multipotent Stem Cells", Coriell

Tailless-like (TLX) protein promotes neuronal differentiation of dermal multipotent stem cells and benefits spinal cord injury in rats.

PubMed

Wang, Tao; Ren, Xiaobao; Xiong, Jianqiong; Zhang, Lei; Qu, Jifu; Xu, Wenyue

2011-04-01

Spinal cord injury (SCI) remains a formidable challenge in the clinic. In the current study, we examined the effects of the TLX gene on the proliferation and neuronal differentiation of dermal multipotent stem cells (DMSCs) in vitro and the potential of these cells to improve SCI in rats in vivo. DMSCs were stably transfected with TLX-expressing plasmid (TLX/DMSCs). Cell proliferation was examined using the MTT assay, and neuronal differentiation was characterized by morphological observation combined with immunocytochemical/immunofluorescent staining. The in vivo functions of these cells were evaluated by transplantation into rats with SCI, followed by analysis of hindlimb locomotion and post-mortem histology. Compared to parental DMSCs, TLX/DMSCs showed enhanced proliferation and preferential differentiation into NF200-positive neurons in contrast to GFAP-positive astrocytes. When the undifferentiated cells were transplanted into rats with SCI injury, TLX/DMSCs led to significant improvement in locomotor recovery and healing of SCI, as evidenced by reduction in scar tissues and cavities, increase in continuous nerve fibers/axons and enrichment of NF200-positive neurons on the histological level. In conclusion, TLX promotes the proliferation and neuronal differentiation of DMSCs and thus, may serve as a promising therapy for SCI in the clinic.

Where will the stem cells lead us? Prospects for dentistry in the 21st century

PubMed Central

Sreenivas, S. Durga; Rao, Akula Sreenivasa; Satyavani, S. Sri; Reddy, Bavigadda Harish; Vasudevan, Sanjay

2011-01-01

It is dentists’ dream to achieve bone repair with predictability, but without donor site morbidity as well as reconstruction of injured or pathologically damaged complex dental structures, however, this will no longer be a dream as these are being made into a reality using stem cell science. Stem cell science is clearly an intriguing and promising area of science. Stem cells have been isolated from a variety of embryonic and adult tissues. Dental stem cells are multipotent mesenchymal stem cells (MSCs) brought new enthusiasm among the researchers because of their easy accessibility, high quality and they don’t pose the same ethical concerns and controversy in comparison with embryonic stem cells. This review article provides brief insights about stem cell basics, the state of art in human dental stem cell research and its possible impact on future dentistry. Even though most of these modalities are still in infancy, it is evident that the 21st century dentist is going to play a critical role in the field of medicine. The aim of this article is to bring awareness among the dentists about the huge potential associated with the use of stem cells in a clinical setting, as well as proper understanding of related problems. PMID:22028504

Human Fetal Keratocytes Have Multipotent Characteristics in the Developing Avian Embryo

PubMed Central

Chao, Jennifer R.; Bronner, Marianne E.

2013-01-01

The human cornea contains stem cells that can be induced to express markers consistent with multipotency in cell culture; however, there have been no studies demonstrating that human corneal keratocytes are multipotent. The objective of this study is to examine the potential of human fetal keratocytes (HFKs) to differentiate into neural crest-derived tissues when challenged in an embryonic environment. HFKs were injected bilaterally into the cranial mesenchyme adjacent to the neural tube and the periocular mesenchyme in chick embryos at embryonic days 1.5 and 3, respectively. The injected keratocytes were detected by immunofluorescence using the human cell-specific marker, HuNu. HuNu-positive keratocytes injected along the neural crest pathway were localized adjacent to HNK-1-positive migratory host neural crest cells and in the cardiac cushion mesenchyme. The HuNu-positive cells transformed into neural crest derivatives such as smooth muscle in cranial blood vessels, stromal keratocytes, and corneal endothelium. However, they failed to form neurons despite their presence in the condensing trigeminal ganglion. These results show that HFKs retain the ability to differentiate into some neural crest-derived tissues. Their ability to respond to embryonic cues and generate corneal endothelium and stromal keratocytes provides a basis for understanding the feasibility of creating specialized cells for possible use in regenerative medicine. PMID:23461574

Are hematopoietic stem cells involved in hepatocarcinogenesis?

PubMed

Facciorusso, Antonio; Antonino, Matteo; Del Prete, Valentina; Neve, Viviana; Scavo, Maria Principia; Barone, Michele

2014-08-01

THE LIVER HAS THREE CELL LINEAGES ABLE TO PROLIFERATE AFTER A HEPATIC INJURY: the mature hepatocyte, the ductular "bipolar" progenitor cell termed "oval cell" and the putative periductular stem cell. Hepatocytes can only produce other hepatocytes whereas ductular progenitor cells are considerate bipolar since they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential and may be multipotent, being this aspect still under investigation. They originate in the bone marrow since their progeny express genetic markers of donor hematopoietic cells after bone marrow transplantation. Since the liver is the hematopoietic organ of the fetus, it is possible that hematopoietic stem cells may reside in the liver of the adult. This assumption is proved by the finding that oval cells express hematopoietic markers like CD34, CD45, CD 109, Thy-1, c-kit, and others, which are also expressed by bone marrow-derived hematopoietic stem cells (BMSCs). Few and discordant studies have evaluated the role of BMSC in hepatocarcinogenesis so far and further studies in vitro and in vivo are warranted in order to definitively clarify such an issue.

Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development.

PubMed

Barker, Nick; Rookmaaker, Maarten B; Kujala, Pekka; Ng, Annie; Leushacke, Marc; Snippert, Hugo; van de Wetering, Marc; Tan, Shawna; Van Es, Johan H; Huch, Meritxell; Poulsom, Richard; Verhaar, Marianne C; Peters, Peter J; Clevers, Hans

2012-09-27

Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage.

PubMed

Yang, Yu; Bolnick, Alan; Shamir, Alexandra; Abdulhasan, Mohammed; Li, Quanwen; Parker, G C; Puscheck, Elizabeth E; Rappolee, D A

2017-08-01

Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.

Laboratory models for central nervous system tumor stem cell research.

PubMed

Khan, Imad Saeed; Ehtesham, Moneeb

2015-01-01

Central nervous system (CNS) tumors are complex organ systems comprising of a neoplastic component with associated vasculature, inflammatory cells, and reactive cellular and extracellular components. Research has identified a subset of cells in CNS tumors that portray defining properties of neural stem cells, namely, that of self-renewal and multi-potency. Growing evidence suggests that these tumor stem cells (TSC) play an important role in the maintenance and growth of the tumor. Furthermore, these cells have also been shown to be refractory to conventional therapy and may be crucial for tumor recurrence and metastasis. Current investigations are focusing on isolating these TSC from CNS tumors to investigate their unique biological processes. This understanding will help identify and develop more effective and comprehensive treatment strategies. This chapter provides an overview of some of the most commonly used laboratory models for CNSTSC research.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells.

PubMed

Liu, Ying; Giannopoulou, Eugenia G; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C David; Rafii, Shahin; Seandel, Marco

2016-04-27

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells

PubMed Central

Liu, Ying; Giannopoulou, Eugenia G.; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C. David; Rafii, Shahin; Seandel, Marco

2016-01-01

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming. PMID:27117588

Mesenchymal stem cells for the treatment of systemic lupus erythematosus: is the cure for connective tissue diseases within connective tissue?

PubMed

Carrion, Flavio A; Figueroa, Fernando E

2011-05-11

Mesenchymal stem cells (MSCs) are now known to display not only adult stem cell multipotency but also robust anti-inflammatory and regenerative properties. After widespread in vitro and in vivo preclinical testing in several autoimmune disease models, allogenic MSCs have been successfully applied in patients with severe treatment-refractory systemic lupus erythematosus. The impressive results of these uncontrolled phase I and II trials - mostly in patients with non-responding renal disease - point to the need to perform controlled multicentric trials. In addition, they suggest that there is much to be learned from the basic and clinical science of MSCs in order to reap the full potential of these multifaceted progenitor cells in the treatment of autoimmune diseases.

Long-Term Effects of Stem Cells on Total-Body Irradiated Mice

NASA Astrophysics Data System (ADS)

Vyalkina, M. V.; Alchinova, I. B.; Yakovenko, E. N.; Medvedeva, Yu S.; Saburina, I. N.; Karganov, M. Yu

2017-01-01

C57Bl/6 mice were exposed to γ-radiation in a sublethal dose of 7.5 Gy. In 3 hours injection 106/mouse of bone marrow multipotent mesenchymal stromal cells stem cells intravenously to experimental group was done. Methods used: body weight measurement, open field behavior, subfraction composition of blood serum (laser correlation spectroscopy, LCS), histological examination of the spleen, liver, and pancreas, count of T and B cells, white blood formula. After 1.5 and 3 months the general trend towards intermediate position of the parameters observed in the experimental between those in intact and irradiated controls attests to partial protective/restorative effects of the injected cells.

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.

Stem cells in drug discovery, tissue engineering, and regenerative medicine: emerging opportunities and challenges.

PubMed

Nirmalanandhan, Victor Sanjit; Sittampalam, G Sitta

2009-08-01

Stem cells, irrespective of their origin, have emerged as valuable reagents or tools in human health in the past 2 decades. Initially, a research tool to study fundamental aspects of developmental biology is now the central focus of generating transgenic animals, drug discovery, and regenerative medicine to address degenerative diseases of multiple organ systems. This is because stem cells are pluripotent or multipotent cells that can recapitulate developmental paths to repair damaged tissues. However, it is becoming clear that stem cell therapy alone may not be adequate to reverse tissue and organ damage in degenerative diseases. Existing small-molecule drugs and biologicals may be needed as "molecular adjuvants" or enhancers of stem cells administered in therapy or adult stem cells in the diseased tissues. Hence, a combination of stem cell-based, high-throughput screening and 3D tissue engineering approaches is necessary to advance the next wave of tools in preclinical drug discovery. In this review, the authors have attempted to provide a basic account of various stem cells types, as well as their biology and signaling, in the context of research in regenerative medicine. An attempt is made to link stem cells as reagents, pharmacology, and tissue engineering as converging fields of research for the next decade.

The expression of cancer stem cell markers in human colorectal carcinoma cells in a microenvironment dependent manner.

PubMed

Stankevicius, Vaidotas; Kunigenas, Linas; Stankunas, Edvinas; Kuodyte, Karolina; Strainiene, Egle; Cicenas, Jonas; Samalavicius, Narimantas E; Suziedelis, Kestutis

2017-03-18

Numerous lines of evidence support the hierarchical model of cancer development and tumor initiation. According to the theory, cancer stem cells play a crucial role in the formation of the tumor and should be targeted for more effective anticancer treatment. However, cancer stem cells quickly loose their characteristics when propagated as 2D cell culture, indicating that the 2D cell culture does not provide the appropriate settings to maintain an in vivo environment. In this study we have investigated the expression of self-renewal, cancer stem cell and epithelial to mesenchymal transition markers after the transfer of human colorectal carcinoma cell DLD1 and HT29 lines from 2D cell cultures to scaffold-attached laminin rich extracellular matrix and scaffold-free multicellular spheroid 3D culture models. Based on the up-regulated expression of multipotency, CSC and EMT markers, our data suggests that human colorectal carcinoma cells grown in 3D exhibit enhanced cancer stem cell characteristics. Therefore, in order to design more efficient targeted therapies, we suggest that 3D cell culture models should be employed in cancer stem cell research. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability, motility, and tropism to brain cancer

PubMed Central

Feng, Y; Zhu, M; Dangelmajer, S; Lee, Y M; Wijesekera, O; Castellanos, C X; Denduluri, A; Chaichana, K L; Li, Q; Zhang, H; Levchenko, A; Guerrero-Cazares, H; Quiñones-Hinojosa, A

2014-01-01

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM. PMID:25501828

Geminin Participates in Differentiation Decisions of Adult Neural Stem Cells Transplanted in the Hemiparkinsonian Mouse Brain.

PubMed

Taouki, Ioanna; Tasiudi, Eve; Lalioti, Maria-Eleni; Kyrousi, Christina; Skavatsou, Eleni; Kaplani, Konstantina; Lygerou, Zoi; Kouvelas, Elias D; Mitsacos, Adamantia; Giompres, Panagiotis; Taraviras, Stavros

2017-08-15

Neural stem cells have been considered as a source of stem cells that can be used for cell replacement therapies in neurodegenerative diseases, as they can be isolated and expanded in vitro and can be used for autologous grafting. However, due to low percentages of survival and varying patterns of differentiation, strategies that will enhance the efficacy of transplantation are under scrutiny. In this article, we have examined whether alterations in Geminin's expression, a protein that coordinates the balance between self-renewal and differentiation, can improve the properties of stem cells transplanted in 6-OHDA hemiparkinsonian mouse model. Our results indicate that, in the absence of Geminin, grafted cells differentiating into dopaminergic neurons were decreased, while an increased number of oligodendrocytes were detected. The number of proliferating multipotent cells was not modified by the absence of Geminin. These findings encourage research related to the impact of Geminin on transplantations for neurodegenerative disorders, as an important molecule in influencing differentiation decisions of the cells composing the graft.

Ablation of Coactivator Med1 Switches the Cell Fate of Dental Epithelia to That Generating Hair

PubMed Central

Nguyen, Thai; Sakai, Kiyoshi; He, Bing; Fong, Chak; Oda, Yuko

2014-01-01

Cell fates are determined by specific transcriptional programs. Here we provide evidence that the transcriptional coactivator, Mediator 1 (Med1), is essential for the cell fate determination of ectodermal epithelia. Conditional deletion of Med1 in vivo converted dental epithelia into epidermal epithelia, causing defects in enamel organ development while promoting hair formation in the incisors. We identified multiple processes by which hairs are generated in Med1 deficient incisors: 1) dental epithelial stem cells lacking Med 1 fail to commit to the dental lineage, 2) Sox2-expressing stem cells extend into the differentiation zone and remain multi-potent due to reduced Notch1 signaling, and 3) epidermal fate is induced by calcium as demonstrated in dental epithelial cell cultures. These results demonstrate that Med1 is a master regulator in adult stem cells to govern epithelial cell fate. PMID:24949995

Critical issues for engineering cord blood stem cells to produce insulin.

PubMed

Denner, Larry; Urban, Randall J

2008-09-01

The objectives of using cord blood stem cells for treating type 1 diabetes are simple in principle yet complex in biological and molecular mechanisms. These are defined by the complexity of the insulin-producing unit of the pancreas, the islet. Islets are composed of various cell types that arise from diverse lineages and communicate by hormones, growth factors and small-molecule mediators. These processes are regulated by integration of signal transduction pathways. While advances have been made to engineer umbilical cord blood stem cells to produce insulin, these studies only illuminate the potential of such cells to fulfil a necessary, but not sufficient, requirement for transplantation. The challenges ahead demand detailed understanding of molecular mechanisms to move from an opportunistic, phenotypic approach to transplantation and amelioration of blood glucose, to an orderly and logical approach to a biologically and medically meaningful solution. The issues include expansion to generate large numbers of cells, self-renewal to regulate the destiny of cord blood stem cells to repopulate the hematopoietic system, and multipotency of stem cells to generate the distinct cell types of an islet.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

PubMed

Li, Yuan-Sheng; Chen, Pao-Jen; Wu, Li-Wei; Chou, Pei-Wen; Sun, Li-Yi; Chiou, Tzyy-Wen

2018-02-01

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

PubMed

Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

2012-08-01

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues.

PubMed

Kosheleva, N V; Saburina, I N; Zurina, I M; Gorkun, A A; Borzenok, S A; Nikishin, D A; Kolokoltsova, T D; Ustinova, E E; Repin, V S

2016-01-01

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

PubMed

Bakkar, Mohammed; Liu, Younan; Fang, Dongdong; Stegen, Camille; Su, Xinyun; Ramamoorthi, Murali; Lin, Li-Chieh; Kawasaki, Takako; Makhoul, Nicholas; Pham, Huan; Sumita, Yoshinori; Tran, Simon D

2017-01-01

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.

Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

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

Ono, Hiromasa; Database Center for Life Science; Oki, Yoshinao

2011-04-15

Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed asmore » well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.« less

Drosophila Perlecan Regulates Intestinal Stem Cell Activity via Cell-Matrix Attachment

PubMed Central

You, Jia; Zhang, Yan; Li, Zhouhua; Lou, Zhefeng; Jin, Longjin; Lin, Xinhua

2014-01-01

Summary Stem cells require specialized local microenvironments, termed niches, for normal retention, proliferation, and multipotency. Niches are composed of cells together with their associated extracellular matrix (ECM). Currently, the roles of ECM in regulating niche functions are poorly understood. Here, we demonstrate that Perlecan (Pcan), a highly conserved ECM component, controls intestinal stem cell (ISC) activities and ISC-ECM attachment in Drosophila adult posterior midgut. Loss of Pcan from ISCs, but not other surrounding cells, causes ISCs to detach from underlying ECM, lose their identity, and fail to proliferate. These defects are not a result of a loss of epidermal growth factor receptor (EGFR) or Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling activity but partially depend on integrin signaling activity. We propose that Pcan secreted by ISCs confers niche properties to the adjacent ECM that is required for ISC maintenance of stem cell identity, activity, and anchorage to the niche. PMID:24936464

Progenitors of the protochordate ocellus as an evolutionary origin of the neural crest

PubMed Central

2013-01-01

The neural crest represents a highly multipotent population of embryonic stem cells found only in vertebrate embryos. Acquisition of the neural crest during the evolution of vertebrates was a great advantage, providing Chordata animals with the first cellular cartilage, bone, dentition, advanced nervous system and other innovations. Today not much is known about the evolutionary origin of neural crest cells. Here we propose a novel scenario in which the neural crest originates from neuroectodermal progenitors of the pigmented ocelli in Amphioxus-like animals. We suggest that because of changes in photoreception needs, these multipotent progenitors of photoreceptors gained the ability to migrate outside of the central nervous system and subsequently started to give rise to neural, glial and pigmented progeny at the periphery. PMID:23575111

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Germline stem cells and sex determination in Hydra.

PubMed

Nishimiya-Fujisawa, Chiemi; Kobayashi, Satoru

2012-01-01

The sex of germline stem cells (GSCs) in Hydra is determined in a cell-autonomous manner. In gonochoristic species like Hydra magnipapillata or H. oligactis, where the sexes are separate, male polyps have sperm-restricted stem cells (SpSCs), while females have egg-restricted stem cells (EgSCs). These GSCs self-renew in a polyp, and are usually transmitted to a new bud from a parental polyp during asexual reproduction. But if these GSCs are lost during subsequent budding or regeneration events, new ones are generated from multipotent stem cells (MPSCs). MPSCs are the somatic stem cells in Hydra that ordinarily differentiate into nerve cells, nematocytes (stinging cells in cnidarians), and gland cells. By means of such a backup system, sexual reproduction is guaranteed for every polyp. Interestingly, Hydra polyps occasionally undergo sex-reversal. This implies that each polyp can produce either type of GSCs, i.e. Hydra are genetically hermaphroditic. Nevertheless a polyp possesses only one type of GSCs at a time. We propose a plausible model for sex-reversal in Hydra. We also discuss so-called germline specific genes, which are expressed in both GSCs and MPSCs, and some future plans to investigate Hydra GSCs.

A stem cell apostasy: A tale of 4 H words

PubMed Central

Quesenberry, Peter J.; Goldberg, Laura R.; Dooner, Mark S.

2014-01-01

The field of hematopoietic stem cell biology has become increasingly dominated by the pursuit and study of highly purified populations of hematopoietic stem cells (HSCs). Such HSCs are typically isolated based on their cell surface marker expression patterns and ultimately defined by their multipotency and capacity for self-generation. However, even with progressively more stringent stem cell separation techniques, the resultant HSC population remains heterogeneous with respect to both self-renewal and differentiation capacity. Critical studies on un-separated whole bone marrow (WBM) have definitively shown that long-term engraftable hematopoietic stem cells are in active cell cycle and thus continually changing phenotype. Therefore, they cannot be purified by current approaches dependent on stable surface epitope expression because the surface markers are continually changing as well. These critical cycling cells are discarded with current stem cell purifications. Despite this, research defining such characteristics as self-renewal capacity, lineage-commitment, bone marrow niches, and proliferative state of HSCs continues to focus predominantly on this small sub-population of purified marrow cells. This review discusses the research leading to the hierarchical model of hematopoiesis and questions the dogmas pertaining to HSC quiescence and purification. PMID:25183450

Harnessing the potential of lung stem cells for regenerative medicine.

PubMed

McQualter, Jonathan L; Anthony, Desiree; Bozinovski, Steven; Prêle, Cecilia M; Laurent, Geoffrey J

2014-11-01

In response to recurrent exposure to environmental insults such as allergens, pollution, irritants, smoke and viral/bacterial infection, the epithelium of the lung is continually damaged. Homeostasis of the lung requires a balance between immune regulation and promotion of tissue regeneration, which requires the co-ordinated proliferation and differentiation of stem and progenitor cells. In this review we reflect on the current understanding of lung epithelial stem and progenitor cells and advocate a model hierarchy in which self-renewing multipotent lung epithelial stem cells give rise to lineage restricted progenitor cells that repopulate airway and alveolar epithelial cell lineages during homeostasis and repair. We also discuss the role of mesenchymal progenitor cells in maintaining the structural integrity of the lung and propose a model in which mesenchymal cells act as the quintessential architects of lung regeneration by providing molecular signals, such as FGF-10, to regulate the fate and specificity of epithelial stem and progenitor cells. Moreover, we discuss the current status and future prospects for translating lung stem cell therapies to the clinic to replace, repair, or regenerate diseased lung tissue. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

PubMed

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

2016-11-17

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

PubMed Central

Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

2016-01-01

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

18F-FDG labeling of mesenchymal stem cells and multipotent adult progenitor cells for PET imaging: effects on ultrastructure and differentiation capacity.

PubMed

Wolfs, Esther; Struys, Tom; Notelaers, Tineke; Roberts, Scott J; Sohni, Abhishek; Bormans, Guy; Van Laere, Koen; Luyten, Frank P; Gheysens, Olivier; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2013-03-01

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

PubMed

Lewallen, Eric A; Jones, Dakota L; Dudakovic, Amel; Thaler, Roman; Paradise, Christopher R; Kremers, Hilal M; Abdel, Matthew P; Kakar, Sanjeev; Dietz, Allan B; Cohen, Robert C; Lewallen, David G; van Wijnen, Andre J

2016-05-01

Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant. Copyright © 2016 Elsevier B.V. All rights reserved.

Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium.

PubMed

Chen, Gecai; Yue, Aihuan; Ruan, Zhongbao; Yin, Yigang; Wang, Ruzhu; Ren, Yin; Zhu, Li

2014-12-01

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology, immunophenotype and multi-lineage potential were analyzed at passages 3, 5, 10, 15, 20, and 25 (P3, P5, P10, P15, P20, and P25, respectively). The cell cycle distribution, apoptosis, and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3, P5, P10, P15, P20, and P25. From P3 to P25, the three defining biological properties of hUC-MSCs [adherence to plastic, specific surface antigen expression, multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased, compared with the cells at P3 (P < 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %), compared to those at P3 (P < 0.01). Within subculture generations 3-20 (P3-P20), the differences between the cell apoptotic rates were not statistically significant (P > 0.05). There were no detectable chromosome eliminations, displacements, or chromosomal imbalances, as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations.

Ectodermal Differentiation of Wharton's Jelly Mesenchymal Stem Cells for Tissue Engineering and Regenerative Medicine Applications.

PubMed

Jadalannagari, Sushma; Aljitawi, Omar S

2015-06-01

Mesenchymal stem cells (MSCs) from Wharton's jelly (WJ) of the human umbilical cord are perinatal stem cells that have self-renewal ability, extended proliferation potential, immunosuppressive properties, and are accordingly excellent candidates for tissue engineering. These MSCs are unique, easily accessible, and a noncontroversial cell source of regeneration in medicine. Wharton's jelly mesenchymal stem cells (WJMSCs) are multipotent and capable of multilineage differentiation into cells like adipocytes, bone, cartilage, and skeletal muscle upon exposure to appropriate conditions. The ectoderm is one of the three primary germ layers found in the very early embryo that differentiates into the epidermis, nervous system (spine, peripheral nerves, brain), and exocrine glands (mammary, sweat, salivary, and lacrimal glands). Accumulating evidence shows that MSCs obtained from WJ have an ectodermal differentiation potential. The current review examines this differentiation potential of WJMSC into the hair follicle, skin, neurons, and sweat glands along with discussing the potential utilization of such differentiation in regenerative medicine.

The promising potential of menstrual stem cells for antenatal diagnosis and cell therapy.

PubMed

Khoury, Maroun; Alcayaga-Miranda, Francisca; Illanes, Sebastián E; Figueroa, Fernando E

2014-01-01

Menstrual-derived stem cells (MenSCs) are a new source of mesenchymal stem cells isolated from the menstrual fluid. Currently, there is a growing interest in their clinical potential due to fact that they are multipotent, highly proliferative, and easy to obtain in a non-invasive manner. Sampling can be repeated periodically in a simplified and reproducible manner devoid of complications that no existing cell source can match. MenSCs are also free of ethical dilemmas, and display novel properties with regard to presently known adult derived stem cells. This review details their distinctive biological properties regarding immunophenotype and function, proliferation rate, differentiation potential, and paracrine effects mediated by secreted factors. Their possible role in antenatal diagnosis is also discussed. While more insight on their immunomodulatory and diagnostic properties is needed, the impact of clinical and epidemiological factors, such as age, use of contraceptives, or hormonal status still requires further investigations to properly assess their current and future use in clinical application and diagnosis.

The role of tumor microenvironment in development and progression of malignant melanomas - a systematic review.

PubMed

Gurzu, Simona; Beleaua, Marius Alexandru; Jung, Ioan

2018-01-01

To reveal the particular aspects of the tumor microenvironment of malignant melanomas, a systematic review including 34 representative papers was performed. The review took into account the aspects related the Wnt/β-catenin pathway-related epithelial-mesenchymal transition (EMT) versus mesenchymal-epithelial transition (MET) of keratinocytes, fibroblasts and melanoma cells, as possible tools for understanding genesis and evolution of malignant melanoma. The possible reversible features of EMT and the role of tumor microenvironment in the metastatic process were also analyzed. A particular issue was related on the cancer stem cells that include melanocyte stem cells (McSCs) and multipotent mesenchymal stem/stromal cells (MSCs). As the McSCs embryological development in mouse is not similar to human development, the role of stem cells in genesis and development of human melanoma should be proved in human melanoma cells only. For further development of targeted therapy, a better understanding of melanomagenesis pathways and its microenvironment particularities is necessary.

The variable role of SIRT1 in the maintenance and differentiation of mesenchymal stem cells.

PubMed

Zainabadi, Kayvan

2018-04-01

SIRT1 is an NAD + -dependent deacetylase that acts as a nutrient sensitive regulator of longevity. SIRT1 also acts as a key regulator of mesenchymal stem cells (MSCs), adult stem cells that give rise to tissues such as bone, fat, muscle and cartilage. This review focuses on how SIRT1 regulates the self-renewal, multipotency and differentiation of MSCs. The variable role of SIRT1 in promoting the differentiation of MSCs towards certain lineages, while repressing others, will be examined within the broader context of aging, calorie restriction, and regenerative medicine. Finally, recent animal and human studies will be highlighted which paint an overall salutary role for SIRT1 in protecting MSCs (and resulting tissues) from age-related atrophy and dysfunction.

New factors controlling the balance between osteoblastogenesis and adipogenesis.

PubMed

Abdallah, Basem M; Kassem, Moustapha

2012-02-01

The majority of conditions associated with bone loss, including aging, are accompanied by increased marrow adiposity possibly due to shifting of the balance between osteoblast and adipocyte differentiation in bone marrow stromal (skeletal) stem cells (MSC). In order to study the relationship between osteoblastogenesis and adipogenesis in bone marrow, we have characterized cellular models of multipotent MSC as well as pre-osteoblastic and pre-adipocytic cell populations. Using these models, we identified two secreted factors in the bone marrow microenviroment: secreted frizzled-related protein 1 (sFRP-1) and delta-like1 (preadipocyte factor 1) (Dlk1/Pref-1). Both exert regulatory effects on osteoblastogenesis and adipogenesis. Our studies suggest a model for lineage fate determination of MSC that is regulated through secreted factors in the bone marrow microenvironment that mediate a cross-talk between lineage committed cell populations in addition to controlling differentiation choices of multipotent MSC. Copyright © 2011 Elsevier Inc. All rights reserved.

PSA-NCAM-Negative Neural Crest Cells Emerging during Neural Induction of Pluripotent Stem Cells Cause Mesodermal Tumors and Unwanted Grafts

PubMed Central

Lee, Dongjin R.; Yoo, Jeong-Eun; Lee, Jae Souk; Park, Sanghyun; Lee, Junwon; Park, Chul-Yong; Ji, Eunhyun; Kim, Han-Soo; Hwang, Dong-Youn; Kim, Dae-Sung; Kim, Dong-Wook

2015-01-01

Summary Tumorigenic potential of human pluripotent stem cells (hPSCs) is an important issue in clinical applications. Despite many efforts, PSC-derived neural precursor cells (NPCs) have repeatedly induced tumors in animal models even though pluripotent cells were not detected. We found that polysialic acid-neural cell adhesion molecule (PSA-NCAM)− cells among the early NPCs caused tumors, whereas PSA-NCAM+ cells were nontumorigenic. Molecular profiling, global gene analysis, and multilineage differentiation of PSA-NCAM− cells confirm that they are multipotent neural crest stem cells (NCSCs) that could differentiate into both ectodermal and mesodermal lineages. Transplantation of PSA-NCAM− cells in a gradient manner mixed with PSA-NCAM+ cells proportionally increased mesodermal tumor formation and unwanted grafts such as PERIPHERIN+ cells or pigmented cells in the rat brain. Therefore, we suggest that NCSCs are a critical target for tumor prevention in hPSC-derived NPCs, and removal of PSA-NCAM− cells eliminates the tumorigenic potential originating from NCSCs after transplantation. PMID:25937368

Two sides of the same coin: stem cells in cancer and regenerative medicine.

PubMed

Ilmer, Matthias; Vykoukal, Jody; Recio Boiles, Alejandro; Coleman, Michael; Alt, Eckhard

2014-07-01

Multipotent stromal cells (MSCs) derived from bone marrow, adipose tissue, cord blood, and other origins have recently received much attention as potential therapeutic agents with beneficial immunomodulatory and regenerative properties. In their native tissue environment, however, such cells also appear to have essential functions in building and supporting tumor microenvironments, providing metastatic niches, and maintaining cancer hallmarks. Here, we consider the varied roles of these tissue-resident stroma-associated cells, synthesize recent and emerging discoveries, and discuss the role, potential, and clinical applications of MSCs in cancer and regenerative medicine.-Ilmer, M., Vykoukal, J., Recio Boiles, A., Coleman, M., Alt, E. Two sides of the same coin: stem cells in cancer and regenerative medicine. © FASEB.

In Vitro Generation of Vascular Wall-Resident Multipotent Stem Cells of Mesenchymal Nature from Murine Induced Pluripotent Stem Cells.

PubMed

Steens, Jennifer; Zuk, Melanie; Benchellal, Mohamed; Bornemann, Lea; Teichweyde, Nadine; Hess, Julia; Unger, Kristian; Görgens, André; Klump, Hannes; Klein, Diana

2017-04-11

The vascular wall (VW) serves as a niche for mesenchymal stem cells (MSCs). In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs), based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs) to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs).

PubMed

Hu, Xue; Li, Li; Yu, Xinyi; Zhang, Ruyi; Yan, Shujuan; Zeng, Zongyue; Shu, Yi; Zhao, Chen; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; An, Liping; Huang, Shifeng; Ji, Xiaojuan; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Liu, Wei; Huang, Bo; Feng, Yixiao; Zhang, Bo; Haydon, Rex C; Luu, Hue H; Reid, Russell R; Lee, Michael J; Wolf, Jennifer Moriatis; Yu, Zebo; He, Tong-Chuan

2017-12-19

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo . Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine.

CRISPR/Cas9-mediated reversibly immortalized mouse bone marrow stromal stem cells (BMSCs) retain multipotent features of mesenchymal stem cells (MSCs)

PubMed Central

Hu, Xue; Li, Li; Yu, Xinyi; Zhang, Ruyi; Yan, Shujuan; Zeng, Zongyue; Shu, Yi; Zhao, Chen; Wu, Xingye; Lei, Jiayan; Li, Yasha; Zhang, Wenwen; Yang, Chao; Wu, Ke; Wu, Ying; An, Liping; Huang, Shifeng; Ji, Xiaojuan; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Liu, Wei; Huang, Bo; Feng, Yixiao; Zhang, Bo; Haydon, Rex C.; Luu, Hue H.; Reid, Russell R.; Lee, Michael J.; Wolf, Jennifer Moriatis; Yu, Zebo; He, Tong-Chuan

2017-01-01

Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo. Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine. PMID:29340096

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

PubMed Central

Xie, Yufen; Zhou, Sichang; Jiang, Zhongliang; Dai, Jing; Puscheck, Elizabeth E; Lee, Icksoo; Parker, Graham; Hüttemann, Maik; Rappolee, Daniel A

2014-01-01

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. Implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor (FGF)4 enabled highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 hr despite FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential, maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited differentiation at the differentiation optimum at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation. PMID:25239494

Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

PubMed Central

Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

2014-01-01

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial

PubMed Central

2013-01-01

Background The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. Methods In an open-label, phase 1/phase 2 study, patients (N = 36) with long-standing T2D were divided into three groups (Group A, oral medications, n = 18; Group B, oral medications + insulin injections, n = 11; Group C having impaired β-cell function with oral medications + insulin injections, n = 7). All patients received one treatment with the Stem Cell Educator therapy in which a patient’s blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient’s circulation. Results Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61% ± 1.12 at baseline to 7.25% ± 0.58 at 12 weeks (P = 2.62E-06), and 7.33% ± 1.02 at one year post-treatment (P = 0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. Conclusions Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. Trial registration ClinicalTrials.gov number, NCT01415726 PMID:23837842

In search of adult renal stem cells.

PubMed

Anglani, F; Forino, M; Del Prete, D; Tosetto, E; Torregrossa, R; D'Angelo, A

2004-01-01

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidney's specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes - together with a knowledge of the embryonic origin of renal cells - should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.

Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases.

PubMed

Ding, Suet Lee Shirley; Kumar, Suresh; Mok, Pooi Ling

2017-07-28

The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action.

Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases

PubMed Central

Ding, Suet Lee Shirley; Kumar, Suresh; Mok, Pooi Ling

2017-01-01

The use of multipotent mesenchymal stem cells (MSCs) has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action. PMID:28788088

Myelodysplastic syndromes are propagated by rare and distinct human cancer stem cells in vivo.

PubMed

Woll, Petter S; Kjällquist, Una; Chowdhury, Onima; Doolittle, Helen; Wedge, David C; Thongjuea, Supat; Erlandsson, Rikard; Ngara, Mtakai; Anderson, Kristina; Deng, Qiaolin; Mead, Adam J; Stenson, Laura; Giustacchini, Alice; Duarte, Sara; Giannoulatou, Eleni; Taylor, Stephen; Karimi, Mohsen; Scharenberg, Christian; Mortera-Blanco, Teresa; Macaulay, Iain C; Clark, Sally-Ann; Dybedal, Ingunn; Josefsen, Dag; Fenaux, Pierre; Hokland, Peter; Holm, Mette S; Cazzola, Mario; Malcovati, Luca; Tauro, Sudhir; Bowen, David; Boultwood, Jacqueline; Pellagatti, Andrea; Pimanda, John E; Unnikrishnan, Ashwin; Vyas, Paresh; Göhring, Gudrun; Schlegelberger, Brigitte; Tobiasson, Magnus; Kvalheim, Gunnar; Constantinescu, Stefan N; Nerlov, Claus; Nilsson, Lars; Campbell, Peter J; Sandberg, Rickard; Papaemmanuil, Elli; Hellström-Lindberg, Eva; Linnarsson, Sten; Jacobsen, Sten Eirik W

2014-06-16

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation. Copyright © 2014 Elsevier Inc. All rights reserved.

microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells.

PubMed

Hamam, Dana; Ali, Dalia; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

2015-02-15

microRNAs (miRNAs) constitute complex regulatory network, fine tuning the expression of a myriad of genes involved in different biological and physiological processes, including stem cell differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells present in the bone marrow stroma, and the stroma of many other tissues, and can give rise to a number of mesoderm-type cells including adipocytes and osteoblasts, which form medullary fat and bone tissues, respectively. The role of bone marrow fat in bone mass homeostasis is an area of intensive investigation with the aim of developing novel approaches for enhancing osteoblastic bone formation through inhibition of bone marrow fat formation. A number of recent studies have reported several miRNAs that enhance or inhibit adipogenic differentiation of MSCs and with potential use in microRNA-based therapy to regulate adipogenesis in the context of treating bone diseases and metabolic disorders. The current review focuses on miRNAs and their role in regulating adipogenic differentiation of MSCs.

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells

PubMed Central

Prescott, Hilary M. A.; Manning, Craig; Gardner, Aaron; Ritchie, William A.; Pizzi, Romain; Girling, Simon; Valentine, Iain; Wang, Chengdong; Jahoda, Colin A. B.

2015-01-01

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation. PMID:26398672

Giant Panda (Ailuropoda melanoleuca) Buccal Mucosa Tissue as a Source of Multipotent Progenitor Cells.

PubMed

Prescott, Hilary M A; Manning, Craig; Gardner, Aaron; Ritchie, William A; Pizzi, Romain; Girling, Simon; Valentine, Iain; Wang, Chengdong; Jahoda, Colin A B

2015-01-01

Since the first mammal was cloned, the idea of using this technique to help endangered species has aroused considerable interest. However, several issues limit this possibility, including the relatively low success rate at every stage of the cloning process, and the dearth of usable tissues from these rare animals. iPS cells have been produced from cells from a number of rare mammalian species and this is the method of choice for strategies to improve cloning efficiency and create new gametes by directed differentiation. Nevertheless information about other stem cell/progenitor capabilities of cells from endangered species could prove important for future conservation approaches and adds to the knowledge base about cellular material that can be extremely limited. Multipotent progenitor cells, termed skin-derived precursor (SKP) cells, can be isolated directly from mammalian skin dermis, and human cheek tissue has also been shown to be a good source of SKP-like cells. Recently we showed that structures identical to SKPs termed m-SKPs could be obtained from monolayer/ two dimensional (2D) skin fibroblast cultures. Here we aimed to isolate m-SKPs from cultured cells of three endangered species; giant panda (Ailuropoda melanoleuca); red panda (Ailurus fulgens); and Asiatic lion (Panthera leo persica). m-SKP-like spheres were formed from the giant panda buccal mucosa fibroblasts; whereas dermal fibroblast (DF) cells cultured from abdominal skin of the other two species were unable to generate spheres. Under specific differentiation culture conditions giant panda spheres expressed neural, Schwann, adipogenic and osteogenic cell markers. Furthermore, these buccal mucosa derived spheres were shown to maintain expression of SKP markers: nestin, versican, fibronectin, and P75 and switch on expression of the stem cell marker ABCG2. These results demonstrate that giant panda cheek skin can be a useful source of m-SKP multipotent progenitors. At present lack of sample numbers means that we can only postulate why we were unable to obtain m-SKPs from the lion and red panda cultures. However the giant panda observations point to the value of archiving cells from rare species, and the possibilities for later progenitor cell derivation.

Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century

PubMed Central

Stoltz, J.-F.; de Isla, N.; Li, Y. P.; Bensoussan, D.; Zhang, L.; Huselstein, C.; Chen, Y.; Decot, V.; Magdalou, J.; Li, N.; Reppel, L.; He, Y.

2015-01-01

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton's Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ's reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC. PMID:26300923

Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-Thrombospondin-1 axis

PubMed Central

Lee, Joo-Hyeon; Bhang, Dong Ha; Beede, Alexander; Huang, Tian Lian; Stripp, Barry R.; Bloch, Kenneth D.; Wagers, Amy J.; Tseng, Yu-Hua; Ryeom, Sandra; Kim, Carla F.

2014-01-01

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level. PMID:24485453

Are hematopoietic stem cells involved in hepatocarcinogenesis?

PubMed Central

Antonino, Matteo; Del Prete, Valentina; Neve, Viviana; Scavo, Maria Principia; Barone, Michele

2014-01-01

The liver has three cell lineages able to proliferate after a hepatic injury: the mature hepatocyte, the ductular “bipolar” progenitor cell termed “oval cell” and the putative periductular stem cell. Hepatocytes can only produce other hepatocytes whereas ductular progenitor cells are considerate bipolar since they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential and may be multipotent, being this aspect still under investigation. They originate in the bone marrow since their progeny express genetic markers of donor hematopoietic cells after bone marrow transplantation. Since the liver is the hematopoietic organ of the fetus, it is possible that hematopoietic stem cells may reside in the liver of the adult. This assumption is proved by the finding that oval cells express hematopoietic markers like CD34, CD45, CD 109, Thy-1, c-kit, and others, which are also expressed by bone marrow-derived hematopoietic stem cells (BMSCs). Few and discordant studies have evaluated the role of BMSC in hepatocarcinogenesis so far and further studies in vitro and in vivo are warranted in order to definitively clarify such an issue. PMID:25202697

Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells.

PubMed

Balikov, Daniel A; Crowder, Spencer W; Boire, Timothy C; Lee, Jung Bok; Gupta, Mukesh K; Fenix, Aidan M; Lewis, Holley N; Ambrose, Caitlyn M; Short, Philip A; Kim, Chang Soo; Burnette, Dylan T; Reilly, Matthew A; Murthy, N Sanjeeva; Kang, Mi-Lan; Kim, Won Shik; Sung, Hak-Joon

2017-07-12

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress-two indicators of naïve, healthy stem cells-in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell-cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.

High-resolution molecular validation of self-renewal and spontaneous differentiation in adipose-tissue derived human mesenchymal stem cells cultured in human platelet lysate

PubMed Central

Dudakovic, Amel Dudakovic; Camilleri, Emily; Riester, Scott M.; Lewallen, Eric A.; Kvasha, Sergiy; Chen, Xiaoyue; Radel, Darcie J.; Anderson, Jarett M.; Nair, Asha A.; Evans, Jared M.; Krych, Aaron J.; Smith, Jay; Deyle, David R.; Stein, Janet L.; Stein, Gary S.; Im, Hee-Jeong; Cool, Simon M.; Westendorf, Jennifer J.; Kakar, Sanjeev; Dietz, Allan B.; van Wijnen, Andre J.

2014-01-01

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1 and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10 fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while up-regulating WNT-related genes (WISP2, SFRP2 and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility. PMID:24905804

Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction.

PubMed

Yoon, Young-Sup; Park, Jong-Seon; Tkebuchava, Tengiz; Luedeman, Corinne; Losordo, Douglas W

2004-06-29

There has been a rapid increase in the number of clinical trials using unselected bone marrow (BM) cells or the mononuclear fraction of BM cells for treating ischemic heart diseases. Thus far, no significant deleterious effects or complications have been reported in any studies using BM-derived cells for treatment of various cardiac diseases. Seven-week-old female Fisher-344 rats underwent surgery to induce acute myocardial infarction and were randomized into 3 groups of 16 rats, each receiving intramyocardial injection of either 7x10(5) DiI-labeled total BM cells (TBMCs), the same number of DiI-labeled, clonally expanded BM multipotent stem cells, or the same volume of phosphate-buffered saline in the peri-infarct area. Echocardiography 2 weeks after cell transplantation indicated intramyocardial calcification in 4 of 14 surviving rats (28.5%) in the TBMC group. Histological examination with hematoxylin and eosin staining and von Kossa staining confirmed the presence of extensive intramyocardial calcification. Alkaline phosphatase staining revealed strong positivity surrounding the calcified area suggestive of ongoing osteogenic activity. Fluorescent microscopic examination revealed that acellular calcific areas were surrounded by DiI-labeled TBMCs, suggesting the direct involvement of transplanted TBMCs in myocardial calcification. In contrast, in hearts receiving equal volumes of saline or BM multipotent stem cells delivered in the same manner, there was no evidence of calcification. These results demonstrate that direct transplantation of unselected BM cells into the acutely infarcted myocardium may induce significant intramyocardial calcification.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A new Postdoctoral Fellow position is immediately available in the laboratory of Dr. Terry Yamaguchi at the National Cancer Institute. Dr.Yamaguchi's lab investigates how secreted growth factors regulate the gene regulatory networks that control the fate of embryonic and adult stem cells. Current projects focus on understanding how Wnts and Fgfs regulate the formation and differentiation of the neuromesodermal progenitor (NMP), a multipotent embryonic cell that generates the spinal cord neurons and musculoskeletal system of the body. Using a combination of mouse genetics, mouse and human embryonic stem cell in vitro differentiation, and genomic, proteomic and biochemical approaches, Dr. Yamaguchi’s lab is investigating the molecular mechanisms underlying the activity of key transcriptional determinants of NMP development.

Label retention identifies a multipotent mesenchymal stem cell-like population in the postnatal thymus.

PubMed

Osada, Masako; Singh, Varan J; Wu, Kenmin; Sant'Angelo, Derek B; Pezzano, Mark

2013-01-01

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.

Are nestin-positive mesenchymal stromal cells a better source of cells for CNS repair?

PubMed

Lindsay, Susan L; Barnett, Susan C

2017-06-01

In recent years there has been a great deal of research within the stem cell field which has led to the definition and classification of a range of stem cells from a plethora of tissues and organs. Stem cells, by classification, are considered to be pluri- or multipotent and have both self-renewal and multi-differentiation capabilities. Presently there is a great deal of interest in stem cells isolated from both embryonic and adult tissues in the hope they hold the therapeutic key to restoring or treating damaged cells in a number of central nervous system (CNS) disorders. In this review we will discuss the role of mesenchymal stromal cells (MSCs) isolated from human olfactory mucosa, with particular emphasis on their potential role as a candidate for transplant mediated repair in the CNS. Since nestin expression defines the entire population of olfactory mucosal derived MSCs, we will compare these cells to a population of neural crest derived nestin positive population of bone marrow-MSCs. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Maintenance of sweat glands by stem cells located in the acral epithelium.

PubMed

Ohe, Shuichi; Tanaka, Toshihiro; Yanai, Hirotsugu; Komai, Yoshihiro; Omachi, Taichi; Kanno, Shohei; Tanaka, Kiyomichi; Ishigaki, Kazuhiko; Saiga, Kazuho; Nakamura, Naohiro; Ohsugi, Haruyuki; Tokuyama, Yoko; Atsumi, Naho; Hisha, Hiroko; Yoshida, Naoko; Kumano, Keiki; Yamazaki, Fumikazu; Okamoto, Hiroyuki; Ueno, Hiroo

2015-10-23

The skin is responsible for a variety of physiological functions and is critical for wound healing and repair. Therefore, the regenerative capacity of the skin is important. However, stem cells responsible for maintaining the acral epithelium had not previously been identified. In this study, we identified the specific stem cells in the acral epithelium that participate in the long-term maintenance of sweat glands, ducts, and interadnexal epidermis and that facilitate the regeneration of these structures following injury. Lgr6-positive cells and Bmi1-positive cells were found to function as long-term multipotent stem cells that maintained the entire eccrine unit and the interadnexal epidermis. However, while Lgr6-positive cells were rapidly cycled and constantly supplied differentiated cells, Bmi1-positive cells were slow to cycle and occasionally entered the cell cycle under physiological conditions. Upon irradiation-induced injury, Bmi1-positive cells rapidly proliferated and regenerated injured epithelial tissue. Therefore, Bmi1-positive stem cells served as reservoir stem cells. Lgr5-positive cells were rapidly cycled and maintained only sweat glands; therefore, we concluded that these cells functioned as lineage-restricted progenitors. Taken together, our data demonstrated the identification of stem cells that maintained the entire acral epithelium and supported the different roles of three cellular classes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Prolonged Growth Hormone/Insulin/Insulin-like Growth Factor Nutrient Response Signaling Pathway as a Silent Killer of Stem Cells and a Culprit in Aging.

PubMed

Ratajczak, Mariusz Z; Bartke, Andrzej; Darzynkiewicz, Zbigniew

2017-08-01

The dream of slowing down the aging process has always inspired mankind. Since stem cells are responsible for tissue and organ rejuvenation, it is logical that we should search for encoded mechanisms affecting life span in these cells. However, in adult life the hierarchy within the stem cell compartment is still not very well defined, and evidence has accumulated that adult tissues contain rare stem cells that possess a broad trans-germ layer differentiation potential. These most-primitive stem cells-those endowed with pluripotent or multipotent differentiation ability and that give rise to other cells more restricted in differentiation, known as tissue-committed stem cells (TCSCs) - are of particular interest. In this review we present the concept supported by accumulating evidence that a population of so-called very small embryonic-like stem cells (VSELs) residing in adult tissues positively impacts the overall survival of mammals, including humans. These unique cells are prevented in vertebrates from premature depletion by decreased sensitivity to growth hormone (GH), insulin (INS), and insulin-like growth factor (IGF) signaling, due to epigenetic changes in paternally imprinted genes that regulate their resistance to these factors. In this context, we can envision nutrient response GH/INS/IGF signaling pathway as a lethal factor for these most primitive stem cells and an important culprit in aging.

Stem Cell-based Tissue Engineering Approaches for Musculoskeletal Regeneration

PubMed Central

Brown, Patrick T.; Handorf, Andrew M.; Jeon, Won Bae; Li, Wan-Ju

2014-01-01

The field of regenerative medicine and tissue engineering is an ever evolving field that holds promise in treating numerous musculoskeletal diseases and injuries. An important impetus in the development of the field was the discovery and implementation of stem cells. The utilization of mesenchymal stem cells, and later embryonic and induced pluripotent stem cells, opens new arenas for tissue engineering and presents the potential of developing stem cell-based therapies for disease treatment. Multipotent and pluripotent stem cells can produce various lineage tissues, and allow for derivation of a tissue that may be comprised of multiple cell types. As the field grows, the combination of biomaterial scaffolds and bioreactors provides methods to create an environment for stem cells that better represent their microenvironment for new tissue formation. As technologies for the fabrication of biomaterial scaffolds advance, the ability of scaffolds to modulate stem cell behavior advances as well. The composition of scaffolds could be of natural or synthetic materials and could be tailored to enhance cell self-renewal and/or direct cell fates. In addition to biomaterial scaffolds, studies of tissue development and cellular microenvironments have determined other factors, such as growth factors and oxygen tension, that are crucial to the regulation of stem cell activity. The overarching goal of stem cell-based tissue engineering research is to precisely control differentiation of stem cells in culture. In this article, we review current developments in tissue engineering, focusing on several stem cell sources, induction factors including growth factors, oxygen tension, biomaterials, and mechanical stimulation, and the internal and external regulatory mechanisms that govern proliferation and differentiation. PMID:23432679

Dynamic methylation and expression of Oct4 in early neural stem cells.

PubMed

Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

2010-09-01

Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form 'induced pluripotent stem cells' (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages.

Co-Culturing of Multipotent Mesenchymal Stromal Cells with Autological and Allogenic Lymphocytes.

PubMed

Kapranov, N M; Davydova, Yu O; Gal'tseva, I V; Petinati, N A; Bakshinskaitė, M V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

2018-03-01

We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

Advances in tissue engineering through stem cell-based co-culture.

PubMed

Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

2015-05-01

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

Multipotency of skeletal muscle stem cells on their native substrate and the expression of Connexin 43 during adoption of adipogenic and osteogenic fate.

PubMed

Elashry, Mohamed I; Heimann, Manuela; Wenisch, Sabine; Patel, Ketan; Arnhold, Stefan

2017-10-01

Muscle regeneration is performed by resident muscle stem cells called satellite cells (SC). However they are multipotent, being able to adopt adipogenic and osteogenic fate under the correct stimuli. Since SC behavior can be regulated by the extra-cellular matrix, we examined the robustness of the myogenic programme of SC on their native substrate-the surface of a myofiber. We show that the native substrate supports myogenic differentiation judged by the expression of members of the Myogenic Determination Factor (MRF) family. However SC even on their native substrate can be induced into adopting adipogenic or osteogenic fate. Furthermore conditions that support adipose or bone formation inhibit the proliferation of SC progeny as well as their migration. We show that Connexin43 (Cx43), a gap junction complex protein, is only expressed by activated and not quiescent SC. Furthermore, it is not expressed by SC that are in the process of changing their fate. Lastly we show that intact adult mouse muscle contains numerous cells expressing Cx43 and that the density of these cells seems to be related to capillary density. We suggest the Cx43 expression is localized to angioblasts and is more prominent in oxidative slow muscle compared to glycolytic fast muscle. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

Conservation of the Nucleotide Excision Repair Pathway: Characterization of Hydra Xeroderma Pigmentosum Group F Homolog

PubMed Central

Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

2013-01-01

Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5′ endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra. PMID:23577191

Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

PubMed

Barve, Apurva; Ghaskadbi, Saroj; Ghaskadbi, Surendra

2013-01-01

Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5' endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges.

PubMed

Yong, Kar Wey; Wan Safwani, Wan Kamarul Zaman; Xu, Feng; Wan Abas, Wan Abu Bakar; Choi, Jane Ru; Pingguan-Murphy, Belinda

2015-08-01

Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells

PubMed Central

Takeda, Norifumi; Jain, Rajan; LeBoeuf, Matthew R.; Padmanabhan, Arun; Wang, Qiaohong; Li, Li; Lu, Min Min; Millar, Sarah E.; Epstein, Jonathan A.

2013-01-01

The mammalian hair follicle relies on adult resident stem cells and their progeny to fuel and maintain hair growth throughout the life of an organism. The cyclical and initially synchronous nature of hair growth makes the hair follicle an ideal system with which to define homeostatic mechanisms of an adult stem cell population. Recently, we demonstrated that Hopx is a specific marker of intestinal stem cells. Here, we show that Hopx specifically labels long-lived hair follicle stem cells residing in the telogen basal bulge. Hopx+ cells contribute to all lineages of the mature hair follicle and to the interfollicular epidermis upon epidermal wounding. Unexpectedly, our analysis identifies a previously unappreciated progenitor population that resides in the lower hair bulb of anagen-phase follicles and expresses Hopx. These cells co-express Lgr5, do not express Shh and escape catagen-induced apoptosis. They ultimately differentiate into the cytokeratin 6-positive (K6) inner bulge cells in telogen, which regulate the quiescence of adjacent hair follicle stem cells. Although previous studies have suggested that K6+ cells arise from Lgr5-expressing lower outer root sheath cells in anagen, our studies indicate an alternative origin, and a novel role for Hopx-expressing lower hair bulb progenitor cells in contributing to stem cell homeostasis. PMID:23487314

Nature vs. nurture: gold perpetuates "stemness".

PubMed

Paul, Willi; Sharma, Chandra P; Deb, Kaushik Dilip

2011-01-01

Adult tissues contain quiescent reservoirs of multipotent somatic stem cells and pluripotent embryonic-like stem cells (ELSCs). Credited with regenerative properties gold is used across both -contemporary and -ancient medicines. Here, we show that gold exerted these effects by enhancing the pool of pluripotent ELSC while improving their stemness. We used hESCs as an in-vitro model to understand if gold could enhance self-renewal and pluripotency. Swarna-bhasma (SB), an ancient Indian gold microparticulate (41.1 nm), preparation, reduced spontaneous-differentiation, improved self-renewal, pluripotency and proliferation of hESCs. Colloidal gold-nanoparticles (GNP) (15.59 nm) were tested to confirm that the observations were attributable to nanoparticulate-gold. SB and GNP exposure: maintained -stemness, -karyotypic stability, enhanced pluripotency till day-12, increased average colony-sizes, and reduced the number of autonomously-derived differentiated FGFR1 positive fibroblast-niche-cells/colony. Particulate-gold induced upregulation of FGFR1 and IGF2 expression, and decrease in IGF1 secretion indicates IGF1/2 mediated support for enhanced pluripotency and self-renewal in hESCs.

The activation of directional stem cell motility by green light-emitting diode irradiation.

PubMed

Ong, Wei-Kee; Chen, How-Foo; Tsai, Cheng-Ting; Fu, Yun-Ju; Wong, Yi-Shan; Yen, Da-Jen; Chang, Tzu-Hao; Huang, Hsien-Da; Lee, Oscar Kuang-Sheng; Chien, Shu; Ho, Jennifer Hui-Chun

2013-03-01

Light-emitting diode (LED) irradiation is potentially a photostimulator to manipulate cell behavior by opsin-triggered phototransduction and thermal energy supply in living cells. Directional stem cell motility is critical for the efficiency and specificity of stem cells in tissue repair. We explored that green LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to migrate away from the LED light source through activation of extracellular signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of these kinases as well as green LED irradiation-induced cell migration was facilitated by increasing adenosine triphosphate (ATP) production in OFSCs after green LED exposure, and which was thermal stress-independent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from human orbital fat tissue, constitutionally express three opsins, i.e. retinal pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin (OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to green LED irradiation-induced OFSC migration. In conclusion, stem cells are sensitive to green LED irradiation-induced directional cell migration through activation of ERK signaling pathway via a wavelength-dependent phototransduction. Copyright © 2012 Elsevier Ltd. All rights reserved.

Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

PubMed

Nakagomi, Takayuki; Kubo, Shuji; Nakano-Doi, Akiko; Sakuma, Rika; Lu, Shan; Narita, Aya; Kawahara, Maiko; Taguchi, Akihiko; Matsuyama, Tomohiro

2015-06-01

Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries. © 2015 AlphaMed Press.

Periodontal tissue engineering strategies based on nonoral stem cells.

PubMed

Requicha, João Filipe; Viegas, Carlos Alberto; Muñoz, Fernando; Reis, Rui Luís; Gomes, Manuela Estima

2014-01-01

Periodontal disease is an inflammatory disease which constitutes an important health problem in humans due to its enormous prevalence and life threatening implications on systemic health. Routine standard periodontal treatments include gingival flaps, root planning, application of growth/differentiation factors or filler materials and guided tissue regeneration. However, these treatments have come short on achieving regeneration ad integrum of the periodontium, mainly due to the presence of tissues from different embryonic origins and their complex interactions along the regenerative process. Tissue engineering (TE) aims to regenerate damaged tissue by providing the repair site with a suitable scaffold seeded with sufficient undifferentiated cells and, thus, constitutes a valuable alternative to current therapies for the treatment of periodontal defects. Stem cells from oral and dental origin are known to have potential to regenerate these tissues. Nevertheless, harvesting cells from these sites implies a significant local tissue morbidity and low cell yield, as compared to other anatomical sources of adult multipotent stem cells. This manuscript reviews studies describing the use of non-oral stem cells in tissue engineering strategies, highlighting the importance and potential of these alternative stem cells sources in the development of advanced therapies for periodontal regeneration. Copyright © 2013 Wiley Periodicals, Inc.

Pharmacologic and genetic strategies to enhance cell therapy for cardiac regeneration.

PubMed

Kanashiro-Takeuchi, Rosemeire M; Schulman, Ivonne Hernandez; Hare, Joshua M

2011-10-01

Cell-based therapy is emerging as an exciting potential therapeutic approach for cardiac regeneration following myocardial infarction (MI). As heart failure (HF) prevalence increases over time, development of new interventions designed to aid cardiac recovery from injury are crucial and should be considered more broadly. In this regard, substantial efforts to enhance the efficacy and safety of cell therapy are continuously growing along several fronts, including modifications to improve the reprogramming efficiency of inducible pluripotent stem cells (iPS), genetic engineering of adult stem cells, and administration of growth factors or small molecules to activate regenerative pathways in the injured heart. These interventions are emerging as potential therapeutic alternatives and/or adjuncts based on their potential to promote stem cell homing, proliferation, differentiation, and/or survival. Given the promise of therapeutic interventions to enhance the regenerative capacity of multipotent stem cells as well as specifically guide endogenous or exogenous stem cells into a cardiac lineage, their application in cardiac regenerative medicine should be the focus of future clinical research. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure." Copyright © 2011 Elsevier Ltd. All rights reserved.

Long Noncoding RNAs: New Players in the Osteogenic Differentiation of Bone Marrow- and Adipose-Derived Mesenchymal Stem Cells.

PubMed

Yang, Qiaolin; Jia, Lingfei; Li, Xiaobei; Guo, Runzhi; Huang, Yiping; Zheng, Yunfei; Li, Weiran

2018-06-01

Mesenchymal stem cells (MSCs) are an important population of multipotent stem cells that differentiate into multiple lineages and display great potential in bone regeneration and repair. Although the role of protein-coding genes in the osteogenic differentiation of MSCs has been extensively studied, the functions of noncoding RNAs in the osteogenic differentiation of MSCs are unclear. The recent application of next-generation sequencing to MSC transcriptomes has revealed that long noncoding RNAs (lncRNAs) are associated with the osteogenic differentiation of MSCs. LncRNAs are a class of non-coding transcripts of more than 200 nucleotides in length. Noncoding RNAs are thought to play a key role in osteoblast differentiation through various regulatory mechanisms including chromatin modification, transcription factor binding, competent endogenous mechanism, and other post-transcriptional mechanisms. Here, we review the roles of lncRNAs in the osteogenic differentiation of bone marrow- and adipose-derived stem cells and provide a theoretical foundation for future research.

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

PubMed Central

Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

2015-01-01

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

PubMed

Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

2014-09-09

Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Body Management: Mesenchymal Stem Cells Control the Internal Regenerator

PubMed Central

Hariri, Robert

2015-01-01

Summary It has been assumed that adult tissues cannot regenerate themselves. With the current understanding that every adult tissue has its own intrinsic progenitor or stem cell, it is now clear that almost all tissues have regenerative potential partially related to their innate turnover dynamics. Moreover, it appears that a separate class of local cells originating as perivascular cells appears to provide regulatory oversight for localized tissue regeneration. The management of this regeneration oversight has a profound influence on the use of specific cells for cell therapies as a health care delivery tool set. The multipotent mesenchymal stem cell (MSC), now renamed the medicinal signaling cell, predominantly arises from pericytes released from broken and inflamed blood vessels and appears to function as both an immunomodulatory and a regeneration mediator. MSCs are being tested for their management capabilities to produce therapeutic outcomes in more than 480 clinical trials for a wide range of clinical conditions. Local MSCs function by managing the body’s primary repair and regeneration activities. Supplemental MSCs can be provided from either endogenous or exogenous sources of either allogeneic or autologous origin. This MSC-based therapy has the potential to change how health care is delivered. These medicinal cells are capable of sensing their surroundings. Also, by using its complex signaling circuitry, these cells organize site-specific regenerative responses as if these therapeutic cells were well-programmed modern computers. Given these facts, it appears that we are entering a new age of cellular medicine. Significance This report is a perspective from an active scientist and an active entrepreneur and commercial leader. It is neither a comprehensive review nor a narrowly focused treatise. The broad themes and the analogy to the working component of a computer and that of a cell are meant to draw several important scientific principles and health care themes together into the thesis that regenerative medicine is a constant throughout life and its management is the next frontier of health care. Mesenchymal stem cells are used as the central connection in the broad theme, not as multipotent progenitors but rather as an important control element in the natural local regeneration process. PMID:26019227

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development.

PubMed

Mansouri, Ladan; Xie, Yufen; Rappolee, Daniel A

2012-12-10

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies.

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development

PubMed Central

Mansouri, Ladan; Xie, Yufen; Rappolee, Daniel A

2012-01-01

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies. PMID:24710551

Human Oral Mucosa and Gingiva

PubMed Central

Zhang, Q.Z.; Nguyen, A.L.; Yu, W.H.; Le, A.D.

2012-01-01

Mesenchymal stem cells (MSCs) represent a heterogeneous population of progenitor cells with self-renewal and multipotent differentiation potential. Aside from their regenerative role, extensive in vitro and in vivo studies have demonstrated that MSCs are capable of potent immunomodulatory effects on a variety of innate and adaptive immune cells. In this article, we will review recent experimental studies on the characterization of a unique population of MSCs derived from human oral mucosa and gingiva, especially their immunomodulatory and anti-inflammatory functions and their application in the treatment of several in vivo models of inflammatory diseases. The ease of isolation, accessible tissue source, and rapid ex vivo expansion, with maintenance of stable stem-cell-like phenotypes, render oral mucosa- and gingiva-derived MSCs a promising alternative cell source for MSC-based therapies. PMID:22988012

Microenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia

PubMed Central

Wei, Junping; Wunderlich, Mark; Fox, Catherine; Alvarez, Sara; Cigudosa, Juan C.; Wilhelm, Jamie S.; Zheng, Yi; Cancelas, Jose A.; Gu, Yi; Jansen, Michael; DiMartino, Jorge F.; Mulloy, James C.

2008-01-01

Summary Faithful modeling of mixed lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid or mixed lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenviromental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML. PMID:18538732

miR-300 mediates Bmi1 function and regulates differentiation in primitive cardiac progenitors

PubMed Central

Cruz, F M; Tomé, M; Bernal, J A; Bernad, A

2015-01-01

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a polycomb-family transcriptional factor critical for self-renewal in many adult stem cells and human neoplasia. We sought to identify microRNAs regulated by Bmi1 that could play a role in multipotent cardiac progenitor cell (CPC) decisions. We found that miR-300, a poorly characterized microRNA mapping in the Dlk1-Dio3 microRNA cluster, was positively regulated by Bmi1 in CPCs. Forced expression of miR-300 in CPCs promoted an improved stemness signature with a significant increase in Oct4 levels, a reduction in senescence progression and an enhanced proliferative status via p19 activation and inhibition of p16 accumulation. Endothelial and cardiogenic differentiation were clearly compromised by sustained miR-300 expression. Additionally, RNA and protein analysis revealed a significant reduction in key cardiac transcription factors, including Nkx2.5 and Tbx5. Collectively, these results suggest that some functions attributed to Bmi1 are due to induction of miR-300, which decreases the cardiogenic differentiation potential of multipotent CPCs in vitro and promotes self-renewal. PMID:26512961

Allogenic banking of dental pulp stem cells for innovative therapeutics.

PubMed

Collart-Dutilleul, Pierre-Yves; Chaubron, Franck; De Vos, John; Cuisinier, Frédéric J

2015-08-26

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.

Allogenic banking of dental pulp stem cells for innovative therapeutics

PubMed Central

Collart-Dutilleul, Pierre-Yves; Chaubron, Franck; De Vos, John; Cuisinier, Frédéric J

2015-01-01

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat. PMID:26328017

Amniotic Fluid-Derived Stem Cells for Cardiovascular Tissue Engineering Applications

PubMed Central

Petsche Connell, Jennifer; Camci-Unal, Gulden; Khademhosseini, Ali

2013-01-01

Recent research has demonstrated that a population of stem cells can be isolated from amniotic fluid removed by amniocentesis that are broadly multipotent and nontumorogenic. These amniotic fluid-derived stem cells (AFSC) could potentially provide an autologous cell source for treatment of congenital defects identified during gestation, particularly cardiovascular defects. In this review, the various methods of isolating, sorting, and culturing AFSC are compared, along with techniques for inducing differentiation into cardiac myocytes and endothelial cells. Although research has not demonstrated complete and high-yield cardiac differentiation, AFSC have been shown to effectively differentiate into endothelial cells and can effectively support cardiac tissue. Additionally, several tissue engineering and regenerative therapeutic approaches for the use of these cells in heart patches, injection after myocardial infarction, heart valves, vascularized scaffolds, and blood vessels are summarized. These applications show great promise in the treatment of congenital cardiovascular defects, and further studies of isolation, culture, and differentiation of AFSC will help to develop their use for tissue engineering, regenerative medicine, and cardiovascular therapies. PMID:23350771

Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

PubMed

Bharadwaj, Shantaram; Liu, Guihua; Shi, Yingai; Wu, Rongpei; Yang, Bin; He, Tongchuan; Fan, Yuxin; Lu, Xinyan; Zhou, Xiaobo; Liu, Hong; Atala, Anthony; Rohozinski, Jan; Zhang, Yuanyuan

2013-09-01

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction. © AlphaMed Press.

Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.

PubMed

Suzuki, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-08-01

During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

Maintenance of sweat glands by stem cells located in the acral epithelium

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

Ohe, Shuichi; Department of Dermatology, Kansai Medical University, Osaka 573-1010; Tanaka, Toshihiro

The skin is responsible for a variety of physiological functions and is critical for wound healing and repair. Therefore, the regenerative capacity of the skin is important. However, stem cells responsible for maintaining the acral epithelium had not previously been identified. In this study, we identified the specific stem cells in the acral epithelium that participate in the long-term maintenance of sweat glands, ducts, and interadnexal epidermis and that facilitate the regeneration of these structures following injury. Lgr6-positive cells and Bmi1-positive cells were found to function as long-term multipotent stem cells that maintained the entire eccrine unit and the interadnexalmore » epidermis. However, while Lgr6-positive cells were rapidly cycled and constantly supplied differentiated cells, Bmi1-positive cells were slow to cycle and occasionally entered the cell cycle under physiological conditions. Upon irradiation-induced injury, Bmi1-positive cells rapidly proliferated and regenerated injured epithelial tissue. Therefore, Bmi1-positive stem cells served as reservoir stem cells. Lgr5-positive cells were rapidly cycled and maintained only sweat glands; therefore, we concluded that these cells functioned as lineage-restricted progenitors. Taken together, our data demonstrated the identification of stem cells that maintained the entire acral epithelium and supported the different roles of three cellular classes. - Highlights: • The acral epithelium have two types of stem cells. • Lgr6-positive cells are rapid-cycling, short-term stem cells. • Bmi1-positive cells are slow-cycling stem cells that act as reserver stem cells. • Lgr5 may be a useful sweat gland marker in mice.« less

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

PubMed Central

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-01-01

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. PMID:25908840

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

PubMed

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-06-03

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Stem cells: a revolution in therapeutics-recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies.

PubMed

Mimeault, M; Hauke, R; Batra, S K

2007-09-01

Basic and clinical research accomplished during the last few years on embryonic, fetal, amniotic, umbilical cord blood, and adult stem cells has constituted a revolution in regenerative medicine and cancer therapies by providing the possibility of generating multiple therapeutically useful cell types. These new cells could be used for treating numerous genetic and degenerative disorders. Among them, age-related functional defects, hematopoietic and immune system disorders, heart failures, chronic liver injuries, diabetes, Parkinson's and Alzheimer's diseases, arthritis, and muscular, skin, lung, eye, and digestive disorders as well as aggressive and recurrent cancers could be successfully treated by stem cell-based therapies. This review focuses on the recent advancements in adult stem cell biology in normal and pathological conditions. We describe how these results have improved our understanding on critical and unique functions of these rare sub-populations of multipotent and undifferentiated cells with an unlimited self-renewal capacity and high plasticity. Finally, we discuss some major advances to translate the experimental models on ex vivo and in vivo expanded and/or differentiated stem cells into clinical applications for the development of novel cellular therapies aimed at repairing genetically altered or damaged tissues/organs in humans. A particular emphasis is made on the therapeutic potential of different tissue-resident adult stem cell types and their in vivo modulation for treating and curing specific pathological disorders.

Characteristics, applications and prospects of mesenchymal stem cells in cell therapy.

PubMed

Guadix, Juan A; Zugaza, José L; Gálvez-Martín, Patricia

2017-05-10

Recent advances in the field of cell therapy and regenerative medicine describe mesenchymal stem cells (MSCs) as potential biological products due to their ability to self-renew and differentiate. MSCs are multipotent adult cells with immunomodulatory and regenerative properties, and, given their therapeutic potential, they are being widely studied in order to evaluate their viability, safety and efficacy. In this review, we describe the main characteristics and cellular sources of MSCs, in addition to providing an overview of their properties and current clinical applications, as well offering updated information on the regulatory aspects that define them as somatic cell therapy products. Cell therapy based on MSCs is offered nowadays as a pharmacological alternative, although there are still challenges to be addressed in this regard. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Fetal Membranes-Derived Stem Cells Microenvironment.

PubMed

Favaron, Phelipe Oliveira; Miglino, Maria Angelica

2017-01-01

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

Morphological changes in paraurethral area after introduction of tissue engineering construct on the basis of adipose tissue stromal cells.

PubMed

Makarov, A V; Arutyunyan, I V; Bol'shakova, G B; Volkov, A V; Gol'dshtein, D V

2009-10-01

We studied morphological changes in the paraurethral area of Wistar rats after introduction of tissue engineering constructs on the basis of multipotent mesenchymal stem cells and gelatin sponge. The tissue engineering construct containing autologous culture of the stromal fraction of the adipose tissue was most effective. After introduction of this construct we observed more rapid degradation of the construct matrix and more intensive formation of collagen fibers.

Automated mitosis detection of stem cell populations in phase-contrast microscopy images.

PubMed

Huh, Seungil; Ker, Dai Fei Elmer; Bise, Ryoma; Chen, Mei; Kanade, Takeo

2011-03-01

Due to the enormous potential and impact that stem cells may have on regenerative medicine, there has been a rapidly growing interest for tools to analyze and characterize the behaviors of these cells in vitro in an automated and high throughput fashion. Among these behaviors, mitosis, or cell division, is important since stem cells proliferate and renew themselves through mitosis. However, current automated systems for measuring cell proliferation often require destructive or sacrificial methods of cell manipulation such as cell lysis or in vitro staining. In this paper, we propose an effective approach for automated mitosis detection using phase-contrast time-lapse microscopy, which is a nondestructive imaging modality, thereby allowing continuous monitoring of cells in culture. In our approach, we present a probabilistic model for event detection, which can simultaneously 1) identify spatio-temporal patch sequences that contain a mitotic event and 2) localize a birth event, defined as the time and location at which cell division is completed and two daughter cells are born. Our approach significantly outperforms previous approaches in terms of both detection accuracy and computational efficiency, when applied to multipotent C3H10T1/2 mesenchymal and C2C12 myoblastic stem cell populations.

Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

PubMed Central

Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

2016-01-01

This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells*

PubMed Central

Meyer, Mark B.; Benkusky, Nancy A.; Sen, Buer; Rubin, Janet; Pike, J. Wesley

2016-01-01

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies. PMID:27402842

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

PubMed

Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

2015-05-01

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

PubMed

Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

2015-10-01

Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.

PubMed

Wu, Xiuwen; Ren, Jianan; Li, Jieshou

2012-05-01

The use of tissue-engineering techniques such as stem-cell therapy to renew injured tissues is a promising strategy in regenerative medicine. As a cell-delivery vehicle, fibrin glues (FG) facilitate cell attachment, growth and differentiation and, ultimately, tissue formation and organization by its three-dimensional structure. Numerous studies have provided evidence that stromal cells derived from bone marrow (bone marrow stromal cells; BMSC) and adipose tissue (adipose-derived stromal cells; ADSC) contain a population of adult multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells that can differentiate into several lineages. By combining MSC with FG, the implantation could take advantage of the mutual benefits. Researchers and physicians have pinned their hopes on stem cells for developing novel approaches in regenerative medicine. This review focuses on the therapeutic potential of MSC with FG in bone defect reconstruction, cartilage and tendon injury repair, ligament, heart and nerve regeneration, and, furthermore, wound healing.

Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells

PubMed Central

Qiu, Juhui; Fan, Xiaoying; Wang, Yixia; Jin, Hongbin; Song, Yixiao; Han, Yang; Huang, Shenghong; Meng, Yaping; Tang, Fuchou; Meng, Anming

2016-01-01

Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM. PMID:27252540

Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

PubMed Central

Kondo, Toru; Raff, Martin

2004-01-01

We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm. PMID:15574597

Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction.

PubMed

Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia; Torrente, Yvan; Cetin, Irene

2016-04-01

Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. ©AlphaMed Press.

Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction

PubMed Central

Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia

2016-01-01

Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. Significance This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. PMID:26956210

Combating Osteoarthritis through Stem Cell Therapies by Rejuvenating Cartilage: A Review

PubMed Central

Dubey, Navneet Kumar; Mishra, Viraj Krishna; Dubey, Rajni; Syed-Abdul, Shabbir; Wang, Joseph R.; Wang, Peter D.

2018-01-01

Knee osteoarthritis (OA) is a chronic degenerative disorder which could be distinguished by erosion of articular cartilage, pain, stiffness, and crepitus. Not only aging-associated alterations but also the metabolic factors such as hyperglycemia, dyslipidemia, and obesity affect articular tissues and may initiate or exacerbate the OA. The poor self-healing ability of articular cartilage due to limited regeneration in chondrocytes further adversely affects the osteoarthritic microenvironment. Traditional and current surgical treatment procedures for OA are limited and incapable to reverse the damage of articular cartilage. To overcome these limitations, cell-based therapies are currently being employed to repair and regenerate the structure and function of articular tissues. These therapies not only depend upon source and type of stem cells but also on environmental conditions, growth factors, and chemical and mechanical stimuli. Recently, the pluripotent and various multipotent mesenchymal stem cells have been employed for OA therapy, due to their differentiation potential towards chondrogenic lineage. Additionally, the stem cells have also been supplemented with growth factors to achieve higher healing response in osteoarthritic cartilage. In this review, we summarized the current status of stem cell therapies in OA pathophysiology and also highlighted the potential areas of further research needed in regenerative medicine. PMID:29765416

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

PubMed

Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Brain mesenchymal stem cells: physiology and pathological implications.

PubMed

Pombero, Ana; Garcia-Lopez, Raquel; Martinez, Salvador

2016-06-01

Mesenchymal stem cells (MSCs) are defined as progenitor cells that give rise to a number of unique, differentiated mesenchymal cell types. This concept has progressively evolved towards an all-encompassing concept including multipotent perivascular cells of almost any tissue. In central nervous system, pericytes are involved in blood-brain barrier, and angiogenesis and vascular tone regulation. They form the neurovascular unit (NVU) together with endothelial cells, astrocytes and neurons. This functional structure provides an optimal microenvironment for neural proliferation in the adult brain. Neurovascular niche include both diffusible signals and direct contact with endothelial and pericytes, which are a source of diffusible neurotrophic signals that affect neural precursors. Therefore, MSCs/pericyte properties such as differentiation capability, as well as immunoregulatory and paracrine effects make them a potential resource in regenerative medicine. © 2016 Japanese Society of Developmental Biologists.

Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells.

PubMed

Ebeid, Michael; Sripal, Prashanth; Pecka, Jason; Beisel, Kirk W; Kwan, Kelvin; Soukup, Garrett A

2017-01-01

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.

Separation technologies for stem cell bioprocessing.

PubMed

Diogo, Maria Margarida; da Silva, Cláudia Lobato; Cabral, Joaquim M S

2012-11-01

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell-derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato-oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost-effective. Important examples are the need for high-resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity-based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical-based methods requiring no cell labeling and integrated with microscale technologies. Copyright © 2012 Wiley Periodicals, Inc.

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

Dynamic methylation and expression of Oct4 in early neural stem cells

PubMed Central

Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

2010-01-01

Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form ‘induced pluripotent stem cells’ (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages. PMID:20646110

Stem Cells for Osteochondral Regeneration.

PubMed

Canadas, Raphaël F; Pirraco, Rogério P; Oliveira, J Miguel; Reis, Rui L; Marques, Alexandra P

2018-01-01

Stem cell research plays a central role in the future of medicine, which is mainly dependent on the advances on regenerative medicine (RM), specifically in the disciplines of tissue engineering (TE) and cellular therapeutics. All RM strategies depend upon the harnessing, stimulation, or guidance of endogenous developmental or repair processes in which cells have an important role. Among the most clinically challenging disorders, cartilage degeneration, which also affects subchondral bone becoming an osteochondral (OC) defect, is one of the most demanding. Although primary cells have been clinically applied, stem cells are currently seen as the promising tool of RM-related research because of its availability, in vitro proliferation ability, pluri- or multipotency, and immunosuppressive features. Being the OC unit, a transition from the bone to cartilage, mesenchymal stem cells (MSCs) are the main focus for OC regeneration. Promising alternatives, which can also be obtained from the patient or at banks and have great differentiation potential toward a wide range of specific cell types, have been reported. Still, ethical concerns and tumorigenic risk are currently under discussion and assessment. In this book chapter, we revise the existing stem cell-based approaches for engineering bone and cartilage, focusing on cell therapy and TE. Furthermore, 3D OC composites based on cell co-cultures are described. Finally, future directions and challenges still to be faced are critically discussed.

Amnion: a potent graft source for cell therapy in stroke.

PubMed

Yu, Seong Jin; Soncini, Maddalena; Kaneko, Yuji; Hess, David C; Parolini, Ornella; Borlongan, Cesar V

2009-01-01

Regenerative medicine is a new field primarily based on the concept of transplanting exogenous or stimulating endogenous stem cells to generate biological substitutes and improve tissue functions. Recently, amnion-derived cells have been reported to have multipotent differentiation ability, and these cells have attracted attention as a novel cell source for cell transplantation therapy. Cells isolated from amniotic membrane can differentiate into all three germ layers, have low immunogenicity and anti-inflammatory function, and do not require the destruction of human embryos for their isolation, thus circumventing the ethical debate commonly associated with the use of human embryonic stem cells. Accumulating evidence now suggests that the amnion, which had been discarded after parturition, is a highly potent transplant material in the field of regenerative medicine. In this report, we review the current progress on the characterization of MSCs derived from the amnion as a remarkable transplantable cell population with therapeutic potential for multiple CNS disorders, especially stroke.

Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro.

PubMed

Langenbach, Fabian; Handschel, Jörg

2013-01-01

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization.

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium.

PubMed

Seeberger, Karen L; Dufour, Jannette M; Shapiro, Andrew M James; Lakey, Jonathan R T; Rajotte, Ray V; Korbutt, Gregory S

2006-02-01

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.

Induction of the neural crest state: Control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions

PubMed Central

Prasad, Maneeshi S.; Sauka-Spengler, Tatjana; LaBonne, Carole

2012-01-01

Neural crest cells are a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates, migrate extensively, and give rise to diverse derivatives such as melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The neural crest gene regulatory network (NC-GRN) includes a number of key factors that are used reiteratively to control multiple steps in the development of neural crest cells, including the acquisition of stem cell attributes. It is therefore essential to understand the mechanisms that control the distinct functions of such reiteratively used factors in different cellular contexts. The context-dependent control of neural crest specification is achieved through combinatorial interaction with other factors, post-transcriptional and post-translational modifications, and the epigenetic status and chromatin state of target genes. Here we review the current understanding of the NC-GRN, including the role of the neural crest specifiers, their links to the control of “stemness,” and their dynamic context-dependent regulation during the formation of neural crest progenitors. PMID:22583479

Receptor for macrophage colony-stimulating factor transduces a signal decreasing erythroid potential in the multipotent hematopoietic EML cell line.

PubMed

Pawlak, G; Grasset, M F; Arnaud, S; Blanchet, J P; Mouchiroud, G

2000-10-01

To test the hypothesis that hematopoietic growth factors may influence lineage choice in pluripotent progenitor cells, we investigated the effects of macrophage colony-stimulating factor (M-CSF) on erythroid and myeloid potentials of multipotent EML cells ectopically expressing M-CSF receptor (M-CSFR). EML cells are stem cell factor (SCF)-dependent murine cells that give rise spontaneously to pre-B cells, burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte macrophage (CFU-GM). We determined BFU-E and CFU-GM frequencies among EML cells transduced with murine M-CSFR, human M-CSFR, or chimeric receptors, and cultivated in the presence of SCF, M-CSF, or both growth factors. Effects of specific inhibitors of signaling molecules were investigated. EML cells transduced with murine M-CSFR proliferated in response to M-CSF but also exhibited a sharp and rapid decrease in BFU-E frequency associated with an increase in CFU-GM frequency. In contrast, EML cells expressing human M-CSFR proliferated in response to M-CSF without any changes in erythroid or myeloid potential. Using chimeric receptors between human and murine M-CSFR, we showed that the effects of M-CSF on EML cell differentiation potential are mediated by a large region in the intracellular domain of murine M-CSFR. Furthermore, phospholipase C (PLC) inhibitor U73122 interfered with the negative effects of ligand-activated murine M-CSFR on EML cell erythroid potential. We propose that signaling pathways activated by tyrosine kinase receptors may regulate erythroid potential and commitment decisions in multipotent progenitor cells and that PLC may play a key role in this process.

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate.

PubMed

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min; Chung, Tae Nyoung; Suh, Sang Won

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30  μ M and 100  μ M of ZnCl 2 . Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.

Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

PubMed Central

Foster, Barbara A.; Gangavarapu, Kalyan J.; Mathew, Grinu; Azabdaftari, Gissou; Morrison, Carl D.; Miller, Austin; Huss, Wendy J.

2013-01-01

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population. PMID:23383057

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.

Origin of Complexity in Multicellular Organisms

NASA Astrophysics Data System (ADS)

Furusawa, Chikara; Kaneko, Kunihiko

2000-06-01

Through extensive studies of dynamical system modeling cellular growth and reproduction, we find evidence that complexity arises in multicellular organisms naturally through evolution. Without any elaborate control mechanism, these systems can exhibit complex pattern formation with spontaneous cell differentiation. Such systems employ a ``cooperative'' use of resources and maintain a larger growth speed than simple cell systems, which exist in a homogeneous state and behave ``selfishly.'' The relevance of the diversity of chemicals and reaction dynamics to the growth of a multicellular organism is demonstrated. Chaotic biochemical dynamics are found to provide the multipotency of stem cells.

Recent progress on normal and malignant pancreatic stem/progenitor cell research: therapeutic implications for the treatment of type 1 or 2 diabetes mellitus and aggressive pancreatic cancer

PubMed Central

Mimeault, M; Batra, S K

2010-01-01

Recent progress on pancreatic stem/progenitor cell research has revealed that the putative multipotent pancreatic stem/progenitor cells and/or more committed beta cell precursors may persist in the pancreatic gland in adult life. The presence of immature pancreatic cells with stem cell-like properties offers the possibility of stimulating their in vivo expansion and differentiation or to use their ex vivo expanded progenies for beta cell replacement-based therapies for type 1 or 2 diabetes mellitus in humans. In addition, the transplantation of either insulin-producing beta cells derived from embryonic, fetal and other tissue-resident adult stem/progenitor cells or genetically modified adult stem/progenitor cells may also constitute alternative promising therapies for treating diabetic patients. The genetic and/or epigenetic alterations in putative pancreatic adult stem/progenitor cells and/or their early progenies may, however, contribute to their acquisition of a dysfunctional behaviour as well as their malignant transformation into pancreatic cancer stem/progenitor cells. More particularly, the activation of distinct tumorigenic signalling cascades, including the hedgehog, epidermal growth factor–epidermal growth factor receptor (EGF–EGFR) system, wingless ligand (Wnt)/β-catenin and/or stromal cell-derived factor-1 (SDF-1)–CXC chemokine receptor 4 (CXCR4) pathways may play a major role in the sustained growth, survival, metastasis and/or drug resistance of pancreatic cancer stem/progenitor cells and their further differentiated progenies. The combination of drugs that target the oncogenic elements in pancreatic cancer stem/progenitor cells and their microenvironment, with the conventional chemotherapeutic regimens, could represent promising therapeutic strategies. These novel targeted therapies should lead to the development of more effective treatments of locally advanced and metastatic pancreatic cancers, which remain incurable with current therapies. PMID:18791122

Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

PubMed

Eggenhofer, Elke; Popp, Felix C; Mendicino, Michael; Silber, Paula; Van't Hof, Wouter; Renner, Philipp; Hoogduijn, Martin J; Pinxteren, Jef; van Rooijen, Nico; Geissler, Edward K; Deans, Robert; Schlitt, Hans J; Dahlke, Marc H

2013-08-01

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

PubMed

Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

2015-10-01

Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology. © 2015 Society for Laboratory Automation and Screening.

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

PubMed

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels.

PubMed

Kim, Hong Sun; Chen, Yu-Chih; Nör, Felipe; Warner, Kristy A; Andrews, April; Wagner, Vivian P; Zhang, Zhaocheng; Zhang, Zhixiong; Martins, Manoela D; Pearson, Alexander T; Yoon, Euisik; Nör, Jacques E

2017-11-21

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDH high CD44 high ) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p<0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo . Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants

PubMed Central

Koelle, Samson J.

2017-01-01

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell–biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies. PMID:28087539

Femtosecond laser pulses for chemical-free embryonic and mesenchymal stem cell differentiation

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; Dholakia, Kishan; Gunn-Moore, Frank

2011-10-01

Owing to their self renewal and pluripotency properties, stem cells can efficiently advance current therapies in tissue regeneration and/or engineering. Under appropriate culture conditions in vitro, pluripotent stem cells can be primed to differentiate into any cell type some examples including neural, cardiac and blood cells. However, there still remains a pressing necessity to answer the biological questions concerning how stem cell renewal and how differentiation programs are operated and regulated at the genetic level. In stem cell research, an urgent requirement on experimental procedures allowing non-invasive, marker-free observation of growth, proliferation and stability of living stem cells under physiological conditions exists. Femtosecond (fs) laser pulses have been reported to non-invasively deliver exogenous materials, including foreign genetic species into both multipotent and pluripotent stem cells successfully. Through this multi-photon facilitated technique, directly administering fs laser pulses onto the cell plasma membrane induces transient submicrometer holes, thereby promoting cytosolic uptake of the surrounding extracellular matter. To display a chemical-free cell transfection procedure that utilises micro-litre scale volumes of reagents, we report for the first time on 70 % transfection efficiency in ES-E14TG2a cells using the enhanced green fluorescing protein (EGFP) DNA plasmid. We also show how varying the average power output during optical transfection influences cell viability, proliferation and cytotoxicity in embryonic stem cells. The impact of utilizing objective lenses of different numerical aperture (NA) on the optical transfection efficiency in ES-E14TG2a cells is presented. Finally, we report on embryonic and mesenchymal stem cell differentiation. The produced specialized cell types could thereafter be characterized and used for cell based therapies.

An update clinical application of amniotic fluid-derived stem cells (AFSCs) in cancer cell therapy and tissue engineering.

PubMed

Gholizadeh-Ghaleh Aziz, Shiva; Fathi, Ezzatollah; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Fardyazar, Zahra; Pashaiasl, Maryam

2017-06-01

Recent studies have elucidated that cell-based therapies are promising for cancer treatments. The human amniotic fluid stem (AFS) cells are advantageous cells for such therapeutic schemes that can be innately changed to express therapeutic proteins. HAFSCs display a natural tropism to cancer cells in vivo. They can be useful in cancer cells targeting. Moreover, they are easily available from surplus diagnostic samples during pregnancy and less ethical and legal concern are associated with the collection and application than other putative cells are subjected. This review will designate representatives of amniotic fluid and stem cell derived from amniotic fluid. For this propose, we collect state of human AFS cells data applicable in cancer therapy by dividing this approach into two main classes (nonengineered and engineered based approaches). Our study shows the advantage of AFS cells over other putative cells types in terms differentiation ability to a wide range of cells by potential and effective use in preclinical studies for a variety of diseases. This study has shown the elasticity of human AFS cells and their favorable potential as a multipotent cell source for regenerative stem cell therapy and capable of giving rise to multiple lineages including such as osteoblasts and adipocyte.

Cellular Analysis of Adult Neural Stem Cells for Investigating Prion Biology.

PubMed

Haigh, Cathryn L

2017-01-01

Traditional primary and secondary cell cultures have been used for the investigation of prion biology and disease for many years. While both types of cultures produce highly valid and immensely valuable results, they also have their limitations; traditional cell lines are often derived from cancers, therefore subject to numerous DNA changes, and primary cultures are labor-intensive and expensive to produce requiring sacrifice of many animals. Neural stem cell (NSC) cultures are a relatively new technology to be used for the study of prion biology and disease. While NSCs are subject to their own limitations-they are generally cultured ex vivo in environments that artificially force their growth-they also have their own unique advantages. NSCs retain the ability for self-renewal and can therefore be propagated in culture similarly to secondary cultures without genetic manipulation. In addition, NSCs are multipotent; they can be induced to differentiate into mature cells of central nervous system (CNS) linage. The combination of self-renewal and multipotency allows NSCs to be used as a primary cell line over multiple generations saving time, costs, and animal harvests, thus providing a valuable addition to the existing cell culture repertoire used for investigation of prion biology and disease. Furthermore, NSC cultures can be generated from mice of any genotype, either by embryonic harvest or harvest from adult brain, allowing gene expression to be studied without further genetic manipulation. This chapter describes a standard method of culturing adult NSCs and assays for monitoring NSC growth, migration, and differentiation and revisits basic reactive oxygen species detection in the context of NSC cultures.

Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.

PubMed

Cordey, Myriam; Limacher, Monika; Kobel, Stefan; Taylor, Verdon; Lutolf, Matthias P

2008-10-01

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

PubMed

Acuna-Mendoza, Soledad; Martin, Sabrina; Kuchler-Bopp, Sabine; Ribes, Sandy; Thalgott, Jérémy; Chaussain, Catherine; Creuzet, Sophie; Lesot, Hervé; Lebrin, Franck; Poliard, Anne

2017-12-01

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

The Holy Grail of Orthopedic Surgery: Mesenchymal Stem Cells—Their Current Uses and Potential Applications

PubMed Central

Berebichez-Fridman, Roberto; Gómez-García, Ricardo; Berebichez-Fastlicht, Enrique; Olivos-Meza, Anell; Granados, Julio; Velasquillo, Cristina

2017-01-01

Only select tissues and organs are able to spontaneously regenerate after disease or trauma, and this regenerative capacity diminishes over time. Human stem cell research explores therapeutic regenerative approaches to treat various conditions. Mesenchymal stem cells (MSCs) are derived from adult stem cells; they are multipotent and exert anti-inflammatory and immunomodulatory effects. They can differentiate into multiple cell types of the mesenchyme, for example, endothelial cells, osteoblasts, chondrocytes, fibroblasts, tenocytes, vascular smooth muscle cells, and sarcomere muscular cells. MSCs are easily obtained and can be cultivated and expanded in vitro; thus, they represent a promising and encouraging treatment approach in orthopedic surgery. Here, we review the application of MSCs to various orthopedic conditions, namely, orthopedic trauma; muscle injury; articular cartilage defects and osteoarthritis; meniscal injuries; bone disease; nerve, tendon, and ligament injuries; spinal cord injuries; intervertebral disc problems; pediatrics; and rotator cuff repair. The use of MSCs in orthopedics may transition the practice in the field from predominately surgical replacement and reconstruction to bioregeneration and prevention. However, additional research is necessary to explore the safety and effectiveness of MSC treatment in orthopedics, as well as applications in other medical specialties. PMID:28698718

Multipotent Mesenchymal Stromal Cells for the Prophylaxis of Acute Graft-versus-Host Disease—A Phase II Study

PubMed Central

Kuzmina, Larisa A.; Petinati, Natalia A.; Parovichnikova, Elena N.; Lubimova, Lidia S.; Gribanova, Elena O.; Gaponova, Tatjana V.; Shipounova, Irina N.; Zhironkina, Oxana A.; Bigildeev, Alexey E.; Svinareva, Daria A.; Drize, Nina J.; Savchenko, Valery G.

2012-01-01

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9–1.3 × 106/kg when the blood counts indicated recovery. aGVHD of stage II–IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, (P = 0.002). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study. PMID:22242033

Multipotent Mesenchymal Stromal Cells for the Prophylaxis of Acute Graft-versus-Host Disease-A Phase II Study.

PubMed

Kuzmina, Larisa A; Petinati, Natalia A; Parovichnikova, Elena N; Lubimova, Lidia S; Gribanova, Elena O; Gaponova, Tatjana V; Shipounova, Irina N; Zhironkina, Oxana A; Bigildeev, Alexey E; Svinareva, Daria A; Drize, Nina J; Savchenko, Valery G

2012-01-01

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9-1.3 × 10(6)/kg when the blood counts indicated recovery. aGVHD of stage II-IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, (P = 0.002). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study.

Mesenchymal Stem Cell Preparation and Transfection-free Ferumoxytol Labeling for MRI Cell Tracking.

PubMed

Liu, Li; Ho, Chien

2017-11-15

Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA-approved superparamagnetic iron-oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme ® ), the only FDA-approved SPIO particle, needs transfection agents. This unit describes a new "bio-mimicry" protocol to prepare more native MSCs by using more "in vivo environment" of MSCs, so that the phagocytic activity of cultured MSCs is restored and expanded MSCs can be labeled with Ferumoxytol, without the need for transfection agents and/or electroporation. Moreover, MSCs re-size to a more native size, reducing from 32.0 to 19.5 μm. The MSCs prepared from this protocol retain more native properties and would be useful for biomedical applications and MSC-tracking studies by MRI. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs

PubMed Central

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A.

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial–mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues. PMID:22539926

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs.

PubMed

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

PubMed

Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

2017-04-01

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064. © 2016 AlphaMed Press.

Fibromodulin reprogrammed cells: A novel cell source for bone regeneration.

PubMed

Li, Chen-Shuang; Yang, Pu; Ting, Kang; Aghaloo, Tara; Lee, Soonchul; Zhang, Yulong; Khalilinejad, Kambiz; Murphy, Maxwell C; Pan, Hsin Chuan; Zhang, Xinli; Wu, Benjamin; Zhou, Yan-Heng; Zhao, Zhihe; Zheng, Zhong; Soo, Chia

2016-03-01

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However, currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. Previously, we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan, fibromodulin (FMOD), circumventing gene transduction. In this study, we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence, engraftment, and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fibromodulin Reprogrammed Cells: A Novel Cell Source for Bone Regeneration

PubMed Central

Li, Chen-Shuang; Yang, Pu; Ting, Kang; Aghaloo, Tara; Lee, Soonchul; Zhang, Yulong; Khalilinejad, Kambiz; Murphy, Maxwell C.; Pan, Hsin Chuan; Zhang, Xinli; Wu, Benjamin; Zhou, Yan-Heng; Zhao, Zhihe; Zheng, Zhong; Soo, Chia

2016-01-01

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However, currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. Previously, we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan, fibromodulin (FMOD), circumventing gene transduction. In this study, we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the ‘molecular blueprint’ of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence, engraftment, and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration. PMID:26774565

Aminopropyl carbazole analogues as potent enhancers of neurogenesis.

PubMed

Yoon, Hye Jin; Kong, Sun-Young; Park, Min-Hye; Cho, Yongsung; Kim, Sung-Eun; Shin, Jae-Yeon; Jung, Sunghye; Lee, Jiyoun; Farhanullah; Kim, Hyun-Jung; Lee, Jeewoo

2013-11-15

Neural stem cells are multipotent and self-renewing cells that can differentiate into new neurons and hold great promise for treating various neurological disorders including multiple sclerosis, Parkinson's disease, and Alzheimer's disease. Small molecules that can trigger neurogenesis and neuroprotection are particularly useful not only because of their therapeutic implications but also because they can provide an invaluable tool to study the mechanisms of neurogenesis. In this report, we have developed and screened 25 aminopropyl carbazole derivatives that can enhance neurogenesis of cultured neural stem cells. Among these analogues, compound 9 demonstrated an excellent proneurogenic and neuroprotective activity with no apparent toxicity. We believe that compound 9 can serve as an excellent lead to develop various analogues and to study the underlying mechanisms of neurogenesis. Copyright © 2013 Elsevier Ltd. All rights reserved.

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).

Mesenchymal stem cell-derived microparticles: a promising therapeutic strategy.

PubMed

Tan, Xi; Gong, Yong-Zhen; Wu, Ping; Liao, Duan-Fang; Zheng, Xi-Long

2014-08-18

Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs.

Mesenchymal Stem Cell-Derived Microparticles: A Promising Therapeutic Strategy

PubMed Central

Tan, Xi; Gong, Yong-Zhen; Wu, Ping; Liao, Duan-Fang; Zheng, Xi-Long

2014-01-01

Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs. PMID:25196436

Adult mesenchymal stem cells and women's health.

PubMed

Caplan, Arnold I

2015-02-01

Adult mesenchymal stem cells (MSCs) were previously described as multipotent cells that could differentiate into bone, cartilage, muscle, and other mesenchymal tissues. New information suggests that MSCs can be found in every tissue of the body because they function as perivascular cells--pericytes--found outside all blood vessels. When these vessels break or are inflamed, pericytes are detached and form MSCs, which are activated by their local microenvironment of injury. Such MSCs function to secrete powerful immune-modulatory and regenerative agents; more than 450 clinical trials are now ongoing, covering a huge spectrum of clinical conditions. How such activated MSCs affect menstrual cycle, menopause, or osteotrophic cancers has only recently been studied. This article outlines these issues and challenges the scientific and medical community to use this newfound knowledge to uncover new clinical logics and medial solutions for women.

Gene delivery in tissue engineering and regenerative medicine.

PubMed

Fang, Y L; Chen, X G; W T, Godbey

2015-11-01

As a promising strategy to aid or replace tissue/organ transplantation, gene delivery has been used for regenerative medicine applications to create or restore normal function at the cell and tissue levels. Gene delivery has been successfully performed ex vivo and in vivo in these applications. Excellent proliferation capabilities and differentiation potentials render certain cells as excellent candidates for ex vivo gene delivery for regenerative medicine applications, which is why multipotent and pluripotent cells have been intensely studied in this vein. In this review, gene delivery is discussed in detail, along with its applications to tissue engineering and regenerative medicine. A definition of a stem cell is compared to a definition of a stem property, and both provide the foundation for an in-depth look at gene delivery investigations from a germ lineage angle. © 2014 Wiley Periodicals, Inc.

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice

PubMed Central

Ogawa, Yasuhiro; Eto, Akira; Miyake, Chisato; Tsuchida, Nana; Miyake, Haruka; Takaku, Yasuhiro; Hagiwara, Hiroaki; Oishi, Kazuhiko

2015-01-01

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases. PMID:26382630

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice.

PubMed

Ogawa, Yasuhiro; Eto, Akira; Miyake, Chisato; Tsuchida, Nana; Miyake, Haruka; Takaku, Yasuhiro; Hagiwara, Hiroaki; Oishi, Kazuhiko

2015-01-01

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases.

BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

PubMed Central

Schemionek, Mirle; Elling, Christian; Steidl, Ulrich; Bäumer, Nicole; Hamilton, Ashley; Spieker, Tilmann; Göthert, Joachim R.; Stehling, Martin; Wagers, Amy; Huettner, Claudia S.; Tenen, Daniel G.; Tickenbrock, Lara; Berdel, Wolfgang E.; Serve, Hubert; Holyoake, Tessa L.; Müller-Tidow, Carsten

2010-01-01

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity. PMID:20053753

Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers.

PubMed

Macias, Maria I; Grande, Jesús; Moreno, Ana; Domínguez, Irene; Bornstein, Rafael; Flores, Ana I

2010-11-01

The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes. We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immunological and differentiation properties were analyzed. The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability. The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration. Copyright © 2010 Mosby, Inc. All rights reserved.

Tracing the destiny of mesenchymal stem cells from embryo to adult bone marrow and white adipose tissue via Pdgfrα expression.

PubMed

Miwa, Hiroyuki; Era, Takumi

2018-01-29

Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α ( Pdgfra ) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1 ) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions. © 2018. Published by The Company of Biologists Ltd.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial.

PubMed

Delgado, Elias; Perez-Basterrechea, Marcos; Suarez-Alvarez, Beatriz; Zhou, Huimin; Revuelta, Eva Martinez; Garcia-Gala, Jose Maria; Perez, Silvia; Alvarez-Viejo, Maria; Menendez, Edelmiro; Lopez-Larrea, Carlos; Tang, Ruifeng; Zhu, Zhenlong; Hu, Wei; Moss, Thomas; Guindi, Edward; Otero, Jesus; Zhao, Yong

2015-12-01

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the "educated" lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4(+) T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4(+) central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4(+) effector memory T cells (TEM) and CD8(+) TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C-C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Obra Social "La Caixa", Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial

PubMed Central

Delgado, Elias; Perez-Basterrechea, Marcos; Suarez-Alvarez, Beatriz; Zhou, Huimin; Revuelta, Eva Martinez; Garcia-Gala, Jose Maria; Perez, Silvia; Alvarez-Viejo, Maria; Menendez, Edelmiro; Lopez-Larrea, Carlos; Tang, Ruifeng; Zhu, Zhenlong; Hu, Wei; Moss, Thomas; Guindi, Edward; Otero, Jesus; Zhao, Yong

2015-01-01

Background Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. Methods In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the “educated” lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Findings Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C–C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Interpretation Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Funding Obra Social “La Caixa”, Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation. PMID:26844283

4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila

NASA Astrophysics Data System (ADS)

Yadav, Jhillu S.; Lavanya, Madugula P.; Das, Pragna P.; Bag, Indira; Krishnan, Anita; Jagannadh, Bulusu; Mohapatra, Debendra K.; Pal Bhadra, Manika; Bhadra, Utpal

2010-04-01

p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.

Human Urine-Derived Stem Cells: Potential for Cell-Based Therapy of Cartilage Defects

PubMed Central

Chen, Long; Li, Lang; Xing, Fei; Peng, Jing; Peng, Kun; Wang, Yuanzheng

2018-01-01

Stem cell therapy is considered an optimistic approach to replace current treatments for cartilage defects. Recently, human urine-derived stem cells (hUSCs), which are isolated from the urine, are studied as a promising candidate for many tissue engineering therapies due to their multipotency and sufficient proliferation activities. However, it has not yet been reported whether hUSCs can be employed in cartilage defects. In this study, we revealed that induced hUSCs expressed chondrogenic-related proteins, including aggrecan and collagen II, and their gene expression levels were upregulated in vitro. Moreover, we combined hUSCs with hyaluronic acid (HA) and injected hUSCs-HA into a rabbit knee joint with cartilage defect. Twelve weeks after the injection, the histologic analyses (HE, toluidine blue, and Masson trichrome staining), immunohistochemistry (aggrecan and collagen II), and histologic grade of the sample indicated that hUSCs-HA could stimulate much more neocartilage formation compared with hUSCs alone, pure HA, and saline, which only induced the modest cartilage regeneration. In this study, we demonstrated that hUSCs could be a potential cell source for stem cell therapies to treat cartilage-related defects in the future. PMID:29765413

Adipose-derived stem cells and periodontal tissue engineering.

PubMed

Tobita, Morikuni; Mizuno, Hiroshi

2013-01-01

Innovative developments in the multidisciplinary field of tissue engineering have yielded various implementation strategies and the possibility of functional tissue regeneration. Technologic advances in the combination of stem cells, biomaterials, and growth factors have created unique opportunities to fabricate tissues in vivo and in vitro. The therapeutic potential of human multipotent mesenchymal stem cells (MSCs), which are harvested from bone marrow and adipose tissue, has generated increasing interest in a wide variety of biomedical disciplines. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve tissue. Adipose-derived stem cells have some advantages compared with other sources of stem cells, most notably that a large number of cells can be easily and quickly isolated from adipose tissue. In current clinical therapy for periodontal tissue regeneration, several methods have been developed and applied either alone or in combination, such as enamel matrix proteins, guided tissue regeneration, autologous/allogeneic/xenogeneic bone grafts, and growth factors. However, there are various limitations and shortcomings for periodontal tissue regeneration using current methods. Recently, periodontal tissue regeneration using MSCs has been examined in some animal models. This method has potential in the regeneration of functional periodontal tissues because the various secreted growth factors from MSCs might not only promote the regeneration of periodontal tissue but also encourage neovascularization of the damaged tissues. Adipose-derived stem cells are especially effective for neovascularization compared with other MSC sources. In this review, the possibility and potential of adipose-derived stem cells for regenerative medicine are introduced. Of particular interest, periodontal tissue regeneration with adipose-derived stem cells is discussed.

Investigation into the cellular origins of posterior regeneration in the annelid Capitella teleta

PubMed Central

de Jong, Danielle M.

2017-01-01

Abstract Many animals can regenerate, although there is great diversity in regenerative capabilities. A major question in regenerative biology is determining the cellular source of newly formed tissue. The polychaete annelid, Capitella teleta, can regenerate posterior segments following transverse amputation. However, the source, behavior and molecular characteristics of the cells that form new tissue during regeneration are largely unknown. Using an indirect cell tracking method involving 5′‐ethynyl‐2′‐deoxyuridine (EdU) incorporation, we show that cell migration occurs during C. teleta posterior regeneration. Expression of the multipotency/germ line marker CapI‐vasa led us to hypothesize that stem cells originate from a multipotent progenitor cell (MPC) cluster, migrate through the coelomic cavity, and contribute to regeneration of tissue. We show that the capacity for posterior regeneration and segment formation is greater with than without the MPC cluster. Finally, we propose a working model of posterior regeneration in C. teleta. This work is the first in C. teleta that addresses the potential source of cells contributing to posterior regeneration, and may provide clues as to why some animals are highly successful regenerators. PMID:29721327

Cellular network entropy as the energy potential in Waddington's differentiation landscape

PubMed Central

Banerji, Christopher R. S.; Miranda-Saavedra, Diego; Severini, Simone; Widschwendter, Martin; Enver, Tariq; Zhou, Joseph X.; Teschendorff, Andrew E.

2013-01-01

Differentiation is a key cellular process in normal tissue development that is significantly altered in cancer. Although molecular signatures characterising pluripotency and multipotency exist, there is, as yet, no single quantitative mark of a cellular sample's position in the global differentiation hierarchy. Here we adopt a systems view and consider the sample's network entropy, a measure of signaling pathway promiscuity, computable from a sample's genome-wide expression profile. We demonstrate that network entropy provides a quantitative, in-silico, readout of the average undifferentiated state of the profiled cells, recapitulating the known hierarchy of pluripotent, multipotent and differentiated cell types. Network entropy further exhibits dynamic changes in time course differentiation data, and in line with a sample's differentiation stage. In disease, network entropy predicts a higher level of cellular plasticity in cancer stem cell populations compared to ordinary cancer cells. Importantly, network entropy also allows identification of key differentiation pathways. Our results are consistent with the view that pluripotency is a statistical property defined at the cellular population level, correlating with intra-sample heterogeneity, and driven by the degree of signaling promiscuity in cells. In summary, network entropy provides a quantitative measure of a cell's undifferentiated state, defining its elevation in Waddington's landscape. PMID:24154593

Functionalized scaffolds to control dental pulp stem cell fate

PubMed Central

Piva, Evandro; Silva, Adriana F.; Nör, Jacques E.

2014-01-01

Emerging understanding about interactions between stem cells, scaffolds and morphogenic factors has accelerated translational research in the field of dental pulp tissue engineering. Dental pulp stem cells constitute a sub-population of cells endowed with self-renewal and multipotency. Dental pulp stem cells seeded in biodegradable scaffolds and exposed to dentin-derived morphogenic signals give rise to a pulp-like tissue capable of generating new dentin. Notably, dentin-derived proteins are sufficient to induce dental pulp stem cell differentiation into odontoblasts. Ongoing work is focused on developing ways of mobilizing dentin-derived proteins and disinfecting the root canal of necrotic teeth without compromising the morphogenic potential of these signaling molecules. On the other hand, dentin by itself does not appear to be capable of inducing endothelial differentiation of dental pulp stem cells, despite the well known presence of angiogenic factors in dentin. This is particularly relevant in the context of dental pulp tissue engineering in full root canals, where access to blood supply is limited to the apical foramina. To address this challenge, scientists are looking at ways to use the scaffold as a controlled release device for angiogenic factors. The aim of this manuscript is to present and discuss current strategies to functionalize injectable scaffolds and customize them for dental pulp tissue engineering. The long-term goal of this work is to develop stem cell-based therapies that enable the engineering of functional dental pulps capable of generating new tubular dentin in humans. PMID:24698691

Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells.

PubMed

Hamano, Sayuri; Tomokiyo, Atsushi; Hasegawa, Daigaku; Yoshida, Shinichiro; Sugii, Hideki; Mitarai, Hiromi; Fujino, Shoko; Wada, Naohisa; Maeda, Hidefumi

2018-01-15

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

PubMed Central

George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

2016-01-01

The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

Human neural crest cells display molecular and phenotypic hallmarks of stem cells

PubMed Central

Thomas, Sophie; Thomas, Marie; Wincker, Patrick; Babarit, Candice; Xu, Puting; Speer, Marcy C.; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Etchevers, Heather C.

2008-01-01

The fields of both developmental and stem cell biology explore how functionally distinct cell types arise from a self-renewing founder population. Multipotent, proliferative human neural crest cells (hNCC) develop toward the end of the first month of pregnancy. It is assumed that most differentiate after migrating throughout the organism, although in animal models neural crest stem cells reportedly persist in postnatal tissues. Molecular pathways leading over time from an invasive mesenchyme to differentiated progeny such as the dorsal root ganglion, the maxillary bone or the adrenal medulla are altered in many congenital diseases. To identify additional components of such pathways, we derived and maintained self-renewing hNCC lines from pharyngulas. We show that, unlike their animal counterparts, hNCC are able to self-renew ex vivo under feeder-free conditions. While cross species comparisons showed extensive overlap between human, mouse and avian NCC transcriptomes, some molecular cascades are only active in the human cells, correlating with phenotypic differences. Furthermore, we found that the global hNCC molecular profile is highly similar to that of pluripotent embryonic stem cells when compared with other stem cell populations or hNCC derivatives. The pluripotency markers NANOG, POU5F1 and SOX2 are also expressed by hNCC, and a small subset of transcripts can unambiguously identify hNCC among other cell types. The hNCC molecular profile is thus both unique and globally characteristic of uncommitted stem cells. PMID:18689800

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

PubMed

Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

2016-01-01

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

The Use of Mesenchymal Stem Cells for the Treatment of Autoimmunity: From Animals Models to Human Disease.

PubMed

Fierabracci, Alessandra; Del Fattore, Andrea; Muraca, Marta; Delfino, Domenico Vittorio; Muraca, Maurizio

2016-01-01

Mesenchymal stem cells are multipotent progenitors able to differentiate into osteoblasts, chondrocytes and adipocytes. These cells also exhibit remarkable immune regulatory properties, which stimulated both in vitro and in vivo experimental studies to unravel the underlying mechanisms as well as extensive clinical applications. Here, we describe the effects of MSCs on immune cells and their application in animal models as well as in clinical trials of autoimmune diseases. It should be pointed out that, while the number of clinical applications is increasing steadily, results should be interpreted with caution, in order to avoid rising false expectations. Major issues conditioning clinical application are the heterogeneity of MSCs and their unpredictable behavior following therapeutic administration. However, increasing knowledge on the interaction between exogenous cell and host tissue, as well as some encouraging clinical observations suggest that the therapeutic applications of MSCs will be further expanded on firmer grounds in the near future.

BMP2 repression and optimized culture conditions promote human bone marrow-derived mesenchymal stem cell isolation.

PubMed

Kay, Alasdair Gawain; Dale, Tina Patricia; Akram, Khondoker Mehedi; Mohan, Param; Hampson, Karen; Maffulli, Nicola; Spiteri, Monica A; El Haj, Alicia Jennifer; Forsyth, Nicholas Robert

2015-01-01

Human mesenchymal stem cells (hMSC) are multipotent progenitor cells. We propose the optimization of hMSC isolation and recovery using the application of a controlled hypoxic environment. We evaluated oxygen, glucose and serum in the recovery of hMSC from bone marrow (BMhMSC). Colony forming units-fibroblastic, cell numbers, tri-lineage differentiation, immunofluorescence and microarray were used to confirm and characterize BMhMSC. In an optimized (2% O(2), 4.5 g/l glucose and 5% serum) environment both colony forming units-fibroblastic (p = 0.01) and cell numbers (p = 0.0001) were enhanced over standard conditions. Transcriptional analysis identified differential expression of bone morphogenetic protein 2 (BMP2) and, putatively, chemokine (C-X-C motif) receptor 2 (CXCR2) signaling pathways. We have detailed a potential milestone in the process of refinement of the BMhMSC isolation process.

Immunoregulation by Mesenchymal Stem Cells: Biological Aspects and Clinical Applications

PubMed Central

Castro-Manrreza, Marta E.; Montesinos, Juan J.

2015-01-01

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro. Currently, MSCs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases. Specifically, because of their high immunoregulatory capacity, MSCs are used as tools in cellular therapies for clinical protocols involving immune system alterations. In this review, we discuss the current knowledge about the capacity of MSCs for the immunoregulation of immunocompetent cells and emphasize the effects of MSCs on T cells, principal effectors of the immune response, and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules. We also describe the mechanisms of MSC immunoregulatory modulation and the participation of MSCs as immune response regulators in several autoimmune diseases, and we emphasize the clinical application in graft versus host disease (GVHD). PMID:25961059

Mesenchymal stem cell-like properties of CD133+ glioblastoma initiating cells

PubMed Central

Pavon, Lorena Favaro; Sibov, Tatiana Tais; de Oliveira, Daniela Mara; Marti, Luciana C.; Cabral, Francisco Romero; de Souza, Jean Gabriel; Boufleur, Pamela; Malheiros, Suzana M.F.; de Paiva Neto, Manuel A.; da Cruz, Edgard Ferreira; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

2016-01-01

Glioblastoma is composed of dividing tumor cells, stromal cells and tumor initiating CD133+ cells. Recent reports have discussed the origin of the glioblastoma CD133+ cells and their function in the tumor microenvironment. The present work sought to investigate the multipotent and mesenchymal properties of primary highly purified human CD133+ glioblastoma-initiating cells. To accomplish this aim, we used the following approaches: i) generation of tumor subspheres of CD133+ selected cells from primary cell cultures of glioblastoma; ii) analysis of the expression of pluripotency stem cell markers and mesenchymal stem cell (MSC) markers in the CD133+ glioblastoma-initiating cells; iii) side-by-side ultrastructural characterization of the CD133+ glioblastoma cells, MSC and CD133+ hematopoietic stem cells isolated from human umbilical cord blood (UCB); iv) assessment of adipogenic differentiation of CD133+ glioblastoma cells to test their MSC-like in vitro differentiation ability; and v) use of an orthotopic glioblastoma xenograft model in the absence of immune suppression. We found that the CD133+ glioblastoma cells expressed both the pluripotency stem cell markers (Nanog, Mush-1 and SSEA-3) and MSC markers. In addition, the CD133+ cells were able to differentiate into adipocyte-like cells. Transmission electron microscopy (TEM) demonstrated that the CD133+ glioblastoma-initiating cells had ultrastructural features similar to those of undifferentiated MSCs. In addition, when administered in vivo to non-immunocompromised animals, the CD133+ cells were also able to mimic the phenotype of the original patient's tumor. In summary, we showed that the CD133+ glioblastoma cells express molecular signatures of MSCs, neural stem cells and pluripotent stem cells, thus possibly enabling differentiation into both neural and mesodermal cell types. PMID:27244897

Hematopoietic stem cells can differentiate into restricted myeloid progenitors before cell division in mice.

PubMed

Grinenko, Tatyana; Eugster, Anne; Thielecke, Lars; Ramasz, Beáta; Krüger, Anja; Dietz, Sevina; Glauche, Ingmar; Gerbaulet, Alexander; von Bonin, Malte; Basak, Onur; Clevers, Hans; Chavakis, Triantafyllos; Wielockx, Ben

2018-05-15

Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps and repeated cell divisions that involve the generation of lineage-committed progenitors. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell-tracing approach and Ki67 RFP knock-in mice, in a non-conditioned transplantation model, to assess divisional history, cell cycle progression, and differentiation of adult HSCs. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid, megakaryocyte-erythroid and pre-megakaryocyte progenitors, without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with the expression of lineage-specific genes and loss of multipotency. Thus HSC fate decisions can be uncoupled from physical cell division. These results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells.

Human mesenchymal stem cells - current trends and future prospective

PubMed Central

Ullah, Imran; Subbarao, Raghavendra Baregundi; Rho, Gyu Jin

2015-01-01

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials. PMID:25797907

Identification of senescence-associated genes in human bone marrow mesenchymal stem cells

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

Ryu, Eunsook; Hong, Su; Kang, Jaeku

2008-07-04

Human bone marrow mesenchymal stem cells (hBMMSCs) are multipotent stem cells that can differentiate into several specialized cell types, including bone, cartilage, and fat cells. The proliferative capacity of hBMMSCs paves the way for the development of regenerative medicine and tissue engineering. However, long-term in vitro culture of hBMMSCs leads to a reduced life span of the cells due to senescence, which leads eventually to growth arrest. To investigate the molecular mechanism behind the cellular senescence of hBMMSCs, microarray analysis was used to compare the expression profiles of early passage hBMMSCs, late passage hBMMSCs and hBMMSCs ectopically expressing human telomerasemore » reverse transcriptase (hTERT). Using an intersection analysis of 3892 differentially expressed genes (DEGs) out of 27,171 total genes analyzed, we identified 338 senescence-related DEGs. GO term categorization and pathway network analysis revealed that the identified genes are strongly related to known senescence pathways and mechanisms. The genes identified using this approach will facilitate future studies of the mechanisms underlying the cellular senescence of hBMMSCs.« less

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique.

PubMed

Bartosh, Thomas J; Ylostalo, Joni H

2014-02-06

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3-D culture without addition of exogenous chemicals or gene-transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, the reported lag time for activation in experimental models has prompted investigations on pre-activating the cells prior to their administration. In this protocol, standard 2-D culture-expanded MSCs are activated by aggregation into 3-D spheres using hanging-drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Further, we elucidate methods to prepare MSC-sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. Copyright © 2014 John Wiley & Sons, Inc.

Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique

PubMed Central

Bartosh, Thomas J.

2014-01-01

Herein, we describe a protocol for preparation of pre-activated anti-inflammatory human mesenchymal stem/precursor cells (MSCs) in 3D culture without addition of exogenous chemicals or gene transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, reported lag time for activation in experimental models have prompted investigations to pre-activate the cells prior to their administration. In this protocol, standard 2D culture expanded MSCs are activated by aggregation into 3D spheres using hanging drop cultures. MSC activation is evaluated by real-time PCR and/or ELISA for anti-inflammatory factors (TSG-6, STC-1, PGE2), and by a functional assay using lipopolysaccharide-stimulated macrophage cultures. Furthermore, we elucidate methods to prepare MSC sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. PMID:24510769

Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

PubMed

Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

2013-11-01

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway

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

Fung, Elizabeth-sharon

2008-01-01

Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expressionmore » of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.« less

A safe and efficient method to retrieve mesenchymal stem cells from three-dimensional fibrin gels.

PubMed

Carrion, Bita; Janson, Isaac A; Kong, Yen P; Putnam, Andrew J

2014-03-01

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.

A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels

PubMed Central

Carrion, Bita; Janson, Isaac A.; Kong, Yen P.

2014-01-01

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation. PMID:23808842

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate

PubMed Central

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30 μM and 100 μM of ZnCl2. Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth. PMID:29765417

The role of undifferentiated adipose-derived stem cells in peripheral nerve repair.

PubMed

Zhang, Rui; Rosen, Joseph M

2018-05-01

Peripheral nerve injuries impose significant health and economic consequences, yet no surgical repair can deliver a complete recovery of sensory or motor function. Traditional methods of repair are less than ideal: direct coaptation can only be performed when tension-free repair is possible, and transplantation of nerve autograft can cause donor-site morbidity and neuroma formation. Cell-based therapy delivered via nerve conduits has thus been explored as an alternative method of nerve repair in recent years. Stem cells are promising sources of the regenerative core material in a nerve conduit because stem cells are multipotent in function, abundant in supply, and more accessible than the myelinating Schwann cells. Among different types of stem cells, undifferentiated adipose-derived stem cell (uASC), which can be processed from adipose tissue in less than two hours, is a promising yet underexplored cell type. Studies of uASC have emerged in the past decade and have shown that autologous uASCs are non-immunogenic, easy to access, abundant in supply, and efficacious at promoting nerve regeneration. Two theories have been proposed as the primary regenerative mechanisms of uASC: in situ trans-differentiation towards Schwann cells, and secretion of trophic and anti-inflammatory factors. Future studies need to fully elucidate the mechanisms, side effects, and efficacy of uASC-based nerve regeneration so that uASCs can be utilized in clinical settings.

Recruitment of host's progenitor cells to sites of human amniotic fluid stem cells implantation.

PubMed

Mirabella, Teodelinda; Poggi, Alessandro; Scaranari, Monica; Mogni, Massimo; Lituania, Mario; Baldo, Chiara; Cancedda, Ranieri; Gentili, Chiara

2011-06-01

The amniotic fluid is a new source of multipotent stem cells with a therapeutic potential for human diseases. Cultured at low cell density, human amniotic fluid stem cells (hAFSCs) were still able to generate colony-forming unit-fibroblast (CFU-F) after 60 doublings, thus confirming their staminal nature. Moreover, after extensive in vitro cell expansion hAFSCs maintained a stable karyotype. The expression of genes, such as SSEA-4, SOX2 and OCT3/4 was confirmed at early and later culture stage. Also, hAFSCs showed bright expression of mesenchymal lineage markers and immunoregulatory properties. hAFSCs, seeded onto hydroxyapatite scaffolds and subcutaneously implanted in nude mice, played a pivotal role in mounting a response resulting in the recruitment of host's progenitor cells forming tissues of mesodermal origin such as fat, muscle, fibrous tissue and immature bone. Implanted hAFSCs migrated from the scaffold to the skin overlying implant site but not to other organs. Given their in vivo: (i) recruitment of host progenitor cells, (ii) homing towards injured sites and (iii) multipotentiality in tissue repair, hAFSCs are a very appealing reserve of stem cells potentially useful for clinical application in regenerative medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Tissue Regeneration and Biomineralization in Sea Urchins: Role of Notch Signaling and Presence of Stem Cell Markers

PubMed Central

Reinardy, Helena C.; Emerson, Chloe E.; Manley, Jason M.; Bodnar, Andrea G.

2015-01-01

Echinoderms represent a phylum with exceptional regenerative capabilities that can reconstruct both external appendages and internal organs. Mechanistic understanding of the cellular pathways involved in regeneration in these animals has been hampered by the limited genomic tools and limited ability to manipulate regenerative processes. We present a functional assay to investigate mechanisms of tissue regeneration and biomineralization by measuring the regrowth of amputated tube feet (sensory and motor appendages) and spines in the sea urchin, Lytechinus variegatus. The ability to manipulate regeneration was demonstrated by concentration-dependent inhibition of regrowth of spines and tube feet by treatment with the mitotic inhibitor, vincristine. Treatment with the gamma-secretase inhibitor DAPT resulted in a concentration-dependent inhibition of regrowth, indicating that both tube feet and spine regeneration require functional Notch signaling. Stem cell markers (Piwi and Vasa) were expressed in tube feet and spine tissue, and Vasa-positive cells were localized throughout the epidermis of tube feet by immunohistochemistry, suggesting the existence of multipotent progenitor cells in these highly regenerative appendages. The presence of Vasa protein in other somatic tissues (e.g. esophagus, radial nerve, and a sub-population of coelomocytes) suggests that multipotent cells are present throughout adult sea urchins and may contribute to normal homeostasis in addition to regeneration. Mechanistic insight into the cellular pathways governing the tremendous regenerative capacity of echinoderms may reveal processes that can be modulated for regenerative therapies, shed light on the evolution of regeneration, and enable the ability to predict how these processes will respond to changing environmental conditions. PMID:26267358

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

PubMed Central

Reichman, Sacha; Terray, Angélique; Slembrouck, Amélie; Nanteau, Céline; Orieux, Gaël; Habeler, Walter; Nandrot, Emeline F.; Sahel, José-Alain; Monville, Christelle; Goureau, Olivier

2014-01-01

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease. PMID:24912154

Stem cell therapies for age-related macular degeneration: the past, present, and future.

PubMed

Dang, Yalong; Zhang, Chun; Zhu, Yu

2015-01-01

In the developed world, age-related macular degeneration (AMD) is one of the major causes of irreversible blindness in the elderly. Although management of neovascular AMD (wet AMD) has dramatically progressed, there is still no effective treatment for nonneovascular AMD (dry AMD), which is characterized by retinal pigment epithelial (RPE) cell death (or dysfunction) and microenvironmental disruption in the retina. Therefore, RPE replacement and microenvironmental regulation represent viable treatments for dry AMD. Recent advances in cell biology have demonstrated that RPE cells can be easily generated from several cell types (pluripotent stem cells, multipotent stem cells, or even somatic cells) by spontaneous differentiation, coculturing, defined factors or cell reprogramming, respectively. Additionally, in vivo studies also showed that the restoration of visual function could be obtained by transplanting functional RPE cells into the subretinal space of recipient. More importantly, clinical trials approved by the US government have shown promising prospects in RPE transplantation. However, key issues such as implantation techniques, immune rejection, and xeno-free techniques are still needed to be further investigated. This review will summarize recent advances in cell transplantation for dry AMD. The obstacles and prospects in this field will also be discussed.

Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development

PubMed Central

Vacaru, Andrei M.; Vitale, Joseph; Nieves, Johnathan; Baron, Margaret H.

2015-01-01

During the development of the hematopoietic system, at least 8 distinct lineages are generated in the mouse embryo. Transgenic mice expressing fluorescent proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to multipotent progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and methods for confirming and analyzing transgene expression in the hematopoietic systems of the embryo, fetus, and postnatal/adult animal. PMID:25064110

Mesenchymal Stem Cells of Dental Origin for Inducing Tissue Regeneration in Periodontitis: A Mini-Review

PubMed Central

Hernández-Monjaraz, Beatriz; Santiago-Osorio, Edelmiro; Monroy-García, Alberto; Ledesma-Martínez, Edgar; Mendoza-Núñez, Víctor Manuel

2018-01-01

Periodontitis is a chronic disease that begins with a period of inflammation of the supporting tissues of the teeth table and then progresses, destroying the tissues until loss of the teeth occurs. The restoration of the damaged dental support apparatus is an extremely complex process due to the regeneration of the cementum, the periodontal ligament, and the alveolar bone. Conventional treatment relies on synthetic materials that fill defects and replace lost dental tissue, but these approaches are not substitutes for a real regeneration of tissue. To address this, there are several approaches to tissue engineering for regenerative dentistry, among them, the use of stem cells. Mesenchymal stem cells (MSC) can be obtained from various sources of adult tissues, such as bone marrow, adipose tissue, skin, and tissues of the orofacial area. MSC of dental origin, such as those found in the bone marrow, have immunosuppressive and immunotolerant properties, multipotency, high proliferation rates, and the capacity for tissue repair. However, they are poorly used as sources of tissue for therapeutic purposes. Their accessibility makes them an attractive source of mesenchymal stem cells, so this review describes the field of dental stem cell research and proposes a potential mechanism involved in periodontal tissue regeneration induced by dental MSC. PMID:29565801

Neural Stem Cell or Human Induced Pluripotent Stem Cell-derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy

PubMed Central

Upadhya, Dinesh; Hattiangady, Bharathi; Shetty, Geetha A.; Zanirati, Gabriele; Kodali, Maheedhar; Shetty, Ashok K.

2016-01-01

Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. PMID:27532817

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells

PubMed Central

Gholizadeh-Ghaleh Aziz, Shiva; Pashaei-Asl, Fatima; Fardyazar, Zahra; Pashaiasl, Maryam

2016-01-01

Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs), one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method), and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR. PMID:27434028

Evaluation of mechanical properties of human mesenchymal stem cells during differentiation to smooth muscle cells.

PubMed

Khani, Mohammad-Mehdi; Tafazzoli-Shadpour, Mohammad; Rostami, Mostafa; Peirovi, Habibollah; Janmaleki, Mohsen

2014-07-01

Human mesenchymal stem cells (hMSCs) are multipotent cells appropriate for a variety of tissue engineering and cell therapy applications. Mechanical properties of hMSCs during differentiation are associated with their particular metabolic activity and regulate cell function due to alternations in cytoskeleton and structural elements. The objective of this study is to evaluate elastic and viscoelastic properties of hMSCs during long term cultivation in control and transforming growth factor-β1 treatment groups using micropipette aspiration technique. The mean Young's modulus (E) of the control samples remained nearly unchanged during 6 days of cultivation, but that of the test samples showed an initial reduction compared to its relevant control sample after 2 days of treatment by biological growth factor, followed by a significant rise after 4 and 6 days. The viscoelastic creep tests showed that both instantaneous and equilibrium moduli significantly increased with the treatment time and reached to maximum values of 622.9 ± 114.2 and 144.3 ± 11.6 Pa at the sixth day, respectively, while increase in apparent viscosity was not statistically significant. Such change of mechanical properties of hMSCs during specific lineage commitment contributes to regenerative medicine as well as stem-cell-based therapy in which biophysical signals regulate stem cell fate.

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells

PubMed Central

Sats, Natalia; Risinskaya, Natalya; Sudarikov, Andrey; Dubniak, Daria; Kraizman, Alina

2018-01-01

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival. PMID:29760731

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells.

PubMed

Petinati, Nataliya; Drize, Nina; Sats, Natalia; Risinskaya, Natalya; Sudarikov, Andrey; Drokov, Michail; Dubniak, Daria; Kraizman, Alina; Nareyko, Maria; Popova, Natalia; Firsova, Maya; Kuzmina, Larisa; Parovichnikova, Elena; Savchenko, Valeriy

2018-01-01

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival.

Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro

PubMed Central

2013-01-01

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization. PMID:24073831

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.

Requirement for Foxd3 in Maintenance of Neural Crest Progenitors

PubMed Central

Teng, Lu; Mundell, Nathan A.; Frist, Audrey Y.; Wang, Qiaohong; Labosky, Patricia A.

2008-01-01

Summary Understanding the molecular mechanisms of stem cell maintenance is critical for the ultimate goal of manipulating stem cells for treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3−/− embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue specific deletion of Foxd3 in the neural crest, we show that Foxd3flox/−; Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo. PMID:18367558

Concise review: genetic dissection of hypoxia signaling pathways in normal and leukemic stem cells.

PubMed

Gezer, Deniz; Vukovic, Milica; Soga, Tomoyoshi; Pollard, Patrick J; Kranc, Kamil R

2014-06-01

Adult hematopoiesis depends on rare multipotent hematopoietic stem cells (HSCs) that self-renew and give rise to progenitor cells, which differentiate to all blood lineages. The strict regulation of the fine balance between self-renewal and differentiation is essential for normal hematopoiesis and suppression of leukemia development. HSCs and progenitor cells are commonly assumed to reside within the hypoxic BM microenvironment, however, there is no direct evidence supporting this notion. Nevertheless, HSCs and progenitors do exhibit a hypoxic profile and strongly express Hif-1α. Although hypoxia signaling pathways are thought to play important roles in adult HSC maintenance and leukemogenesis, the precise function of Hif-dependent signaling in HSCs remains to be uncovered. Here we discuss recent gain-of-function and loss-of-function studies that shed light on the complex roles of hypoxia-signaling pathways in HSCs and their niches in normal and malignant hematopoiesis. Importantly, we comment on the current and often contrasting interpretations of the role of Hif-dependent signaling in stem cell functions. © 2014 AlphaMed Press.

Requirement for Foxd3 in the maintenance of neural crest progenitors.

PubMed

Teng, Lu; Mundell, Nathan A; Frist, Audrey Y; Wang, Qiaohong; Labosky, Patricia A

2008-05-01

Understanding the molecular mechanisms of stem cell maintenance is crucial for the ultimate goal of manipulating stem cells for the treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3(-/-) embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue-specific deletion of Foxd3 in the neural crest, we show that Foxd3(flox/-); Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo.

Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

PubMed

Cooper, Tyler T; Sherman, Stephen E; Kuljanin, Miljan; Bell, Gillian I; Lajoie, Gilles A; Hess, David A

2018-05-01

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDH hi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDH hi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDH hi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDH hi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDH hi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDH hi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDH hi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736. © AlphaMed Press 2018.

Epigenetic and Epitranscriptomic Factors Make a Mark on Hematopoietic Stem Cell Development.

PubMed

Kasper, Dionna M; Nicoli, Stefania

2018-03-01

Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between 'epi'-machineries. Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis.

PubMed

Zhang, Qunzhou; Nguyen, Andrew L; Shi, Shihong; Hill, Colin; Wilder-Smith, Petra; Krasieva, Tatiana B; Le, Anh D

2012-04-10

Mesenchymal stem cells (MSCs) are capable of regenerative and immunomodulatory functions in cell-based therapies in a variety of human diseases and injuries; however, their therapeutic efficacy and potential side effects remain major obstacles in clinical applications. We report here a 3D spheroid culture approach to optimize stem cell properties and therapeutic effects of human gingiva-derived mesenchymal stem cells (GMSCs) in mitigation of experimental oral mucositis. Under growth condition of ultra-low attachment, GMSCs spontaneously aggregated into 3D spheroids and exhibited distinct early stem cell phenotype characterized by elevated expression Stro-1 and CXC chemokine receptor 4 (CXCR-4) as well as OCT-4 and Nanog, 2 important transcriptional factors relevant to stem cell properties, and decreased expression of MSC-associated markers, including CD29, CD90, and CD105. Functionally, spheroid GMSCs are capable of enhanced multipotency and augmented secretion of several chemokines and cytokines relevant to cell migration, survival, and angiogenesis. More importantly, spheroid GMSCs expressed increased levels of reactive oxygen species, hypoxia-inducible factor (HIF)-1 and -2α, and manganese superoxide dismutase, which correlated with improved resistance to oxidative stress-induced apoptosis. Using an in vivo murine model of chemotherapy-induced oral mucositis, we demonstrated that spheroid-derived GMSCs possessed better therapeutic efficacy than their adherent cells in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues. These findings suggest that 3D spheroid culture allows early stemness preservation and potentially precondition GMSCs for enhanced mitigation of oral mucositis. © Mary Ann Liebert, Inc.

Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

PubMed Central

López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

2016-01-01

The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating efficiency to the level of unfrozen controls. Moreover, ASCs cryopreserved in this defined medium retained their multipotency and chromosomal normality. These results are of significance for tissue engineering and clinical applications of stem cells. PMID:27010403

Mesenchymal Stem Cells: Time to Change the Name!

PubMed

Caplan, Arnold I

2017-06-01

Mesenchymal stem cells (MSCs) were officially named more than 25 years ago to represent a class of cells from human and mammalian bone marrow and periosteum that could be isolated and expanded in culture while maintaining their in vitro capacity to be induced to form a variety of mesodermal phenotypes and tissues. The in vitro capacity to form bone, cartilage, fat, etc., became an assay for identifying this class of multipotent cells and around which several companies were formed in the 1990s to medically exploit the regenerative capabilities of MSCs. Today, there are hundreds of clinics and hundreds of clinical trials using human MSCs with very few, if any, focusing on the in vitro multipotential capacities of these cells. Unfortunately, the fact that MSCs are called "stem cells" is being used to infer that patients will receive direct medical benefit, because they imagine that these cells will differentiate into regenerating tissue-producing cells. Such a stem cell treatment will presumably cure the patient of their medically relevant difficulties ranging from osteoarthritic (bone-on-bone) knees to various neurological maladies including dementia. I now urge that we change the name of MSCs to Medicinal Signaling Cells to more accurately reflect the fact that these cells home in on sites of injury or disease and secrete bioactive factors that are immunomodulatory and trophic (regenerative) meaning that these cells make therapeutic drugs in situ that are medicinal. It is, indeed, the patient's own site-specific and tissue-specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs. Stem Cells Translational Medicine 2017;6:1445-1451. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

2014-03-01

Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

Adipose tissue-derived stem cells in neural regenerative medicine.

PubMed

Yeh, Da-Chuan; Chan, Tzu-Min; Harn, Horng-Jyh; Chiou, Tzyy-Wen; Chen, Hsin-Shui; Lin, Zung-Sheng; Lin, Shinn-Zong

2015-01-01

Adipose tissue-derived stem cells (ADSCs) have two essential characteristics with regard to regenerative medicine: the convenient and efficient generation of large numbers of multipotent cells and in vitro proliferation without a loss of stemness. The implementation of clinical trials has prompted widespread concern regarding safety issues and has shifted research toward the therapeutic efficacy of stem cells in dealing with neural degeneration in cases such as stroke, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, cavernous nerve injury, and traumatic brain injury. Most existing studies have reported that cell therapies may be able to replenish lost cells and promote neuronal regeneration, protect neuronal survival, and play a role in overcoming permanent paralysis and loss of sensation and the recovery of neurological function. The mechanisms involved in determining therapeutic capacity remain largely unknown; however, this concept can still be classified in a methodical manner by citing current evidence. Possible mechanisms include the following: 1) the promotion of angiogenesis, 2) the induction of neuronal differentiation and neurogenesis, 3) reductions in reactive gliosis, 4) the inhibition of apoptosis, 5) the expression of neurotrophic factors, 6) immunomodulatory function, and 7) facilitating neuronal integration. In this study, several human clinical trials using ADSCs for neuronal disorders were investigated. It is suggested that ADSCs are one of the choices among various stem cells for translating into clinical application in the near future.

Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

PubMed Central

Magnusson, Mattias; Sierra, Maria I.; Sasidharan, Rajkumar; Prashad, Sacha L.; Romero, Melissa; Saarikoski, Pamela; Van Handel, Ben; Huang, Andy; Li, Xinmin; Mikkola, Hanna K. A.

2013-01-01

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC. PMID:23342037

Bone Marrow-Derived Mesenchymal Stem Cell Therapy as a Candidate Disease-Modifying Strategy in Parkinson's Disease and Multiple System Atrophy

PubMed Central

Park, Hyun Jung

2009-01-01

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases representative of α-synucleinopathies characterized pathologically by α-synuclein-abundant Lewy bodies and glial cytoplasmic inclusions, respectively. Embryonic stem cells, fetal mesencephalic neurons, and neural stem cells have been introduced as restorative strategies in PD animals and patients, but ethical and immunological problems as well as the serious side effects of tumorigenesis and disabling dyskinesia have limited clinical application of these stem cells. Meanwhile, cell therapy using mesenchymal stem cells (MSCs) is attractive clinically because these cells are free from ethical and immunological problems. MSCs are present in adult bone marrow and represent <0.01% of all nucleated bone marrow cells. MSCs are themselves capable of multipotency, differentiating under appropriate conditions into chondrocytes, skeletal myocytes, and neurons. According to recent studies, the neuroprotective effect of MSCs is mediated by their ability to produce various trophic factors that contribute to functional recovery, neuronal cell survival, and stimulation of endogenous regeneration and by immunoregulatory properties that not only inhibit nearly all cells participating in the immune response cell-cell-contact-dependent mechanism, but also release various soluble factors associated with immunosuppressive activity. However, the use of MSCs as neuroprotectives in PD and MSA has seldom been studied. Here we comprehensively review recent advances in the therapeutic roles of MSCs in PD and MSA, especially focusing on their neuroprotective properties and use in disease-modifying therapeutic strategies. PMID:19513327

Stem cell death and survival in heart regeneration and repair.

PubMed

Abdelwahid, Eltyeb; Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

2016-03-01

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function.

Stem cell death and survival in heart regeneration and repair

PubMed Central

Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

2016-01-01

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function. PMID:26687129

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells.

PubMed

Buravkova, L B; Rylova, Y V; Andreeva, E R; Kulikov, A V; Pogodina, M V; Zhivotovsky, B; Gogvadze, V

2013-10-01

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs. Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide. We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs. Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state. These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells.

PubMed

Hong, Seung Hyun; Gang, Eun Ji; Jeong, Ju Ah; Ahn, Chiyoung; Hwang, Soo Han; Yang, Il Ho; Park, Hwon Kyum; Han, Hoon; Kim, Hoeon

2005-05-20

In addition to long-term self-renewal capability, human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. In this study, we have investigated whether human umbilical cord blood (UCB)-derived MSCs are also able to differentiate into hepatocyte-like cells. MSCs isolated from UCB were cultured under the pro-hepatogenic condition similar to that for bone marrow (BM)-derived MSCs. Expression of a variety of hepatic lineage markers was analyzed by flow cytometry, RT-PCR, Western blot, and immunofluorescence. The functionality of differentiated cells was assessed by their ability to incorporate DiI-acetylated low-density lipoprotein (DiI-Ac-LDL). As the cells were morphologically transformed into hepatocyte-like cells, they expressed Thy-1, c-Kit, and Flt-3 at the cell surface, as well as albumin, alpha-fetoprotein, and cytokeratin-18 and 19 in the interior. Moreover, about a half of the cells were found to acquire the capability to transport DiI-Ac-LDL. Based on these observations, and taking into account immense advantages of UCB over other stem cell sources, we conclude that UCB-derived MSCs retain hepatogenic potential suitable for cell therapy and transplantation against intractable liver diseases.

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

PubMed

Saito, Kosuke; Tamaki, Tetsuro; Hirata, Maki; Hashimoto, Hiroyuki; Nakazato, Kenei; Nakajima, Nobuyuki; Kazuno, Akihito; Sakai, Akihiro; Iida, Masahiro; Okami, Kenji

2015-01-01

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

Generation of Regionally Specific Neural Progenitor Cells (NPCs) and Neurons from Human Pluripotent Stem Cells (hPSCs).

PubMed

Cutts, Josh; Brookhouser, Nicholas; Brafman, David A

2016-01-01

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population capable of long-term expansion and differentiation into a variety of neuronal subtypes. As such, NPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine. Current methods for the generation of NPCs results in cell populations homogenous for pan-neural markers such as SOX1 and SOX2 but heterogeneous with respect to regional identity. In order to use NPCs and their neuronal derivatives to investigate mechanisms of neurological disorders and develop more physiologically relevant disease models, methods for generation of regionally specific NPCs and neurons are needed. Here, we describe a protocol in which exogenous manipulation of WNT signaling, through either activation or inhibition, during neural differentiation of hPSCs, promotes the formation of regionally homogenous NPCs and neuronal cultures. In addition, we provide methods to monitor and characterize the efficiency of hPSC differentiation to these regionally specific cell identities.

Treatment of type 1 diabetes with adipose tissue-derived stem cells expressing pancreatic duodenal homeobox 1.

PubMed

Lin, Guiting; Wang, Guifang; Liu, Gang; Yang, Li-Jun; Chang, Lung-Ji; Lue, Tom F; Lin, Ching-Shwun

2009-12-01

Due to the limited supply of donor pancreas, it is imperative that we identify alternative cell sources that can be used to treat diabetes mellitus (DM). Multipotent adipose tissue-derived stem cells (ADSC) can be abundantly and safely isolated for autologous transplantation and therefore are an ideal candidate. Here, we report the derivation of insulin-producing cells from human or rat ADSC by transduction with the pancreatic duodenal homeobox 1 (Pdx1) gene. RT-PCR analyses showed that native ADSC expressed insulin, glucagon, and NeuroD genes that were up-regulated following Pdx1 transduction. ELISA analyses showed that the transduced cells secreted increasing amount of insulin in response to increasing concentration of glucose. Transplantation of these cells under the renal capsule of streptozotocin-induced diabetic rats resulted in lowered blood glucose, higher glucose tolerance, smoother fur, and less cataract. Histological examination showed that the transplanted cells formed tissue-like structures and expressed insulin. Thus, ADSC-expressing Pdx1 appear to be suitable for treatment of DM.

Quercetin potentiates transdifferentiation of bone marrow mesenchymal stem cells into the beta cells in vitro.

PubMed

Miladpour, B; Rasti, M; Owji, A A; Mostafavipour, Z; Khoshdel, Z; Noorafshan, A; Zal, F

2017-05-01

Type 1 diabetes is an autoimmune disease caused by the destruction of β-cells in the pancreas. Bone marrow mesenchymal stem cells are multipotent and easy accessible adult stem cells that may provide options in the treatment of type 1 diabetes. Injured pancreatic extract can promote the differentiation of rat bone marrow mesenchymal stem cells into β-cells. We aimed to observe the effect of quercetin in differentiation and insulin secretion in β-cells. Bone marrow mesenchymal stem cells were obtained from the tibiae of rats. Cell surface markers were analyzed by flow cytometry. The cells were treated with rat injured pancreatic extract and quercetin for 2 weeks. Insulin secretion was measured by ELISA. Insulin expression and some islet factors were evaluated by RT-PCR. PDX1, a marker for β-cell function and differentiation, was evaluated by both immunocytochemistry and Western blot. β-cell count was determined by stereology and cell count assay. ELISA showed significant differences in insulin secretion in the cells treated with RIPE + 20 μM quercetin (0.55 ± 0.01 µg/L) compared with the cells treated with RIPE alone (0.48 ± 0.01 µg/L) (P = 0.026). RT-PCR results confirmed insulin expression in both groups. PDX1 protein was detected in both groups by Western blot and immunocytochemistry. Stereology results showed a significant increase in β-cell number in the RIPE + quercetin-treated cells (47 ± 2.0) when compared with RIPE treatment alone (44 ± 2.5) (P = 0.015). Quercetin has a strengthening effect on the differentiation of rat bone marrow mesenchymal stem cells into β-cells and increases insulin secretion from the differentiated β-cells in vitro.

Comparison of Immunological Characteristics of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells and Bone Marrow

PubMed Central

Fu, Xin; Chen, Yao; Xie, Fang-Nan; Dong, Ping; Liu, Wen-bo; Cao, Yilin

2015-01-01

Mesenchymal stem cell (MSC) has great potential for both regenerative medicine and immunotherapy due to its multipotency and immunomodulatory property. The derivation of MSCs from human tissues involves an invasive procedure and the obtained MSCs often suffer from inconsistent quality. To overcome these issues, the approaches of deriving a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs) were established. However, few studies compared the immunological characteristics of MSCs derived from hESCs with tissue-derived MSCs or demonstrated differences and the underlying mechanisms. Here, we differentiated H9 hESCs into MSC-like cells (H9-MSCs) through an embryoid body outgrowth method and compared the immunological characteristics of H9-MSCs with bone marrow-derived MSCs (BMSCs). Both sources of derived cells exhibited typical MSC morphologies and surface marker expressions, as well as multipotency to differentiate into osteogenic and adipogenic lineages. A immunological characterization study showed that H9-MSCs and BMSCs had similar immunoprivileged properties without triggering allogeneic lymphocyte proliferation as well as equivalent immunosuppressive effects on T-cell proliferation induced by either cellular or mitogenic stimuli. Flow cytometry analysis revealed a lower expression of human major histocompatability complex class II molecule human lymphocyte antigen (HLA)-DR and a higher expression of coinhibitory molecule B7-H1 in H9-MSCs than in BMSCs. Interferon gamma (IFN-γ) is a proinflammatory cytokine that can induce the expression of HLA class II molecules in many cell types. Our results showed that pretreatment of H9-MSCs and BMSCs with IFN-γ did not change their immunogenicity and immunosuppressive abilities, but increased the difference between H9-MSCs and BMSCs for their expression of HLA-DR. Further detection of expression of molecules involved in IFN-γ signaling pathways suggested that the lower expression of HLA-DR in H9-MSCs could be partially attributed to the lower expression and the less nuclear translocation of its transcriptional factor CIITA. The present study provides evidence that the hESC-derived MSCs share similar immunogenicity and immunosuppressive abilities with BMSCs, but differ in the expression profile of immunological markers and the responsiveness to certain inflammatory cytokines, which suggests that H9-MSCs could be a safe and efficient candidate for MSC treatment in patients with inflammatory disorders. PMID:25256849

Chondrogenesis, osteogenesis and adipogenesis of canine mesenchymal stem cells: a biochemical, morphological and ultrastructural study.

PubMed

Csaki, C; Matis, U; Mobasheri, A; Ye, H; Shakibaei, M

2007-12-01

Musculoskeletal diseases with osteochondrotic articular cartilage defects, such as osteoarthritis, are an increasing problem for humans and companion animals which necessitates the development of novel and improved therapeutic strategies. Canine mesenchymal stem cells (cMSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no comprehensive analysis has been undertaken to characterize the ultrastructure of in vitro differentiated cMSCs. The main goal of this paper was to focus on cMSCs and to analyse their differentiation capacity. To achieve this aim, bone marrow cMSCs from three canine patients were isolated, expanded in monolayer culture and characterized with respect to their ability for osteogenic, adipogenic and chondrogenic differentiation capacities. cMSCs showed proliferative potential and were capable of osteogenic, adipogenic and chondrogenic differentiation. cMSCs treated with the osteogenic induction medium differentiated into osteoblasts, produced typical bone matrix components, beta1-integrins and upregulated the osteogenic specific transcription factor Cbfa-1. cMSCs treated with the adipogenic induction medium showed typical adipocyte morphology, produced adiponectin, collagen type I and beta1-integrins, and upregulated the adipogenic specific transcription factor PPAR-gamma. cMSCs treated with the chondrogenic induction medium exhibited a round to oval shape, produced a cartilage-specific extracellular matrix, beta1-integrins and upregulated the chondrogenic specific transcription factor Sox9. These results demonstrate, at the biochemical, morphological and ultrastructural levels, the multipotency of cMSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

Current and future regenerative medicine — Principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine

PubMed Central

Koch, Thomas G.; Berg, Lise C.; Betts, Dean H.

2009-01-01

This paper provides a bird’s-eye perspective of the general principles of stem-cell therapy and tissue engineering; it relates comparative knowledge in this area to the current and future status of equine regenerative medicine. The understanding of equine stem cell biology, biofactors, and scaffolds, and their potential therapeutic use in horses are rudimentary at present. Mesenchymal stem cell isolation has been proclaimed from several equine tissues in the past few years. Based on the criteria of the International Society for Cellular Therapy, most of these cells are more correctly referred to as multipotent mesenchymal stromal cells, unless there is proof that they exhibit the fundamental in vivo characteristics of pluripotency and the ability to self-renew. That said, these cells from various tissues hold great promise for therapeutic use in horses. The 3 components of tissue engineering — cells, biological factors, and biomaterials — are increasingly being applied in equine medicine, fuelled by better scaffolds and increased understanding of individual biofactors and cell sources. The effectiveness of stem cell-based therapies and most tissue engineering concepts has not been demonstrated sufficiently in controlled clinical trials in equine patients to be regarded as evidence-based medicine. In the meantime, the medical mantra “do no harm” should prevail, and the application of stem cell-based therapies in the horse should be done critically and cautiously, and treatment outcomes (good and bad) should be recorded and reported. Stem cell and tissue engineering research in the horse has exciting comparative and equine specific perspectives that most likely will benefit the health of horses and humans. Controlled, well-designed studies are needed to move this new equine research field forward. PMID:19412395

Testing stem cell therapy in a rat model of inflammatory bowel disease: role of bone marrow stem cells and stem cell factor in mucosal regeneration.

PubMed

Qu, Bo; Xin, Guo-Rong; Zhao, Li-Xia; Xing, Hui; Lian, Li-Ying; Jiang, Hai-Yan; Tong, Jia-Zhao; Wang, Bei-Bei; Jin, Shi-Zhu

2014-01-01

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD). BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls. PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group. BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.

Stable chromosomal aberrations in haemopoietic stem cells in the blood of radiation accident victims.

PubMed

Kreja, L; Greulich, K M; Fliedner, T M; Heinze, B

1999-10-01

The detection of long-term persistent chromosome aberrations in circulating haemopoietic stem cells after accidental radiation exposure. Peripheral blood samples from highly exposed persons were collected 7-25 years after the radiation accidents in Moscow (1971), Kazan (1975) and Chernobyl (1996). Haemopoietic blood stem cells were analysed when investigating individual colonies derived from haemopoietic progenitor cells: burst-forming units-erythroid (BFU-E), granulocyte-macrophage-colony-forming cells (GM-CFC) and multipotent granulocyte-erythrocyte-macrophage- megakaryocyte-colony-forming cells (GEMM-CFC). Colony formation was obtained in methylcellulose cultures. Chromosome preparations in single colonies were performed using a microtechnique. Nine patients were investigated at 1 to 4 follow-up time points after radiation exposure. Three hundred and thirty-four single colonies were analyzed resulting in 1375 mitoses. It was found that colonies showed chromosome aberrations (ChA) up to 25 years after radiation exposure by classical cytogenetics and by fluorescence in situ hybridization (FISH). Stable aberrations were detected in 21% of colonies. They were clonal in 19% of colonies, i.e. the same abnormality was found in all cells derived from a single colony. In 2% of colonies ChA were stable but non-clonal; unstable ChA were not observed. The results indicate that blood-derived haemopoietic stem cells may serve as a biological indicator to detect radiation-induced ChA. Since they are considered to be in dynamic and functional exchange with stem cells in the medullary sites of blood cell formation such as bone marrow, the use of blood stem cells as a marker of radiation effects should be explored to assess the repair status of the stem cell pool as such.

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

PubMed

Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Isolation and characterization of 2 new human rotator cuff and long head of biceps tendon cells possessing stem cell-like self-renewal and multipotential differentiation capacity.

PubMed

Randelli, Pietro; Conforti, Erika; Piccoli, Marco; Ragone, Vincenza; Creo, Pasquale; Cirillo, Federica; Masuzzo, Pamela; Tringali, Cristina; Cabitza, Paolo; Tettamanti, Guido; Gagliano, Nicoletta; Anastasia, Luigi

2013-07-01

Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Controlled laboratory study. Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells. This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells.

PubMed

Karimzadeh, Alborz; Scarfone, Vanessa M; Varady, Erika; Chao, Connie; Grathwohl, Karin; Fathman, John W; Fruman, David A; Serwold, Thomas; Inlay, Matthew A

2018-06-01

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. Stem Cells Translational Medicine 2018;7:468-476. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Identification of multipotent stem cells from adult dog periodontal ligament.

PubMed

Wang, Wen-Jun; Zhao, Yu-Ming; Lin, Bi-Chen; Yang, Jie; Ge, Li-Hong

2012-08-01

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease. © 2012 Eur J Oral Sci.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

PubMed

Yalvac, M E; Ramazanoglu, M; Rizvanov, A A; Sahin, F; Bayrak, O F; Salli, U; Palotás, A; Kose, G T

2010-04-01

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

Biomaterial-mesenchymal stem cell constructs for immunomodulation in composite tissue engineering.

PubMed

Hanson, Summer; D'Souza, Rena N; Hematti, Peiman

2014-08-01

Cell-based treatments are being developed as a novel approach for the treatment of many diseases in an effort to repair injured tissues and regenerate lost tissues. Interest in the potential use of multipotent progenitor or stem cells has grown significantly in recent years, specifically the use of mesenchymal stem cells (MSCs), for tissue engineering in combination with extracellular matrix-based scaffolds. An area that warrants further attention is the local or systemic host responses toward the implanted cell-biomaterial constructs. Such immunological responses could play a major role in determining the clinical efficacy of the therapeutic device or biomaterials used. MSCs, due to their unique immunomodulatory properties, hold great promise in tissue engineering as they not only directly participate in tissue repair and regeneration but also modulate the host foreign body response toward the engineered constructs. The purpose of this review was to summarize the current state of knowledge and applications of MSC-biomaterial constructs as a potential immunoregulatory tool in tissue engineering. Better understanding of the interactions between biomaterials and cells could translate to the development of clinically relevant and novel cell-based therapeutics for tissue reconstruction and regenerative medicine.

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering.

PubMed

James, Aaron W; Zhang, Xinli; Crisan, Mihaela; Hardy, Winters R; Liang, Pei; Meyers, Carolyn A; Lobo, Sonja; Lagishetty, Venu; Childers, Martin K; Asatrian, Greg; Ding, Catherine; Yen, Yu-Hsin; Zou, Erin; Ting, Kang; Peault, Bruno; Soo, Chia

2017-01-01

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

Mammalian cell models to advance our understanding of wound healing: a review.

PubMed

Vidmar, Jerneja; Chingwaru, Constance; Chingwaru, Walter

2017-04-01

Rapid and efficient healing of damaged tissue is critical for the restoration of tissue function and avoidance of tissue defects. Many in vitro cell models have been described for wound healing studies; however, the mechanisms that underlie the process, especially in chronic or complicated wounds, are not fully understood. The identification of cell culture systems that closely simulate the physiology of damaged tissue in vivo is necessary. We describe the cell culture models that have enhanced our understanding, this far, of the wound healing process or have been used in drug discovery. Cell cultures derived from the epithelium, including corneal, renal, intestinal (IEC-8 cells and IEC-6), skin epithelial cells (keratinocytes, fibroblasts, and multipotent mesenchymal stem cells), and the endothelium (human umbilical vein endothelial cells, primary mouse endothelial cells, endodermal stem cells, human mesenchymal stem cells, and corneal endothelial cells) have played a pivotal role toward our understanding of the mechanisms of wound healing. More studies are necessary to develop co-culture cell models which closely simulate the environment of a wound in vivo. Cell culture models are invaluable tools to promote our understanding of the mechanisms that regulate the wound healing process and provide a platform for drug discovery. Copyright © 2016 Elsevier Inc. All rights reserved.

A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application.

PubMed

Martinelli, Daniela; Pereira, Rui Cruz; Mogni, Massimo; Benelli, Roberto; Mastrogiacomo, Maddalena; Coviello, Domenico; Cancedda, Ranieri; Gentili, Chiara

2016-03-01

The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors. We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset). We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity. Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Low-Oxygen Culture Conditions Extend the Multipotent Properties of Human Retinal Progenitor Cells

PubMed Central

Tucker, Budd A.; Young, Michael J.

2014-01-01

Purpose: Development of an effective cell-based therapy is highly dependent upon having a reproducible cell source suitable for transplantation. One potential source, isolated from the developing fetal neural retina, is the human retinal progenitor cell (hRPC). One limiting factor for the use of hRPCs is their in vitro expansion limit. As such, the aim of this study was to determine whether culturing hRPCs under 3% O2 would support their proliferative capacity while maintaining multipotency. Methods: To determine the effect of low oxygen on the ability of hRPCs to self-renew, rates of proliferation and apoptosis, telomerase activity, and expression of proliferative, stemness, and differentiation markers were assessed for hRPCs cultured in 3% and 20% oxygen conditions. Results: Culture under 3% oxygen increases the proliferation rate and shifts the proliferation limit of hRPCs to greater 40 divisions. This increased capacity for proliferation is correlated with an upregulation of Ki67, CyclinD1, and telomerase activity and a decrease in p53 expression and apoptosis. Increased expression of cMyc, Klf4, Oct4, and Sox2 in 3% O2 is correlated with stabilization of both HIF1α and HIF2α. The eye field development markers Pax6, Sox2, and Otx2 are present in hRPCs up to passage 16 in 3% O2. Following in vitro differentiation hRPCs expanded in the 3% O2 were able to generate specialized retinal cells, including rods and cones. Conclusions: Low-oxygen culture conditions act to maintain both multipotency and self-renewal properties of hRPCs in vitro. The extended expansion limits permit the development of a clinical-grade reagent for transplantation. PMID:24320879

Mesenchymal stem cell mechanobiology and emerging experimental platforms

PubMed Central

MacQueen, Luke; Sun, Yu; Simmons, Craig A.

2013-01-01

Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms. PMID:23635493

Adipose-Derived Stem Cells Enhance Cancer Stem Cell Property and Tumor Formation Capacity in Lewis Lung Carcinoma Cells Through an Interleukin-6 Paracrine Circuit.

PubMed

Lu, Jui-Hua; Wei, Hong-Jian; Peng, Bou-Yue; Chou, Hsin-Hua; Chen, Wei-Hong; Liu, Hen-Yu; Deng, Win-Ping

2016-12-01

Adipose-derived stem cells (ADSCs) are multipotent cells that have attracted much recent attention and emerged as therapeutic approaches in several medical fields. Although current knowledge of the biological impacts of ADSCs in cancer research is greatly improved, the underlying effects of ADSCs in tumor development remain controversial and cause the safety concerns in clinical utilization. Hence, we isolated primary ADSCs from the abdominal fat of mice and conducted interaction of ADSCs with Lewis lung carcinoma cells in culture and in mice to investigate the impacts of ADSCs on tumor development. Cytokine array and neutralizing antibody were further utilized to identify the key regulator and downstream signaling pathway. In this study, we demonstrated that ADSCs enhance the malignant characteristics of LLC1 cells, including cell growth ability and especially cancer stem cell property. ADSCs were then identified to promote tumor formation and growth in mice. We further determined that ADSC interaction with LLC1 cells stimulates increased secretion of interleukin-6 mainly from ADSCs, which then act in a paracrine manner on LLC1 cells to enhance their malignant characteristics. Interleukin-6 was also identified to regulate genes related to cell proliferation and cancer stem cell, as well as to activate JAK2/STAT3, a predominant interleukin-6-activated pathway, in LLC1 cells. Collectively, we demonstrated that ADSCs play a pro-malignant role in tumor development of Lewis lung carcinoma cells by particularly promoting cancer stem cell property through interleukin-6 paracrine circuit, which is important for safety considerations regarding the clinical application of ADSCs.

miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

PubMed Central

Costantino, Vincenzo; Curci, Claudia; Cox, Sharon N.; De Palma, Giuseppe; Schena, Francesco P.

2013-01-01

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity. PMID:23861881

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization

PubMed Central

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-01-01

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations. PMID:26311223

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization.

PubMed

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-08-27

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

PubMed

Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

2017-06-01

During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDH hi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553. © 2017 AlphaMed Press.

Engineering micropatterned surfaces to modulate the function of vascular stem cells.

PubMed

Li, Jennifer; Wu, Michelle; Chu, Julia; Sochol, Ryan; Patel, Shyam

2014-02-21

Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces. Copyright © 2014 Elsevier Inc. All rights reserved.

β2-Microglobulin as a potential factor for the expansion of mesenchymal stem cells

PubMed Central

Zhu, Ying; Su, Yongping; Cheng, Tianmin; Chung, Leland W. K.

2010-01-01

Multipotent mesenchymal stem cells (MSCs) hold great promise in regenerative medicine, but one of the biggest challenges facing for their application is the ex vivo expansion to obtain enough undifferentiated cells. Fetal bovine serum (FBS), which can elicit possible contaminations of prion, virus, zoonosis or immunological reaction against xenogenic serum antigens, still remains essential to the culture formulations. There is an urgent need to identify potential factors for the undifferentiated expansion of MSCs to reduce the use of FBS or eventually replace it. A previously recognized housekeeping gene, β2-microglobulin (β2M), is demonstrated to act as a novel growth factor to stimulate the undifferentiated ex vivo expansion and preserve the pluripotency of adult MSCs from various sources. The use of β2M might have promising implications for future clinical application of MSCs. PMID:19466557

Transplantation of human umbilical cord-derived mesenchymal stems cells for the treatment of Becker muscular dystrophy in affected pedigree members.

PubMed

Li, Pang; Cui, Kai; Zhang, Bo; Wang, Zhendan; Shen, Yangyang; Wang, Xiangyu; Zhang, Jianbo; Tong, Feng; Li, Sheng

2015-04-01

The regeneration of muscle tissue has been achieved using multipotent mesenchymal stem cells in mouse models of injured skeletal muscle. In the present study, the utility of multipotent human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in the treatment of Becker muscular dystrophy (BMD), a genetic disease where muscle tissue fails to regenerate, was examined in members from a pedigree affected by BMD. The disease status was evaluated in 4 affected pedigree members (II1, II2, II3 and III2; aged 50, 46, 42 and 6 years, respectively). The transplantation of the hUC‑MSCs (performed on 3 patients, I2, II3 and III2) was performed by infusion with an intravenous drip over a 30‑min period, and the patients were evaluated at 1, 3, 4 and 12 weeks following the procedure. The evaluation was based on physical characteristics, as well as on molecular testing for serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels and a histological examination of muscle biopsies. The patients suffered no adverse reactions in response to the transplantation of the hUC‑MSCs. At 1 week following transplantation all 3 patients showed improvement in the muscle force of the limbs, muscle size and daily activity. The walking gait of patient III2 had improved by 1 week post-transplantation and reached a normal status by 12 weeks. Serum CK and LDH levels were decreased relative to the baseline levels. A histological examination of muscle biopsies displayed no obvious tissue regeneration. In conclusion, the treatment of patients with BMD using hUC-MSCs was safe and of therapeutic benefit that lasted for up to 12 weeks. hUC-MSCs are, therefore, a potential cell therapy-based treatment option for patients with muscular dystrophies.

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

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

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

PubMed Central

Jang, Sumin; Choubey, Sandeep; Furchtgott, Leon; Zou, Ling-Nan; Doyle, Adele; Menon, Vilas; Loew, Ethan B; Krostag, Anne-Rachel; Martinez, Refugio A; Madisen, Linda; Levi, Boaz P; Ramanathan, Sharad

2017-01-01

The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development. DOI: http://dx.doi.org/10.7554/eLife.20487.001 PMID:28296635

Gene therapy with mesenchymal stem cells expressing IFN-ß ameliorates neuroinflammation in experimental models of multiple sclerosis.

PubMed

Marin-Bañasco, C; Benabdellah, K; Melero-Jerez, C; Oliver, B; Pinto-Medel, M J; Hurtado-Guerrero, I; de Castro, F; Clemente, D; Fernández, O; Martin, F; Leyva, L; Suardíaz, M

2017-02-01

Recombinant IFN-ß is one of the first-line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose-derived MSCs (AdMSCs), transduced with the IFN-β gene, into mice with experimental autoimmune encephalomyelitis (EAE). Relapsing-remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN-ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro-inflammation. Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN-β. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN-ß treatment, by providing long-term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose-limiting side effects. © 2016 The British Pharmacological Society.

Gene therapy with mesenchymal stem cells expressing IFN‐ß ameliorates neuroinflammation in experimental models of multiple sclerosis

PubMed Central

Marin‐Bañasco, C; Benabdellah, K; Melero‐Jerez, C; Oliver, B; Pinto‐Medel, M J; Hurtado‐Guerrero, I; de Castro, F; Clemente, D; Fernández, O; Martin, F; Leyva, L

2017-01-01

Background and Purpose Recombinant IFN‐ß is one of the first‐line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose‐derived MSCs (AdMSCs), transduced with the IFN‐β gene, into mice with experimental autoimmune encephalomyelitis (EAE). Experimental Approach Relapsing–remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively. Key Results Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN‐ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro‐inflammation. Conclusion and Implications Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN‐β. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN‐ß treatment, by providing long‐term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose‐limiting side effects. PMID:27882538

Combined effects of pericytes in the tumor microenvironment.

PubMed

Ribeiro, Aline Lopes; Okamoto, Oswaldo Keith

2015-01-01

Pericytes are multipotent perivascular cells whose involvement in vasculature development is well established. Evidences in the literature also suggest that pericytes display immune properties and that these cells may serve as an in vivo reservoir of stem cells, contributing to the regeneration of diverse tissues. Pericytes are also capable of tumor homing and are important cellular components of the tumor microenvironment (TME). In this review, we highlight the contribution of pericytes to some classical hallmarks of cancer, namely, tumor angiogenesis, growth, metastasis, and evasion of immune destruction, and discuss how collectively these hallmarks could be tackled by therapies targeting pericytes, providing a rationale for cancer drugs aiming at the TME.

Extrinsic and intrinsic mechanisms by which mesenchymal stem cells suppress the immune system

PubMed Central

Coulson-Thomas, Vivien J.; Coulson-Thomas, Yvette M.; Gesteira, Tarsis F.; Kao, Winston W.-Y.

2016-01-01

Mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells that have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Recent studies have demonstrated that MSCs possess a unique ability to exert suppressive and regulatory effects on both adaptive and innate immunity in an autologous and allogeneic manner. A vital step in stem cell transplantation is overcoming the potential graft-versus-host disease, which is a limiting factor to transplantation success. Given that MSCs attain powerful differentiation capabilities and also present immunosuppressive properties, which enable them to survive host immune rejection, MSCs are of great interest. Due to their ability to differentiate into different cell types and to suppress and modulate the immune system, MSCs are being developed for treating a plethora of diseases, including immune disorders. Moreover, in recent years, MSCs have been genetically engineered to treat and sometimes even cure some diseases, and the use of MSCs for cell therapy presents new perspectives for overcoming tissue rejection. In this review, we discuss the potential extrinsic and intrinsic mechanisms that underlie MSCs’ unique ability to modulate inflammation, and both innate and adaptive immunity. PMID:26804815

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.

PubMed

McCully, Mark; Conde, João; V Baptista, Pedro; Mullin, Margaret; Dalby, Matthew J; Berry, Catherine C

2018-01-01

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine.

Notch1 stimulation induces a vascularization switch with pericyte-like cell differentiation of glioblastoma stem cells.

PubMed

Guichet, Pierre-Olivier; Guelfi, Sophie; Teigell, Marisa; Hoppe, Liesa; Bakalara, Norbert; Bauchet, Luc; Duffau, Hugues; Lamszus, Katrin; Rothhut, Bernard; Hugnot, Jean-Philippe

2015-01-01

Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. © 2014 AlphaMed Press.

Isolation and clonal characterization of hematopoietic and liver stem cells.

PubMed

Nakauchi, Hiromitsu

2004-11-01

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

PubMed

Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

2015-06-01

It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

PubMed

Yang, Sufang; Pilgaard, Linda; Chase, Lucas G; Boucher, Shayne; Vemuri, Mohan C; Fink, Trine; Zachar, Vladimir

2012-08-01

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells

PubMed Central

Pitrone, Maria; Pizzolanti, Giuseppe; Tomasello, Laura; Coppola, Antonina; Morini, Lorenzo; Pantuso, Gianni; Ficarella, Romina; Guarnotta, Valentina; Perrini, Sebastio; Giorgino, Francesco; Giordano, Carla

2017-01-01

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self-renewal potential and differentiation capacity. Our aim was to study the different expression of the embryonic stem cell markers NANOG (homeobox protein NANOG), SOX2 (SRY (sex determining region Y)-box 2) and OCT4 (octamer-binding transcription factor 4) and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 ± 5 and 30 ± 5 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90, CD105, CD29, CD31, CD45 and CD146 were analyzed by flow cytometry, and the stem cell transcription factors NANOG, SOX2 and OCT4 were detected by immunoblotting and real-time PCR. NANOG, SOX2 and OCT4 interplay was explored by gene silencing. ASCs from VAT and SAT confirmed their mesenchymal stem cell (MSC) phenotype expressing the specific MSC markers CD90, CD105, NANOG, SOX2 and OCT4. NANOG silencing induced a significant OCT4 (70 ± 0.05%) and SOX2 (75 ± 0.03%) downregulation, whereas SOX2 silencing did not affect NANOG gene expression. Adipose tissue is an important source of MSC, and siRNA experiments endorse a hierarchical role of NANOG in the complex transcription network that regulates pluripotency. PMID:28545230

Laser processing of polymer constructs from poly(3-hydroxybutyrate).

PubMed

Volova, T G; Tarasevich, A A; Golubev, A I; Boyandin, A N; Shumilova, A A; Nikolaeva, E D; Shishatskaya, E I

2015-01-01

CO2 laser radiation was used to process poly(3-hydroxybutyrate) constructs - films and 3D pressed plates. Laser processing increased the biocompatibility of unperforated films treated with moderate uniform radiation, as estimated by the number and degree of adhesion of NIH 3T3 mouse fibroblast cells. The biocompatibility of perforated films modified in the pulsed mode did not change significantly. At the same time, pulsed laser processing of the 3D plates produced perforated scaffolds with improved mechanical properties and high biocompatibility with bone marrow-derived multipotent, mesenchymal stem cells, which show great promise for bone regeneration.

Alginate Hydrogel Microencapsulation Inhibits Devitrification and Enables Large-Volume Low-CPA Cell Vitrification

PubMed Central

Huang, Haishui; Choi, Jung Kyu; Rao, Wei; Zhao, Shuting; Agarwal, Pranay; Zhao, Gang

2015-01-01

Cryopreservation of stem cells is important to meet their ever-increasing demand by the burgeoning cell-based medicine. The conventional slow freezing for stem cell cryopreservation suffers from inevitable cell injury associated with ice formation and the vitrification (i.e., no visible ice formation) approach is emerging as a new strategy for cell cryopreservation. A major challenge to cell vitrification is intracellular ice formation (IIF, a lethal event to cells) induced by devitrification (i.e., formation of visible ice in previously vitrified solution) during warming the vitrified cells at cryogenic temperature back to super-zero temperatures. Consequently, high and toxic concentrations of penetrating cryoprotectants (i.e., high CPAs, up to ~8 M) and/or limited sample volumes (up to ~2.5 μl) have been used to minimize IIF during vitrification. We reveal that alginate hydrogel microencapsulation can effectively inhibit devitrification during warming. Our data show that if ice formation were minimized during cooling, IIF is negligible in alginate hydrogel-microencapsulated cells during the entire cooling and warming procedure of vitrification. This enables vitrification of pluripotent and multipotent stem cells with up to ~4 times lower concentration of penetrating CPAs (up to 2 M, low CPA) in up to ~100 times larger sample volume (up to ~250 μl, large volume). PMID:26640426

Alginate Hydrogel Microencapsulation Inhibits Devitrification and Enables Large-Volume Low-CPA Cell Vitrification.

PubMed

Huang, Haishui; Choi, Jung Kyu; Rao, Wei; Zhao, Shuting; Agarwal, Pranay; Zhao, Gang; He, Xiaoming

2015-11-25

Cryopreservation of stem cells is important to meet their ever-increasing demand by the burgeoning cell-based medicine. The conventional slow freezing for stem cell cryopreservation suffers from inevitable cell injury associated with ice formation and the vitrification ( i.e. , no visible ice formation) approach is emerging as a new strategy for cell cryopreservation. A major challenge to cell vitrification is intracellular ice formation (IIF, a lethal event to cells) induced by devitrification ( i.e. , formation of visible ice in previously vitrified solution) during warming the vitrified cells at cryogenic temperature back to super-zero temperatures. Consequently, high and toxic concentrations of penetrating cryoprotectants ( i.e. , high CPAs, up to ~8 M) and/or limited sample volumes (up to ~2.5 μl) have been used to minimize IIF during vitrification. We reveal that alginate hydrogel microencapsulation can effectively inhibit devitrification during warming. Our data show that if ice formation were minimized during cooling, IIF is negligible in alginate hydrogel-microencapsulated cells during the entire cooling and warming procedure of vitrification. This enables vitrification of pluripotent and multipotent stem cells with up to ~4 times lower concentration of penetrating CPAs (up to 2 M, low CPA) in up to ~100 times larger sample volume (up to ~250 μl, large volume).

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

Mesenchymal Stem Cells: Time to Change the Name!

PubMed Central

2017-01-01

Summary Mesenchymal stem cells (MSCs) were officially named more than 25 years ago to represent a class of cells from human and mammalian bone marrow and periosteum that could be isolated and expanded in culture while maintaining their in vitro capacity to be induced to form a variety of mesodermal phenotypes and tissues. The in vitro capacity to form bone, cartilage, fat, etc., became an assay for identifying this class of multipotent cells and around which several companies were formed in the 1990s to medically exploit the regenerative capabilities of MSCs. Today, there are hundreds of clinics and hundreds of clinical trials using human MSCs with very few, if any, focusing on the in vitro multipotential capacities of these cells. Unfortunately, the fact that MSCs are called “stem cells” is being used to infer that patients will receive direct medical benefit, because they imagine that these cells will differentiate into regenerating tissue‐producing cells. Such a stem cell treatment will presumably cure the patient of their medically relevant difficulties ranging from osteoarthritic (bone‐on‐bone) knees to various neurological maladies including dementia. I now urge that we change the name of MSCs to Medicinal Signaling Cells to more accurately reflect the fact that these cells home in on sites of injury or disease and secrete bioactive factors that are immunomodulatory and trophic (regenerative) meaning that these cells make therapeutic drugs in situ that are medicinal. It is, indeed, the patient's own site‐specific and tissue‐specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs. Stem Cells Translational Medicine 2017;6:1445–1451 PMID:28452204

Robust G2 pausing of adult stem cells in Hydra.

PubMed

Buzgariu, Wanda; Crescenzi, Marco; Galliot, Brigitte

2014-01-01

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Changes of neural markers expression during late neurogenic differentiation of human adipose-derived stem cells

PubMed Central

Razavi, Shahnaz; Khosravizadeh, Zahra; Bahramian, Hamid; Kazemi, Mohammad

2015-01-01

Background: Different studies have been done to obtain sufficient number of neural cells for treatment of neurodegenerative diseases, spinal cord, and traumatic brain injury because neural stem cells are limited in central nerves system. Recently, several studies have shown that adipose-derived stem cells (ADSCs) are the appropriate source of multipotent stem cells. Furthermore, these cells are found in large quantities. The aim of this study was an assessment of proliferation and potential of neurogenic differentiation of ADSCs with passing time. Materials and Methods: Neurosphere formation was used for neural induction in isolated human ADSCs (hADSCs). The rate of proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and potential of neural differentiation of induced hADSCs was evaluated by immunocytochemical and real-time reverse transcription polymerase chain reaction analysis after 10 and 14 days post-induction. Results: The rate of proliferation of induced hADSCs increased after 14 days while the expression of nestin, glial fibrillary acidic protein, and microtubule-associated protein 2 was decreased with passing time during neurogenic differentiation. Conclusion: These findings showed that the proliferation of induced cells increased with passing time, but in early neurogenic differentiation of hADSCs, neural expression was higher than late of differentiation. Thus, using of induced cells in early differentiation may be suggested for in vivo application. PMID:26605238

Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux.

PubMed

Ahmed, Raya; Roger, Laureline; Costa Del Amo, Pedro; Miners, Kelly L; Jones, Rhiannon E; Boelen, Lies; Fali, Tinhinane; Elemans, Marjet; Zhang, Yan; Appay, Victor; Baird, Duncan M; Asquith, Becca; Price, David A; Macallan, Derek C; Ladell, Kristin

2016-12-13

Adaptive immunity requires the generation of memory T cells from naive precursors selected in the thymus. The key intermediaries in this process are stem cell-like memory T (T SCM ) cells, multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells. In theory, antigen specificity within the T SCM pool may be imprinted statically as a function of largely dormant cells and/or retained dynamically by more transitory subpopulations. To explore the origins of immunological memory, we measured the turnover of T SCM cells in vivo using stable isotope labeling with heavy water. The data indicate that T SCM cells in both young and elderly subjects are maintained by ongoing proliferation. In line with this finding, T SCM cells displayed limited telomere length erosion coupled with high expression levels of active telomerase and Ki67. Collectively, these observations show that T SCM cells exist in a state of perpetual flux throughout the human lifespan. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Complex interactions in EML cell stimulation by stem cell factor and IL-3.

PubMed

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M

2011-03-22

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.

Complex interactions in EML cell stimulation by stem cell factor and IL-3

PubMed Central

Ye, Zhi-jia; Gulcicek, Erol; Stone, Kathryn; Lam, Tukiet; Schulz, Vincent; Weissman, Sherman M.

2011-01-01

Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34− cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone. PMID:21383156

Mesenchymal stem cells in tumor development

PubMed Central

Cuiffo, Benjamin G.; Karnoub, Antoine E.

2012-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that participate in the structural and functional maintenance of connective tissues under normal homeostasis. They also act as trophic mediators during tissue repair, generating bioactive molecules that help in tissue regeneration following injury. MSCs serve comparable roles in cases of malignancy and are becoming increasingly appreciated as critical components of the tumor microenvironment. MSCs home to developing tumors with great affinity, where they exacerbate cancer cell proliferation, motility, invasion and metastasis, foster angiogenesis, promote tumor desmoplasia and suppress anti-tumor immune responses. These multifaceted roles emerge as a product of reciprocal interactions occurring between MSCs and cancer cells and serve to alter the tumor milieu, setting into motion a dynamic co-evolution of both tumor and stromal tissues that favors tumor progression. Here, we summarize our current knowledge about the involvement of MSCs in cancer pathogenesis and review accumulating evidence that have placed them at the center of the pro-malignant tumor stroma. PMID:22863739

Cell Encapsulating Biomaterial Regulates Mesenchymal Stromal/Stem Cell Differentiation and Macrophage Immunophenotype

PubMed Central

Cantu, David Antonio; Hematti, Peiman

2012-01-01

Bone marrow mesenchymal stromal/stem cell (MSC) encapsulation within a biomatrix could improve cellular delivery and extend survival and residence time over conventional intravenous administration. Although MSCs modulate monocyte/macrophage (Mø) immunophenotypic properties, little is known about how such interactions are influenced when MSCs are entrapped within a biomaterial. Furthermore, the impact of the cell-encapsulating matrix on MSC multipotency and on Møs, which infiltrate biomaterials, remains poorly understood. Here we elucidate this three-way interaction. The Mø immunophenotype and MSC differentiation were examined with regard to established and experimental collagen-based biomaterials for MSC entrapment. Tumor necrosis factor-α secretion was acutely inhibited at 4 days. MSCs cocultured with Møs demonstrated attenuated chondrocyte differentiation, whereas osteoblast differentiation was enhanced. Adipocyte differentiation was considerably enhanced for MSCs entrapped within the gelatin/polyethylene glycol-based matrix. A better understanding of the effect of cell encapsulation on differentiation potency and immunomodulation of MSCs is essential for MSC-based, biomaterial-enabled therapies. PMID:23197666

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

Th17 Pathway As a Target for Multipotent Stromal Cell Therapy in Dogs: Implications for Translational Research

PubMed Central

Kol, A.; Walker, N. J.; Nordstrom, M.; Borjesson, D. L.

2016-01-01

Detrimental Th17 driven inflammatory and autoimmune disease such as Crohn’s disease, graft versus host disease and multiple sclerosis remain a significant cause of morbidity and mortality worldwide. Multipotent stromal/stem cell (MSC) inhibit Th17 polarization and activation in vitro and in rodent models. As such, MSC based therapeutic approaches are being investigated as novel therapeutic approaches to treat Th17 driven diseases in humans. The significance of naturally occurring diseases in dogs is increasingly recognized as a realistic platform to conduct pre-clinical testing of novel therapeutics. Full characterization of Th17 cells in dogs has not been completed. We have developed and validated a flow-cytometric method to detect Th17 cells in canine blood. We further demonstrate that Th17 and other IL17 producing cells are present in tissues of dogs with naturally occurring chronic inflammatory diseases. Finally, we have determined the kinetics of a canine specific Th17 polarization in vitro and demonstrate that canine MSC inhibit Th17 polarization in vitro, in a PGE2 independent mechanism. Our findings provide fundamental research tools and suggest that naturally occurring diseases in dogs, such as inflammatory bowel disease, may be harnessed to translate novel MSC based therapeutic strategies that target the Th17 pathway. PMID:26872054

Epithelial morphogenesis of germline-derived pluripotent stem cells on organotypic skin equivalents in vitro.

PubMed

van de Kamp, Julia; Kramann, Rafael; Anraths, Julia; Schöler, Hans R; Ko, Kinarm; Knüchel, Ruth; Zenke, Martin; Neuss, Sabine; Schneider, Rebekka K

2012-03-01

For tissue engineering, cultivation of pluripotent stem cells on three-dimensional scaffolds allows the generation of organ-like structures. Previously, we have established an organotypic culture system of skin to induce epidermal differentiation in adult stem cells. Multipotent stem cells are not able to differentiate across germinal boundaries. In contrast, pluripotent stem cells readily differentiate into tissues of all three germ layers. Germline-derived pluripotent stem cells (gPS cells) can be generated by induction of pluripotency in mouse unipotent germline stem cells without the introduction of exogenous transcription factors. In the current study, we analyzed the influence of organotypic culture conditions of skin on the epithelial differentiation of gPS cells in comparison to the well-established HM1 ES cell line. Quantitative RT-PCR data of the pluripotency gene Oct4 showed that gPS cells are characterized by an accelerated Oct4-downregulation compared to HM1 ES cells. When subjected to the organotypic culture conditions of skin, gPS cells formed tubulocystic structures lined by stratified (CK5/6(+), CK14(+), CK8/18(-)) epithelia. HM1 ES cells formed only small tubulocystic structures lined by simple, CK8/18(+) epithelia. BMP-4, an epidermal morphogen, significantly enhanced the expression of epithelial markers in HM1 ES cells, but did not significantly affect the formation of complex (squamous) epithelia in gPS cells. In HM1 ES cells the differentiation into squamous epithelium was only inducible in the presence of mature dermal fibroblasts. Both pluripotent stem cell types spontaneously differentiated into mesodermal, endodermal and into neuroectodermal cells at low frequency, underlining their pluripotent differentiation capacity. Concluding, the organotypic culture conditions of skin induce a multilayered, stratified epithelium in gPS cells, in HM1 ES cells only in the presence of dermal fibroblasts. Thus, our data show that differentiation protocols strongly depend on the stem cell type and have to be modified for each specific stem cell type. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

PubMed Central

Song, Bing; Jiang, Wenkai; Alraies, Amr; Liu, Qian; Gudla, Vijay; Oni, Julia; Wei, Xiaoqing; Sloan, Alastair; Ni, Longxing; Agarwal, Meena

2016-01-01

Dental pulp stem cells (DPSCs) are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC) regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1). After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin) increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering. PMID:26880982

Isolation of canine mesenchymal stem cells from amniotic fluid and differentiation into hepatocyte-like cells.

PubMed

Choi, Seon-A; Choi, Hoon-Sung; Kim, Keun Jung; Lee, Dong-Soo; Lee, Ji Hey; Park, Jie Yeun; Kim, Eun Young; Li, Xiaoxia; Oh, Hyun-Yang; Lee, Dong-Seok; Kim, Min Kyu

2013-01-01

Recent findings have demonstrated that amniotic fluid cells are an interesting and potential source of mesenchymal stem cells (MSCs). In this study, we isolated MSCs from canine amniotic fluid and then characterized their multilineage differentiation ability. Canine amniotic fluid stem (cAFS) cells at passage 5 had a fibroblast-like morphology instead of forming colonies and were positive for pluripotent stem cell markers such as OCT4, NANOG, and SOX2. Flow cytometry analysis showed the expression of MSC surface markers CD44, CD29, and CD90 on the cAFS cells. In addition, these cells were cultured under conditions favorable for adipogenic, chondrogenic, and osteogenic induction. The results of this experiment confirmed the mesenchymal nature of cAFS cells and their multipotent potential. Interestingly, although the cells exhibited a fibroblast-like morphology after hepatogenic induction, reverse transcription-polymerase chain reaction revealed that the expression of several hepatic genes, such as albumin, tyrosine aminotransferase, and alpha-1 antiproteinase, increased following maturation and differentiation. These findings indicated that cAFS cells have functional properties similar to those of hepatocytes. Taken together, the results of our study demonstrated that cAFS cells with mesenchymal characteristics can be successfully isolated from canine amniotic fluid and possess functional properties characteristic of hepatocytes. The findings of our work suggest that cAFS cells have the potential to be a resource for cell-based therapies in a canine model of hepatic disease.

Effect of Diabetes Mellitus on Adipocyte-Derived Stem Cells in Rat.

PubMed

Jumabay, Medet; Moon, Jeremiah H; Yeerna, Huwate; Boström, Kristina I

2015-11-01

Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. © 2015 Wiley Periodicals, Inc.

Quiescent gastric stem cells maintain the adult Drosophila stomach.

PubMed

Strand, Marie; Micchelli, Craig A

2011-10-25

The adult Drosophila copper cell region or "stomach" is a highly acidic compartment of the midgut with pH < 3. In this region, a specialized group of acid-secreting cells similar to mammalian gastric parietal cells has been identified by a unique ultrastructure and by copper-metallothionein fluorescence. However, the homeostatic mechanism maintaining the acid-secreting "copper cells" of the adult midgut has not been examined. Here, we combine cell lineage tracing and genetic analysis to investigate the mechanism by which the gastric epithelium is maintained. Our investigation shows that a molecularly identifiable population of multipotent, self-renewing gastric stem cells (GSSCs) produces the acid-secreting copper cells, interstitial cells, and enteroendocrine cells of the stomach. Our assays demonstrate that GSSCs are largely quiescent but can be induced to regenerate the gastric epithelium in response to environmental challenge. Finally, genetic analysis reveals that adult GSSC maintenance depends on Wnt signaling. Characterization of the GSSC lineage in Drosophila, with striking similarities to mammals, will advance the study of both homeostatic and pathogenic processes in the stomach.

Glioma stem cells targeted by oncolytic virus carrying endostatin-angiostatin fusion gene and the expression of its exogenous gene in vitro.

PubMed

Zhu, Guidong; Su, Wei; Jin, Guishan; Xu, Fujian; Hao, Shuyu; Guan, Fangxia; Jia, William; Liu, Fusheng

2011-05-16

The development of the cancer stem cell (CSCs) niche theory has provided a new target for the treatment of gliomas. Gene therapy using oncolytic viral vectors has shown great potential for the therapeutic targeting of CSCs. To explore whether a viral vector carrying an exogenous Endo-Angio fusion gene (VAE) can infect and kill glioma stem cells (GSCs), as well as inhibit their vascular niche in vitro, we have collected surgical specimens of human high-grade glioma (world health organization, WHO Classes III-VI) from which we isolated and cultured GSCs under conditions originally designed for the selective expansion of neural stem cells. Our results demonstrate the following: (1) Four lines of GSCs (isolated from 20 surgical specimens) could grow in suspension, were multipotent, had the ability to self-renew and expressed the neural stem cell markers, CD133 and nestin. (2) VAE could infect GSCs and significantly inhibit their viability. (3) The Endo-Angio fusion gene was expressed in GSCs 48 h after VAE infection and could inhibit the proliferation of human brain microvascular endothelial cells (HBMEC). (4) Residual viable cells lose the ability of self-renewal and adherent differentiation. In conclusion, VAE can significantly inhibit the activity of GSCs in vitro and the expression of exogenous Endo-Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novel virus-gene therapy strategy for glioma. Copyright © 2011 Elsevier B.V. All rights reserved.

Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT

PubMed Central

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle. PMID:25602959

Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

PubMed

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4(+) cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4(+) cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4(+) cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4(+) cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4(-) cells. Moreover, blastocysts derived from SSEA-4(+) cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4(-) derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4(+) cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

Prenatal alcohol exposure increases the susceptibility to develop aggressive prolactinomas in the pituitary gland.

PubMed

Jabbar, Shaima; Reuhl, Kenneth; Sarkar, Dipak K

2018-05-16

Excess alcohol use is known to promote development of aggressive tumors in various tissues in human patients, but the cause of alcohol promotion of tumor aggressiveness is not clearly understood. We used an animals model of fetal alcohol exposure that is known to promote tumor development and determined if alcohol programs the pituitary to acquire aggressive prolactin-secreting tumors. Our results show that pituitaries of fetal alcohol-exposed rats produced increased levels of intra-pituitary aromatase protein and plasma estrogen, enhanced pituitary tissue growth, and upon estrogen challenge developed prolactin-secreting tumors (prolactinomas) that were hemorrhagic and often penetrated into the surrounding tissue. Pituitary tumors of fetal alcohol-exposed rats produced higher levels of hemorrhage-associated genes and proteins and multipotency genes and proteins. Cells of pituitary tumor of fetal alcohol exposed rat grew into tumor spheres in ultra-low attachment plate, expressed multipotency genes, formed an increased number of colonies, showed enhanced cell migration, and induced solid tumors following inoculation in immunodeficient mice. These data suggest that fetal alcohol exposure programs the pituitary to develop aggressive prolactinoma after estrogen treatment possibly due to increase in stem cell niche within the tumor microenvironment.

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

PubMed Central

Hosseinzadeh Shirzeily, Maryam; Pasbakhsh, Parichehr; Amidi, Fardin; Mehrannia, Kobra; Sobhani, Aligholi

2013-01-01

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs. This article extracted from M.Sc. thesis. (Maryam Hosseinzadeh Shirzeily) PMID:24639722

Endogenous GFAP-Positive Neural Stem/Progenitor Cells in the Postnatal Mouse Cortex Are Activated following Traumatic Brain Injury

PubMed Central

Ahmed, Aminul I.; Shtaya, Anan B.; Zaben, Malik J.; Owens, Emma V.; Kiecker, Clemens

2012-01-01

Abstract Interest in promoting regeneration of the injured nervous system has recently turned toward the use of endogenous stem cells. Elucidating cues involved in driving these precursor cells out of quiescence following injury, and the signals that drive them toward neuronal and glial lineages, will help to harness these cells for repair. Using a biomechanically validated in vitro organotypic stretch injury model, cortico-hippocampal slices from postnatal mice were cultured and a stretch injury equivalent to a severe traumatic brain injury (TBI) applied. In uninjured cortex, proliferative potential under in vitro conditions is virtually absent in older slices (equivalent postnatal day 15 compared to 8). However, following a severe stretch injury, this potential is restored in injured outer cortex. Using slices from mice expressing a fluorescent reporter on the human glial fibrillary acidic protein (GFAP) promoter, we show that GFAP+ cells account for the majority of proliferating neurospheres formed, and that these cells are likely to arise from the cortical parenchyma and not from the subventricular zone. Moreover, we provide evidence for a correlation between upregulation of sonic hedgehog signaling, a pathway known to regulate stem cell proliferation, and this restoration of regenerative potential following TBI. Our results indicate that a source of quiescent endogenous stem cells residing in the cortex and subcortical tissue proliferate in vitro following TBI. Moreover, these proliferating cells are multipotent and are derived mostly from GFAP-expressing cells. This raises the possibility of using this endogenous source of stem cells for repair following TBI. PMID:21895532

The Role of Mesenchymal Stem Cells in the Regenerative Wound Healing Phenotype.

PubMed

Balaji, Swathi; Keswani, Sundeep G; Crombleholme, Timothy M

2012-08-01

Mesenchymal stem cells (MSCs) are key to regenerative wound healing. MSCs have spatial memory and respond to local environment. MSCs orchestrate wound repair by: (1) structural repair via cellular differentiation; (2) immune-modulation; (3) secretion of growth factors that drive neovascularization and re-epithelialization; and (4) mobilization of resident stem cells. Autologous bone-marrow-derived cells and MSCs demonstrate improved healing and tissue-integrity in animal models and clinical trials. However, the effects are variable and the mechanisms of MSC-mediated wound healing are not fully understood. The mammalian MSC niche and signaling sequences and factors affecting their homing, differentiation, viability, and safety need to be characterized to get full benefits of MSC cellular therapy. MSCs can be isolated from bone-marrow, and less-invasive tissues such as adipose, gingiva, muscle, and umbilical cord, with similar functional effects. However, isolation, culture conditions, and markers used to identify and trace the lineage of these MSCs have not been standardized, which is crucial to determine the extent to which MSCs act as multipotent stem cells or sources of secreted factors in wounds. In chronic nonhealing wounds, where efficacy of conventional therapies is unsatisfactory, autotransplantation of MSCs could accelerate wound healing, promote regeneration and restoration of tissue integrity, and reduce recurrence of wounds at characteristically predisposed sites. Regenerative medicine and novel wound therapies using autologous stem cells holds great promise for clinical management of difficult wounds. The ideal candidate stem cells can be used to repopulate the wound bed to mediate appropriate epidermal and dermal regeneration and promote efficient wound repair, while modulating the immune system to prevent infection.

Time related variations in stem cell harvesting of umbilical cord blood

NASA Astrophysics Data System (ADS)

Mazzoccoli, Gianluigi; Miscio, Giuseppe; Fontana, Andrea; Copetti, Massimiliano; Francavilla, Massimo; Bosi, Alberto; Perfetto, Federico; Valoriani, Alice; de Cata, Angelo; Santodirocco, Michele; Totaro, Angela; Rubino, Rosa; di Mauro, Lazzaro; Tarquini, Roberto

2016-02-01

Umbilical cord blood (UCB) contains hematopoietic stem cells and multipotent mesenchymal cells useful for treatment in malignant/nonmalignant hematologic-immunologic diseases and regenerative medicine. Transplantation outcome is correlated with cord blood volume (CBV), number of total nucleated cells (TNC), CD34+ progenitor cells and colony forming units in UCB donations. Several studies have addressed the role of maternal/neonatal factors associated with the hematopoietic reconstruction potential of UCB, including: gestational age, maternal parity, newborn sex and birth weight, placental weight, labor duration and mode of delivery. Few data exist regarding as to how time influences UCB collection and banking patterns. We retrospectively analyzed 17.936 cord blood donations collected from 1999 to 2011 from Tuscany and Apulia Cord Blood Banks. Results from generalized multivariable linear mixed models showed that CBV, TNC and CD34+ cell were associated with known obstetric and neonatal parameters and showed rhythmic patterns in different time domains and frequency ranges. The present findings confirm that volume, total nucleated cells and stem cells of the UCB donations are hallmarked by rhythmic patterns in different time domains and frequency ranges and suggest that temporal rhythms in addition to known obstetric and neonatal parameters influence CBV, TNC and CD34+ cell content in UBC units.

Expression of Pluripotency Markers in Nonpluripotent Human Neural Stem and Progenitor Cells.

PubMed

Vincent, Per Henrik; Benedikz, Eirikur; Uhlén, Per; Hovatta, Outi; Sundström, Erik

2017-06-15

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface.

PubMed

Cha, Kyoung Je; Kong, Sun-Young; Lee, Ji Soo; Kim, Hyung Woo; Shin, Jae-Yeon; La, Moonwoo; Han, Byung Woo; Kim, Dong Sung; Kim, Hyun-Jung

2017-10-12

Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

The CD11a and Endothelial Protein C Receptor Marker Combination Simplifies and Improves the Purification of Mouse Hematopoietic Stem Cells

PubMed Central

Karimzadeh, Alborz; Scarfone, Vanessa M.; Varady, Erika; Chao, Connie; Grathwohl, Karin; Fathman, John W.; Fruman, David A.; Serwold, Thomas

2018-01-01

Abstract Hematopoietic stem cells (HSCs) are the self‐renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two‐color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. stem cells translational medicine 2018;7:468–476 PMID:29543389

Changing the Properties of Multipotent Mesenchymal Stromal Cells by IFNγ Administration.

PubMed

Petinati, N A; Kapranov, N M; Bigil'deev, A E; Popova, M D; Davydova, Yu O; Gal'tseva, I V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

2017-06-01

We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

PubMed

Bourkoula, Evgenia; Mangoni, Damiano; Ius, Tamara; Pucer, Anja; Isola, Miriam; Musiello, Daniela; Marzinotto, Stefania; Toffoletto, Barbara; Sorrentino, Marisa; Palma, Anita; Caponnetto, Federica; Gregoraci, Giorgia; Vindigni, Marco; Pizzolitto, Stefano; Falconieri, Giovanni; De Maglio, Giovanna; Pecile, Vanna; Ruaro, Maria Elisabetta; Gri, Giorgia; Parisse, Pietro; Casalis, Loredana; Scoles, Giacinto; Skrap, Miran; Beltrami, Carlo Alberto; Beltrami, Antonio Paolo; Cesselli, Daniela

2014-05-01

Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma. © 2013 AlphaMed Press.

Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

PubMed

Khorrami, Samaneh; Zavaran Hosseini, Ahmad; Mowla, Seyed Javad; Malekzadeh, Reza

2015-10-01

Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation.

PubMed

Chu, Ling; Jiang, Yuehua; Hao, Hong; Xia, Yong; Xu, Jian; Liu, Zehao; Verfaillie, Catherine M; Zweier, Jay L; Liu, Zhenguo

2008-09-04

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.