Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

PubMed Central

Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

2016-01-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

An albumin nanocomplex-based endosomal pH-activatable on/off probe system.

PubMed

Lee, Changkyu; Lee, Seunghyun; Thao, Le Quang; Hwang, Ha Shin; Kim, Jong Oh; Lee, Eun Seong; Oh, Kyung Taek; Shin, Beom Soo; Choi, Han-Gon; Youn, Yu Seok

2016-08-01

Albumin has gained considerable interest as a material for fabricating nanoparticulate systems due to its biomedical advantages, such as biocompatibility and chemical functionality. Here, we report a new pH-sensitive albumin nanocomplex prototype with a zinc-imidazole coordination bond. Albumin was conjugated with 1-(3-aminopropyl)imidazole and mPEG10kDa-NHS, and the resulting albumin conjugate (PBI) was then modified with either Cy5.5 or BHQ-3. The newly formed albumin nanocomplex (C/BQ-PBI Zn NCs: ∼116nm) system was facilely self-assembled around pH 7.4 in the presence of Zn(2+), but it quickly disassembled in an acidic environment (∼pH 5.0). Based on this pH-sensitivity, C/BQ-PBI Zn NCs emitted strong near-infrared fluorescence and released Zn(2+), turning "off" at pH ∼7.4 (e.g., plasma) and "on" at pH ∼5.0 (e.g., endo/lysosomes in tumor cells) on account of fluorescence resonance energy transfer. C/BQ-PBI Zn NCs displayed significant cytotoxicity due to an increase in cellular Zn(2+) in response to endosomal pH (∼5.0) in breast cancer MCF-7 cells and lung adenocarcinoma A549 cells. Particularly, confocal laser scanning microscopic images showed a strong fluorescence signal caused by the disassembly of C/BQ-PBI Zn NCs in the endosomal region of MCF-7 cells. Based on these results, we believe that this albumin nanocomplex is an attractive biocompatible tumor targeting probe carrier for the theranostic purpose. Copyright © 2016 Elsevier B.V. All rights reserved.

Anakoinosis: Communicative Reprogramming of Tumor Systems - for Rescuing from Chemorefractory Neoplasia.

PubMed

Hart, Christina; Vogelhuber, Martin; Wolff, Daniel; Klobuch, Sebastian; Ghibelli, Lina; Foell, Jürgen; Corbacioglu, Selim; Rehe, Klaus; Haegeman, Guy; Thomas, Simone; Herr, Wolfgang; Reichle, Albrecht

2015-08-01

Disruptive technologies, such as communicative reprogramming (anakoinosis) with cellular therapies in situ for treating refractory metastatic cancer allow patient care to accelerate along a totally new trajectory and highlight what may well become the next sea change in the care of patients with many types of advanced neoplasia. Cellular therapy in situ consisted of repurposed drugs, pioglitazone plus all-trans retinoic acid or dexamethasone or interferon-alpha (dual transcriptional modulation) combined with metronomic low-dose chemotherapy or low-dose 5-azacytidine, plus/minus classic targeted therapy. The novel therapeutic tools for specifically designing communication processes within tumor diseases focus on redirecting (1) rationalizations of cancer hallmarks (constitution of single cancer hallmarks), (2) modular events, (3) the 'metabolism' of evolutionary processes (the sum of therapeutically and intrinsically inducible evolutionary processes) and (4) the holistic communicative context, which determines validity and denotation of tumor promoting communication lines. Published data on cellular therapies in situ (6 histologic tumor types, 144 patients, age 0.9-83 years) in castration-resistant prostate cancer, pretreated renal clear cell carcinoma, chemorefractory acute myelocytic leukemia, multiple myeloma > second-line, chemorefractory Hodgkin lymphoma or multivisceral Langerhans cell histiocytosis, outline the possibility for treating refractory metastatic cancer with the hope that this type of reprogrammed communication will be scalable with minimal toxicity. Accessibility to anakoinosis is a tumor inherent feature, and cellular therapy in situ addresses extrinsic and intrinsic drug resistance, by redirecting convergent organized communication tools, while been supported by quite different pattern of (molecular-)genetic aberrations.

CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models

PubMed Central

Zhu, Yu; Knolhoff, Brett L.; Meyer, Melissa A.; Nywening, Timothy M.; West, Brian L.; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C.; DeNardo, David G.

2014-01-01

Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC), which not only mediate immune suppression but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive anti-tumor T cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. PMID:25082815

Biomimetic RNA-silencing nanocomplexes: overcoming multidrug resistance in cancer cells.

PubMed

Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang; Yang, Min; Chen, Xiaoyuan

2014-02-10

RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by an RNA-induced silencing complex (RISC). Herein, we present a synthetic RISC-mimic nanocomplex, which can actively cleave its target RNA in a sequence-specific manner. With high enzymatic stability and efficient self-delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. These nanocomplexes, which target multidrug resistance, are not only able to bypass the P-glycoprotein (Pgp) transporter, due to their nano-size effect, but also effectively suppress Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp-transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Throughput Fabrication of Nanocomplexes Using 3D-Printed Micromixers.

PubMed

Bohr, Adam; Boetker, Johan; Wang, Yingya; Jensen, Henrik; Rantanen, Jukka; Beck-Broichsitter, Moritz

2017-03-01

3D printing allows a rapid and inexpensive manufacturing of custom made and prototype devices. Micromixers are used for rapid and controlled production of nanoparticles intended for therapeutic delivery. In this study, we demonstrate the fabrication of micromixers using computational design and 3D printing, which enable a continuous and industrial scale production of nanocomplexes formed by electrostatic complexation, using the polymers poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate). Several parameters including polymer concentration, flow rate, and flow ratio were systematically varied and their effect on the properties of nanocomplexes was studied and compared with nanocomplexes prepared by bulk mixing. Particles fabricated using this cost effective device were equally small and homogenous but more consistent and controllable in size compared with those prepared manually via bulk mixing. Moreover, each micromixer could process more than 2 liters per hour with unaffected performance and the setup could easily be scaled-up by aligning several micromixers in parallel. This demonstrates that 3D printing can be used to prepare disposable high-throughput micromixers for production of therapeutic nanoparticles. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Reprogramming tumor-infiltrating dendritic cells for CD103+ CD8+ mucosal T-cell differentiation and breast cancer rejection.

PubMed

Wu, Te-Chia; Xu, Kangling; Banchereau, Romain; Marches, Florentina; Yu, Chun I; Martinek, Jan; Anguiano, Esperanza; Pedroza-Gonzalez, Alexander; Snipes, G Jackson; O'Shaughnessy, Joyce; Nishimura, Stephen; Liu, Yong-Jun; Pascual, Virginia; Banchereau, Jacques; Oh, Sangkon; Palucka, Karolina

2014-05-01

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+ CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+ CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection.

Reprogramming tumor-infiltrating dendritic cells for CD103+CD8+ mucosal T cell differentiation and breast cancer rejection

PubMed Central

Wu, Te-Chia; Xu, Kangling; Banchereau, Romain; Marches, Florentina; Yu, Chun I; Martinek, Jan; Anguiano, Esperanza; Pedroza-Gonzalez, Alexander; Snipes, G. Jackson; O’Shaughnessy, Joyce; Nishimura, Stephen; Liu, Yong-Jun; Pascual, Virginia; Banchereau, Jacques; Oh, Sangkon; Palucka, Karolina

2014-01-01

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory T helper 2 (iTh2) cells and protumor inflammation. Here we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells, and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DC via the ligation of dectin-1, enabling the DC to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL12p70, and to favor the generation of T helper 1 (Th1) cells. DC activated via dectin-1, but not those activated with TLR-7/8 ligand or poly IC, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+CD8+ mucosal T cells accumulate in the tumors thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+CD8+ mucosal T cells elicited by reprogrammed DC can reject established cancer. Thus, reprogramming tumor-infiltrating DC represents a new strategy for cancer rejection. PMID:24795361

Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery.

PubMed

Lee, Dongwon; Zhang, Weidong; Shirley, Shawna A; Kong, Xiaoyuan; Hellermann, Gary R; Lockey, Richard F; Mohapatra, Shyam S

2007-01-01

Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo.

PubMed

Bardhan, Rizia; Chen, Wenxue; Bartels, Marc; Perez-Torres, Carlos; Botero, Maria F; McAninch, Robin Ward; Contreras, Alejandro; Schiff, Rachel; Pautler, Robia G; Halas, Naomi J; Joshi, Amit

2010-12-08

Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.

Tracking of Multimodal Therapeutic Nanocomplexes Targeting Breast Cancer in Vivo

PubMed Central

Bardhan, Rizia; Chen, Wenxue; Bartels, Marc; Perez-Torres, Carlos; Botero, Maria F.; McAninch, Robin Ward; Contreras, Alejandro; Schiff, Rachel; Pautler, Robia G.; Halas, Naomi J.; Joshi, Amit

2014-01-01

Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions. PMID:21090693

Safety and Efficacy in Advanced Solid Tumors of a Targeted Nanocomplex Carrying the p53 Gene Used in Combination with Docetaxel: A Phase 1b Study

PubMed Central

Pirollo, Kathleen F; Nemunaitis, John; Leung, Po Ki; Nunan, Robert; Adams, Jana; Chang, Esther H

2016-01-01

Loss of p53 suppressor function, through mutations or inactivation of the p53 pathway, occurs in most human cancers. SGT-53 is a liposomal nanocomplex designed for systemic, tumor-targeting delivery of the wt p53 gene. In this nanodelivery system, an anti-transferrin receptor single-chain antibody fragment serves as the targeting moiety. In an initial phase 1 trial in patients with advanced solid tumors, SGT-53 demonstrated tumor-specific targeting, was shown to be well tolerated, and was associated with an antitumor effect in several patients. Our preclinical studies have also demonstrated enhanced antitumor activity with the combination of SGT-53 and docetaxel. Thus, this dose-escalation trial was undertaken to assess the combination of SGT-53 and docetaxel for safety and potential efficacy in 14 advanced cancer patients. Results reveal that the combination of SGT-53 (maximum dose, 3.6 mg DNA/infusion) and docetaxel (75 mg/m2/infusion) was well tolerated. Moreover, clinical activity involving 12 evaluable patients was observed. Three of these patients achieved RECIST-verified partial responses with tumor reductions of −47%, −51%, and −79%. Two others had stable disease with significant shrinkage (−25% and −16%). These results support phase 2 testing of SGT-53 in combination with docetaxel. PMID:27357628

Hyaluronic acid-green tea catechin micellar nanocomplexes: Fail-safe cisplatin nanomedicine for the treatment of ovarian cancer without off-target toxicity.

PubMed

Bae, Ki Hyun; Tan, Susi; Yamashita, Atsushi; Ang, Wei Xia; Gao, Shu Jun; Wang, Shu; Chung, Joo Eun; Kurisawa, Motoichi

2017-12-01

The green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant to enhance the antitumor efficacy of cisplatin while mitigating its harmful side effects. Herein we report the development of a fail-safe cisplatin nanomedicine constructed with hyaluronic acid-EGCG conjugate for ovarian cancer therapy. A simple mixing of this conjugate and cisplatin induces spontaneous self-assembly of micellar nanocomplexes having a spherical core-shell structure. The surface-exposed hyaluronic acid enables efficient delivery of cisplatin into CD44-overexpressing cancer cells via receptor-mediated endocytosis whereas the internally packed EGCG moieties offer an environment favorable for the encapsulation of cisplatin. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity originating from cisplatin-evoked oxidative stress. Pharmacokinetic and biodistribution studies reveal the prolonged blood circulation and preferential tumor accumulation of intravenously administered nanocomplexes. Moreover, the nanocomplexes exhibit superior antitumor efficacy over free cisplatin while displaying no toxicity in both a subcutaneous xenograft model and peritoneal metastatic model of human ovarian cancer. Our findings demonstrate proof of concept for the feasibility of green tea catechin-based micellar nanocomplexes as a safe and effective cisplatin nanomedicine for ovarian cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reprogramming multipotent tumor cells with the embryonic neural crest microenvironment

PubMed Central

Kasemeier-Kulesa, Jennifer C.; Teddy, Jessica M.; Postovit, Lynne-Marie; Seftor, Elisabeth A.; Seftor, Richard E.B.; Hendrix, Mary J.C.; Kulesa, Paul M.

2008-01-01

The embryonic microenvironment is an important source of signals that program multipotent cells to adopt a particular fate and migratory path, yet its potential to reprogram and restrict multipotent tumor cell fate and invasion is unrealized. Aggressive tumor cells share many characteristics with multipotent, invasive embryonic progenitors, contributing to the paradigm of tumour cell plasticity. In the vertebrate embryo, multiple cell types originate from a highly invasive cell population called the neural crest. The neural crest and the embryonic microenvironments they migrate through represent an excellent model system to study cell diversification during embryogenesis and phenotype determination. Recent exciting studies of tumor cells transplanted into various embryo models, including the neural crest rich chick microenvironment, have revealed the potential to control and revert the metastatic phenotype, suggesting further work may help to identify new targets for therapeutic intervention derived from a convergence of tumorigenic and embryonic signals. In this mini-review, we summarize markers that are common to the neural crest and highly aggressive human melanoma cells. We highlight advances in our understanding of tumor cell behaviors and plasticity studied within the chick neural crest rich microenvironment. In so doing, we honor the tremendous contributions of Professor Elizabeth D. Hay towards this important interface of developmental and cancer biology. PMID:18629870

Hydrothermal preparation and physicochemical studies of new copper nano-complexes for antitumor application

NASA Astrophysics Data System (ADS)

Saif, M.; El-Shafiy, Hoda F.; Mashaly, Mahmoud M.; Eid, Mohamed F.; Nabeel, A. I.; Fouad, R.

2018-03-01

Two novel nano-complexes [(Cu)2(L) (NO3)2(OH2)] (CuH) and [Cu(HL) (OH2)2(NO3)] (CuCTH)were synthesized by hydrothermal method at 200 °C for 48 h in absence and presence of surfactant (CTAB), respectively. Introducing surfactant (CTAB) leads to changing stoichiometric metal/ligand ratio from binuclear (CuH) to mononuclear (CuCTH) nano-complexes. CuH shows irregular nano-flake shape while CuCTH have separately uniform nano-spherical morphology. Thermal analysis revealed that CuCTH is thermally stable in comparison with CuH Nano-complex. CuCTH absorption peak shifted to shorter wavelength (blue shift) and sharpness of the peak also decreased in presence of CTAB. The role of CTAB in the crystal growth is discussed. CuH and CuCTH nano-complexes were tested for their in vitro cytotoxicity against Ehrlich Ascites Carcinoma cell line (E.A.C.). Both nano-complexes effectively inhibited E.A.C. growth with IC50value of 37 and 25 μM for CuH and CuCTH, respectively. The high antitumor activity of CuCTH was attributed to several factors such as spherical morphology, smaller size, chemical structure, and geometry. The LD50 for high cytotoxic CuCTH nano-complex on mice was found to be 100 mg/kg with strong abscess in abdomen side effect. To overcome this side effect, different molar ratio of CuCTH and previously prepared ZnNano-complexes were tested for their in vitrocytotoxicity and in vivo toxicity. Obtained results show that the 2:8 M ratio between CuCTH and Zn nano-complexes gives very low toxicity without any side effects. Also, geometric optimization and conformational analysis were performed using semi-empirical PM3 method. Energy gap (ΔE), dipole moment, and structure activity relationship were performed and discussed.

Rational assembly of a biointerfaced core@shell nanocomplex towards selective and highly efficient synergistic photothermal/photodynamic therapy

NASA Astrophysics Data System (ADS)

Qin, Chenchen; Fei, Jinbo; Wang, Anhe; Yang, Yang; Li, Junbai

2015-11-01

To optimize synergistic cancer therapy, we rationally assemble an inorganic-organic nanocomplex using a folate-modified lipid bilayer spread on photosensitizer-entrapped mesoporous silica nanoparticle (MSN) coated gold nanorods (AuNRs). In this hybrid bioconjugate, the large specific surface area and pore size of AuNR@MSN guarantee a high loading capacity of small photosensitive molecules. The modification with selective mixed liposomes on the surface of AuNR@MSN enables faster cellular internalization and enhancement of endocytosis. Under one-time NIR two-photon illumination, AuNR-mediated hyperthermia can kill cancer cells directly. Meanwhile, the loaded photosensitizer, hypocrellin B, generates two kinds of reactive oxygen species (ROS) to induce cell apoptosis. Remarkably, hyperthermia can improve the yield of ROS. After intravenous injection of this bioconjugate into female BALB/c nude mice followed by laser irradiation (808 nm, 1.3 W cm-2, 6 min), the tumor growth is suppressed completely. The tumors are not recurrent within the observation time (19 days), and the normal or main organs are not obviously pathological. Thus, such a simplified and selective cancer treatment, combining photothermal and photodynamic therapy in a synergistic manner, provides outstanding efficiency in vivo. This nanocomplex with well-defined core@shell nanostructures integrated with a two-photon technique holds great promise to improve cancer phototherapy with a high efficiency in the clinic.To optimize synergistic cancer therapy, we rationally assemble an inorganic-organic nanocomplex using a folate-modified lipid bilayer spread on photosensitizer-entrapped mesoporous silica nanoparticle (MSN) coated gold nanorods (AuNRs). In this hybrid bioconjugate, the large specific surface area and pore size of AuNR@MSN guarantee a high loading capacity of small photosensitive molecules. The modification with selective mixed liposomes on the surface of AuNR@MSN enables faster cellular

Encapsulation of temozolomide in a tumor-targeting nanocomplex enhances anti-cancer efficacy and reduces toxicity in a mouse model of glioblastoma.

PubMed

Kim, Sang-Soo; Rait, Antonina; Kim, Eric; DeMarco, James; Pirollo, Kathleen F; Chang, Esther H

2015-12-01

Although temozolomide (TMZ) is the current first-line chemotherapy for glioblastoma multiforme (GBM), most patients either do not respond or ultimately fail TMZ treatment. Both intrinsic tumor resistance and limited access of TMZ to brain tumors as a result of the blood-brain barrier (BBB) contribute to poor response and ultimately to poor prognosis for GBM patients. We have developed a "dual-targeting" nanomedicine that both actively crosses the BBB and actively targets cancer cells once in the brain parenchyma. This nanomedicine (termed scL-TMZ) is sized ~40 nm and comprised of a cationic liposome (DOTAP:DOPE) encapsulating TMZ. The surface of liposome is decorated with anti-transferrin receptor single-chain antibody fragments to facilitate the crossing of the BBB by the scL-TMZ in addition to targeting GBM in the brain. This novel formulation was found to be markedly more effective than standard TMZ in both TMZ-resistant and TMZ-sensitive GBM. Encapsulation of TMZ also markedly enhanced its efficacy in killing a variety of non-GBM tumor cells. The scL-TMZ nanocomplex was shown to target cancer stem cells, which have been linked to both drug resistance and recurrence in GBM. Most significantly, systemically administered scL-TMZ significantly prolonged survival in mice bearing intracranial GBM tumors. The improved efficacy of scL-TMZ compared to standard TMZ was accompanied by reduced toxicity, so we conclude that the scL-TMZ nanomedicine holds great promise as a more effective therapy for GBM and other tumor types. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Targeting tumor-associated macrophages by anti-tumor Chinese materia medica.

PubMed

Pu, Wei-Ling; Sun, Li-Kang; Gao, Xiu-Mei; Rüegg, Curzio; Cuendet, Muriel; Hottiger, Micheal O; Zhou, Kun; Miao, Lin; Zhang, Yun-Sha; Gebauer, Margaret

2017-10-01

Tumor-associated macrophages (TAMs) play a key role in all stages of tumorigenesis and tumor progression. TAMs secrete different kinds of cytokines, chemokines, and enzymes to affect the progression, metastasis, and resistance to therapy depending on their state of reprogramming. Therapeutic benefit in targeting TAMs suggests that macrophages are attractive targets for cancer treatment. Chinese materia medica (CMM) is an important approach for treating cancer in China and in the Asian region. According to the theory of Chinese medicine (CM) and its practice, some prescriptions of CM regulate the body's internal environment possibly including the remodeling the tumor microenvironment (TME). Here we briefly summarize the pivotal effects of TAMs in shaping the TME and promoting tumorigenesis, invasion, metastasis and immunosuppression. Furthermore, we illustrate the effects and mechanisms of CMM targeting TAMs in antitumor therapy. Finally, we reveal the CMM's dual-regulatory and multi-targeting functions on regulating TAMs, and hopefully, provide the theoretical basis for CMM clinical practice related to cancer therapy.

Cellular reprogramming in skin cancer.

PubMed

Song, Ihn Young; Balmain, Allan

2015-06-01

Early primitive stem cells have long been viewed as the cancer cells of origin (tumor initiating target cells) due to their intrinsic features of self-renewal and longevity. However, emerging evidence suggests a surprising capacity for normal committed cells to function as reserve stem cells upon reprogramming as a consequence of tissue damage resulting in inflammation and wound healing. This results in an alternative concept positing that tumors may originate from differentiated cells that can re-acquire stem cell properties due to genetic or epigenetic reprogramming. It is likely that both models are correct, and that a continuum of potential cells of origin exists, ranging from early primitive stem cells to committed progenitor or even terminally differentiated cells. A combination of the nature of the target cell and the specific types of gene mutations introduced determine tumor cell lineage, as well as potential for malignant conversion. Evidence from mouse skin models of carcinogenesis suggests that initiated cells at different stages within a stem cell hierarchy have varying degrees of requirement for reprogramming (e.g. inflammation stimuli), depending on their degree of differentiation. This article will present evidence in favor of these concepts that has been developed from studies of several mouse models of skin carcinogenesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combining QD-FRET and microfluidics to monitor DNA nanocomplex self-assembly in real-time.

PubMed

Ho, Yi-Ping; Chen, Hunter H; Leong, Kam W; Wang, Tza-Huei

2009-08-26

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be

Targeting the Metabolic Reprogramming That Controls Epithelial-to-Mesenchymal Transition in Aggressive Tumors

PubMed Central

Morandi, Andrea; Taddei, Maria Letizia; Chiarugi, Paola; Giannoni, Elisa

2017-01-01

The epithelial-to-mesenchymal transition (EMT) process allows the trans-differentiation of a cell with epithelial features into a cell with mesenchymal characteristics. This process has been reported to be a key priming event for tumor development and therefore EMT activation is now considered an established trait of malignancy. The transcriptional and epigenetic reprogramming that governs EMT has been extensively characterized and reviewed in the last decade. However, increasing evidence demonstrates a correlation between metabolic reprogramming and EMT execution. The aim of the current review is to gather the recent findings that illustrate this correlation to help deciphering whether metabolic changes are causative or just a bystander effect of EMT activation. The review is divided accordingly to the catabolic and anabolic pathways that characterize carbohydrate, aminoacid, and lipid metabolism. Moreover, at the end of each part, we have discussed a series of potential metabolic targets involved in EMT promotion and execution for which drugs are either available or that could be further investigated for therapeutic intervention. PMID:28352611

Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer

NASA Astrophysics Data System (ADS)

Grigsby, Christopher L.; Ho, Yi-Ping; Lin, Chao; Engbersen, Johan F. J.; Leong, Kam W.

2013-11-01

As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Reductive nanocomplex encapsulation of cRGD-siRNA conjugates for enhanced targeting to cancer cells

PubMed Central

Zhang, Yanfen; Yang, Xiantao; Ma, Yuan; Guan, Zhu; Wu, Yun; Zhang, Lihe; Yang, Zhenjun

2017-01-01

In this study, through covalent conjugation and lipid material entrapment, a combined modification strategy was established for effective delivery of small interfering RNA (siRNA). Single strands of siRNA targeting to BRAFV600E gene (siMB3) conjugated with cRGD peptide at 3′-terminus or 5′-terminus via cleavable disulfide bond was synthesized and then annealed with corresponding strands to obtain single and bis-cRGD-siRNA conjugates. A cationic lipid material (CLD) developed by our laboratory was mixed with the conjugates to generate nanocomplexes; their uniformity and electrical property were revealed by particle size and zeta potential measurement. Compared with CLD/siBraf, CLD/cRGD-siBraf achieved higher cell uptake and more excellent tumor-targeting ability, especially 21 (sense-5′/antisense-3″-cRGD-congjugate) nanocomplex. Moreover, they can regulate multiple pathways to varying degree and reduce acidification of endosome. Compared with the gene silencing of different conjugates, single or bis-cRGD-conjugated siRNA showed little differences except 22 (5/5) which cRGD was conjugated at 5′-terminus of antisense strand and sense strand. However bis-cRGD conjugate 21 nanocomplex exhibited better specific target gene silencing at multiple time points. Furthermore, the serum stabilities of the bis-cRGD conjugates were higher than those of the single-cRGD conjugates. In conclusion, all these data suggested that CLD/bis-conjugates, especially CLD/21, can be an effective system for delivery of siRNA to target BRAFV600E gene for therapy of melanoma. PMID:29042774

Imprints and DPPA3 are bypassed during pluripotency- and differentiation-coupled methylation reprogramming in testicular germ cell tumors

PubMed Central

Killian, J. Keith; Dorssers, Lambert C.J.; Trabert, Britton; Gillis, Ad J.M.; Cook, Michael B.; Wang, Yonghong; Waterfall, Joshua J.; Stevenson, Holly; Smith, William I.; Noyes, Natalia; Retnakumar, Parvathy; Stoop, J. Hans; Oosterhuis, J. Wolter; Meltzer, Paul S.; McGlynn, Katherine A.; Looijenga, Leendert H.J.

2016-01-01

Testicular germ cell tumors (TGCTs) share germline ancestry but diverge phenotypically and clinically as seminoma (SE) and nonseminoma (NSE), the latter including the pluripotent embryonal carcinoma (EC) and its differentiated derivatives, teratoma (TE), yolk sac tumor (YST), and choriocarcinoma. Epigenomes from TGCTs may illuminate reprogramming in both normal development and testicular tumorigenesis. Herein we investigate pure-histological forms of 130 TGCTs for conserved and subtype-specific DNA methylation, including analysis of relatedness to pluripotent stem cell (ESC, iPSC), primordial germ cell (PGC), and differentiated somatic references. Most generally, TGCTs conserve PGC-lineage erasure of maternal and paternal genomic imprints and DPPA3 (also known as STELLA); however, like ESCs, TGCTs show focal recurrent imprinted domain hypermethylation. In this setting of shared physiologic erasure, NSEs harbor a malignancy-associated hypermethylation core, akin to that of a diverse cancer compendium. Beyond these concordances, we found subtype epigenetic homology with pluripotent versus differentiated states. ECs demonstrate a striking convergence of both CpG and CpH (non-CpG) methylation with pluripotent states; the pluripotential methyl-CpH signature crosses species boundaries and is distinct from neuronal methyl-CpH. EC differentiation to TE and YST entails reprogramming toward the somatic state, with loss of methyl-CpH but de novo methylation of pluripotency loci such as NANOG. Extreme methyl-depletion among SE reflects the PGC methylation nadir. Adjacent to TGCTs, benign testis methylation profiles are determined by spermatogenetic proficiency measured by Johnsen score. In sum, TGCTs share collective entrapment in a PGC-like state of genomic-imprint and DPPA3 erasure, recurrent hypermethylation of cancer-associated targets, and subtype-dependent pluripotent, germline, or somatic methylation. PMID:27803193

Tumoral stem cell reprogramming as a driver of cancer: Theory, biological models, implications in cancer therapy.

PubMed

Vicente-Dueñas, Carolina; Hauer, Julia; Ruiz-Roca, Lucía; Ingenhag, Deborah; Rodríguez-Meira, Alba; Auer, Franziska; Borkhardt, Arndt; Sánchez-García, Isidro

2015-06-01

Cancer is a clonal malignant disease originated in a single cell and characterized by the accumulation of partially differentiated cells that are phenotypically reminiscent of normal stages of differentiation. According to current models, therapeutic strategies that block oncogene activity are likely to selectively target tumor cells. However, recent evidences have revealed that cancer stem cells could arise through a tumor stem cell reprogramming mechanism, suggesting that genetic lesions that initiate the cancer process might be dispensable for tumor progression and maintenance. This review addresses the impact of these results toward a better understanding of cancer development and proposes new approaches to treat cancer in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exploration of hydrophobic modification degree of chitosan-based nanocomplexes on the oral delivery of enoxaparin.

PubMed

Wang, Linlin; Li, Liang; Sun, Yujiao; Tian, Ye; Li, Ying; Li, Conghao; Junyaprasert, Varaporn B; Mao, Shirui

2013-11-20

The objective of this paper is to elucidate the influence of lipophilic modification degree of chitosan on the peroral absorption of enoxaparin. A series of novel chitosan grafted glyceryl monostearate (GM) copolymers with different GM substitution degree were synthesized and the successful synthesis was confirmed by (1)H NMR, FTIR and X-ray diffraction. Enoxaparin loaded nanocomplexes with different carriers were prepared by self-assembly process. Influence of GM substitution degree and chitosan molecular weight in the copolymer on the properties of the nanocomplexes was investigated. Morphology of the nanocomplexes was observed by atomic force microscopy. Mucoadhesive properties of the nanocomplexes were characterized using mucin particle method. Initially, mucoadhesion of the nanocomplexes increased with the increase of GM substitution degree and it started to decrease when the substitution degree was up to 18.6%. A good linear relationship between GM substitution degree and in vivo absorption of enoxaparin in fasted rats was established in the substitution degree range of 0-11.1%. In agreement with mucoadhesion data, further increasing GM substitution degree to 18.6% caused a decrease in oral absorption. In conclusion, oral bioavailability of enoxaparin can be enhanced by structure modification of the carriers and the bioavailability is hydrophobic modification degree dependent. Copyright © 2013 Elsevier B.V. All rights reserved.

Metabolic reprogramming: a hallmark of viral oncogenesis.

PubMed

Lévy, P; Bartosch, B

2016-08-11

More than 1 in 10 cases of cancer in the world are due to chronic viral infections. Viruses induce oncogenesis by targeting the same pathways known to be responsible for neoplasia in tumor cells, such as control of cell cycle progression, cell migration, proliferation and evasion from cell death and the host's immune defense. In addition, metabolic reprogramming has been identified over a century ago as a requirement for growth of transformed cells. Renewed interest in this topic has emerged recently with the discovery that basically all metabolic changes in tumor cells are finely orchestrated by oncogenes and tumor suppressors. Indeed, cancer cells activate biosynthetic pathways in order to provide them with sufficient levels of energy and building blocks to proliferate. Interestingly, viruses introduce into their host cells similar metabolic adaptations, and importantly, it seems that they depend on these changes for their persistence and amplification. The central carbon metabolism, for example, is not only frequently altered in tumor cells but also modulated by human papillomavirus, hepatitis B and C viruses, Epstein-Barr virus and Kaposi's Sarcoma-associated virus. Moreover, adenoviruses (Ad) and human cytomegalovirus, which are not directly oncogenic but present oncomodulatory properties, also divert cellular metabolism in a tumor cell-like mnner. Thus, metabolic reprogramming appears to be a hallmark of viral infection and provides an interesting therapeutic target, in particular, for oncogenic viruses. Therapeutic targeting of metabolic pathways may not only allow to eliminate or control the viral infection but also to prevent virus-induced carcinogenesis.

Phase-assisted synthesis and DNA unpacking evaluation of biologically inspired metallo nanocomplexes using peptide as unique building block.

PubMed

Raman, N; Sudharsan, S

2011-12-01

The goal of nanomaterials' surface modification using a biomaterial is to preserve the materials' bulk properties while modifying only their surface to possess desired recognition and specificity. Here, we have developed a phase-assisted, modified Brust-Schiffrin methodological synthesis of metallo nanocomplexes anchored by a peptide, N,N'-(1,3-propylene)-bis-hippuricamide. The spectral, thermal and morphological characterizations assure the formation of nanocomplexes. Therapeutic behavior of all the nanocomplexes has been well sighted by evaluating their DNA unpacking skills. In addition, we demonstrate their biological inspiration by targeting few bacterial and fungal strains. The in vitro antimicrobial investigation reports that all the nanocomplexes disrupt microbial cell walls/membranes efficiently and inhibit the growth of microbes. These sorts of nanocomplexes synthesized in large quantities and at low cost, deliver versatile biomedical applications, and can be used to treat various diseases which may often cause high mortality. Copyright © 2011 Elsevier Inc. All rights reserved.

Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication

NASA Astrophysics Data System (ADS)

Liu, Yang; Du, Juanjuan; Yan, Ming; Lau, Mo Yin; Hu, Jay; Han, Hui; Yang, Otto O.; Liang, Sheng; Wei, Wei; Wang, Hui; Li, Jianmin; Zhu, Xinyuan; Shi, Linqi; Chen, Wei; Ji, Cheng; Lu, Yunfeng

2013-03-01

Organisms have sophisticated subcellular compartments containing enzymes that function in tandem. These confined compartments ensure effective chemical transformation and transport of molecules, and the elimination of toxic metabolic wastes. Creating functional enzyme complexes that are confined in a similar way remains challenging. Here we show that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomplexes. These nanocomplexes exhibit improved catalytic efficiency and enhanced stability when compared with free enzymes. Furthermore, the co-localized enzymes display complementary functions, whereby toxic intermediates generated by one enzyme can be promptly eliminated by another enzyme. We show that nanocomplexes containing alcohol oxidase and catalase could reduce blood alcohol levels in intoxicated mice, offering an alternative antidote and prophylactic for alcohol intoxication.

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Microstructure and dielectric properties of silver-barium titanate nanocomplex materials by wet chemical approach

NASA Astrophysics Data System (ADS)

Ueno, Shintaro; Sakamoto, Yasunao; Nakashima, Kouichi; Wada, Satoshi

2014-09-01

To develop ceramic capacitors with a high effective dielectric constant, we attempted to fabricate BaTiO3 (BT) complexes with embedded Ag nanoparticles by wet chemical processes. Ag nanoparticle-adsorbed dendritic BT particles, Ag-BT hybrid particles, were synthesized from the sol-gel-derived precursor gel powders containing Ag, Ba, and Ti by hydrothermal treatment. These particles were pressed with BT fillers and TiO2 precursor nanoparticles into green compacts, and then, the green compacts were chemically converted into the Ag/BT nanocomplex compacts in Ba(OH)2 aqueous solution under the hydrothermal condition at 160 °C. The effective dielectric constant of the resultant Ag/BT nanocomplexes increases with an increase in Ag content. The maximal effective dielectric constant of approximately 900 was recorded for the nanocomplex with the Ag content of 10.7 vol %.

An Assembled Nanocomplex for Improving both Therapeutic Efficiency and Treatment Depth in Photodynamic Therapy.

PubMed

Cao, Hongqian; Wang, Lei; Yang, Yang; Li, Juan; Qi, Yanfei; Li, Yue; Li, Ying; Wang, Hao; Li, Junbai

2018-06-25

Photodynamic therapy (PDT) shows unique selectivity and irreversible destruction toward treated tissues or cells, but still has several problems in clinical practice. One is limited therapeutic efficiency, which is attributed to hypoxia in tumor sites. Another is the limited treatment depth because traditional photosensitizes are excited by short wavelength light (<700 nm). An assembled nano-complex system composed of oxygen donor, two-photon absorption (TPA) species, and photosensitizer (PS) was synthesized to address both problems. The photosensitizer is excited indirectly by two-photon laser through intraparticle FRET mechanism for improving treatment depth. The oxygen donor, hemoglobin, can supply extra oxygen into tumor location through targeting effect for enhanced PDT efficiency. The mechanism and PDT effect were verified through both in vitro and in vivo experiments. The simple system is promising to promote two-photon PDT for clinical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A nanocomplex of Cu(II) with theophylline drug; synthesis, characterization, and anticancer activity against K562 cell line

NASA Astrophysics Data System (ADS)

Sahlabadi, Maryam; Daryanavard, Marzieh; Hadadzadeh, Hassan; Amirghofran, Zahra

2018-03-01

A new mononuclear of copper (II), [Cu(theophylline)2(H2O)3]·2H2O, has been synthesized by reaction of theophylline (1,3-dimethyl-7H-purine-2,6-dione) with copper (II) nitrate in water. Further, its nanocomplex has been prepared through the three different methods including sonication, grinding, and a combination thereof, sonication-grinding. The prepared nanocomplex was characterized using different techniques including FT-IR, UV-Vis, X-ray diffraction (XRD) analysis, and field-emission scanning electron microscopy (FE-SEM). Moreover, the anticancer activity of the precursor complex, nanocomplex, free theophylline ligand, and the starting copper salt (Cu(NO3)2·3H2O) was investigated against the K562 cell line. The results show that the nanocomplex is an effective nano metal-based anticancer agent with IC50 = 11.7 μM.

Metabolic reprogramming in glioblastoma: the influence of cancer metabolism on epigenetics and unanswered questions

PubMed Central

Agnihotri, Sameer; Zadeh, Gelareh

2016-01-01

A defining hallmark of glioblastoma is altered tumor metabolism. The metabolic shift towards aerobic glycolysis with reprogramming of mitochondrial oxidative phosphorylation, regardless of oxygen availability, is a phenomenon known as the Warburg effect. In addition to the Warburg effect, glioblastoma tumor cells also utilize the tricarboxylic acid cycle/oxidative phosphorylation in a different capacity than normal tissue. Altered metabolic enzymes and their metabolites are oncogenic and not simply a product of tumor proliferation. Here we highlight the advantages of why tumor cells, including glioblastoma cells, require metabolic reprogramming and how tumor metabolism can converge on tumor epigenetics and unanswered questions in the field. PMID:26180081

Epigenetic reprogramming in plant sexual reproduction.

PubMed

Kawashima, Tomokazu; Berger, Frédéric

2014-09-01

Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation.

Chitosan-based nanocomplexes for simultaneous loading, burst reduction and controlled release of doxorubicin and 5-fluorouracil.

PubMed

Di Martino, Antonio; Kucharczyk, Pavel; Capakova, Zdenka; Humpolicek, Petr; Sedlarik, Vladimir

2017-09-01

In this work, nanocomplexes based on chitosan grafted by carboxy-modified polylactic acid (SPLA) were prepared with the aim of loading simultaneously two anticancer drugs - doxorubicin and 5-fluorouracil, as well as to control their release, reduce the initial burst and boost cytotoxicity. The SPLA was prepared by a polycondensation reaction, using pentetic acid as the core molecule, and linked to the chitosan backbone through a coupling reaction. Nanocomplexes loaded with both drugs were formulated by the polyelectrolyte complexation method. The structure of the SPLA was characterized by 1 H NMR, while the product CS-SPLA was analyzed by FTIR-ATR to prove the occurrence of the reaction. Results showed that the diameters and ζ-potential of the nanocomplexes fall in the range 120-200nm and 20-37mV, respectively. SEM and TEM analysis confirmed the spherical shape and dimensions of the nanocomplexes. The presence of hydrophobic side chain SPLA did not influence the encapsulation efficiency of the drugs but strongly reduced the initial burst and prolonged release over time compared to unmodified chitosan. MS analysis showed that no degradation or interactions between the drugs and carrier were exhibited after loading or 24h of release had taken place, confirming the protective role of the nanocomplexes. In vitro tests demonstrated an increase in the cytotoxicity of the drugs when loaded in the prepared carriers. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages.

PubMed

Ball, Michael S; Shipman, Emilie P; Kim, Hyunjung; Liby, Karen T; Pioli, Patricia A

2016-01-01

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.

CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

PubMed Central

Ball, Michael S.; Shipman, Emilie P.; Kim, Hyunjung; Liby, Karen T.; Pioli, Patricia A.

2016-01-01

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer. PMID:26918785

A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

NASA Astrophysics Data System (ADS)

Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

2015-05-01

Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au

Reprogramming of human cancer cells to pluripotency for models of cancer progression

PubMed Central

Kim, Jungsun; Zaret, Kenneth S

2015-01-01

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

Reprogramming Enhancers to Drive Metastasis.

PubMed

Mostoslavsky, Raul; Bardeesy, Nabeel

2017-08-24

Acquired molecular changes can promote the spreading of primary tumor cells to distant tissues. In this issue of Cell, Roe et al. show that metastatic progression of pancreatic cancer involves large-scale enhancer reprogramming by Foxa1, which activates transcriptional program specifying early endodermal stem cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Epigenomic Reprogramming of the Developing Reproductive Tract and Disease Susceptibility in Adulthood

PubMed Central

Walker, Cheryl Lyn

2014-01-01

During development, epigenetic programs are “installed” on the genome that direct differentiation and normal tissue and organ function in adulthood. Consequently, development is also a period of susceptibility to reprogramming of the epigenome. Developmental reprogramming occurs when an adverse stimulus or insult interrupts the proper “install” of epigenetic programs during development, reprogramming normal physiological responses in such a way as to promote disease later in life. Some of the best examples of developmental reprogramming involve the reproductive tract, where early life exposures to environmental estrogens can increase susceptibility to benign and malignant tumors in adulthood including leiomyoma (fibroids), endometrial and prostate cancer. Although specific mechanism(s) by which environmental estrogens reprogram the developing epigenome were unknown, both DNA and histone methylation were considered likely targets for epigenetic reprogramming. We have now identified a mechanism by which developmental exposures to environmental estrogens reprogram the epigenome by inducing inappropriate activation of nongenomic estrogen receptor (ER) signaling. Activation of non-genomic ER signaling via the PI3K pathway activates the kinase AKT/PKB in the developing reproductive tract, which phosphorylates the histone lysine methyltransferase (HKMT) EZH2, the key “installer” of epigenetic histone H3 lysine 27 trimethylation (H3K27me3). AKT phosphorylation inactivates EZH2, decreasing levels of H3K27 methylation, a repressive mark that inhibits gene expression, in the developing uterus. As a result of this developmental reprogramming, many estrogen-responsive genes become hypersensitive to estrogen in adulthood, exhibiting elevated expression throughout the estrus cycle, and resulting in a “hyper-estrogenized” phenotype in the adult uterus that promotes development of hormone dependent tumors. PMID:21656660

SAXS characterization of the interactions among digested food compounds and the anti-oxidant and anti-inflammatory activities of the formed nanocomplexes.

PubMed

Yang, Yingkang; Wang, Xiaoqi; Chen, Guijie; Zhou, Wenhua; Zeng, Xiaoxiong; Hu, Bing; Li, Yunqi; Huang, Qingrong

2018-06-06

In the present study, small angle X-ray scattering (SAXS) is applied to investigate the interaction between caseinophosphopeptides (CPP), the major digested product of milk protein, and chitosan (CS) under simulated gastrointestinal (GI) pH conditions. The change in pH value from the gastric to small intestinal environment induces complexation between CPP and CS, which is mainly driven by electrostatic interactions. The fractal dimension (Df) value of the domains inside the CPP-CS complexes is greater than 3, indicating the formation of dense particles/aggregates at the nanoscale. The Df value generally increases with an increase in the CS/CPP mass ratio. As a representative of polyphenols, (-)-epigallocatechin gallate (EGCG) associates with CS and CPP, forming nanocomplexes with a spherical shape and average particle size of around 208 nm. The formed CS-EGCG-CPP nanocomplexes do not affect the antioxidant activity of EGCG in the in vitro assays. Moreover, in the cellular assay, the nanocomplexes protect the RAW264.7 cells against H2O2-induced oxidative injury. In addition, the nanocomplexes significantly inhibit the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), TNF-α, IL-1β and IL-6. Furthermore, they inhibit the expression of iNOS, phosphorylation and degradation of IκB, as well as the translocation of NF-κB p65; this indicates that the mechanism for their anti-inflammatory activity is via the mediation of the NF-κB signaling pathway.

Polyanionic carbohydrate doxorubicin–dextran nanocomplex as a delivery system for anticancer drugs: in vitro analysis and evaluations

PubMed Central

Yousefpour, Parisa; Atyabi, Fatemeh; Farahani, Ebrahim Vashegani; Sakhtianchi, Ramin; Dinarvand, Rassoul

2011-01-01

This study deals with the preparation and investigation of a nanoscale delivery system for the anticancer drug doxorubicin (DOX) using its complexation with polyanionic carbohydrate dextran sulfate (DS). Dynamic light scattering, SEM, and zeta potential determination were used to characterize nanocomplexes. DOX-DS complexation was studied in the presence of ethanol as a hydrogen-bond disrupting agent, NaCl as an electrostatic shielding agent, and chitosan as a positively charged polymer. Thermodynamics of DOX-DS interaction was studied using isothermal titration calorimetry (ITC). A dialysis method was applied to investigate the release profile of DOX from DOX-DS nanocomplexes. Spherical and smooth-surfaced DOX-DS nanocomplexes (250–500 nm) with negative zeta potential were formed at a DS/DOX (w/w) ratio of 0.4–0.6, with over 90% drug encapsulation efficiency. DOX when complexed with DS showed lower fluorescence emission and 480 nm absorbance plus a 15 nm bathometric shift in its visible absorbance spectrum. Electrostatic hydrogen bonding and π-π stacking interactions are the main contributing interactions in DOX-DS complexation. Thermal analysis of DOX-DS complexation by ITC revealed that each DOX molecule binds with 3 DS glycosyl monomers. Drug release profile of nanocomplexes showed a fast DOX release followed by a slow sustained release, leading to release of 32% of entrapped DOX within 15 days. DOX-DS nanocomplexes may serve as a drug delivery system with efficient drug encapsulation and also may be taken into consideration in designing DOX controlled-release systems. PMID:21796249

Metabolic Reprogramming in Thyroid Carcinoma

PubMed Central

Coelho, Raquel Guimaraes; Fortunato, Rodrigo S.; Carvalho, Denise P.

2018-01-01

Among all the adaptations of cancer cells, their ability to change metabolism from the oxidative to the glycolytic phenotype is a hallmark called the Warburg effect. Studies on tumor metabolism show that improved glycolysis and glutaminolysis are necessary to maintain rapid cell proliferation, tumor progression, and resistance to cell death. Thyroid neoplasms are common endocrine tumors that are more prevalent in women and elderly individuals. The incidence of thyroid cancer has increased in the Past decades, and recent findings describing the metabolic profiles of thyroid tumors have emerged. Currently, several drugs are in development or clinical trials that target the altered metabolic pathways of tumors are undergoing. We present a review of the metabolic reprogramming in cancerous thyroid tissues with a focus on the factors that promote enhanced glycolysis and the possible identification of promising metabolic targets in thyroid cancer. PMID:29629339

Obesity-Associated Alterations in Inflammation, Epigenetics, and Mammary Tumor Growth Persist in Formerly Obese Mice.

PubMed

Rossi, Emily L; de Angel, Rebecca E; Bowers, Laura W; Khatib, Subreen A; Smith, Laura A; Van Buren, Eric; Bhardwaj, Priya; Giri, Dilip; Estecio, Marcos R; Troester, Melissa A; Hair, Brionna Y; Kirk, Erin L; Gong, Ting; Shen, Jianjun; Dannenberg, Andrew J; Hursting, Stephen D

2016-05-01

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339-48. ©2016 AACR. ©2016 American Association for Cancer Research.

Self-assembled micellar nanocomplexes comprising green tea catechin derivatives and protein drugs for cancer therapy

NASA Astrophysics Data System (ADS)

Chung, Joo Eun; Tan, Susi; Gao, Shu Jun; Yongvongsoontorn, Nunnarpas; Kim, Soon Hee; Lee, Jeong Heon; Choi, Hak Soo; Yano, Hirohisa; Zhuo, Lang; Kurisawa, Motoichi; Ying, Jackie Y.

2014-11-01

When designing drug carriers, the drug-to-carrier ratio is an important consideration, because the use of high quantities of carriers can result in toxicity as a consequence of poor metabolism and elimination of the carriers. However, these issues would be of less concern if both the drug and carrier had therapeutic effects. (-)-Epigallocatechin-3-O-gallate (EGCG), a major ingredient of green tea, has been shown, for example, to possess anticancer effects, anti-HIV effects, neuroprotective effects and DNA-protective effects. Here, we show that sequential self-assembly of the EGCG derivative with anticancer proteins leads to the formation of stable micellar nanocomplexes, which have greater anticancer effects in vitro and in vivo than the free protein. The micellar nanocomplex is obtained by complexation of oligomerized EGCG with the anticancer protein Herceptin to form the core, followed by complexation of poly(ethylene glycol)-EGCG to form the shell. When injected into mice, the Herceptin-loaded micellar nanocomplex demonstrates better tumour selectivity and growth reduction, as well as longer blood half-life, than free Herceptin.

A new chemotherapy agent-free theranostic system composed of graphene oxide nano-complex and aptamers for treatment of cancer cells.

PubMed

Bahreyni, Amirhossein; Yazdian-Robati, Rezvan; Hashemitabar, Shirin; Ramezani, Mohammad; Ramezani, Pouria; Abnous, Khalil; Taghdisi, Seyed Mohammad

2017-06-30

The common cancer treatment strategies like chemotherapy and radiotherapy are nonspecific and can trigger severe side effects by damaging normal cells. So, targeted cancer therapies, such as apoptosis induction, have attracted great attention in recent years. In this project, two nano-complexes, MUC1 aptamer-NAS-24 aptamer-Graphene oxide (GO) and MUC1 aptamer-Cytochrome C aptamer-GO, were designed to induce cell programmed death in MDA-MB-231 and MCF-7 cells (breast cancer cell lines) and to verify the level of apoptosis in both cell lines. MUC1 aptamer was a molecular recognition probe that led the internalization of two nano-complexes into MDA-MB-231 and MCF-7 cells (MUC1 positive cells) but not into HepG2 cell (liver cancer cell line, MUC1 negative cells). The apoptosis induction relied on binding of NAS-24 aptamer to its target, vimentin, in MDA-MB-231 and MCF-7 (target cells) with different levels of vimentin content. The function of first nano-complex was confirmed by binding of FAM-labeled cytochrome C aptamer to its target (cytochrome C) which was released from mitochondria, based on the function of the first nano-complex. Fluorometric analysis and gel retardation assay proved the formation of nano-complexes. The results of flow cytometry and fluorescence microscopy indicated efficient apoptosis induction just in target cells (MDA-MB-231 and MCF-7 cells) but not in non-target cells (HepG2 cell). The results of MTT assay also confirmed cell death process. Overall, our results proved excellent targeted apoptosis in breast cancer cells by designed nano-complexes which can be applied as an efficient cancer therapy method. Copyright © 2017 Elsevier B.V. All rights reserved.

Thiolated trimethyl chitosan nanocomplexes as gene carriers with high in vitro and in vivo transfection efficiency.

PubMed

Zhao, Xin; Yin, Lichen; Ding, Jieying; Tang, Cui; Gu, Shaohua; Yin, Chunhua; Mao, Yumin

2010-05-21

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was evaluated as non-viral gene carriers to combine the advantages of TMC and thiolated chitosan. TMC-Cys with various molecular weights (30, 100, and 200 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanocomplexes with plasmid encoding enhanced green fluorescence protein (pEGFP), which demonstrated preferable diameters of below 200 nm and zeta potentials of +15 to +20 mV. Cell binding and uptake of TMC-Cys/pEGFP nanocomplexes (TMC-Cys NC) were enhanced 2.4-3.0 and 1.4-3.0 folds, respectively, compared to TMC/pEGFP nanocomplexes (TMC NC). pEGFP could be easily released from TMC-Cys NC at the intracellular glutathione concentration, which promoted its nuclear transport and accumulation. Consequently, TMC-Cys NC showed a 1.4 to 3.2-fold increase in the transfection efficiency in HEK293 cells as compared to TMC NC and the optimal TMC-Cys(100,30) NC showed a 1.5-fold enhancement than Lipofectamine2000. Such results were further confirmed by in vivo transfection with a 2.3-fold and 4.1-fold higher transfection efficiency of TMC-Cys(100,30) NC than TMC(100,30) NC and Lipofectamine2000, respectively. Therefore, TMC-Cys/DNA nanocomplexes could be a promising gene delivery system with in vitro and in vivo superiority to Lipofectamine2000. Copyright 2010 Elsevier B.V. All rights reserved.

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells.

PubMed

Vazquez-Martin, Alejandro; Cufí, Sílvia; López-Bonet, Eugeni; Corominas-Faja, Bruna; Cuyàs, Elisabet; Vellon, Luciano; Iglesias, Juan Manuel; Leis, Olatz; Martín, Angel G; Menendez, Javier A

2013-11-15

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E 2/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E 2 signaling that leads to

Reprogramming cancer cells: a novel approach for cancer therapy or a tool for disease-modeling?

PubMed

Yilmazer, Açelya; de Lázaro, Irene; Taheri, Hadiseh

2015-12-01

Chromatin dynamics have been the major focus of many physiological and pathological processes over the past 20 years. Epigenetic mechanisms have been shown to be reshaped during both cellular reprogramming and tumorigenesis. For this reason, cancer cell reprogramming can provide a powerful tool to better understand both regenerative and cancer-fate processes, with a potential to develop novel therapeutic approaches. Recent studies showed that cancer cells can be reprogrammed to a pluripotent state by the overexpression of reprogramming transcription factors. Activation of transcription factors and modification of chromatin regulators may result in the remodeling of epigenetic status and refueling of tumorigenicity in these reprogrammed cancer cells. However, studies focusing on cancer cell reprogramming are contradictory; some studies reported increased tumor progression whereas others showed that cellular reprogramming has a treatment potential for cancer. In this review, first, the current knowledge on the epigenetic mechanisms involved during cancer development and cellular reprogramming will be presented. Later, different reports and key factors about pluripotency-based reprogramming of cancer cells will be reviewed in detail. New insights will be provided on cancer biology and therapy in the light of cellular reprogramming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Reprogramming of Normal Fibroblasts into Cancer-Associated Fibroblasts by miRNAs-Mediated CCL2/VEGFA Signaling

PubMed Central

Shen, Hua; Yu, Xiaobo; Yang, Fengming; Zhang, Zhihua; Shen, Jianxin; Sun, Jin; Choksi, Swati; Jitkaew, Siriporn; Shu, Yongqian

2016-01-01

Cancer-associated fibroblasts (CAFs), the most common constituent of the tumor stoma, are known to promote tumor initiation, progression and metastasis. However, the mechanism of how cancer cells transform normal fibroblasts (NFs) into CAFs is largely unknown. In this study, we determined the contribution of miRNAs in the transformation of NFs into CAFs. We found that miR-1 and miR-206 were down-regulated, whereas miR-31 was up-regulated in lung CAFs when compared with matched NFs. Importantly, modifying the expression of these three deregulated miRNAs induced a functional conversion of NFs into CAFs and vice versa. When the miRNA-reprogrammed NFs and CAFs were co-cultured with lung cancer cells (LCCs), a similar pattern of cytokine expression profiling were observed between two groups. Using a combination of cytokine expression profiling and miRNAs algorithms, we identified VEGFA/CCL2 and FOXO3a as direct targets of miR-1, miR-206 and miR-31, respectively. Importantly, systemic delivery of anti-VEGFA/CCL2 or pre-miR-1, pre-miR-206 and anti-miR-31 significantly inhibited tumor angiogenesis, TAMs accumulation, tumor growth and lung metastasis. Our results show that miRNAs-mediated FOXO3a/VEGF/CCL2 signaling plays a prominent role in LCCs-mediated NFs into CAFs, which may have clinical implications for providing novel biomarker(s) and potential therapeutic target(s) of lung cancer in the future. PMID:27541266

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

PubMed Central

Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

2011-01-01

Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

Metabolic reprogramming and dependencies associated with epithelial cancer stem cells independent of the epithelial-mesenchymal transition program

PubMed Central

Aguilar, Esther; de Mas, Igor Marin; Zodda, Erika; Marin, Silvia; Morrish, Fionnuala; Selivanov, Vitaly; Meca-Cortés, Óscar; Delowar, Hossain; Pons, Mònica; Izquierdo, Inés; Celià-Terrassa, Toni; de Atauri, Pedro; Centelles, Josep J; Hockenbery, David; Thomson, Timothy M; Cascante, Marta

2016-01-01

In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial-mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e-CSCs) from non-CSCs expressing a stable EMT. We have found that the e-CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e-CSCs consistent with the metabolomics and fluxomics analysis that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics and transcriptomics analysis allowed us to identify key metabolic players regulated at the post-transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis. PMID:27146024

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

PubMed

Gallego-Perez, Daniel; Otero, Jose J; Czeisler, Catherine; Ma, Junyu; Ortiz, Cristina; Gygli, Patrick; Catacutan, Fay Patsy; Gokozan, Hamza Numan; Cowgill, Aaron; Sherwood, Thomas; Ghatak, Subhadip; Malkoc, Veysi; Zhao, Xi; Liao, Wei-Ching; Gnyawali, Surya; Wang, Xinmei; Adler, Andrew F; Leong, Kam; Wulff, Brian; Wilgus, Traci A; Askwith, Candice; Khanna, Savita; Rink, Cameron; Sen, Chandan K; Lee, L James

2016-02-01

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined methionine deprivation and chloroethylnitrosourea have time-dependent therapeutic synergy on melanoma tumors that NMR spectroscopy-based metabolomics explains by methionine and phospholipid metabolism reprogramming.

PubMed

Guénin, Samuel; Morvan, Daniel; Thivat, Emilie; Stepien, Georges; Demidem, Aicha

2009-01-01

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met-sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

PubMed

Tani, Tetsuya; Shimada, Hiroaki; Kato, Yoko; Tsunoda, Yukio

2007-01-01

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Biomimetic remineralization of demineralized dentine using scaffold of CMC/ACP nanocomplexes in an in vitro tooth model of deep caries.

PubMed

Chen, Zhen; Cao, Shansong; Wang, Haorong; Li, Yanqiu; Kishen, Anil; Deng, Xuliang; Yang, Xiaoping; Wang, Yinghui; Cong, Changhong; Wang, Huajun; Zhang, Xu

2015-01-01

Currently, it is still a tough task for dentists to remineralize dentine in deep caries. The aim of this study was to remineralize demineralized dentine in a tooth model of deep caries using nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) based on mimicking the stabilizing effect of dentine matrix protein 1 (DMP1) on ACP in the biomineralization of dentine. The experimental results indicate that CMC can stabilize ACP to form nanocomplexes of CMC/ACP, which is able to be processed into scaffolds by lyophilization. In the single-layer collagen model, ACP nanoparticles are released from scaffolds of CMC/ACP nanocomplexes dissolved and then infiltrate into collagen fibrils via the gap zones (40 nm) to accomplish intrafibrillar mineralization of collagen. With this method, the completely demineralized dentine was partially remineralized in the tooth mode. This is a bottom-up remineralizing strategy based on non-classical crystallization theory. Since nanocomplexes of CMC/ACP show a promising effect of remineralization on demineralized dentine via biomimetic strategy, thereby preserving dentinal tissue to the maximum extent possible, it would be a potential indirect pulp capping (IPC) material for the management of deep caries during vital pulp therapy based on the concept of minimally invasive dentistry (MID).

Biomimetic Remineralization of Demineralized Dentine Using Scaffold of CMC/ACP Nanocomplexes in an In Vitro Tooth Model of Deep Caries

PubMed Central

Chen, Zhen; Cao, Shansong; Wang, Haorong; Li, Yanqiu; Kishen, Anil; Deng, Xuliang; Yang, Xiaoping; Wang, Yinghui; Cong, Changhong; Wang, Huajun; Zhang, Xu

2015-01-01

Currently, it is still a tough task for dentists to remineralize dentine in deep caries. The aim of this study was to remineralize demineralized dentine in a tooth model of deep caries using nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) based on mimicking the stabilizing effect of dentine matrix protein 1 (DMP1) on ACP in the biomineralization of dentine. The experimental results indicate that CMC can stabilize ACP to form nanocomplexes of CMC/ACP, which is able to be processed into scaffolds by lyophilization. In the single-layer collagen model, ACP nanoparticles are released from scaffolds of CMC/ACP nanocomplexes dissolved and then infiltrate into collagen fibrils via the gap zones (40 nm) to accomplish intrafibrillar mineralization of collagen. With this method, the completely demineralized dentine was partially remineralized in the tooth mode. This is a bottom-up remineralizing strategy based on non-classical crystallization theory. Since nanocomplexes of CMC/ACP show a promising effect of remineralization on demineralized dentine via biomimetic strategy, thereby preserving dentinal tissue to the maximum extent possible, it would be a potential indirect pulp capping (IPC) material for the management of deep caries during vital pulp therapy based on the concept of minimally invasive dentistry (MID). PMID:25587986

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer *

PubMed Central

Hutton, Josiah E.; Wang, Xiaojing; Zimmerman, Lisa J.; Slebos, Robbert J. C.; Trenary, Irina A.; Young, Jamey D.; Li, Ming; Liebler, Daniel C.

2016-01-01

Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25–twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

Nebulisation of receptor-targeted nanocomplexes for gene delivery to the airway epithelium.

PubMed

Manunta, Maria D I; McAnulty, Robin J; Tagalakis, Aristides D; Bottoms, Stephen E; Campbell, Frederick; Hailes, Helen C; Tabor, Alethea B; Laurent, Geoffrey J; O'Callaghan, Christopher; Hart, Stephen L

2011-01-01

Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.

Metabolic Reprogramming and Oncogenesis: One Hallmark, Many Organelles.

PubMed

Costa, A S H; Frezza, C

2017-01-01

The process of tumorigenesis can be described by a series of molecular features, among which alteration of cellular metabolism has recently emerged. This metabolic rewiring fulfills the energy and biosynthetic demands of fast proliferating cancer cells and amplifies their metabolic repertoire to survive and proliferate in the poorly oxygenated and nutrient-deprived tumor microenvironment. During the last decade, the complex reprogramming of cancer cell metabolism has been widely investigated, revealing cancer-specific metabolic alterations. These include dysregulation of glucose and glutamine metabolism, alterations of lipid synthesis and oxidation, and a complex rewiring of mitochondrial function. However, mitochondria are not the only metabolically active organelles within the cell, and other organelles, including lysosomes, peroxisomes, and endoplasmic reticulum, harbor components of the metabolic network. Of note, dysregulation of the function of these organelles is increasingly recognized in cancer cells. However, to what extent these organelles contribute to the metabolic reprogramming of cancer is not fully understood. In this review, we describe the main metabolic functions of these organelles and provide insights into how they communicate to orchestrate a coordinated metabolic reprogramming during transformation. © 2017 Elsevier Inc. All rights reserved.

NF-κB activation impairs somatic cell reprogramming in ageing.

PubMed

Soria-Valles, Clara; Osorio, Fernando G; Gutiérrez-Fernández, Ana; De Los Angeles, Alejandro; Bueno, Clara; Menéndez, Pablo; Martín-Subero, José I; Daley, George Q; Freije, José M P; López-Otín, Carlos

2015-08-01

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

System-Wide Hypersensitive Response-Associated Transcriptome and Metabolome Reprogramming in Tomato1[W][OA

PubMed Central

Etalo, Desalegn W.; Stulemeijer, Iris J.E.; Peter van Esse, H.; de Vos, Ric C.H.; Bouwmeester, Harro J.; Joosten, Matthieu H.A.J.

2013-01-01

The hypersensitive response (HR) is considered to be the hallmark of the resistance response of plants to pathogens. To study HR-associated transcriptome and metabolome reprogramming in tomato (Solanum lycopersicum), we used plants that express both a resistance gene to Cladosporium fulvum and the matching avirulence gene of this pathogen. In these plants, massive reprogramming occurred, and we found that the HR and associated processes are highly energy demanding. Ubiquitin-dependent protein degradation, hydrolysis of sugars, and lipid catabolism are used as alternative sources of amino acids, energy, and carbon skeletons, respectively. We observed strong accumulation of secondary metabolites, such as hydroxycinnamic acid amides. Coregulated expression of WRKY transcription factors and genes known to be involved in the HR, in addition to a strong enrichment of the W-box WRKY-binding motif in the promoter sequences of the coregulated genes, point to WRKYs as the most prominent orchestrators of the HR. Our study has revealed several novel HR-related genes, and reverse genetics tools will allow us to understand the role of each individual component in the HR. PMID:23719893

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

PubMed

Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

2014-01-01

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

Application of Nanoparticle Technology to Reduce the Anti-Microbial Resistance through β-Lactam Antibiotic-Polymer Inclusion Nano-Complex.

PubMed

Salamanca, Constain H; Yarce, Cristhian J; Roman, Yony; Davalos, Andrés F; Rivera, Gustavo R

2018-02-10

Biocompatible polymeric materials with potential to form functional structures in association with different therapeutic molecules have a high potential for biological, medical and pharmaceutical applications. Therefore, the capability of the inclusion of nano-Complex formed between the sodium salt of poly(maleic acid- alt -octadecene) and a β-lactam drug (ampicillin trihydrate) to avoid the chemical and enzymatic degradation and enhance the biological activity were evaluated. PAM-18Na was produced and characterized, as reported previously. The formation of polymeric hydrophobic aggregates in aqueous solution was determined, using pyrene as a fluorescent probe. Furthermore, the formation of polymer-drug nano-complexes was characterized by Differential Scanning Calorimetry-DSC, viscometric, ultrafiltration/centrifugation assays, zeta potential and size measurements were determined by dynamic light scattering-DLS. The PAM-18Na capacity to avoid the chemical degradation was studied through stress stability tests. The enzymatic degradation was evaluated from a pure β-lactamase, while the biological degradation was determined by different β-lactamase producing Staphylococcus aureus strains. When ampicillin was associated with PAM-18Na, the half-life time in acidic conditions increased, whereas both the enzymatic degradation and the minimum inhibitory concentration decreased to a 90 and 75%, respectively. These results suggest a promissory capability of this polymer to protect the β-lactam drugs against chemical, enzymatic and biological degradation.

Application of Nanoparticle Technology to Reduce the Anti-Microbial Resistance through β-Lactam Antibiotic-Polymer Inclusion Nano-Complex

PubMed Central

Yarce, Cristhian J.; Roman, Yony; Davalos, Andrés F.; Rivera, Gustavo R.

2018-01-01

Biocompatible polymeric materials with potential to form functional structures in association with different therapeutic molecules have a high potential for biological, medical and pharmaceutical applications. Therefore, the capability of the inclusion of nano-Complex formed between the sodium salt of poly(maleic acid-alt-octadecene) and a β-lactam drug (ampicillin trihydrate) to avoid the chemical and enzymatic degradation and enhance the biological activity were evaluated. PAM-18Na was produced and characterized, as reported previously. The formation of polymeric hydrophobic aggregates in aqueous solution was determined, using pyrene as a fluorescent probe. Furthermore, the formation of polymer-drug nano-complexes was characterized by Differential Scanning Calorimetry-DSC, viscometric, ultrafiltration/centrifugation assays, zeta potential and size measurements were determined by dynamic light scattering-DLS. The PAM-18Na capacity to avoid the chemical degradation was studied through stress stability tests. The enzymatic degradation was evaluated from a pure β-lactamase, while the biological degradation was determined by different β-lactamase producing Staphylococcus aureus strains. When ampicillin was associated with PAM-18Na, the half-life time in acidic conditions increased, whereas both the enzymatic degradation and the minimum inhibitory concentration decreased to a 90 and 75%, respectively. These results suggest a promissory capability of this polymer to protect the β-lactam drugs against chemical, enzymatic and biological degradation. PMID:29439391

Continuous Production of Discrete Plasmid DNA-Polycation Nanoparticles Using Flash Nanocomplexation

PubMed Central

Santos, Jose Luis; Ren, Yong; Vandermark, John; Archang, Maani M.; Williford, John-Michael; Liu, Heng-wen; Lee, Jason; Wang, Tza-Huei; Mao, Hai-Quan

2016-01-01

Despite successful demonstration of linear polyethyleneimine (lPEI) as an effective carrier for a wide range of gene medicine, including DNA plasmids, small interfering RNAs, mRNAs, etc., and continuous improvement of the physical properties and biological performance of the polyelectrolyte complex nanoparticles prepared from lPEI and nucleic acids, there still exist major challenges to produce these nanocomplexes in a scalable manner, particularly for lPEI/DNA nanoparticles. This has significantly hindered the progress towards clinical translation of these nanoparticle-based gene medicine. Here we report a flash nanocomplexation (FNC) method that achieves continuous production of lPEI/plasmid DNA nanoparticles with narrow size distribution using a confined impinging jet device. The method involves the complex coacervation of negatively charged DNA plasmid and positive charged lPEI under rapid, highly dynamic, and homogeneous mixing conditions, producing polyelectrolyte complex nanoparticles with narrow distribution of particle size and shape. The average number of plasmid DNA packaged per nanoparticles and its distribution are similar between the FNC method and the small-scale batch mixing method. In addition, the nanoparticles prepared by these two methods exhibit similar cell transfection efficiency. These results confirm that FNC is an effective and scalable method that can produce well-controlled lPEI/plasmid DNA nanoparticles. PMID:27717227

A case of cellular alchemy: lineage reprogramming and its potential in regenerative medicine

PubMed Central

Asuelime, Grace E.; Shi, Yanhong

2012-01-01

The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting. PMID:22371436

Biomarkers of Coordinate Metabolic Reprogramming in Colorectal Tumors in Mice and Humans

PubMed Central

Manna, Soumen K.; Tanaka, Naoki; Krausz, Kristopher W.; Haznadar, Majda; Xue, Xiang; Matsubara, Tsutomu; Bowman, Elise D.; Fearon, Eric R.; Harris, Curtis C.; Shah, Yatrik M.; Gonzalez, Frank J.

2014-01-01

BACKGROUND & AIMS There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis. METHODS Mass spectrometry-based metabolomic analyses of urine and tissues from wild-type C57BL/6J and ApcMin/+ mice, as well as from mice with azoxymethane-induced tumors, was employed in tandem with gene expression analysis. Metabolomics profiles were also determined on colon tumor and adjacent non-tumor tissues from 39 patients. The effects of β-catenin activity on metabolic profiles were assessed in mice with colon-specific disruption of Apc. RESULTS Thirteen markers were found in urine associated with development of colorectal tumors in ApcMin/+ mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice reflected the observed changes in metabolic products detected in urine; similar changes were observed in mice with azoxymethane-induced tumors and mice with colon-specific activation of β-catenin. The metabolic alterations indicated by markers in urine therefore appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 12 metabolites associated with the same metabolic pathways identified in mice (including amino acid metabolism and polyamine metabolism). Ten metabolites that were increased in tumor tissues, compared with non-tumor tissues (proline, threonine, glutamic acid, arginine, N1-acetylspermidine, xanthine, uracil, betaine, symmetric dimethylarginine, and asymmetric-dimethylarginine), were also increased in urine from tumor-bearing mice. CONCLUSIONS Gene expression and metabolomic profiles of urine and tissue samples from

Multispectroscopic DNA-Binding studies and antimicrobial evaluation of new mixed-ligand Silver(I) complex and nanocomplex: A comparative study

NASA Astrophysics Data System (ADS)

Movahedi, Elaheh; Rezvani, Ali Reza

2018-05-01

A novel mixed-ligand Ag(I) complex, , has been synthesized and characterized by the elemental analysis, IR spectroscopy and 1HNMR. In the formula, dian and phen are N-(4,5-diazafluoren-9-ylidene)aniline and 1,10-phenanthroline, respectively. This complex also has been prepared at nano size by sonochemical technique and characterized by the FTIR and scanning electron microscopy (SEM). To evaluate the biological preferences of the Ag(I) complex and nanocomplex and verify the relationships between the structure and biological function, in vitro DNA binding and antibacterial experiments have been carried out. DNA-complex interaction has been pursued by electronic absorption titration, luminescence titration, competitive binding experiment, effect of ionic strength, thermodynamic studies, viscometric evaluation and circular dichroism spectroscopy in the physiological pH. Each compound displays significant binding trend to the CT-DNA. The mode of binding to the CT-DNA probably is a moderate intercalation mode with the partial insertion of the planar ligands between the base stacks of double-stranded DNA. The relative viscosities and circular dichroism spectra of the CT-DNA with the complex solutions, confirm the intense interactions of the Ag(I) complex and nanocomplex with DNA. An in vitro antibacterial test of the complex and nanocomplex on a series of the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) shows a remarkable antibacterial feature of the Ag(I) complex. The MIC values (minimum inhibitory concentration) of the compounds compare with silver nitrate and silver sulfadiazine. The bacterial inhibitions of the Ag(I) complex and nanocomplex are agreed to their DNA binding affinities.

Treatment-Induced Autophagy Associated with Tumor Dormancy and Relapse

DTIC Science & Technology

2016-07-01

Payne KK. Cancer immunotherapy: re-programming cells of the innate and adaptive immune systems. Oncoimmunology 1(2):201-204, 2012. Mast EE, Margolis HS...2012) Innate IFN-g is essential for programmed death ligand-1-mediated T cell stimulation following Listeria monocytogenes infection. J. Immunol. 189...immunoediting and escape. Adoptive  immunotherapy (AIT) was also found to support regression of ADR‐induced dormant tumor  cells .  15. SUBJECT TERMS Autophagy

Coactivator SRC-2–dependent metabolic reprogramming mediates prostate cancer survival and metastasis

PubMed Central

Dasgupta, Subhamoy; Putluri, Nagireddy; Long, Weiwen; Zhang, Bin; Wang, Jianghua; Kaushik, Akash K.; Arnold, James M.; Bhowmik, Salil K.; Stashi, Erin; Brennan, Christine A.; Rajapakshe, Kimal; Coarfa, Cristian; Mitsiades, Nicholas; Ittmann, Michael M.; Chinnaiyan, Arul M.; Sreekumar, Arun; O’Malley, Bert W.

2015-01-01

Metabolic pathway reprogramming is a hallmark of cancer cell growth and survival and supports the anabolic and energetic demands of these rapidly dividing cells. The underlying regulators of the tumor metabolic program are not completely understood; however, these factors have potential as cancer therapy targets. Here, we determined that upregulation of the oncogenic transcriptional coregulator steroid receptor coactivator 2 (SRC-2), also known as NCOA2, drives glutamine-dependent de novo lipogenesis, which supports tumor cell survival and eventual metastasis. SRC-2 was highly elevated in a variety of tumors, especially in prostate cancer, in which SRC-2 was amplified and overexpressed in 37% of the metastatic tumors evaluated. In prostate cancer cells, SRC-2 stimulated reductive carboxylation of α-ketoglutarate to generate citrate via retrograde TCA cycling, promoting lipogenesis and reprogramming of glutamine metabolism. Glutamine-mediated nutrient signaling activated SRC-2 via mTORC1-dependent phosphorylation, which then triggered downstream transcriptional responses by coactivating SREBP-1, which subsequently enhanced lipogenic enzyme expression. Metabolic profiling of human prostate tumors identified a massive increase in the SRC-2–driven metabolic signature in metastatic tumors compared with that seen in localized tumors, further implicating SRC-2 as a prominent metabolic coordinator of cancer metastasis. Moreover, SRC-2 inhibition in murine models severely attenuated the survival, growth, and metastasis of prostate cancer. Together, these results suggest that the SRC-2 pathway has potential as a therapeutic target for prostate cancer. PMID:25664849

A tumor-targeting p53 nanodelivery system limits chemoresistance to temozolomide prolonging survival in a mouse model of glioblastoma multiforme.

PubMed

Kim, Sang-Soo; Rait, Antonina; Kim, Eric; Pirollo, Kathleen F; Chang, Esther H

2015-02-01

Development of temozolomide (TMZ) resistance contributes to the poor prognosis for glioblastoma multiforme (GBM) patients. It was previously demonstrated that delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex (SGT-53) which crosses the blood-brain barrier could sensitize highly TMZ-resistant GBM tumors to TMZ. Here we assessed whether SGT-53 could inhibit development of TMZ resistance. SGT-53 significantly chemosensitized TMZ-sensitive human GBM cell lines (U87 and U251), in vitro and in vivo. Furthermore, in an intracranial GBM tumor model, two cycles of concurrent treatment with systemically administered SGT-53 and TMZ inhibited tumor growth, increased apoptosis and most importantly, significantly prolonged median survival. In contrast TMZ alone had no significant effect on median survival compared to a single cycle of TMZ. These results suggest that combining SGT-53 with TMZ appears to limit development of TMZ resistance, prolonging its anti-tumor effect and could be a more effective therapy for GBM. Using human glioblastoma multiforma cell lines, this research team demonstrated that the delivery of exogenous wild-type tumor suppressor gene p53 via a tumor-targeted nanocomplex limited the development of temozolomide resistance and prolonged its anti-tumor effect, which may enable future human application of this or similar techniques. Copyright © 2015 Elsevier Inc. All rights reserved.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer

PubMed Central

Timofeeva, Olga A.; Palechor-Ceron, Nancy; Li, Guanglei; Yuan, Hang; Krawczyk, Ewa; Zhong, Xiaogang; Liu, Geng; Upadhyay, Geeta; Dakic, Aleksandra; Yu, Songtao; Fang, Shuang; Choudhury, Sujata; Zhang, Xueping; Ju, Andrew; Lee, Myeong-Seon; Dan, Han C.; Ji, Youngmi; Hou, Yong; Zheng, Yun-Ling; Albanese, Chris; Rhim, Johng; Schlegel, Richard; Dritschilo, Anatoly; Liu, Xuefeng

2017-01-01

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. PMID:28009986

Reprogramming cancer cells: overview & current progress.

PubMed

Lim, Kian Lam; Teoh, Hoon Koon; Choong, Pei Feng; Teh, Hui Xin; Cheong, Soon Keng; Kamarul, Tunku

2016-07-01

Cancer is a disease with genetic and epigenetic origins, and the possible effects of reprogramming cancer cells using the defined sets of transcription factors remain largely uninvestigated. In the handful of publications available so far, findings have shown that reprogramming cancer cells changed the characteristics of the cells to differ from the parental cancer cells. These findings indicated the possibility of utilizing reprogramming technology to create a disease model in the laboratory to be used in studying the molecular pathogenesis or for drug screening of a particular cancer model. Despite numerous methods employed in generating induced pluripotent stem cells (iPSCs) from cancer cells only a few studies have successfully reprogrammed malignant human cells. In this review we will provide an overview on i) methods to reprogram cancer cells, ii) characterization of the reprogrammed cancer cells, and iii) the differential effects of reprogramming on malignancy, epigenetics and response of the cancer cells to chemotherapeutic agents. Continued technical progress in cancer cell reprogramming technology will be instrumental for more refined in vitro disease models and ultimately for the development of directed and personalized therapy for cancer patients in the future.

Biocompatible mannosylated endosomal-escape nanoparticles enhance selective delivery of short nucleotide sequences to tumor associated macrophages

NASA Astrophysics Data System (ADS)

Ortega, Ryan A.; Barham, Whitney J.; Kumar, Bharat; Tikhomirov, Oleg; McFadden, Ian D.; Yull, Fiona E.; Giorgio, Todd D.

2014-12-01

Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Manipulation of the TAM phenotype is a novel, potential therapeutic approach to engage anti-cancer immunity. siRNA is a molecular tool for knockdown of specific mRNAs that is tunable in both strength and duration. The use of siRNA to reprogram TAMs to adopt an immunogenic, anti-tumor phenotype is an attractive alternative to ablation of this cell population. One current difficulty with this approach is that TAMs are difficult to specifically target and transfect. We report here successful utilization of novel mannosylated polymer nanoparticles (MnNP) that are capable of escaping the endosomal compartment to deliver siRNA to TAMs in vitro and in vivo. Transfection with MnNP-siRNA complexes did not significantly decrease TAM cell membrane integrity in culture, nor did it create adverse kidney or liver function in mice, even at repeated doses of 5 mg kg-1. Furthermore, MnNP effectively delivers labeled nucleotides to TAMs in mice with primary mammary tumors. We also confirmed TAM targeting in the solid tumors disseminated throughout the peritoneum of ovarian tumor bearing mice following injection of fluorescently labeled MnNP-nucleotide complexes into the peritoneum. Finally, we show enhanced uptake of MnNP in lung metastasis associated macrophages compared to untargeted particles when using an intubation delivery method. In summary, we have shown that MnNP specifically and effectively deliver siRNA to TAMs in vivo.

Discovery and progress of direct cardiac reprogramming.

PubMed

Kojima, Hidenori; Ieda, Masaki

2017-06-01

Cardiac disease remains a major cause of death worldwide. Direct cardiac reprogramming has emerged as a promising approach for cardiac regenerative therapy. After the discovery of MyoD, a master regulator for skeletal muscle, other single cardiac reprogramming factors (master regulators) have been sought. Discovery of cardiac reprogramming factors was inspired by the finding that multiple, but not single, transcription factors were needed to generate induced pluripotent stem cells (iPSCs) from fibroblasts. We first reported a combination of cardiac-specific transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that could convert mouse fibroblasts into cardiomyocyte-like cells, which were designated as induced cardiomyocyte-like cells (iCMs). Following our first report of cardiac reprogramming, many researchers, including ourselves, demonstrated an improvement in cardiac reprogramming efficiency, in vivo direct cardiac reprogramming for heart regeneration, and cardiac reprogramming in human cells. However, cardiac reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. We believe that future research elucidating epigenetic barriers and molecular mechanisms of direct cardiac reprogramming will improve the reprogramming efficiency, and that this new technology has great potential for clinical applications.

Fabrication of peptide stabilized fluorescent gold nanocluster/graphene oxide nanocomplex and its application in turn-on detection of metalloproteinase-9.

PubMed

Nguyen, Phuong-Diem; Cong, Vu Thanh; Baek, Changyoon; Min, Junhong

2017-03-15

This study introduces the double-ligands stabilizing gold nanoclusters and the fabrication of gold nanocluster/graphene nanocomplex as a "turn-on" fluorescent probe for the detection of cancer-related enzyme matrix metalloproteinase-9. A facile, one-step approach was developed for the synthesis of fluorescent gold nanoclusters using peptides and mercaptoundecanoic acid as co-templating ligands. The peptide was designed to possess a metalloproteinase-9 cleavage site and to act not only as a stabilizer but also as a targeting ligand for the enzyme detection. The prepared gold nanoclusters show an intense red fluorescence with a broad adsorption spectrum. In the presence of the enzyme, due to the excellent quenching properties and the negligible background of graphene oxide, the developed peptide-gold nanocluster/graphene nanocomplex yielded an intense "turn-on" fluorescent response, which strongly correlated with the enzyme concentration. The limit of detection of the nanocomplex was 0.15nM. The sensor was successfully applied for "turn-on" detection of metalloproteinase-9 secreted from human breast adenocarcinoma MCF-7 cells with high sensitivity, selectivity, significant improvement in terms of detection time and simplicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Continuous Production of Discrete Plasmid DNA-Polycation Nanoparticles Using Flash Nanocomplexation.

PubMed

Santos, Jose Luis; Ren, Yong; Vandermark, John; Archang, Maani M; Williford, John-Michael; Liu, Heng-Wen; Lee, Jason; Wang, Tza-Huei; Mao, Hai-Quan

2016-12-01

Despite successful demonstration of linear polyethyleneimine (lPEI) as an effective carrier for a wide range of gene medicine, including DNA plasmids, small interfering RNAs, mRNAs, etc., and continuous improvement of the physical properties and biological performance of the polyelectrolyte complex nanoparticles prepared from lPEI and nucleic acids, there still exist major challenges to produce these nanocomplexes in a scalable manner, particularly for lPEI/DNA nanoparticles. This has significantly hindered the progress toward clinical translation of these nanoparticle-based gene medicine. Here the authors report a flash nanocomplexation (FNC) method that achieves continuous production of lPEI/plasmid DNA nanoparticles with narrow size distribution using a confined impinging jet device. The method involves the complex coacervation of negatively charged DNA plasmid and positive charged lPEI under rapid, highly dynamic, and homogeneous mixing conditions, producing polyelectrolyte complex nanoparticles with narrow distribution of particle size and shape. The average number of plasmid DNA packaged per nanoparticles and its distribution are similar between the FNC method and the small-scale batch mixing method. In addition, the nanoparticles prepared by these two methods exhibit similar cell transfection efficiency. These results confirm that FNC is an effective and scalable method that can produce well-controlled lPEI/plasmid DNA nanoparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis.

PubMed

McDonald, Oliver G; Li, Xin; Saunders, Tyler; Tryggvadottir, Rakel; Mentch, Samantha J; Warmoes, Marc O; Word, Anna E; Carrer, Alessandro; Salz, Tal H; Natsume, Sonoko; Stauffer, Kimberly M; Makohon-Moore, Alvin; Zhong, Yi; Wu, Hao; Wellen, Kathryn E; Locasale, Jason W; Iacobuzio-Donahue, Christine A; Feinberg, Andrew P

2017-03-01

During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.

Aldolase B-Mediated Fructose Metabolism Drives Metabolic Reprogramming of Colon Cancer Liver Metastasis.

PubMed

Bu, Pengcheng; Chen, Kai-Yuan; Xiang, Kun; Johnson, Christelle; Crown, Scott B; Rakhilin, Nikolai; Ai, Yiwei; Wang, Lihua; Xi, Rui; Astapova, Inna; Han, Yan; Li, Jiahe; Barth, Bradley B; Lu, Min; Gao, Ziyang; Mines, Robert; Zhang, Liwen; Herman, Mark; Hsu, David; Zhang, Guo-Fang; Shen, Xiling

2018-06-05

Cancer metastasis accounts for the majority of cancer-related deaths and remains a clinical challenge. Metastatic cancer cells generally resemble cells of the primary cancer, but they may be influenced by the milieu of the organs they colonize. Here, we show that colorectal cancer cells undergo metabolic reprogramming after they metastasize and colonize the liver, a key metabolic organ. In particular, via GATA6, metastatic cells in the liver upregulate the enzyme aldolase B (ALDOB), which enhances fructose metabolism and provides fuel for major pathways of central carbon metabolism during tumor cell proliferation. Targeting ALDOB or reducing dietary fructose significantly reduces liver metastatic growth but has little effect on the primary tumor. Our findings suggest that metastatic cells can take advantage of reprogrammed metabolism in their new microenvironment, especially in a metabolically active organ such as the liver. Manipulation of involved pathways may affect the course of metastatic growth. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of metabolic reprogramming in γ-herpesvirus-associated oncogenesis.

PubMed

Lo, Angela Kwok-Fung; Dawson, Christopher W; Young, Lawrence S; Lo, Kwok-Wai

2017-10-15

The γ-herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ-herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ-herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ-herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ-herpesviruses alter metabolic phenotypes as a means to support viral infection and long-term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ-herpesviruses provide new opportunities for therapeutic intervention. © 2017 UICC.

In Utero Exposure to Low-Dose Alcohol Induces Reprogramming of Mammary Development and Tumor Risk in MMTV-erbB-2 Transgenic Mice

PubMed Central

Ma, Zhikun; Blackwelder, Amanda J.; Lee, Harry; Zhao, Ming; Yang, Xiaohe

2015-01-01

There is increasing evidence that prenatal exposure to environmental factors may modify breast cancer risk later in life. This study aimed to investigate the effects of in utero exposure to low-dose alcohol on mammary development and tumor risk. Pregnant MMTV-erbB-2 mice were exposed to alcohol (6 g/kg/day) between day 13 and day 19 of gestation, and the female offspring were examined for tumor risk. Whole mount analysis indicated that in utero exposure to low-dose alcohol induced significant increases in ductal extension at 10 weeks of age. Molecular analysis showed that in utero alcohol exposure induced upregulation of ERα signaling and activation of Akt and Erk1/2 in pubertal mammary glands. However, enhanced signaling in the EGFR/erbB-2 pathway appeared to be more prominent in 10-week-old glands than did signaling in the other pathways. Interestingly, tumor development in mice with in utero exposure to low-dose alcohol was slightly delayed compared to control mice, but tumor multiplicity was increased. The results indicate that in utero exposure to low-dose alcohol induces the reprogramming of mammary development by mechanisms that include altered signaling in the estrogen receptor (ER) and erbB-2 pathways. The intriguing tumor development pattern might be related to alcohol dose and exposure conditions, and warrants further investigation. PMID:25853264

Spin glass model for cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Castillo, Horacio E.

2014-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor state to another attractor state. We use a simple model based on spin glass theory that can construct a simulated epigenetic landscape starting from the experimental genomic data. We modify the model to incorporate experimental reprogramming protocols. Our simulations successfully reproduce several reprogramming experiments. We probe the robustness of the results against random changes in the model, explore the importance of asymmetric interactions between transcription factors and study the importance of histone modification errors in reprogramming.

Reprogramming cells with synthetic proteins

PubMed Central

Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

2015-01-01

Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

Boosters and barriers for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Zonooz, Elmira Rezaei; Baharvand, Hossein

2017-06-01

Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFβ signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming. Copyright © 2017 Elsevier Inc. All rights reserved.

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

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

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellularmore » response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.« less

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.

PubMed

Mohamed, Tamer M A; Stone, Nicole R; Berry, Emily C; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N; Srivastava, Deepak

2017-03-07

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin. Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. © 2016 American Heart Association, Inc.

Inclusion complex from cyclodextrin-grafted hyaluronic acid and pseudo protein as biodegradable nano-delivery vehicle for gambogic acid.

PubMed

Ji, Ying; Shan, Shuo; He, Mingyu; Chu, Chih-Chang

2017-10-15

β-Cyclodextrin can form inclusion complex with a series of guest molecules including phenyl moieties, and has gained considerable popularity in the study of supramolecular nanostructure. In this study, a biodegradable nanocomplex (HA(CD)-4Phe4 nanocomplex) was developed from β-cyclodextrin grafted hyaluronic acid (HA) and phenylalanine based poly(ester amide). The phenylalanine based poly(ester amide) is a biodegradable pseudo protein which provides the encapsulation capacity for gambogic acid (GA), a naturally-derived chemotherapeutic which has been effectively employed to treat multidrug resistant tumor. The therapeutic potency of free GA is limited due to its poor solubility in water and the lack of tumor-selective toxicity. The nanocomplex carrier enhanced the solubility and availability of GA in aqueous media, and the HA component enabled the targeted delivery to tumor cells with overexpression of CD44 receptors. In the presence of hyaluronidase, the release of GA from the nanocomplex was significantly accelerated, due to the enzymatic biodegradation of the carrier. Compared to free GA, GA-loaded nanocomplex exhibited improved cytotoxicity in MDA-MB-435/MDR multidrug resistant melanoma cells, and induced enhanced level of apoptosis and mitochondrial depolarization, at low concentration of GA (1-2µM). The nanocomplex enhanced the therapeutic potency of GA, especially when diluted in physiological environment. In addition, suppressed matrix metalloproteinase activity was also detected in MDA-MB-435/MDR cells treated by GA-loaded nanocomplex, which demonstrated its potency in the inhibition of tumor metastasis. The in vitro data suggested that HA(CD)-4Phe4 nanocomplex could provide a promising alternative in the treatment of multidrug resistant tumor cells. Gambogic acid (GA), naturally derived from genus Garcinia trees, exhibited significant cytotoxic activity against multiple types of tumors with resistance to traditional chemotherapeutics. Unfortunately

MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients

PubMed Central

Martinelli, Camila Maria da Silva; Lengert, André van Helvoort; Cárcano, Flavio Mavignier; Silva, Eduardo Caetano Albino; Brait, Mariana; Lopes, Luiz Fernando; Vidal, Daniel Onofre

2017-01-01

Testicular germ cell tumors (TGCT) represent the second main cause of cancer-related death in young men. Despite high cure rates, refractory disease results in poor prognosis. Epigenetic reprogramming occurs during the development of seminomas and non-seminomas. Understanding the molecular and genetic basis of these tumors would represent an important advance in the search for new TGCT molecular markers. Hence the frequency of methylation of a gene panel (VGF, MGMT, ADAMTS1, CALCA, HOXA9, CDKN2B, CDO1 and NANOG) was evaluated in 72 primary TGCT by quantitative methylation specific PCR. A high frequency of MGMT (90.9%, 20/22; p=0.019) and CALCA (90.5%, 19/21; p<0.026) methylation was associated with non-seminomatous tumors while CALCA methylation was also associated with refractory disease (47.4%, 09/19; p=0.005). Moreover, promoter methylation of both genes predicts poor clinical outcome for TGCT patients (5-year EFS: 50.5% vs 77.1%; p=0.032 for MGMT and 51.3% vs 77.0%; p=0.029 for CALCA). The findings of this study indicate that methylation of MGMT and CALCA are frequent and could be used as new molecular markers of prognosis in TGCT. PMID:28881587

Ultrasound-enhanced delivery of doxorubicin/all-trans retinoic acid-loaded nanodiamonds into tumors.

PubMed

Li, Huanan; Zeng, Deping; Wang, Zhenyu; Fang, Liaoqiong; Li, Faqi; Wang, Zhibiao

2018-03-14

To build up a combined therapy strategy to address limitations of the enhanced permeability and retention (EPR) effect and improve the efficiency of tumor therapy. A pH-sensitive nanocomplex for co-delivery of doxorubicin (DOX) and all-trans retinoic acid (ATRA) was developed based on nanodiamonds (DOX/ATRA-NDs) to enhance intracellular retention of drugs. Meanwhile, ultrasound was employed to enhance tumor vascular penetration of DOX-ATRA-NDs. The distribution of DOX/ATRA-NDs in the tumor tissues increased threefold when ultrasound was applied at 1 MHz and 0.6 W/cm 2 . Comparing with unmodified chemotherapeutics, the combined therapy induced more tumor cells apoptosis and greater tumor growth inhibition in both liver and breast tumor models. DOX-ATRA-NDs demonstrate great potential in clinical applications.

Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

PubMed

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

PubMed Central

Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

2016-01-01

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor

PubMed Central

Chaudhri, Virendra K.; Salzler, Gregory G.; Dick, Salihah A.; Buckman, Melanie S.; Sordella, Raffaella; Karoly, Edward D.; Mohney, Robert; Stiles, Brendon M.; Elemento, Olivier; Altorki, Nasser K.; McGraw, Timothy E.

2013-01-01

SUMMARY Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung tumor cancer-associated fibroblast lines (CAFs) to “normal” fibroblast lines (NFs) generated from adjacent non-neoplastic lung tissue. CAFs are pro-tumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor’s glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we demonstrate a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer. PMID:23475953

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

PubMed Central

Hu, Gangqing; Yu, Zu-Xi; Liu, Chengyu

2013-01-01

Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. PMID:24100015

Epigenetic reprogramming in mammalian species after SCNT-based cloning.

PubMed

Niemann, Heiner

2016-07-01

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. Copyright © 2016 Elsevier Inc. All rights reserved.

Human papillomavirus oncogenes reprogram the cervical cancer microenvironment independently of and synergistically with estrogen

PubMed Central

Spurgeon, Megan E.; den Boon, Johan A.; Horswill, Mark; Barthakur, Sonalee; Forouzan, Omid; Rader, Janet S.; Beebe, David J.; Roopra, Avtar; Ahlquist, Paul; Lambert, Paul F.

2017-01-01

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment. PMID:29073104

Algorithm for cellular reprogramming.

PubMed

Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

PubMed

Tagalakis, Aristides D; Munye, Mustafa M; Ivanova, Rositsa; Chen, Hanpeng; Smith, Claire M; Aldossary, Ahmad M; Rosa, Luca Z; Moulding, Dale; Barnes, Josephine L; Kafetzis, Konstantinos N; Jones, Stuart A; Baines, Deborah L; Moss, Guy W J; O'Callaghan, Christopher; McAnulty, Robin J; Hart, Stephen L

2018-05-10

Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (V t ), short circuit current (I sc ), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced V t , the amiloride-sensitive I sc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Aging and reprogramming: a two-way street

PubMed Central

Mahmoudi, Salah; Brunet, Anne

2012-01-01

Aging is accompanied by the functional decline of cells, tissues, and organs, as well as a striking increase in a wide range of diseases. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) opens new avenues for the aging field and has important applications for therapeutic treatments of age-related diseases. Here we review emerging studies on how aging and age-related pathways influence iPSC generation and property. We discuss the exciting possibility that reverting to a pluripotent stem cell stage erases several deficits associated with aging and will provide new strategies for rejuvenation. Finally, we argue that reprogramming provides a unique opportunity to model aging and perhaps exceptional longevity. PMID:23146768

Totipotency, Pluripotency and Nuclear Reprogramming

NASA Astrophysics Data System (ADS)

Mitalipov, Shoukhrat; Wolf, Don

Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

Adaptive mitochondrial reprogramming and resistance to PI3K therapy.

PubMed

Ghosh, Jagadish C; Siegelin, Markus D; Vaira, Valentina; Faversani, Alice; Tavecchio, Michele; Chae, Young Chan; Lisanti, Sofia; Rampini, Paolo; Giroda, Massimo; Caino, M Cecilia; Seo, Jae Ho; Kossenkov, Andrew V; Michalek, Ryan D; Schultz, David C; Bosari, Silvano; Languino, Lucia R; Altieri, Dario C

2015-03-01

Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4-5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial

Delayed transition to new cell fates during cellular reprogramming

PubMed Central

Cheng, Xianrui; Lyons, Deirdre C.; Socolar, Joshua E. S.; McClay, David R.

2014-01-01

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues. PMID:24780626

para-Sulfonatocalix[4]arene and polyamidoamine dendrimer nanocomplexes as delivery vehicles for a novel platinum anticancer agent.

PubMed

Pang, Chi Ting; Ammit, Alaina J; Ong, Yu Qing Elysia; Wheate, Nial J

2017-11-01

Novel para-sulfonatocalix[4]arene (sCX[4]) and polyamidoamine (PAMAM) dendrimer nanocomplexes were evaluated as delivery vehicles for the platinum anticancer agent [(1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)] chloride (PHENSS). Different ratios of sCX[4] to PHENSS were tested for their compatibility, with a ratio of 6:1 sCX[4]:PHENSS having the best solubility. The loading of sCX[4], and sCX[4]-bound PHENSS, onto three different generations of PAMAM dendrimers (G3.0-5.0) was examined using UV-visible spectrophotometry. The quantity of sCX[4] bound was found to increase exponentially with dendrimer size: G3, 15 sCX[4] molecules per dendrimer; G4, 37; and G5, 78. Similarly, the loading of sCX[4]-bound PHENSS also increased with increasing dendrimer size: G3, 7 PHENSS molecules per dendrimer; G4, 14; and G5, 28.5. The loading of sCX[4]-bound PHENSS molecules is significantly lower when compared with that of sCX[4], which indicates that less than half of the binding sites were occupied (45, 44, and 44%, respectively). By 1 H NMR and UV-vis analysis, the nanocomplex was found to be stable in NaCl solutions at concentrations up to 150mM. While PHENSS is more active in vitro than cisplatin against the human breast cancer cell line, MCF-7, delivery of PHENSS using the sCX[4]-dendrimer nanocomplexes, regardless of dendrimer generation, had little effect on PHENSS cytotoxicity. The results of this study may have application in the delivery of a variety of small molecule metal-based drugs for which chemical conjugation to a nanoparticle is undesired or not feasible. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct reprogramming and biomaterials for controlling cell fate.

PubMed

Kim, Eunsol; Tae, Giyoong

2016-01-01

Direct reprogramming which changes the fate of matured cell is a very useful technique with a great interest recently. This approach can eliminate the drawbacks of direct usage of stem cells and allow the patient specific treatment in regenerative medicine. Overexpression of diverse factors such as general reprogramming factors or lineage specific transcription factors can change the fate of already differentiated cells. On the other hand, biomaterials can provide physical and topographical cues or biochemical cues on cells, which can dictate or significantly affect the differentiation of stem cells. The role of biomaterials on direct reprogramming has not been elucidated much, but will be potentially significant to improve the efficiency or specificity of direct reprogramming. In this review, the strategies for general direct reprogramming and biomaterials-guided stem cell differentiation are summarized with the addition of the up-to-date progress on biomaterials for direct reprogramming.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

PubMed

Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

2018-01-01

Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

PubMed

Xue, Gang; Ren, Zhenxin; Grabham, Peter W; Chen, Yaxiong; Zhu, Jiayun; Du, Yarong; Pan, Dong; Li, Xiaoman; Hu, Burong

2015-07-01

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Conversion of partially reprogrammed cells to fully pluripotent stem cells is associated with further activation of stem cell maintenance- and gamete generation-related genes.

PubMed

Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Seo, Han Geuk; Moon, Sung-Hwan; Chung, Hyung-Min; Do, Jeong Tae

2014-11-01

Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

PubMed

El Khatib, Moustafa M; Ohmine, Seiga; Jacobus, Egon J; Tonne, Jason M; Morsy, Salma G; Holditch, Sara J; Schreiber, Claire A; Uetsuka, Koji; Fusaki, Noemi; Wigle, Dennis A; Terzic, Andre; Kudva, Yogish C; Ikeda, Yasuhiro

2016-05-01

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature β cell phenotype would lead to

Deterministic direct reprogramming of somatic cells to pluripotency.

PubMed

Rais, Yoach; Zviran, Asaf; Geula, Shay; Gafni, Ohad; Chomsky, Elad; Viukov, Sergey; Mansour, Abed AlFatah; Caspi, Inbal; Krupalnik, Vladislav; Zerbib, Mirie; Maza, Itay; Mor, Nofar; Baran, Dror; Weinberger, Leehee; Jaitin, Diego A; Lara-Astiaso, David; Blecher-Gonen, Ronnie; Shipony, Zohar; Mukamel, Zohar; Hagai, Tzachi; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Tanay, Amos; Amit, Ido; Novershtern, Noa; Hanna, Jacob H

2013-10-03

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Preparation and in vivo evaluation of an orally available enteric-microencapsulated parathyroid hormone (1-34)-deoxycholic acid nanocomplex

PubMed Central

Hwang, Seung Rim; Seo, Dong-Hyun; Byun, Youngro; Park, Jin Woo

2016-01-01

The N-terminal 34-amino-acid peptide fragment of human parathyroid hormone PTH (1-34), is used clinically to treat osteoporosis; however, it is currently administered by a once-daily subcutaneous injection, resulting in poor patient compliance. We have developed enteric microcapsules containing an ionic nanocomplex between PTH (1-34) and lysine-linked deoxycholic acid (LysDOCA) for the oral delivery of PTH (1-34). We measured the particle size of the PTH/LysDOCA complex and assessed its biological activity by determining the cAMP content in MC3T3-E1 cells. We also assessed its permeability across a Caco-2 cell monolayer and the bioavailability of the intrajejunally administered PTH/LysDOCA complex compared with PTH (1-34) in rats. In addition, the antiosteoporotic activity of the PTH/LysDOCA complex, encapsulated in an enteric carrier by coaxial ultrasonic atomization, was evaluated after it was orally administered to ovariectomized (OVX) rats. The formation of an ionic complex between PTH (1-34) and LysDOCA produced nanoparticles of diameter 33.0±3.36 nm, and the bioactivity of the complex was comparable with that of PTH (1-34). The Caco-2 cell permeability and AUClast value of the PTH/LysDOCA (1:10) nanocomplex increased by 2.87- and 16.3-fold, respectively, compared with PTH (1-34) alone. Furthermore, the OVX rats treated with oral PTH/LysDOCA-loaded enteric microcapsules showed an increase in bone mineral density (159%), bone volume fraction (175%), and trabecular number (174%) compared with those in the OVX control group. Therefore, the PTH/LysDOCA nanocomplex oral delivery system is a promising treatment modality for osteoporosis because it improves osteogenesis and trabecular connectivity. PMID:27621618

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

PubMed

Schmitt, Christopher E; Morales, Blanca M; Schmitz, Ellen M H; Hawkins, John S; Lizama, Carlos O; Zape, Joan P; Hsiao, Edward C; Zovein, Ann C

2017-06-05

Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Marine Corps Budgetary Reprogramming Effectiveness

DTIC Science & Technology

2015-03-01

infrastructure (Appropriations Act of Congress, 2008). The environmental restoration is a transfer account controlled by the DOD. Usually in the case of...at an average just over 11 percent and the Marine Corps encircle the backend of the DOD portion of reprogramming with the Marine Corps reprogramming...blue force tracker (BFT), radio systems, high mobility multipurpose wheeled vehicle (HMMWV), medium tactical vehicle replacement (MTVR), and

Small molecule-induced cellular fate reprogramming: promising road leading to Rome.

PubMed

Li, Xiang; Xu, Jun; Deng, Hongkui

2018-05-29

Cellular fate reprogramming holds great promise to generate functional cell types for replenishing new cells and restoring functional loss. Inspired by transcription factor-induced reprogramming, the field of cellular reprogramming has greatly advanced and developed into divergent streams of reprogramming approaches. Remarkably, increasing studies have shown the power and advantages of small molecule-based approaches for cellular fate reprogramming, which could overcome the limitations of conventional transgenic-based reprogramming. In this concise review, we discuss these findings and highlight the future potentiality with particular focus on this new trend of chemical reprogramming. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hodgkin lymphoma: A complex metabolic ecosystem with glycolytic reprogramming of the tumor microenvironment.

PubMed

Mikkilineni, Lekha; Whitaker-Menezes, Diana; Domingo-Vidal, Marina; Sprandio, John; Avena, Paola; Cotzia, Paolo; Dulau-Florea, Alina; Gong, Jerald; Uppal, Guldeep; Zhan, Tingting; Leiby, Benjamin; Lin, Zhao; Pro, Barbara; Sotgia, Federica; Lisanti, Michael P; Martinez-Outschoorn, Ubaldo

2017-06-01

Twenty percent of patients with classical Hodgkin Lymphoma (cHL) have aggressive disease defined as relapsed or refractory disease to initial therapy. At present we cannot identify these patients pre-treatment. The microenvironment is very important in cHL because non-cancer cells constitute the majority of the cells in these tumors. Non-cancer intra-tumoral cells, such as tumor-associated macrophages (TAMs) have been shown to promote tumor growth in cHL via crosstalk with the cancer cells. Metabolic heterogeneity is defined as high mitochondrial metabolism in some tumor cells and glycolysis in others. We hypothesized that there are metabolic differences between cancer cells and non-cancer tumor cells, such as TAMs and tumor-infiltrating lymphocytes in cHL and that greater metabolic differences between cancer cells and TAMs are associated with poor outcomes. A case-control study was conducted with 22 tissue samples of cHL at diagnosis from a single institution. The case samples were from 11 patients with aggressive cHL who had relapsed after standard treatment with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) or were refractory to this treatment. The control samples were from 11 patients with cHL who achieved a remission and never relapsed after ABVD. Reactive non-cancerous lymph nodes from four subjects served as additional controls. Samples were stained by immunohistochemistry for three metabolic markers: translocase of the outer mitochondrial membrane 20 (TOMM20), monocarboxylate transporter 1 (MCT1), and monocarboxylate transporter 4 (MCT4). TOMM20 is a marker of mitochondrial oxidative phosphorylation (OXPHOS) metabolism. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is a marker of OXPHOS. Monocarboxylate transporter 4 (MCT4) is the main lactate exporter out of cells and is a marker of glycolysis. The immunoreactivity for TOMM20, MCT1, and MCT4 was scored based on staining intensity and percentage of positive

Role of human oocyte-enriched factors in somatic cell reprograming.

PubMed

El-Gammal, Zaynab; AlOkda, Abdelrahman; El-Badri, Nagwa

2018-06-08

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Linking tumor glycolysis and immune evasion in cancer: Emerging concepts and therapeutic opportunities.

PubMed

Ganapathy-Kanniappan, Shanmugasundaram

2017-08-01

Metabolic reprogramming and immune evasion are two hallmarks of cancer. Metabolic reprogramming is exemplified by cancer's propensity to utilize glucose at an exponential rate which in turn is linked with "aerobic glycolysis", popularly known as the "Warburg effect". Tumor glycolysis is pivotal for the efficient management of cellular bioenergetics and uninterrupted cancer growth. Mounting evidence suggests that tumor glycolysis also plays a key role in instigating immunosuppressive networks that are critical for cancer cells to escape immune surveillance ("immune evasion"). Recent data show that induction of cellular stress or metabolic dysregulation sensitize cancer cells to antitumor immune cells implying that metabolic reprogramming and immune evasion harmonize during cancer progression. However, the molecular link between these two hallmarks of cancer remains obscure. In this review the molecular intricacies of tumor glycolysis that facilitate immune evasion has been discussed in the light of recent research to explore immunotherapeutic potential of targeting cancer metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

PubMed

Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth

PubMed Central

Brown, Kristin K.; Evason, Kimberley; Beltz, Sebastian; Tsomides, Allison; O'Connor, Keelin; Galli, Giorgio G.; Yimlamai, Dean; Chhangawala, Sagar; Yuan, Min; Lien, Evan C.; Wucherpfennig, Julia; Nissim, Sahar; Minami, Akihiro; Cohen, David E.; Camargo, Fernando D.; Asara, John M.; Houvras, Yariv; Stainier, Didier Y.R.; Goessling, Wolfram

2016-01-01

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signaling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumor formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppresses hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. PMID:27428308

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.

American Brain Tumor Association

MedlinePlus

... Brain Tumor Association Names Leslie M. Stokes Interim Chief Executive Officer and Begins Search for Permanent CEO September 7, ... American Brain Tumor Association Names Kelly Sitkin as Chief Advancement Officer Read More ABTA Live ABTA Facebook Follow @theabta ...

Advances in Reprogramming-Based Study of Neurologic Disorders

PubMed Central

Baldwin, Kristin K.

2015-01-01

The technology to convert adult human non-neural cells into neural lineages, through induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer, and direct lineage reprogramming or transdifferentiation has progressed tremendously in recent years. Reprogramming-based approaches aimed at manipulating cellular identity have enormous potential for disease modeling, high-throughput drug screening, cell therapy, and personalized medicine. Human iPSC (hiPSC)-based cellular disease models have provided proof of principle evidence of the validity of this system. However, several challenges remain before patient-specific neurons produced by reprogramming can provide reliable insights into disease mechanisms or be efficiently applied to drug discovery and transplantation therapy. This review will first discuss limitations of currently available reprogramming-based methods in faithfully and reproducibly recapitulating disease pathology. Specifically, we will address issues such as culture heterogeneity, interline and inter-individual variability, and limitations of two-dimensional differentiation paradigms. Second, we will assess recent progress and the future prospects of reprogramming-based neurologic disease modeling. This includes three-dimensional disease modeling, advances in reprogramming technology, prescreening of hiPSCs and creating isogenic disease models using gene editing. PMID:25749371

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

PubMed

Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

2016-11-22

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

PubMed Central

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

PubMed

Matrka, Marie C; Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F; Lane, Andrew N; Romick-Rosendale, Lindsey E; Wells, Susanne I

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Actin stress in cell reprogramming

PubMed Central

Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

2014-01-01

Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

Characterization of the Tumor Microenvironment and Tumor–Stroma Interaction by Non-invasive Preclinical Imaging

PubMed Central

Ramamonjisoa, Nirilanto; Ackerstaff, Ellen

2017-01-01

Tumors are often characterized by hypoxia, vascular abnormalities, low extracellular pH, increased interstitial fluid pressure, altered choline-phospholipid metabolism, and aerobic glycolysis (Warburg effect). The impact of these tumor characteristics has been investigated extensively in the context of tumor development, progression, and treatment response, resulting in a number of non-invasive imaging biomarkers. More recent evidence suggests that cancer cells undergo metabolic reprograming, beyond aerobic glycolysis, in the course of tumor development and progression. The resulting altered metabolic content in tumors has the ability to affect cell signaling and block cellular differentiation. Additional emerging evidence reveals that the interaction between tumor and stroma cells can alter tumor metabolism (leading to metabolic reprograming) as well as tumor growth and vascular features. This review will summarize previous and current preclinical, non-invasive, multimodal imaging efforts to characterize the tumor microenvironment, including its stromal components and understand tumor–stroma interaction in cancer development, progression, and treatment response. PMID:28197395

Reprogramming cellular identity for regenerative medicine

PubMed Central

Cherry, Anne B.C.; Daley, George Q.

2012-01-01

The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

PubMed

Feng, Dingqing; Yan, Keqin; Zhou, Ying; Liang, Haiyan; Liang, Jing; Zhao, Weidong; Dong, Zhongjun; Ling, Bin

2016-10-04

The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming.

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

PubMed Central

Singhal, Hari; Greene, Marianne E.; Zarnke, Allison L.; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G.; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B.; Nickisch, Klaus J.; Long, Henry W.; Becker, Lev; Brown, Myles; Greene, Geoffrey L.

2018-01-01

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Somatic cell reprogramming informed by the oocyte.

PubMed

Gonzalez-Munoz, Elena; Cibelli, Jose B

2018-05-08

The successful production of animals and embryonic stem cells (ESCs) using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency.

Spin glass model for dynamics of cell reprogramming

NASA Astrophysics Data System (ADS)

Pusuluri, Sai Teja; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2015-03-01

Recent experiments show that differentiated cells can be reprogrammed to become pluripotent stem cells. The possible cell fates can be modeled as attractors in a dynamical system, the ``epigenetic landscape.'' Both cellular differentiation and reprogramming can be described in the landscape picture as motion from one attractor to another attractor. We perform Monte Carlo simulations in a simple model of the landscape. This model is based on spin glass theory and it can be used to construct a simulated epigenetic landscape starting from the experimental genomic data. We re-analyse data from several cell reprogramming experiments and compare with our simulation results. We find that the model can reproduce some of the main features of the dynamics of cell reprogramming.

Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction.

PubMed

Miyamoto, Kazutaka; Akiyama, Mizuha; Tamura, Fumiya; Isomi, Mari; Yamakawa, Hiroyuki; Sadahiro, Taketaro; Muraoka, Naoto; Kojima, Hidenori; Haginiwa, Sho; Kurotsu, Shota; Tani, Hidenori; Wang, Li; Qian, Li; Inoue, Makoto; Ide, Yoshinori; Kurokawa, Junko; Yamamoto, Tsunehisa; Seki, Tomohisa; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2018-01-04

Direct cardiac reprogramming holds great promise for regenerative medicine. We previously generated directly reprogrammed induced cardiomyocyte-like cells (iCMs) by overexpression of Gata4, Mef2c, and Tbx5 (GMT) using retrovirus vectors. However, integrating vectors pose risks associated with insertional mutagenesis and disruption of gene expression and are inefficient. Here, we show that Sendai virus (SeV) vectors expressing cardiac reprogramming factors efficiently and rapidly reprogram both mouse and human fibroblasts into integration-free iCMs via robust transgene expression. SeV-GMT generated 100-fold more beating iCMs than retroviral-GMT and shortened the duration to induce beating cells from 30 to 10 days in mouse fibroblasts. In vivo lineage tracing revealed that the gene transfer of SeV-GMT was more efficient than retroviral-GMT in reprogramming resident cardiac fibroblasts into iCMs in mouse infarct hearts. Moreover, SeV-GMT improved cardiac function and reduced fibrosis after myocardial infarction. Thus, efficient, non-integrating SeV vectors may serve as a powerful system for cardiac regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Senescence-associated reprogramming promotes cancer stemness.

PubMed

Milanovic, Maja; Fan, Dorothy N Y; Belenki, Dimitri; Däbritz, J Henry M; Zhao, Zhen; Yu, Yong; Dörr, Jan R; Dimitrova, Lora; Lenze, Dido; Monteiro Barbosa, Ines A; Mendoza-Parra, Marco A; Kanashova, Tamara; Metzner, Marlen; Pardon, Katharina; Reimann, Maurice; Trumpp, Andreas; Dörken, Bernd; Zuber, Johannes; Gronemeyer, Hinrich; Hummel, Michael; Dittmar, Gunnar; Lee, Soyoung; Schmitt, Clemens A

2018-01-04

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16 INK4a , p21 CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse

Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

NASA Astrophysics Data System (ADS)

Vora, Lalit; Tyagi, Monica; Patel, Ketan; Gupta, Sanjay; Vavia, Pradeep

2014-12-01

The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP-PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX-CMP) via hydrazone and confirmed by FTIR and 1H-NMR. In vitro release studies of pH-sensitive DOX-CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP-PAA NCs or DOX-CMP-PAA NCs self-assembled into a nanosized (<250 nm) spherical shape as confirmed by DLS and TEM. The hemolysis and cytotoxicity study indicated that the CMP-PAA NCs did not show cytotoxicity in comparison with plain polyallylamine. Gel retardation assay showed complete binding of pDNA with CMP-PAA NCs at 1:2 weight ratio. CMP-PAA NCs/pDNA showed significantly higher transfection in HEK293 cells compared to PAA/pDNA complexes. Confocal imaging demonstrated successful cellular uptake of DOX-CMP-PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery.

Somatic cell cloning: the ultimate form of nuclear reprogramming?

PubMed

Piedrahita, Jorge A; Mir, Bashir; Dindot, Scott; Walker, Shawn

2004-05-01

With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

PubMed Central

2014-01-01

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

PubMed

Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

2016-04-14

Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

PubMed

Ikeda, Takashi; Uchiyama, Ikuo; Iwasaki, Mio; Sasaki, Tetsuhiko; Nakagawa, Masato; Okita, Keisuke; Masui, Shinji

2017-10-01

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Therapeutic cloning and cellular reprogramming.

PubMed

Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

2012-01-01

Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use.

Non-stochastic reprogramming from a privileged somatic cell state

PubMed Central

Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P.; Cheng, Jijun; Zhong, Mei; Koochaki, Sebastian H.J.; Megyola, Cynthia M.; Pan, Xinghua; Heydari, Kartoosh; Weissman, Sherman M.; Gallagher, Patrick G.; Krause, Diane S.; Fan, Rong; Lu, Jun

2014-01-01

SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PMID:24486105

Cytoplasmic GPER translocation in cancer-associated fibroblasts mediates cAMP/PKA/CREB/glycolytic axis to confer tumor cells with multidrug resistance.

PubMed

Yu, T; Yang, G; Hou, Y; Tang, X; Wu, C; Wu, X-A; Guo, L; Zhu, Q; Luo, H; Du, Y-E; Wen, S; Xu, L; Yin, J; Tu, G; Liu, M

2017-04-01

Multiple drug resistance is a challenging issue in the clinic. There is growing evidence that the G-protein-coupled estrogen receptor (GPER) is a novel mediator in the development of multidrug resistance in both estrogen receptor (ER)-positive and -negative breast cancers, and that cancer-associated fibroblasts (CAFs) in the tumor microenvironment may be a new agent that promotes drug resistance in tumor cells. However, the role of cytoplasmic GPER of CAFs on tumor therapy remains unclear. Here we first show that the breast tumor cell-activated PI3K/AKT (phosphoinositide 3-kinase/AKT) signaling pathway induces the cytoplasmic GPER translocation of CAFs in a CRM1-dependent pattern, and leads to the activation of a novel estrogen/GPER/cAMP/PKA/CREB signaling axis that triggers the aerobic glycolysis switch in CAFs. The glycolytic CAFs feed the extra pyruvate and lactate to tumor cells for augmentation of mitochondrial activity, and this energy metabolically coupled in a 'host-parasite relationship' between catabolic CAFs and anabolic cancer cells confers the tumor cells with multiple drug resistance to several conventional clinical treatments including endocrine therapy (tamoxifen), Her-2-targeted therapy (herceptin) and chemotherapy (epirubicin). Moreover, the clinical data from 18 F-fluorodeoxyglucose positron emission tomography/computed tomography further present a strong association between the GPER/cAMP/PKA/CREB pathway of stromal fibroblasts with tumor metabolic activity and clinical treatment, suggesting that targeting cytoplasmic GPER in CAFs may rescue the drug sensitivity in patients with breast cancer. Thus, our data define novel insights into the stromal GPER-mediated multiple drug resistance from the point of reprogramming of tumor energy metabolism and provide the rationale for CAFs as a promising target for clinical therapy.

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

PubMed Central

Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

2013-01-01

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance.

PubMed

Kang, Yoo Kyung; Kwon, Kyu; Ryu, Jea Sung; Lee, Ha Neul; Park, Chankyu; Chung, Hyun Jung

2017-04-19

The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.

Five classic articles in somatic cell reprogramming.

PubMed

Park, In-Hyun

2010-09-01

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

PubMed

Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Harnessing the cross-talk between tumor cells and tumor-associated macrophages with a nano-drug for modulation of glioblastoma immune microenvironment.

PubMed

Li, Tong-Fei; Li, Ke; Wang, Chao; Liu, Xin; Wen, Yu; Xu, Yong-Hong; Zhang, Quan; Zhao, Qiu-Ya; Shao, Ming; Li, Yan-Ze; Han, Min; Komatsu, Naoki; Zhao, Li; Chen, Xiao

2017-12-28

Glioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The presence of a large population of macrophages (Mφ) in the tumor microenvironment is a prominent feature of GBM and these so-called tumor-associated Mφ (TAM) closely interact with the GBM cells to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GBM cells or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GBM cells (GC) and TAM for modulation of the GBM microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work on human cell models, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. Nano-DOX was next proven to be a more potent inducer of GC DAMPs emission than doxorubicin. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, the in vivo relevance of the in vitro findings was tested in animal study. Mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with

Deterministic versus stochastic model of reprogramming: new evidence from cellular barcoding technique

PubMed Central

Yunusova, Anastasia M.; Fishman, Veniamin S.; Vasiliev, Gennady V.

2017-01-01

Factor-mediated reprogramming of somatic cells towards pluripotency is a low-efficiency process during which only small subsets of cells are successfully reprogrammed. Previous analyses of the determinants of the reprogramming potential are based on average measurements across a large population of cells or on monitoring a relatively small number of single cells with live imaging. Here, we applied lentiviral genetic barcoding, a powerful tool enabling the identification of familiar relationships in thousands of cells. High-throughput sequencing of barcodes from successfully reprogrammed cells revealed a significant number of barcodes from related cells. We developed a computer model, according to which a probability of synchronous reprogramming of sister cells equals 10–30%. We conclude that the reprogramming success is pre-established in some particular cells and, being a heritable trait, can be maintained through cell division. Thus, reprogramming progresses in a deterministic manner, at least at the level of cell lineages. PMID:28446707

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

PubMed

Qian, Li; Berry, Emily C; Fu, Ji-dong; Ieda, Masaki; Srivastava, Deepak

2013-06-01

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Chromatin reprogramming in breast cancer.

PubMed

Swinstead, Erin E; Paakinaho, Ville; Hager, Gordon

2018-04-24

Reprogramming of the chromatin landscape is a critical component to the transcriptional response in breast cancer. Effects of sex hormones such as estrogens and progesterone have been well described to have a critical impact on breast cancer proliferation. However, the complex network of the chromatin landscape, enhancer regions, and mode of function of steroid receptors (SRs) and other transcription factors (TFs), is an intricate web of signaling and functional processes that is still largely misunderstood at the mechanistic level. In this review, we describe what is currently known about the dynamic interplay between TFs with chromatin and the reprogramming of enhancer elements. Emphasis has been placed on characterizing the different modes of action of TFs in regulating enhancer activity, specifically, how different SRs target enhancer regions and reprogram chromatin in breast cancer cells. In addition, we discuss current techniques employed to study enhancer function at a genome-wide level. Further, we have noted recent advances in live cell imaging technology. These single cell approaches enable the coupling of population based assays with real-time studies to address many unsolved questions about SRs and chromatin dynamics in breast cancer.

Fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C.

PubMed

Lin, Zhaoyu; Liu, Fei; Shi, Peiliang; Song, Anying; Huang, Zan; Zou, Dayuan; Chen, Qin; Li, Jianxin; Gao, Xiang

2018-02-26

Changes in metabolic pathway preferences are key events in the reprogramming process of somatic cells to induced pluripotent stem cells (iPSCs). The optimization of metabolic conditions can enhance reprogramming; however, the detailed underlying mechanisms are largely unclear. By comparing the gene expression profiles of somatic cells, intermediate-phase cells, and iPSCs, we found that carnitine palmitoyltransferase (Cpt)1b, a rate-limiting enzyme in fatty acid oxidation, was significantly upregulated in the early stage of the reprogramming process. Mouse embryonic fibroblasts isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs were used for reprogramming. Various fatty acid oxidation-related metabolites were added during the reprogramming process. Colony counting and fluorescence-activated cell sorting (FACS) were used to calculate reprogramming efficiency. Fatty acid oxidation-related metabolites were measured by liquid chromatography-mass spectrometry. Seahorse was used to measure the level of oxidative phosphorylation. We found that overexpression of cpt1b enhanced reprogramming efficiency. Furthermore, palmitoylcarnitine or acetyl-CoA, the primary and final products of Cpt1-mediated fatty acid oxidation, also promoted reprogramming. In the early reprogramming process, fatty acid oxidation upregulated oxidative phosphorylation and downregulated protein kinase C activity. Inhibition of protein kinase C also promoted reprogramming. We demonstrated that fatty acid oxidation promotes reprogramming by enhancing oxidative phosphorylation and inhibiting protein kinase C activity in the early stage of the reprogramming process. This study reveals that fatty acid oxidation is crucial for the reprogramming efficiency.

Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4

PubMed Central

Corbineau, Sébastien; Lassalle, Bruno; Givelet, Maelle; Souissi-Sarahoui, Inès; Firlej, Virginie; Romeo, Paul Henri; Allemand, Isabelle; Riou, Lydia; Fouchet, Pierre

2017-01-01

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events. PMID:28052023

Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

PubMed

Eguizabal, C; Herrera, L; De Oñate, L; Montserrat, N; Hajkova, P; Izpisua Belmonte, J C

2016-09-01

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428. © 2016 AlphaMed Press.

Elixir of Life: Thwarting Aging With Regenerative Reprogramming.

PubMed

Beyret, Ergin; Martinez Redondo, Paloma; Platero Luengo, Aida; Izpisua Belmonte, Juan Carlos

2018-01-05

All living beings undergo systemic physiological decline after ontogeny, characterized as aging. Modern medicine has increased the life expectancy, yet this has created an aged society that has more predisposition to degenerative disorders. Therefore, novel interventions that aim to extend the healthspan in parallel to the life span are needed. Regeneration ability of living beings maintains their biological integrity and thus is the major leverage against aging. However, mammalian regeneration capacity is low and further declines during aging. Therefore, modalities that reinforce regeneration can antagonize aging. Recent advances in the field of regenerative medicine have shown that aging is not an irreversible process. Conversion of somatic cells to embryonic-like pluripotent cells demonstrated that the differentiated state and age of a cell is not fixed. Identification of the pluripotency-inducing factors subsequently ignited the idea that cellular features can be reprogrammed by defined factors that specify the desired outcome. The last decade consequently has witnessed a plethora of studies that modify cellular features including the hallmarks of aging in addition to cellular function and identity in a variety of cell types in vitro. Recently, some of these reprogramming strategies have been directly used in animal models in pursuit of rejuvenation and cell replacement. Here, we review these in vivo reprogramming efforts and discuss their potential use to extend the longevity by complementing or augmenting the regenerative capacity. © 2017 American Heart Association, Inc.

Generation of stable PDX derived cell lines using conditional reprogramming.

PubMed

Borodovsky, Alexandra; McQuiston, Travis J; Stetson, Daniel; Ahmed, Ambar; Whitston, David; Zhang, Jingwen; Grondine, Michael; Lawson, Deborah; Challberg, Sharon S; Zinda, Michael; Pollok, Brian A; Dougherty, Brian A; D'Cruz, Celina M

2017-12-06

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

Targeting CD147 for T to NK Lineage Reprogramming and Tumor Therapy.

PubMed

Geng, Jie-Jie; Tang, Juan; Yang, Xiang-Min; Chen, Ruo; Zhang, Yang; Zhang, Kui; Miao, Jin-Lin; Chen, Zhi-Nan; Zhu, Ping

2017-06-01

CD147 is highly expressed on the surface of numerous tumor cells to promote invasion and metastasis. Targeting these cells with CD147-specific antibodies has been validated as an effective approach for lung and liver cancer therapy. In the immune system, CD147 is recognized as a co-stimulatory receptor and impacts the outcome of thymic selection. Using T cell-specific deletion, we showed here that in thymus CD147 is indispensable for the stable αβ T cell lineage commitment: loss of CD147 biases both multipotent DN (double negative) and fully committed DP (double positive) cells into innate NK-like lineages. Mechanistically, CD147 deficiency results in impaired Wnt signaling and expression of BCL11b, a master transcription factor in determining T cell identity. In addition, functional blocking of CD147 by antibody phenocopies genetic deletion to enrich NK-like cells in the periphery. Furthermore, using a melanoma model and orthotopic liver cancer transplants, we showed that the augmentation of NK-like cells strongly associates with resistance against tumor growth upon CD147 suppression. Therefore, besides its original function in tumorigenesis, CD147 is also an effective surface target for immune modulation in tumor therapy. Copyright © 2017. Published by Elsevier B.V.

Biomimetic one-pot synthesis of gold nanoclusters/nanoparticles for targeted tumor cellular dual-modality imaging

NASA Astrophysics Data System (ADS)

Lin, Jing; Zhou, Zhijun; Li, Zhiming; Zhang, Chunlei; Wang, Xiansong; Wang, Kan; Gao, Guo; Huang, Peng; Cui, Daxiang

2013-04-01

Biomimetic synthesis has become a promising green pathway to prepare nanomaterials. In this study, bovine serum albumin (BSA)-conjugated gold nanoclusters/nanoparticles were successfully synthesized in water at room temperature by a protein-directed, solution-phase, green synthetic method. The synthesized BSA-Au nanocomplexes have fluorescence emission (588 nm) of gold nanoclusters and surface plasmon resonance of gold nanoparticles. The BSA-Au nanocomplexes display non-cytotoxicity and excellent biocompatibility on MGC803 gastric cancer cells. After conjugation of folic acid molecules, the obtained BSA-Au nanocomplexes showed highly selective targeting for MGC803 cells and dual-modality dark-field and fluorescence imaging.

Reprogramming the tumor microenvironment to enhance adoptive cellular therapy.

PubMed

Beavis, Paul A; Slaney, Clare Y; Kershaw, Michael H; Gyorki, David; Neeson, Paul J; Darcy, Phillip K

2016-02-01

The frontiers of cancer immunotherapy are extending in terms of both the range of cancer types that can potentially be targeted and the types of therapeutics that are in clinical development. The use of adoptive cellular therapy (ACT) and its derivative, chimeric antigen receptor (CAR) T cells, is currently limited to hematological malignancies and immunogenic cancers such as melanoma and renal cell carcinoma. Although ACT utilizing ex vivo expanded tumor-infiltrating lymphocytes (TIL) or engineered CAR/TCR T cells have undergone clinical trials for other solid cancers, their efficacy to date has been limited. This may be due, in part, to the immunosuppressive nature of the tumor microenvironment. The development of novel combination approaches which target the immunosuppressive network engineered by tumors has raised the possibility of using ACT for a broader range of cancers. This review summarizes the potential of such strategies and outlines the clinical relevance of these observations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Local epigenetic reprograming induced by G-quadruplex ligands

PubMed Central

Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-01-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here we demonstrate that impeding replication by small molecule-mediated stabilisation of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterisation of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps, first loss of H3K4me3 and subsequently DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming. PMID:29064488

Local epigenetic reprogramming induced by G-quadruplex ligands

NASA Astrophysics Data System (ADS)

Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-11-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

Cell reprogramming: Therapeutic potential and the promise of rejuvenation for the aging brain.

PubMed

López-León, Micaela; Outeiro, Tiago F; Goya, Rodolfo G

2017-11-01

Aging is associated with a progressive increase in the incidence of neurodegenerative diseases, with Alzheimer's (AD) and Parkinson's (PD) disease being the most conspicuous examples. Within this context, the absence of efficacious therapies for most age-related brain pathologies has increased the interest in regenerative medicine. In particular, cell reprogramming technologies have ushered in the era of personalized therapies that not only show a significant potential for the treatment of neurodegenerative diseases but also promise to make biological rejuvenation feasible. We will first review recent evidence supporting the emerging view that aging is a reversible epigenetic phenomenon. Next, we will describe novel reprogramming approaches that overcome some of the intrinsic limitations of conventional induced-pluripotent-stem-cell technology. One of the alternative approaches, lineage reprogramming, consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific transcription factors (TF). Another strategy, termed pluripotency factor-mediated direct reprogramming, uses universal TF to generate epigenetically unstable intermediates able to differentiate into somatic cell types in response to specific differentiation factors. In the third part we will review studies showing the potential relevance of the above approaches for the treatment of AD and PD. Copyright © 2017 Elsevier B.V. All rights reserved.

Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming.

PubMed

West, Jason A; Cook, April; Alver, Burak H; Stadtfeld, Matthias; Deaton, Aimee M; Hochedlinger, Konrad; Park, Peter J; Tolstorukov, Michael Y; Kingston, Robert E

2014-08-27

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions

PubMed Central

Yamakawa, Hiroyuki; Muraoka, Naoto; Miyamoto, Kazutaka; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Umei, Tomohiko; Akiyama, Mizuha; Kuishi, Yuki; Kurokawa, Junko; Furukawa, Tetsushi; Fukuda, Keiichi; Ieda, Masaki

2015-01-01

Summary Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming. PMID:26626177

Purine synthesis promotes maintenance of brain tumor initiating cells in glioma.

PubMed

Wang, Xiuxing; Yang, Kailin; Xie, Qi; Wu, Qiulian; Mack, Stephen C; Shi, Yu; Kim, Leo J Y; Prager, Briana C; Flavahan, William A; Liu, Xiaojing; Singer, Meromit; Hubert, Christopher G; Miller, Tyler E; Zhou, Wenchao; Huang, Zhi; Fang, Xiaoguang; Regev, Aviv; Suvà, Mario L; Hwang, Tae Hyun; Locasale, Jason W; Bao, Shideng; Rich, Jeremy N

2017-05-01

Brain tumor initiating cells (BTICs), also known as cancer stem cells, hijack high-affinity glucose uptake active normally in neurons to maintain energy demands. Here we link metabolic dysregulation in human BTICs to a nexus between MYC and de novo purine synthesis, mediating glucose-sustained anabolic metabolism. Inhibiting purine synthesis abrogated BTIC growth, self-renewal and in vivo tumor formation by depleting intracellular pools of purine nucleotides, supporting purine synthesis as a potential therapeutic point of fragility. In contrast, differentiated glioma cells were unaffected by the targeting of purine biosynthetic enzymes, suggesting selective dependence of BTICs. MYC coordinated the control of purine synthetic enzymes, supporting its role in metabolic reprogramming. Elevated expression of purine synthetic enzymes correlated with poor prognosis in glioblastoma patients. Collectively, our results suggest that stem-like glioma cells reprogram their metabolism to self-renew and fuel the tumor hierarchy, revealing potential BTIC cancer dependencies amenable to targeted therapy.

Understanding direct neuronal reprogramming-from pioneer factors to 3D chromatin.

PubMed

Ninkovic, Jovica; Götz, Magdalena

2018-06-14

Cell replacement therapies aim at reestablishment of neuronal circuits after brain injury, stroke or neurodegeneration. Recently, direct reprogramming of resident glial cells into the affected neuronal subtypes has become a feasible and promising option for central nervous system regeneration. Direct reprogramming relies on the implementation of a new transcriptional program defining the desired neuronal identity in fully differentiated glial cells implying the more or less complete down-regulation of the program for the former identity of the glial cell. Despite the enormous progress achieved in this regard with highly efficient in vivo reprogramming after injury, a number of hurdles still need to be resolved. One way to further improve direct neuronal reprogramming is to understand the molecular hurdles which we discuss with the focus on chromatin states of the starting versus the reprogrammed cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming

PubMed Central

Mohamed, Tamer M. A.; Stone, Nicole R.; Berry, Emily C.; Radzinsky, Ethan; Huang, Yu; Pratt, Karishma; Ang, Yen-Sin; Yu, Pengzhi; Wang, Haixia; Tang, Shibing; Magnitsky, Sergey; Ding, Sheng; Ivey, Kathryn N.; Srivastava, Deepak

2017-01-01

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Methods We screened 5,500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. Results We found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-β and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Conclusions Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration. PMID:27834668

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells

PubMed Central

Prima, Victor; Kaliberova, Lyudmila N.; Kaliberov, Sergey; Curiel, David T.; Kusmartsev, Sergei

2017-01-01

In recent years, it has been established that programmed cell death protein ligand 1 (PD-L1)–mediated inhibition of activated PD-1+ T lymphocytes plays a major role in tumor escape from immune system during cancer progression. Lately, the anti–PD-L1 and –PD-1 immune therapies have become an important tool for treatment of advanced human cancers, including bladder cancer. However, the underlying mechanisms of PD-L1 expression in cancer are not fully understood. We found that coculture of murine bone marrow cells with bladder tumor cells promoted strong expression of PD-L1 in bone marrow–derived myeloid cells. Tumor-induced expression of PD-L1 was limited to F4/80+ macrophages and Ly-6C+ myeloid-derived suppressor cells. These PD-L1–expressing cells were immunosuppressive and were capable of eliminating CD8 T cells in vitro. Tumor-infiltrating PD-L1+ cells isolated from tumor-bearing mice also exerted morphology of tumor-associated macrophages and expressed high levels of prostaglandin E2 (PGE2)-forming enzymes microsomal PGE2 synthase 1 (mPGES1) and COX2. Inhibition of PGE2 formation, using pharmacologic mPGES1 and COX2 inhibitors or genetic overexpression of PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), resulted in reduced PD-L1 expression. Together, our study demonstrates that the COX2/mPGES1/PGE2 pathway involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells and, therefore, reprogramming of PGE2 metabolism in tumor microenvironment provides an opportunity to reduce immune suppression in tumor host. PMID:28096371

A Blueprint for a Synthetic Genetic Feedback Controller to Reprogram Cell Fate.

PubMed

Del Vecchio, Domitilla; Abdallah, Hussein; Qian, Yili; Collins, James J

2017-01-25

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The AP-1 transcription factor FOSL1 causes melanocyte reprogramming and transformation.

PubMed

Maurus, K; Hufnagel, A; Geiger, F; Graf, S; Berking, C; Heinemann, A; Paschen, A; Kneitz, S; Stigloher, C; Geissinger, E; Otto, C; Bosserhoff, A; Schartl, M; Meierjohann, S

2017-09-07

The MAPK pathway is activated in the majority of melanomas and is the target of therapeutic approaches. Under normal conditions, it initiates the so-called immediate early response, which encompasses the transient transcription of several genes belonging to the AP-1 transcription factor family. Under pathological conditions, such as continuous MAPK pathway overactivation due to oncogenic alterations occurring in melanoma, these genes are constitutively expressed. The consequences of a permanent expression of these genes are largely unknown. Here, we show that FOSL1 is the main immediate early AP-1 member induced by melanoma oncogenes. We first examined its role in established melanoma cells. We found that FOSL1 is involved in melanoma cell migration as well as cell proliferation and anoikis-independent growth, which is mediated by the gene product of its target gene HMGA1, encoding a multipotent chromatin modifier. As FOSL1 expression is increased in patient melanoma samples compared to nevi, we investigated the effect of enhanced FOSL1 expression on melanocytes. Intriguingly, we found that FOSL1 acts oncogenic and transforms melanocytes, enabling subcutaneous tumor growth in vivo. During the process of transformation, FOSL1 reprogrammed the melanocytes and downregulated MITF in a HMGA1-dependent manner. At the same time, AXL was upregulated, leading to a shift in the MITF/AXL balance. Furthermore, FOSL1 re-enforced pro-tumorigenic transcription factors MYC, E2F3 and AP-1. Together, this led to the enhancement of several growth-promoting processes, such as ribosome biogenesis, cellular detachment and pyrimidine metabolism. Overall, we demonstrate that FOSL1 is a novel reprogramming factor for melanocytes with potent tumor transformation potential.

Tumor cell-derived microparticles polarize M2 tumor-associated macrophages for tumor progression.

PubMed

Ma, Ruihua; Ji, Tiantian; Chen, Degao; Dong, Wenqian; Zhang, Huafeng; Yin, Xiaonan; Ma, Jingwei; Liang, Xiaoyu; Zhang, Yi; Shen, Guanxin; Qin, Xiaofeng; Huang, Bo

2016-04-01

Despite identification of macrophages in tumors (tumor-associated macrophages, TAM) as potential targets for cancer therapy, the origin and function of TAM in the context of malignancy remain poorly characterized. Here, we show that microparticles (MPs), as a by-product, released by tumor cells act as a general mechanism to mediate M2 polarization of TAM. Taking up tumor MPs by macrophages is a very efficient process, which in turn results in the polarization of macrophages into M2 type, not only leading to promoting tumor growth and metastasis but also facilitating cancer stem cell development. Moreover, we demonstrate that the underlying mechanism involves the activation of the cGAS/STING/TBK1/STAT6 pathway by tumor MPs. Finally, in addition to murine tumor MPs, we show that human counterparts also possess consistent effect on human M2 polarization. These findings provide new insights into a critical role of tumor MPs in remodeling of tumor microenvironment and better understanding of the communications between tumors and macrophages.

Optical reprogramming with ultrashort femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans G.; Batista, Ana; König, Karsten

2015-03-01

The use of sub-15 femtosecond laser pulses in stem cell research is explored with particular emphasis on the optical reprogramming of somatic cells. The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.

A comparison of non-integrating reprogramming methods

PubMed Central

Schlaeger, Thorsten M; Daheron, Laurence; Brickler, Thomas R; Entwisle, Samuel; Chan, Karrie; Cianci, Amelia; DeVine, Alexander; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen S; Brookes, Emily; Cherry, Anne B C; Demirbas, Didem; Tsankov, Alexander M; Zon, Leonard I; Rubin, Lee L; Feinberg, Andrew P; Meissner, Alexander; Cowan, Chad A; Daley, George Q

2015-01-01

Human induced pluripotent stem cells (hiPSCs1–3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. PMID:25437882

Reprogramming: A Preventive Strategy in Hypertension Focusing on the Kidney

PubMed Central

Tain, You-Lin; Joles, Jaap A.

2015-01-01

Adulthood hypertension can be programmed in response to a suboptimal environment in early life. However, developmental plasticity also implies that one can prevent hypertension in adult life by administrating appropriate compounds during early development. We have termed this reprogramming. While the risk of hypertension has been assessed in many mother-child cohorts of human developmental programming, interventions necessary to prove causation and provide a reprogramming strategy are lacking. Since the developing kidney is particularly vulnerable to environmental insults and blood pressure is determined by kidney function, renal programming is considered key in developmental programming of hypertension. Common pathways, whereby both genetic and acquired developmental programming converge into the same phenotype, have been recognized. For instance, the same reprogramming interventions aimed at shifting nitric oxide (NO)-reactive oxygen species (ROS) balance, such as perinatal citrulline or melatonin supplements, can be protective in both genetic and developmentally programmed hypertension. Furthermore, a significantly increased expression of gene Ephx2 (soluble epoxide hydrolase) was noted in both genetic and acquired animal models of hypertension. Since a suboptimal environment is often multifactorial, such common reprogramming pathways are a practical finding for translation to the clinic. This review provides an overview of potential clinical applications of reprogramming strategies to prevent programmed hypertension. We emphasize the kidney in the following areas: mechanistic insights from human studies and animal models to interpret programmed hypertension; identified risk factors of human programmed hypertension from mother-child cohorts; and the impact of reprogramming strategies on programmed hypertension from animal models. It is critical that the observed effects on developmental reprogramming in animal models are replicated in human studies. PMID

Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis

PubMed Central

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; Bianchi, Cristina; Johnstone, Megan E.; Donohoe, Dallas R.; Trott, Josephine F.; Aboud, Omran Abu; Stirdivant, Steven; Neri, Bruce; Wolfert, Robert; Stewart, Benjamin; Perego, Roberto; Hsieh, James J.; Weiss, Robert H.

2015-01-01

Kidney cancer (or renal cell carcinoma [RCC]) is known as “the internist’s tumor” because it has protean systemic manifestations suggesting it utilizes complex, non-physiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the VHL tumor suppressor, in characterizing higher grade tumors we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, while the β-oxidation pathway is inhibited leading to increased fatty acyl-carnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared to other metabolic pathways. Together, our results offer a rationale to evaluate novel anti-metabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC. PMID:25952651

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions.

PubMed

Yamakawa, Hiroyuki; Muraoka, Naoto; Miyamoto, Kazutaka; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Umei, Tomohiko; Akiyama, Mizuha; Kuishi, Yuki; Kurokawa, Junko; Furukawa, Tetsushi; Fukuda, Keiichi; Ieda, Masaki

2015-12-08

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Developmental Programming of Adult Disease: Reprogramming by Melatonin?

PubMed

Tain, You-Lin; Huang, Li-Tung; Hsu, Chien-Ning

2017-02-16

Adult-onset chronic non-communicable diseases (NCDs) can originate from early life through so-called the "developmental origins of health and disease" (DOHaD) or "developmental programming". The DOHaD concept offers the "reprogramming" strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

PubMed

Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming

PubMed Central

Mitani, Yasuyuki; Vagnozzi, Ronald J.; Millay, Douglas P.

2017-01-01

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non–muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle–specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.—Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. PMID:27825107

On metabolic reprogramming and tumor biology: A comprehensive survey of metabolism in breast cancer

PubMed Central

Penkert, Judith; Ripperger, Tim; Schieck, Maximilian; Schlegelberger, Brigitte; Steinemann, Doris; Illig, Thomas

2016-01-01

Altered metabolism in tumor cells has been a focus of cancer research for as long as a century but has remained controversial and vague due to an inhomogeneous overall picture. Accumulating genomic, metabolomic, and lastly panomic data as well as bioenergetics studies of the past few years enable a more comprehensive, systems-biologic approach promoting deeper insight into tumor biology and challenging hitherto existing models of cancer bioenergetics. Presenting a compendium on breast cancer-specific metabolome analyses performed thus far, we review and compile currently known aspects of breast cancer biology into a comprehensive network, elucidating previously dissonant issues of cancer metabolism. As such, some of the aspects critically discussed in this review include the dynamic interplay or metabolic coupling between cancer (stem) cells and cancer-associated fibroblasts, the intratumoral and intertumoral heterogeneity and plasticity of cancer cell metabolism, the existence of distinct metabolic tumor compartments in need of separate yet simultaneous therapeutic targeting, the reliance of cancer cells on oxidative metabolism and mitochondrial power, and the role of pro-inflammatory, pro-tumorigenic stromal conditioning. Comprising complex breast cancer signaling networks as well as combined metabolomic and genomic data, we address metabolic consequences of mutations in tumor suppressor genes and evaluate their contribution to breast cancer predisposition in a germline setting, reasoning for distinct personalized preventive and therapeutic measures. The review closes with a discussion on central root mechanisms of tumor cell metabolism and rate-limiting steps thereof, introducing essential strategies for therapeutic targeting. PMID:27590516

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

NASA Astrophysics Data System (ADS)

Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

2017-10-01

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.

Intratumoral CpG-B promotes anti-tumoral neutrophil, cDC, and T cell cooperation without reprograming tolerogenic pDC.

PubMed

Humbert, Marion; Guery, Leslie; Brighouse, Dale; Lemeille, Sylvain; Hugues, Stephanie

2018-03-27

Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunological outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated (TA) pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells (cDC), subsequent increased anti-tumor T cell priming in draining lymph nodes, and enhanced effector T cell infiltration in the tumor microenvironment. These results reinforce the concept that intratumoral delivery of TLR9 agonists alters the tumor microenvironment by improving the anti-tumor activity of both innate and adaptive immune cells. Copyright ©2018, American Association for Cancer Research.

The RON Receptor Tyrosine Kinase Promotes Metastasis by Triggering MBD4-Dependent DNA Methylation Reprogramming

PubMed Central

Cunha, Stéphanie; Lin, Yi-Chun; Goossen, Elizabeth A.; DeVette, Christa I.; Albertella, Mark R.; Thomson, Stuart; Mulvihill, Mark J.; Welm, Alana L.

2017-01-01

SUMMARY Metastasis is the major cause of death in cancer patients, yet the genetic and epigenetic programs that drive metastasis are poorly understood. Here, we report an epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by the activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in the misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a pharmacological agent blocks metastasis of patient-derived breast tumor grafts in vivo. PMID:24388747

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

PubMed

Bektik, Emre; Dennis, Adrienne; Pawlowski, Gary; Zhou, Chen; Maleski, Danielle; Takahashi, Satoru; Laurita, Kenneth R; Deschênes, Isabelle; Fu, Ji-Dong

2018-05-04

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFP high iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFP low cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

PubMed Central

Kelton, William; Waindok, Ann Cathrin; Pesch, Theresa; Pogson, Mark; Ford, Kyle; Parola, Cristina; Reddy, Sai T.

2017-01-01

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation. PMID:28374766

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis.

PubMed

Manunta, Maria D I; Tagalakis, Aristides D; Attwood, Martin; Aldossary, Ahmad M; Barnes, Josephine L; Munye, Mustafa M; Weng, Alexander; McAnulty, Robin J; Hart, Stephen L

2017-04-06

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.

Tumor-associated fibrosis as a regulator of tumor immunity and response to immunotherapy.

PubMed

Jiang, Hong; Hegde, Samarth; DeNardo, David G

2017-08-01

Tumor-associated fibrosis is characterized by unchecked pro-fibrotic and pro-inflammatory signaling. The components of fibrosis including significant numbers of cancer-associated fibroblasts, dense collagen deposition, and extracellular matrix stiffness, are well appreciated regulators of tumor progression but may also be critical regulators of immune surveillance. While this suggests that the efficacy of immunotherapy may be limited in highly fibrotic cancers like pancreas, it also suggests a therapeutic opportunity to target fibrosis in these tumor types to reawaken anti-tumor immunity. This review discusses the mechanisms by which fibrosis might subvert tumor immunity and how to overcome these mechanisms.

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

MiR-29b antagonizes the pro-inflammatory tumor-promoting activity of multiple myeloma-educated dendritic cells

PubMed Central

Botta, C; Cucè, M; Pitari, M R; Caracciolo, D; Gullà, A; Morelli, E; Riillo, C; Biamonte, L; Gallo Cantafio, M E; Prabhala, R; Mignogna, C; Di Vito, A; Altomare, E; Amodio, N; Di Martino, M T; Correale, P; Rossi, M; Giordano, A; Munshi, N C; Tagliaferri, P; Tassone, P

2018-01-01

Dendritic cells (DCs) have a key role in regulating tumor immunity, tumor cell growth and drug resistance. We hypothesized that multiple myeloma (MM) cells might recruit and reprogram DCs to a tumor-permissive phenotype by changes within their microRNA (miRNA) network. By analyzing six different miRNA-profiling data sets, miR-29b was identified as the only miRNA upregulated in normal mature DCs and significantly downregulated in tumor-associated DCs. This finding was validated in primary DCs co-cultured in vitro with MM cell lines and in primary bone marrow DCs from MM patients. In DCs co-cultured with MM cells, enforced expression of miR-29b counteracted pro-inflammatory pathways, including signal transducer and activator of transcription 3 and nuclear factor-κB, and cytokine/chemokine signaling networks, which correlated with patients’ adverse prognosis and development of bone disease. Moreover, miR-29b downregulated interleukin-23 in vitro and in the SCID-synth-hu in vivo model, and antagonized a Th17 inflammatory response. All together, these effects translated into strong anti-proliferative activity and reduction of genomic instability of MM cells. Our study demonstrates that MM reprograms the DCs functional phenotype by downregulating miR-29b whose reconstitution impairs DCs ability to sustain MM cell growth and survival. These results underscore miR-29b as an innovative and attractive candidate for miRNA-based immune therapy of MM. PMID:29158557

Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

PubMed

Malinowski, Andrzej R; Fisher, Amanda G

2016-01-01

Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

Cellular trajectories and molecular mechanisms of iPSC reprogramming.

PubMed

Apostolou, Effie; Stadtfeld, Matthias

2018-06-16

The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

PubMed Central

Li, Dong; Secher, Jan O.; Mashayekhi, Kaveh; Nielsen, Troels T.; Hyttel, Poul; Freude, Kristine K.

2017-01-01

ABSTRACT Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore, reprogramming of SSEA-1+ sorted pEFs led to higher reprogramming efficiency. Subsequent transcriptome profiling of the SSEA-1+ vs. the SSEA-1neg cell fraction revealed highly comparable gene signatures. However several genes that were found to be upregulated in the SSEA-1+ cells were similarly expressed in mesenchymal stem cells (MSCs). We therefore termed these cells SSEA-1 Expressing Enhanced Reprogramming (SEER) cells. Interestingly, SEER cells were more effective at differentiating into osteocytes and chondrocytes in vitro. We conclude that SEER cells are more amenable for reprogramming and that the expression of mesenchymal stem cell genes is advantageous in the reprogramming process. This data provides evidence supporting the elite theory and helps to delineate which cell types and specific genes are important for reprogramming in the pig. PMID:28426281

Glucose enhances tilapia against Edwardsiella tarda infection through metabolome reprogramming.

PubMed

Zeng, Zao-hai; Du, Chao-Chao; Liu, Shi-Rao; Li, Hui; Peng, Xuan-Xian; Peng, Bo

2017-02-01

We have recently reported that the survival of tilapia, Oreochromis niloticus, during Edwardsiella tarda infection is tightly associated with their metabolome, where the survived O. niloticus has distinct metabolomic profile to dying O. niloticus. Glucose is the key metabolite to distinguish the survival- and dying-metabolome. More importantly, exogenous administration of glucose to the fish greatly enhances their survival for the infection, indicating the functional roles of glucose in metabolome repurposing, known as reprogramming metabolomics. However, the underlying information for the reprogramming is not yet available. Here, GC/MS based metabolomics is used to understand the mechanisms by which how exogenous glucose elevates O. niloticus, anti-infectious ability to E. tarda. Results showed that exogenous glucose promotes stearic acid and palmitic acid biosynthesis but attenuates TCA cycle to potentiate O. niloticus against bacterial infection, which is confirmed by the fact that exogenous stearic acid increases immune protection in O. niloticus against E. tarda infection in a manner of Mx protein. These results indicate that exogenous glucose reprograms O. niloticus anti-infective metabolome that characterizes elevation of stearic acid and palmitic acid and attenuation of the TCA cycle. Therefore, our results proposed a novel mechanism that glucose promotes unsaturated fatty acid biosynthesis to cope with infection, thereby highlighting a potential way of enhancing fish immunity in aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

The synergistic effect of nitrogen-doped titanium dioxide/mercaptobenzoic acid/silver nanocomplexes for surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Feng, Jun; Bao, Wenyuan; Li, Lijun; Cheng, Hao; Huang, Wenyi; Kong, Hongxing; Li, Yanqing

2018-03-01

We synthesized titanium dioxide (TiO2) and nitrogen-doped TiO2 nanoparticles (N-TiO2 NPs) via a sol-hydrothermal method using ammonium chloride (NH4Cl) as the nitrogen (N) source. Furthermore, an N-TiO2/4-mercaptobenzoic acid (4-MBA)/silver (Ag) nanocomplex served as an active substrate for surface-enhanced Raman scattering (SERS) and was prepared by self-assembly. During SERS, the Raman signals of 4-MBA of the N-TiO2/MBA/Ag nanocomplexes exhibited higher intensity and sensitivity than pure TiO2/MBA/Ag, with 1% N doping in N-TiO2, producing the strongest Raman signals. We characterized the N-TiO2 hybrid materials by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and ultraviolet-visible diffuse reflectance spectra. N doping did not influence the phase of the TiO2 crystal. The doped N entered into the crystal lattice of the TiO2, replacing some oxygen (O) to form Ti-O-N or Ti-N-O linkage. The results indicated that an appropriate amount of N doping could enhance the SERS performance of the TiO2 SERS substrate via N substitution doping. These doping forms were beneficial to the molecular charge transfer (CT), and this resulted in improved SERS performance for N-doped TiO2 NPs. We attributed this improvement to the formation of N-doping energy levels that were beneficial to the process of TiO2 to MBA molecule CT. This work not only enriched the nonmetal-doped CT mechanism in SERS but also provided several reference values for practical applications. [Figure not available: see fulltext.

The association between glomus tumors and neurofibromatosis.

PubMed

Harrison, Bridget; Moore, Amy M; Calfee, Ryan; Sammer, Douglas M

2013-08-01

To determine whether an epidemiologic association exists between glomus tumors and neurofibromatosis. Using a pathology database, we established a study cohort consisting of all patients who had undergone excision of a glomus tumor of the hand between 1995 and 2010. We created a control cohort by randomly selecting 200 patients who had undergone excision of a ganglion cyst over the same period. We reviewed medical records for each cohort to identify patients with a diagnosis of neurofibromatosis. We calculated the odds ratio was calculated and performed Fisher's exact test to determine the significance of the association. We identified 21 patients with glomus tumors of the hand. Six of these patients carried the diagnosis of neurofibromatosis (29%). In contrast, no patients in the control group carried the diagnosis of neurofibromatosis. The odds ratio for a diagnosis of neurofibromatosis in association with a glomus tumor compared with controls was 168:1. This study provides evidence of a strong epidemiologic association between glomus tumors and neurofibromatosis. Glomus tumor should be included in the differential diagnosis in neurofibromatosis patients who present with a painful lesion of the hand or finger. Diagnostic III. Copyright © 2013 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells

PubMed Central

Sharma, Bal Krishan; Kolhe, Ravindra; Black, Stephen M.; Keller, Jonathan R.; Mivechi, Nahid F.; Satyanarayana, Ande

2016-01-01

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50–70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice

PubMed Central

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C.; Zhou, Qiao

2016-01-01

Aims/hypothesis Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Methods Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. Results We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue’s response to clear the pancreas from insufficiently reprogrammed cells. Conclusions/interpretation Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications. PMID:26693711

Hyperglycaemia attenuates in vivo reprogramming of pancreatic exocrine cells to beta cells in mice.

PubMed

Cavelti-Weder, Claudia; Li, Weida; Zumsteg, Adrian; Stemann-Andersen, Marianne; Zhang, Yuemei; Yamada, Takatsugu; Wang, Max; Lu, Jiaqi; Jermendy, Agnes; Bee, Yong Mong; Bonner-Weir, Susan; Weir, Gordon C; Zhou, Qiao

2016-03-01

Reprogramming of pancreatic exocrine to insulin-producing cells by viral delivery of the genes encoding transcription factors neurogenin-3 (Ngn3), pancreas/duodenum homeobox protein 1 (Pdx1) and MafA is an efficient method for reversing diabetes in murine models. The variables that modulate reprogramming success are currently ill-defined. Here, we assess the impact of glycaemia on in vivo reprogramming in a mouse model of streptozotocin-induced beta cell ablation, using subsequent islet transplantation or insulin pellet implantation for creation of groups with differing levels of glycaemia before viral delivery of transcription factors. We observed that hyperglycaemia significantly impaired reprogramming of exocrine to insulin-producing cells in their quantity, differentiation status and function. With hyperglycaemia, the reprogramming of acinar towards beta cells was less complete. Moreover, inflammatory tissue changes within the exocrine pancreas including macrophage accumulation were found, which may represent the tissue's response to clear the pancreas from insufficiently reprogrammed cells. Our findings shed light on normoglycaemia as a prerequisite for optimal reprogramming success in a diabetes model, which might be important in other tissue engineering approaches and disease models, potentially facilitating their translational applications.

Biophysical regulation of epigenetic state and cell reprogramming

NASA Astrophysics Data System (ADS)

Downing, Timothy L.; Soto, Jennifer; Morez, Constant; Houssin, Timothee; Fritz, Ashley; Yuan, Falei; Chu, Julia; Patel, Shyam; Schaffer, David V.; Li, Song

2013-12-01

Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells’ epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PubMed Central

Winiecka-Klimek, Marta; Smolarz, Maciej; Walczak, Maciej P.; Zieba, Jolanta; Hulas-Bigoszewska, Krystyna; Kmieciak, Blazej; Piaskowski, Sylwester; Rieske, Piotr; Grzela, Dawid P.; Stoczynska-Fidelus, Ewelina

2015-01-01

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed

Efficient Direct Lineage Reprogramming of Fibroblasts into Induced Cardiomyocytes Using Nanotopographical Cues.

PubMed

Yoo, Junsang; Chang, Yujung; Kim, Hongwon; Baek, Soonbong; Choi, Hwan; Jeong, Gun-Jae; Shin, Jaein; Kim, Hongnam; Kim, Byung-Soo; Kim, Jongpil

2017-03-01

Induced cardiomyocytes (iCMs) generated via direct lineage reprogramming offer a novel therapeutic target for the study and treatment of cardiac diseases. However, the efficiency of iCM generation is significantly low for therapeutic applications. Here, we show an efficient direct conversion of somatic fibroblasts into iCMs using nanotopographic cues. Compared with flat substrates, the direct conversion of fibroblasts into iCMs on nanopatterned substrates resulted in a dramatic increase in the reprogramming efficiency and maturation of iCM phenotypes. Additionally, enhanced reprogramming by substrate nanotopography was due to changes in the activation of focal adhesion kinase and specific histone modifications. Taken together, these results suggest that nanotopographic cues can serve as an efficient stimulant for direct lineage reprogramming into iCMs.

Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

PubMed

Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P; Schöler, Hans R; Mitalipov, Shoukhrat

2014-05-01

Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative

[Autoimmune Encephalitis Associated with Malignant Tumors].

PubMed

Inuzuka, Takashi

2016-09-01

Autoimmune encephalitis consists of limbic symptoms and signs associated with antibodies against neuronal cell-surface antigens or intracellular antigens. Some cases are known to be associated with anti-channel or anti-receptor-related molecule antibodies. Whether these cases are paraneoplastic depends on the kinds of antigens that the antibodies are produced against. Other cases due to well-characterized onco-neural antibodies are almost always paraneoplastic and are generally resistant to anti-tumor therapy and/or immunotherapy. An exception is anti-Ma2 antibody-positive encephalitis associated with a testicular tumor. Antibodies for intracellular antigens are considered not to be pathogenic. Rather, the T-cell response is thought to be responsible. These antibodies are useful markers for the diagnosis of paraneoplastic disorders and in the search for underlying cancer, as neurological symptoms often precede tumor diagnosis. There is a relationship among onco-neural antibodies, clinical features, tumor types, and response to immunotherapy. Here we describe the characteristics of autoimmune encephalitis cases with antibodies against different intracellular antigens, such as Hu, Ma2, CRMP5, or amphiphysin.

Glycolytic reprogramming through PCK2 regulates tumor initiation of prostate cancer cells

PubMed Central

Zhao, Jiangsha; Li, Jieran; Fan, Teresa W.M.; Hou, Steven X.

2017-01-01

Tumor-initiating cells (TICs) play important roles in tumor progression and metastasis. Identifying the factors regulating TICs may open new avenues in cancer therapy. Here, we show that TIC-enriched prostate cancer cell clones use more glucose and secrete more lactate than TIC-low clones. We determined that elevated levels of phosphoenolpyruvate carboxykinase isoform 2 (PCK2) are critical for the metabolic switch and the maintenance of TICs in prostate cancer. Information from prostate cancer patient databases revealed that higher PCK2 levels correlated with more aggressive tumors and lower survival rates. PCK2 knockdown resulted in low TIC numbers, increased cytosolic acetyl-CoA and cellular protein acetylation. Our data suggest PCK2 promotes tumor initiation by lowering acetyl-CoA level through reducing the mitochondrial tricarboxylic acid (TCA) cycle. Thus, PCK2 is a potential therapeutic target for aggressive prostate tumors. PMID:29137367

Syndrome-Associated Tumors by Organ System

PubMed Central

Gonzalez, Raul S.; Riddle, Nicole D.

2016-01-01

Certain tumors suggest the possibility of a patient harboring a genetic syndrome, particularly in children. Syndrome-associated tumors of the gastrointestinal tract, genitourinary tract, gynecologic tract, heart, lungs, brain, eye, endocrine organs, and hematopoietic system will be briefly discussed. PMID:27617151

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Extensive epigenetic reprogramming during the life cycle of Marchantia polymorpha.

PubMed

Schmid, Marc W; Giraldo-Fonseca, Alejandro; Rövekamp, Moritz; Smetanin, Dmitry; Bowman, John L; Grossniklaus, Ueli

2018-01-25

In plants, the existence and possible role of epigenetic reprogramming has been questioned because of the occurrence of stably inherited epialleles. Evidence suggests that epigenetic reprogramming does occur during land plant reproduction, but there is little consensus on the generality and extent of epigenetic reprogramming in plants. We studied DNA methylation dynamics during the life cycle of the liverwort Marchantia polymorpha. We isolated thalli and meristems from male and female gametophytes, archegonia, antherozoids, as well as sporophytes at early and late developmental stages, and compared their DNA methylation profiles. Of all cytosines tested for differential DNA methylation, 42% vary significantly in their methylation pattern throughout the life cycle. However, the differences are limited to few comparisons between specific stages of the life cycle and suggest four major epigenetic states specific to sporophytes, vegetative gametophytes, antherozoids, and archegonia. Further analyses indicated clear differences in the mechanisms underlying reprogramming in the gametophytic and sporophytic generations, which are paralleled by differences in the expression of genes involved in DNA methylation. Differentially methylated cytosines with a gain in methylation in antherozoids and archegonia are enriched in the CG and CHG contexts, as well as in gene bodies and gene flanking regions. In contrast, gain of DNA methylation during sporophyte development is mostly limited to the CHH context, LTR retrotransposons, DNA transposons, and repeats. We conclude that epigenetic reprogramming occurs at least twice during the life cycle of M. polymorpha and that the underlying mechanisms are likely different between the two events.

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes

PubMed Central

Masserdotti, Giacomo; Gillotin, Sébastien; Sutor, Bernd; Drechsel, Daniela; Irmler, Martin; Jørgensen, Helle F.; Sass, Steffen; Theis, Fabian J.; Beckers, Johannes; Berninger, Benedikt; Guillemot, François; Götz, Magdalena

2015-01-01

Summary Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming. PMID:26119235

Prognostic significance of tumor-associated macrophages in endometrial adenocarcinoma.

PubMed

Kübler, Kirsten; Ayub, Tiyasha H; Weber, Sarah K; Zivanovic, Oliver; Abramian, Alina; Keyver-Paik, Mignon-Denise; Mallmann, Michael R; Kaiser, Christina; Serçe, Nuran Bektas; Kuhn, Walther; Rudlowski, Christian

2014-11-01

Endometrial adenocarcinoma is one of the most common gynecologic malignancies worldwide and in stages confined to the uterus considered to have an excellent prognosis. However, in advanced or recurrent cases when surgery fails to achieve disease control other treatment options are less effective. Thus, new therapeutic avenues are needed. To provide the rationale for the use of novel agents that target immune checkpoints 163 type I endometrial cancer samples were immunohistochemically screened for the presence of CD163(+) tumor-associated macrophages and Foxp3(+) regulatory T cells. Further, a D2-40-based evaluation of lymph vessel density and lymphovascular space invasion was carried out. Correlation analysis with clinicopathological parameters was performed; Kaplan-Meier curves were generated; multivariate analysis was undertaken as appropriate. A substantial amount of tumor-associated macrophages and regulatory T cells was detected in all specimens characterizing endometrial cancer as an immunogenic tumor. However, only the increased infiltration of tumor-associated macrophages was proportionally associated with advanced FIGO stages, high tumor grade, increased lymph vessel density, lymphovascular space invasion and lymph node metastasis. Thus, the presence of tumor-associated macrophages indicates aggressive tumor behavior and appeared to be an independent prognostic factor for recurrence-free survival. Our results make future therapeutic approaches that target tumor-associated macrophages reasonable to improve the outcome of women with advanced or recurrent endometrial adenocarcinoma. Copyright © 2014 Elsevier Inc. All rights reserved.

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

PubMed Central

Qian, Li; Huang, Yu; Spencer, C. Ian; Foley, Amy; Vedantham, Vasanth; Liu, Lei; Conway, Simon J.; Fu, Ji-dong; Srivastava, Deepak

2012-01-01

SUMMARY The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here, we use genetic lineage-tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became bi-nucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast activating peptide, Thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. PMID:22522929

p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

PubMed

Patel, Vivekkumar; Singh, Vivek P; Pinnamaneni, Jaya Pratap; Sanagasetti, Deepthi; Olive, Jacqueline; Mathison, Megumi; Cooney, Austin; Flores, Elsa R; Crystal, Ronald G; Yang, Jianchang; Rosengart, Todd K

2018-04-13

Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. p63 Knockout ( -/- ) and knockdown murine embryonic fibroblasts (MEFs), p63 -/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM). Flow cytometry revealed that a significantly greater number of p63 -/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63 -/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63 -/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

PlGF/VEGFR-1 Signaling Promotes Macrophage Polarization and Accelerated Tumor Progression in Obesity.

PubMed

Incio, Joao; Tam, Josh; Rahbari, Nuh N; Suboj, Priya; McManus, Dan T; Chin, Shan M; Vardam, Trupti D; Batista, Ana; Babykutty, Suboj; Jung, Keehoon; Khachatryan, Anna; Hato, Tai; Ligibel, Jennifer A; Krop, Ian E; Puchner, Stefan B; Schlett, Christopher L; Hoffmman, Udo; Ancukiewicz, Marek; Shibuya, Masabumi; Carmeliet, Peter; Soares, Raquel; Duda, Dan G; Jain, Rakesh K; Fukumura, Dai

2016-06-15

Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity. We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples. We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity. Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. Clin Cancer Res; 22(12); 2993-3004. ©2016 AACR. ©2016 American Association for Cancer Research.

Developmental Programming of Adult Disease: Reprogramming by Melatonin?

PubMed Central

Tain, You-Lin; Huang, Li-Tung; Hsu, Chien-Ning

2017-01-01

Adult-onset chronic non-communicable diseases (NCDs) can originate from early life through so-called the “developmental origins of health and disease” (DOHaD) or “developmental programming”. The DOHaD concept offers the “reprogramming” strategy to shift the treatment from adulthood to early life, before clinical disease is apparent. Melatonin, an endogenous indoleamine produced by the pineal gland, has pleiotropic bioactivities those are beneficial in a variety of human diseases. Emerging evidence support that melatonin is closely inter-related to other proposed mechanisms contributing to the developmental programming of a variety of chronic NCDs. Recent animal studies have begun to unravel the multifunctional roles of melatonin in many experimental models of developmental programming. Even though some progress has been made in research on melatonin as a reprogramming strategy to prevent DOHaD-related NCDs, future human studies should aim at filling the translational gap between animal models and clinical trials. Here, we review several key themes on the reprogramming effects of melatonin in DOHaD research. We have particularly focused on the following areas: mechanisms of developmental programming; the interrelationship between melatonin and mechanisms underlying developmental programming; pathophysiological roles of melatonin in pregnancy and fetal development; and insight provided by animal models to support melatonin as a reprogramming therapy. Rates of NCDs are increasing faster than anticipated all over the world. Hence, there is an urgent need to understand reprogramming mechanisms of melatonin and to translate experimental research into clinical practice for halting a growing list of DOHaD-related NCDs. PMID:28212315

Laterally confined growth of cells induces nuclear reprogramming in the absence of exogenous biochemical factors.

PubMed

Roy, Bibhas; Venkatachalapathy, Saradha; Ratna, Prasuna; Wang, Yejun; Jokhun, Doorgesh Sharma; Nagarajan, Mallika; Shivashankar, G V

2018-05-22

Cells in tissues undergo transdifferentiation programs when stimulated by specific mechanical and biochemical signals. While seminal studies have demonstrated that exogenous biochemical factors can reprogram somatic cells into pluripotent stem cells, the critical roles played by mechanical signals in such reprogramming process have not been well documented. In this paper, we show that laterally confined growth of fibroblasts on micropatterned substrates induces nuclear reprogramming with high efficiency in the absence of any exogenous reprogramming factors. We provide compelling evidence on the induction of stem cell-like properties using alkaline phosphatase assays and expression of pluripotent markers. Early onset of reprogramming was accompanied with enhanced nuclear dynamics and changes in chromosome intermingling degrees, potentially facilitating rewiring of the genome. Time-lapse analysis of promoter occupancy by immunoprecipitation of H3K9Ac chromatin fragments revealed that epithelial, proliferative, and reprogramming gene promoters were progressively acetylated, while mesenchymal promoters were deacetylated by 10 days. Consistently, RNA sequencing analysis showed a systematic progression from mesenchymal to stem cell transcriptome, highlighting pathways involving mechanisms underlying nuclear reprogramming. We then demonstrated that these mechanically reprogrammed cells could be maintained as stem cells and can be redifferentiated into multiple lineages with high efficiency. Importantly, we also demonstrate the induction of cancer stemness properties in MCF7 cells grown in such laterally confined conditions. Collectively, our results highlight an important generic property of somatic cells that, when grown in laterally confined conditions, acquire stemness. Such mechanical reprogramming of somatic cells demonstrated here has important implications in tissue regeneration and disease models. Copyright © 2018 the Author(s). Published by PNAS.

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.

PubMed

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-02-27

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.

Rodlike Supramolecular Nanoassemblies of Degradable Poly(Aspartic Acid) Derivatives and Hydroxyl-Rich Polycations for Effective Delivery of Versatile Tumor-Suppressive ncRNAs.

PubMed

Song, Hai-Qing; Pan, Wenting; Li, Rui-Quan; Yu, Bingran; Liu, Wenjuan; Yang, Ming; Xu, Fu-Jian

2018-03-01

The delivery of tumor-suppressive noncoding RNAs (ncRNAs) including short ncRNAs (i.e., miRNAs) and long ncRNAs (lncRNAs) is put forward to treat tumors. In this work, novel rodlike supramolecular nanoassemblies (CNC @CB[8] @ PGEA) of degradable poly(aspartic acid) (PAsp) derivatives-grafted cellulose nanocrystals (CNCs) and hydroxyl-rich polycations (ethanolamine-functionalized poly(glycidyl methacrylate), PGEA) are proposed via typical cucurbit[8]uril (CB[8])-based host-guest interactions for delivery of different ncRNAs to treat hepatocellular carcinoma (HCC). Spindly CNCs, one kind of natural polysaccharide nanoparticles, possess good biocompatibility and unique physico-chemical properties. PGEA with abundant hydroxyl groups is one promising gene carrier with low cytotoxicity. PAsp can benefit the disassembly and degradability of nanoassemblies within cells. CNC @ CB[8]@PGEA combines the different unique properties of CNC, PGEA, and PAsp. CNC @ CB[8] @ PGEA effectively complexes the expression constructs of miR-101 (plasmid pc3.0-miR-101) and lncRNA MEG3 (plasmid pc3.0-MEG3). CNC @ CB[8] @ PGEA produces much better transfection performances than PGEA-containing assembly units. In addition, the codelivery system of CNC @ CB[8] @ PGEA/(pc3.0-MEG3+pc3.0-miR-101) nanocomplexes demonstrates better efficacy in suppressing HCC than CNC @ CB[8] @ PGEA/pc3.0-MEG3 or CNC @ CB[8] @ PGEA/pc3.0-miR-101 nanocomplexes alone. Such rodlike supramolecular nanoassemblies will provide a promising means to produce efficient delivery vectors of versatile tumor-suppressive nucleic acids. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes in the microarchitecture of the pancreatic cancer stroma are linked to neutrophil-dependent reprogramming of stellate cells and reflected by diffusion-weighted magnetic resonance imaging

PubMed Central

Mayer, Philipp; Dinkic, Christine; Jesenofsky, Ralf; Klauss, Miriam; Schirmacher, Peter; Dapunt, Ulrike; Hackert, Thilo; Uhle, Florian; Hänsch, G. Maria; Gaida, Matthias M.

2018-01-01

activation of the p38-cofilin axis, up-regulation of MMP9, and acquisition of intracellular lipid droplets, which together indicate a phenotype switch of the stellate cells. Conclusion: In PDAC, PMN infiltrates are associated with alterations of the tumor microarchitecture. As a causal relationship, we propose a reprogramming of stellate cells by PMN-derived azurocidin towards a phenotype, which affects the microarchitecture of the tumor. PMID:29290790

The Effect of Substrate Topography on Direct Reprogramming of Fibroblasts to Induced Neurons

PubMed Central

Kulangara, Karina; Adler, Andrew F.; Wang, Hong; Chellappan, Malathi; Hammett, Ellen; Yasuda, Ryohei; Leong, Kam W.

2014-01-01

Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2+ neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming. PMID:24709523

Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons.

PubMed

Broccoli, Vania; Rubio, Alicia; Taverna, Stefano; Yekhlef, Latefa

2015-06-01

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons. © 2015 by the Society for Experimental Biology and Medicine.

Single-Cell RNA-Seq Reveals Dynamic Early Embryonic-like Programs during Chemical Reprogramming.

PubMed

Zhao, Ting; Fu, Yao; Zhu, Jialiang; Liu, Yifang; Zhang, Qian; Yi, Zexuan; Chen, Shi; Jiao, Zhonggang; Xu, Xiaochan; Xu, Junquan; Duo, Shuguang; Bai, Yun; Tang, Chao; Li, Cheng; Deng, Hongkui

2018-06-12

Chemical reprogramming provides a powerful platform for exploring the molecular dynamics that lead to pluripotency. Although previous studies have uncovered an intermediate extraembryonic endoderm (XEN)-like state during this process, the molecular underpinnings of pluripotency acquisition remain largely undefined. Here, we profile 36,199 single-cell transcriptomes at multiple time points throughout a highly efficient chemical reprogramming system using RNA-sequencing and reconstruct their progression trajectories. Through identifying sequential molecular events, we reveal that the dynamic early embryonic-like programs are key aspects of successful reprogramming from XEN-like state to pluripotency, including the concomitant transcriptomic signatures of two-cell (2C) embryonic-like and early pluripotency programs and the epigenetic signature of notable genome-wide DNA demethylation. Moreover, via enhancing the 2C-like program by fine-tuning chemical treatment, the reprogramming process is remarkably accelerated. Collectively, our findings offer a high-resolution dissection of cell fate dynamics during chemical reprogramming and shed light on mechanistic insights into the nature of induced pluripotency. Copyright © 2018 Elsevier Inc. All rights reserved.

Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

PubMed Central

Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

2016-01-01

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive Pluripotency.

PubMed

Adachi, Kenjiro; Kopp, Wolfgang; Wu, Guangming; Heising, Sandra; Greber, Boris; Stehling, Martin; Araúzo-Bravo, Marcos J; Boerno, Stefan T; Timmermann, Bernd; Vingron, Martin; Schöler, Hans R

2018-06-11

Transcription factor (TF)-mediated reprogramming to pluripotency is a slow and inefficient process, because most pluripotency TFs fail to access relevant target sites in a refractory chromatin environment. It is still unclear how TFs actually orchestrate the opening of repressive chromatin during the long latency period of reprogramming. Here, we show that the orphan nuclear receptor Esrrb plays a pioneering role in recruiting the core pluripotency factors Oct4, Sox2, and Nanog to inactive enhancers in closed chromatin during the reprogramming of epiblast stem cells. Esrrb binds to silenced enhancers containing stable nucleosomes and hypermethylated DNA, which are inaccessible to the core factors. Esrrb binding is accompanied by local loss of DNA methylation, LIF-dependent engagement of p300, and nucleosome displacement, leading to the recruitment of core factors within approximately 2 days. These results suggest that TFs can drive rapid remodeling of the local chromatin structure, highlighting the remarkable plasticity of stable epigenetic information. Copyright © 2018 Elsevier Inc. All rights reserved.

Fatty acid metabolic reprogramming via mTOR-mediated inductions of PPARγ directs early activation of T cells

PubMed Central

Angela, Mulki; Endo, Yusuke; Asou, Hikari K.; Yamamoto, Takeshi; Tumes, Damon J.; Tokuyama, Hirotake; Yokote, Koutaro; Nakayama, Toshinori

2016-01-01

To fulfil the bioenergetic requirements for increased cell size and clonal expansion, activated T cells reprogramme their metabolic signatures from energetically quiescent to activated. However, the molecular mechanisms and essential components controlling metabolic reprogramming in T cells are not well understood. Here, we show that the mTORC1–PPARγ pathway is crucial for the fatty acid uptake programme in activated CD4+ T cells. This pathway is required for full activation and rapid proliferation of naive and memory CD4+ T cells. PPARγ directly binds and induces genes associated with fatty acid uptake in CD4+ T cells in both mice and humans. The PPARγ-dependent fatty acid uptake programme is critical for metabolic reprogramming. Thus, we provide important mechanistic insights into the metabolic reprogramming mechanisms that govern the expression of key enzymes, fatty acid metabolism and the acquisition of an activated phenotype during CD4+ T cell activation. PMID:27901044

Restoration of Mitochondrial NAD+ Levels Delays Stem Cell Senescence and Facilitates Reprogramming of Aged Somatic Cells.

PubMed

Son, Myung Jin; Kwon, Youjeong; Son, Taekwon; Cho, Yee Sook

2016-12-01

The fundamental tenet that aging is irreversible has been challenged by the development of reprogramming technology that can restore molecular and cellular age by reversing the progression of aging. The use of cells from aged individuals as sources for reprogramming or transplantation creates a major barrier in stem cell therapy with respect to cell quality and quantity. Here, we investigated the molecular features underlying senescence and rejuvenation during aged cell reprogramming and identified novel factors that can overcome age-associated barriers. Enzymes, such as nicotinamide nucleotide transhydrogenase (NNT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), that control mitochondrial NAD + levels appear to be susceptible to aging. In aged cells, mitochondrial NAD + levels decrease, accompanied by reduced SIRT3 activity; these changes severely impede cell fate transition. However, in cells collected from aged p16 knockout mice, which exhibit delayed cellular senescence, no changes in NNT or NMNAT3 expression were found. Importantly, restoring mitochondrial NAD + levels by overexpressing NNT and NMNAT3 enhanced reprogramming efficiency of aged somatic cells and extended the lifespan of human mesenchymal stem cells by delaying replicative senescence. These results demonstrate that maintenance of mitochondrial NAD + levels is critical for reversing the mechanisms of aging and ensuring that cells collected from aged individuals are of high quality. Stem Cells 2016;34:2840-2851. © 2016 AlphaMed Press.

Reprogramming Human Retinal Pigmented Epithelial Cells to Neurons Using Recombinant Proteins

PubMed Central

Hu, Qirui; Chen, Renwei; Teesalu, Tambet; Ruoslahti, Erkki

2014-01-01

Somatic cells can be reprogrammed to an altered lineage by overexpressing specific transcription factors. To avoid introducing exogenous genetic material into the genome of host cells, cell-penetrating peptides can be used to deliver transcription factors into cells for reprogramming. Position-dependent C-end rule (CendR) cell- and tissue-penetrating peptides provide an alternative to the conventional cell-penetrating peptides, such as polyarginine. In this study, we used a prototypic, already active CendR peptide, RPARPAR, to deliver the transcription factor SOX2 to retinal pigmented epithelial (RPE) cells. We demonstrated that RPE cells can be directly reprogrammed to a neuronal fate by introduction of SOX2. Resulting neuronal cells expressed neuronal marker mRNAs and proteins and downregulated expression of RPE markers. Cells produced extensive neurites and developed synaptic machinery capable of dye uptake after depolarization with potassium. The RPARPAR-mediated delivery of SOX2 alone was sufficient to allow cell lineage reprogramming of both fetal and stem cell-derived RPE cells to become functional neurons. PMID:25298373

The association of pituitary tumors and headache.

PubMed

Levy, Miles J

2011-04-01

Pituitary tumors may give rise to headache via numerous mechanisms. The traditional explanation for headache is dural stretch and cavernous sinus invasion, although this is not borne out by clinical studies. Certain functional pituitary tumors are associated with headache, notably growth hormone and prolactin-secreting tumors. The observation that somatostatin analogues can have a dramatic analgesic effect in acromegaly suggests that biochemical properties of the tumor may be important. The genetic predisposition of the patient to primary headache also determines whether headache occurs with pituitary tumors. This article attempts to outline the clinical features, management strategies, and key academic questions regarding the subject of pituitary tumors and headache.

Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors.

PubMed

Koblas, Tomas; Leontovyc, Ivan; Loukotova, Sarka; Kosinova, Lucie; Saudek, Frantisek

2016-05-17

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

PubMed

Quintana-Bustamante, Oscar; Segovia, Jose C

2016-01-01

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

21 CFR 866.6010 - Tumor-associated antigen immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tumor-associated antigen immunological test system... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Tumor Associated Antigen immunological Test Systems § 866.6010 Tumor-associated antigen immunological test system. (a) Identification. A...

Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

PubMed

Wen, Yan; Wani, Prachi; Zhou, Lu; Baer, Tom; Phadnis, Smruti Madan; Reijo Pera, Renee A; Chen, Bertha

2013-02-01

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated β-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

In vivo reprogramming for heart regeneration: A glance at efficiency, environmental impacts, challenges and future directions.

PubMed

Ebrahimi, Behnam

2017-07-01

Replacing dying or diseased cells of a tissue with new ones that are converted from patient's own cells is an attractive strategy in regenerative medicine. In vivo reprogramming is a novel strategy that can circumvent the hurdles of autologous/allogeneic cell injection therapies. Interestingly, studies have demonstrated that direct injection of cardiac transcription factors or specific miRNAs into the infarct border zone of murine hearts following myocardial infarction converts resident cardiac fibroblasts into functional cardiomyocytes. Moreover, in vivo cardiac reprogramming not only drives cardiac tissue regeneration, but also improves cardiac function and survival rate after myocardial infarction. Thanks to the influence of cardiac microenvironment and the same developmental origin, cardiac fibroblasts seem to be more amenable to reprogramming toward cardiomyocyte fate than other cell sources (e.g. skin fibroblasts). Thus, reprogramming of cardiac fibroblasts to functional induced cardiomyocytes in the cardiac environment holds great promises for induced regeneration and potential clinical purposes. Application of small molecules in future studies may represent a major advancement in this arena and pharmacological reprogramming would convey reprogramming technology to the translational medicine paradigm. This study reviews accomplishments in the field of in vitro and in vivo mouse cardiac reprogramming and then deals with strategies for the enhancement of the efficiency and quality of the process. Furthermore, it discusses challenges ahead and provides suggestions for future research. Human cardiac reprogramming is also addressed as a foundation for possible application of in vivo cardiac reprogramming for human heart regeneration in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

PubMed Central

Lina, Taslima T.; Farris, Tierra; Luo, Tian; Mitra, Shubhajit; Zhu, Bing; McBride, Jere W.

2016-01-01

Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival. PMID:27303657

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming.

PubMed

Gascón, Sergio; Murenu, Elisa; Masserdotti, Giacomo; Ortega, Felipe; Russo, Gianluca L; Petrik, David; Deshpande, Aditi; Heinrich, Christophe; Karow, Marisa; Robertson, Stephen P; Schroeder, Timm; Beckers, Johannes; Irmler, Martin; Berndt, Carsten; Angeli, José P Friedmann; Conrad, Marcus; Berninger, Benedikt; Götz, Magdalena

2016-03-03

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

PubMed Central

Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

2013-01-01

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

Current reprogramming systems in regenerative medicine: from somatic cells to induced pluripotent stem cells.

PubMed

Hu, Chenxia; Li, Lanjuan

2016-01-01

Induced pluripotent stem cells (iPSCs) paved the way for research fields including cell therapy, drug screening, disease modeling and the mechanism of embryonic development. Although iPSC technology has been improved by various delivery systems, direct transduction and small molecule regulation, low reprogramming efficiency and genomic modification steps still inhibit its clinical use. Improvements in current vectors and the exploration of novel vectors are required to balance efficiency and genomic modification for reprogramming. Herein, we set out a comprehensive analysis of current reprogramming systems for the generation of iPSCs from somatic cells. By clarifying advantages and disadvantages of the current reprogramming systems, we are striding toward an effective route to generate clinical grade iPSCs.

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

PubMed

Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

2016-04-01

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hi

Epigenetic regulation leading to induced pluripotency drives cancer development in vivo

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

Ohnishi, Kotaro; Department of Medicine, Gifu University Graduate School of Medicine, Gifu 501-1194; Semi, Katsunori

Highlights: • Epigenetic regulation of failed reprogramming-associated cancer cells is discussed. • Similarity between pediatric cancer and reprogramming-associated cancer is discussed. • Concept for epigenetic cancer is discussed. - Abstract: Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the transient expression of reprogramming factors. During the reprogramming process, somatic cells acquire the ability to undergo unlimited proliferation, which is also an important characteristic of cancer cells, while their underlying DNA sequence remains unchanged. Based on the characteristics shared between pluripotent stem cells and cancer cells, the potential involvement of the factors leading to reprogramming toward pluripotencymore » in cancer development has been discussed. Recent in vivo reprogramming studies provided some clues to understanding the role of reprogramming-related epigenetic regulation in cancer development. It was shown that premature termination of the in vivo reprogramming result in the development of tumors that resemble pediatric cancers. Given that epigenetic modifications play a central role during reprogramming, failed reprogramming-associated cancer development may have provided a proof of concept for epigenetics-driven cancer development in vivo.« less

Targeting DNA Methyltranferases in Urological Tumors

PubMed Central

Marques-Magalhães, Ângela; Graça, Inês; Henrique, Rui; Jerónimo, Carmen

2018-01-01

Urological cancers are a heterogeneous group of malignancies accounting for a considerable proportion of cancer-related morbidity and mortality worldwide. Aberrant epigenetic traits, especially altered DNA methylation patterns constitute a hallmark of these tumors. Nonetheless, these alterations are reversible, and several efforts have been carried out to design and test several epigenetic compounds that might reprogram tumor cell phenotype back to a normal state. Indeed, several DNMT inhibitors are currently under evaluation for therapeutic efficacy in clinical trials. This review highlights the critical role of DNA methylation in urological cancers and summarizes the available data on pre-clinical assays and clinical trials with DNMT inhibitors in bladder, kidney, prostate, and testicular germ cell cancers. PMID:29706891

The environmental carcinogen benzo[a]pyrene induces a Warburg-like metabolic reprogramming dependent on NHE1 and associated with cell survival

PubMed Central

Hardonnière, Kévin; Saunier, Elise; Lemarié, Anthony; Fernier, Morgane; Gallais, Isabelle; Héliès-Toussaint, Cécile; Mograbi, Baharia; Antonio, Samantha; Bénit, Paule; Rustin, Pierre; Janin, Maxime; Habarou, Florence; Ottolenghi, Chris; Lavault, Marie-Thérèse; Benelli, Chantal; Sergent, Odile; Huc, Laurence; Bortoli, Sylvie; Lagadic-Gossmann, Dominique

2016-01-01

Cancer cells display alterations in many cellular processes. One core hallmark of cancer is the Warburg effect which is a glycolytic reprogramming that allows cells to survive and proliferate. Although the contributions of environmental contaminants to cancer development are widely accepted, the underlying mechanisms have to be clarified. Benzo[a]pyrene (B[a]P), the prototype of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, and it is a human carcinogen according to the International Agency for Research on Cancer. In addition to triggering apoptotic signals, B[a]P may induce survival signals, both of which are likely to be involved in cancer promotion. We previously suggested that B[a]P-induced mitochondrial dysfunctions, especially membrane hyperpolarization, might trigger cell survival signaling in rat hepatic epithelial F258 cells. Here, we further characterized these dysfunctions by focusing on energy metabolism. We found that B[a]P promoted a metabolic reprogramming. Cell respiration decreased and lactate production increased. These changes were associated with alterations in the tricarboxylic acid cycle which likely involve a dysfunction of the mitochondrial complex II. The glycolytic shift relied on activation of the Na+/H+ exchanger 1 (NHE1) and appeared to be a key feature in B[a]P-induced cell survival related to changes in cell phenotype (epithelial-to-mesenchymal transition and cell migration). PMID:27488617

[A clinical study of associated bladder tumor in patients with renal pelvic and ureteral tumor].

PubMed

Sugano, O; Shouji, N; Horigome, T; Uchi, K; Katou, H

1995-08-01

We investigated the incidence of associated bladder tumor and prognosis in 101 cases with a pathological diagnosis of transitional cell carcinoma, selected from those with renal pelvic and ureteral tumor whom we had encountered over the 18 years between April 1976 and March 1993. Among these 101 cases, the incidence of associated bladder tumor was noted in 42 (41.6%), 23 (22.8%) with coexistence and 19 (18.8%) with subsequence. As for the primary site of renal pelvis and ureter, the coexistence was 15.4% and subsequence 20.5% in renal pelvis, and the coexistence was 24.6% and subsequence 19.3% in ureter, and the coexistence was 60.0% and subsequence 0.0% in both renal pelvis and ureter. The incidence of coexistent bladder tumor was high in both renal pelvis and ureter, but no significant difference was noted. As for the stage, the incidence of coexistence was high in T1, while subsequence was high in T2, but no significant difference was noted. As for the grade, the incidence of coexistence was high in G2, but no significant difference was noted. The 5 year survival rate was 58.2% in those without, 54.2% with coexistence, and 82.5% with subsequent bladder tumor, with a significant difference (p < 0.05) between the last two groups. The interval of subsequent bladder tumor ranged from 4 to 164 months (mean 27.7 months), with the incidence within 2 years being approximately 70.0%. It was found that the renal pelvic and ureteral tumors are frequently associated bladder tumor while associated bladder tumor dose not appear to have an ill effect on the prognosis. Therefore it is necessary that patients with renal pelvic and ureteral tumor be observed closely for 5 years, especially for the initial 2 years.

Mitochondrial Redox Signaling and Tumor Progression.

PubMed

Chen, Yuxin; Zhang, Haiqing; Zhou, Huanjiao Jenny; Ji, Weidong; Min, Wang

2016-03-25

Cancer cell can reprogram their energy production by switching mitochondrial oxidative phosphorylation to glycolysis. However, mitochondria play multiple roles in cancer cells, including redox regulation, reactive oxygen species (ROS) generation, and apoptotic signaling. Moreover, these mitochondrial roles are integrated via multiple interconnected metabolic and redox sensitive pathways. Interestingly, mitochondrial redox proteins biphasically regulate tumor progression depending on cellular ROS levels. Low level of ROS functions as signaling messengers promoting cancer cell proliferation and cancer invasion. However, anti-cancer drug-initiated stress signaling could induce excessive ROS, which is detrimental to cancer cells. Mitochondrial redox proteins could scavenger basal ROS and function as "tumor suppressors" or prevent excessive ROS to act as "tumor promoter". Paradoxically, excessive ROS often also induce DNA mutations and/or promotes tumor metastasis at various stages of cancer progression. Targeting redox-sensitive pathways and transcriptional factors in the appropriate context offers great promise for cancer prevention and therapy. However, the therapeutics should be cancer-type and stage-dependent.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade

PubMed Central

Lue, Hui-wen; Podolak, Jennifer; Kolahi, Kevin; Cheng, Larry; Rao, Soumya; Garg, Devin; Xue, Chang-Hui; Rantala, Juha K.; Tyner, Jeffrey W.; Thornburg, Kent L.; Martinez-Acevedo, Ann; Liu, Jen-Jane; Amling, Christopher L.; Truillet, Charles; Louie, Sharon M.; Anderson, Kimberly E.; Evans, Michael J.; O'Donnell, Valerie B.; Nomura, Daniel K.; Drake, Justin M.; Ritz, Anna

2017-01-01

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K–AKT–mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5. Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage. PMID:29138276

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

PubMed

Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

2017-06-08

Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities

PubMed Central

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behaviour and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatio-temporal gradients of these endogenous transmembrane voltage potentials (Vmem) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells’ programs towards the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in non-neural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. PMID:23897652

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities.

PubMed

Levin, Michael

2013-01-01

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behavior and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatiotemporal gradients of these endogenous transmembrane voltage potentials (Vmem ) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells' programs toward the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in nonneural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering. Copyright © 2013 Wiley Periodicals, Inc.

Canonical and non-canonical WNT signaling in cancer stem cells and their niches: Cellular heterogeneity, omics reprogramming, targeted therapy and tumor plasticity (Review).

PubMed

Katoh, Masaru

2017-11-01

Cancer stem cells (CSCs), which have the potential for self-renewal, differentiation and de-differentiation, undergo epigenetic, epithelial-mesenchymal, immunological and metabolic reprogramming to adapt to the tumor microenvironment and survive host defense or therapeutic insults. Intra-tumor heterogeneity and cancer-cell plasticity give rise to therapeutic resistance and recurrence through clonal replacement and reactivation of dormant CSCs, respectively. WNT signaling cascades cross-talk with the FGF, Notch, Hedgehog and TGFβ/BMP signaling cascades and regulate expression of functional CSC markers, such as CD44, CD133 (PROM1), EPCAM and LGR5 (GPR49). Aberrant canonical and non-canonical WNT signaling in human malignancies, including breast, colorectal, gastric, lung, ovary, pancreatic, prostate and uterine cancers, leukemia and melanoma, are involved in CSC survival, bulk-tumor expansion and invasion/metastasis. WNT signaling-targeted therapeutics, such as anti-FZD1/2/5/7/8 monoclonal antibody (mAb) (vantictumab), anti-LGR5 antibody-drug conjugate (ADC) (mAb-mc-vc-PAB-MMAE), anti-PTK7 ADC (PF-06647020), anti-ROR1 mAb (cirmtuzumab), anti-RSPO3 mAb (rosmantuzumab), small-molecule porcupine inhibitors (ETC-159, WNT-C59 and WNT974), tankyrase inhibitors (AZ1366, G007-LK, NVP-TNKS656 and XAV939) and β-catenin inhibitors (BC2059, CWP232228, ICG-001 and PRI-724), are in clinical trials or preclinical studies for the treatment of patients with WNT-driven cancers. WNT signaling-targeted therapeutics are applicable for combination therapy with BCR-ABL, EGFR, FLT3, KIT or RET inhibitors to treat a subset of tyrosine kinase-driven cancers because WNT and tyrosine kinase signaling cascades converge to β-catenin for the maintenance and expansion of CSCs. WNT signaling-targeted therapeutics might also be applicable for combination therapy with immune checkpoint blockers, such as atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab and pembrolizumab, to treat cancers

Canonical and non-canonical WNT signaling in cancer stem cells and their niches: Cellular heterogeneity, omics reprogramming, targeted therapy and tumor plasticity (Review)

PubMed Central

Katoh, Masaru

2017-01-01

Cancer stem cells (CSCs), which have the potential for self-renewal, differentiation and de-differentiation, undergo epigenetic, epithelial-mesenchymal, immunological and metabolic reprogramming to adapt to the tumor microenvironment and survive host defense or therapeutic insults. Intra-tumor heterogeneity and cancer-cell plasticity give rise to therapeutic resistance and recurrence through clonal replacement and reactivation of dormant CSCs, respectively. WNT signaling cascades cross-talk with the FGF, Notch, Hedgehog and TGFβ/BMP signaling cascades and regulate expression of functional CSC markers, such as CD44, CD133 (PROM1), EPCAM and LGR5 (GPR49). Aberrant canonical and non-canonical WNT signaling in human malignancies, including breast, colorectal, gastric, lung, ovary, pancreatic, prostate and uterine cancers, leukemia and melanoma, are involved in CSC survival, bulk-tumor expansion and invasion/metastasis. WNT signaling-targeted therapeutics, such as anti-FZD1/2/5/7/8 monoclonal antibody (mAb) (vantictumab), anti-LGR5 antibody-drug conjugate (ADC) (mAb-mc-vc-PAB-MMAE), anti-PTK7 ADC (PF-06647020), anti-ROR1 mAb (cirmtuzumab), anti-RSPO3 mAb (rosmantuzumab), small-molecule porcupine inhibitors (ETC-159, WNT-C59 and WNT974), tankyrase inhibitors (AZ1366, G007-LK, NVP-TNKS656 and XAV939) and β-catenin inhibitors (BC2059, CWP232228, ICG-001 and PRI-724), are in clinical trials or preclinical studies for the treatment of patients with WNT-driven cancers. WNT signaling-targeted therapeutics are applicable for combination therapy with BCR-ABL, EGFR, FLT3, KIT or RET inhibitors to treat a subset of tyrosine kinase-driven cancers because WNT and tyrosine kinase signaling cascades converge to β-catenin for the maintenance and expansion of CSCs. WNT signaling-targeted therapeutics might also be applicable for combination therapy with immune checkpoint blockers, such as atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab and pembrolizumab, to treat cancers

Novel targeted therapy for neuroblastoma: silencing the MXD3 gene using siRNA.

PubMed

Duong, Connie; Yoshida, Sakiko; Chen, Cathy; Barisone, Gustavo; Diaz, Elva; Li, Yueju; Beckett, Laurel; Chung, Jong; Antony, Reuben; Nolta, Jan; Nitin, Nitin; Satake, Noriko

2017-09-01

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide-common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

PubMed

Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression.

PubMed

Fitamant, Julien; Kottakis, Filippos; Benhamouche, Samira; Tian, Helen S; Chuvin, Nicolas; Parachoniak, Christine A; Nagle, Julia M; Perera, Rushika M; Lapouge, Marjorie; Deshpande, Vikram; Zhu, Andrew X; Lai, Albert; Min, Bosun; Hoshida, Yujin; Avruch, Joseph; Sia, Daniela; Campreciós, Genís; McClatchey, Andrea I; Llovet, Josep M; Morrissey, David; Raj, Lakshmi; Bardeesy, Nabeel

2015-03-10

Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

PubMed

Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

2017-08-01

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

PubMed Central

Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

2015-01-01

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

The electric and thermoelectric properties of Cu(II)-Schiff base nano-complexes

NASA Astrophysics Data System (ADS)

Ibrahim, E. M. M.; Abdel-Rahman, Laila H.; Abu-Dief, Ahmed M.; Elshafaie, A.; Hamdan, Samar Kamel; Ahmed, A. M.

2018-05-01

The physical properties, such as electric and optical properties, of metal-Schiff base complexes have been widely investigated. However, their thermoelectric (TE) properties remain unreported. This work presents Cu(II)-Schiff base complexes as promising materials for TE power generation. Therefore, three Cu(II)-Schiff base complexes (namely, [Cu(C32H22N4O2)].3/2H2O, [Cu(C23H17N4O7Br)], and [Cu(C27H22N4O8)].H2O) have been synthesized in nanosized scale. The electric and TE properties have been studied and comprehensive discussions have been presented to promote the nano-complexes (NCs) practical applications in the field of TE power generation. The electrical measurements confirm that the NCs are semiconductors and the electrical conduction process is governed by intermolecular and intramolecular transfer of the charge carriers. The TE measurements reveal that the Cu(II)-Schiff base NCs are nondegenerate P-type semiconductors. The measured Seebeck coefficient values were higher compared to the values reported in previous works for other organic materials indicating that the complexes under study are promising candidates for theremoelectric applications if the electrical conductivity could be enhanced.

Ubiquitous Release Of Exosomal Tumor Suppressor miR-6126 from Ovarian Cancer Cells

PubMed Central

Kanlikilicer, Pinar; Rashed, Mohammed H.; Bayraktar, Recep; Mitra, Rahul; Ivan, Cristina; Aslan, Burcu; Zhang, Xinna; Filant, Justyna; Silva, Andreia M.; Rodriguez-Aguayo, Cristian; Bayraktar, Emine; Pichler, Martin; Ozpolat, Bulent; Calin, George A.; Sood, Anil K.; Lopez-Berestein, Gabriel

2017-01-01

Cancer cells actively promote their tumorigenic behavior by reprogramming gene expression. Loading intraluminal vesicles with specific miRNAs and releasing them into the tumor microenvironment as exosomes is one mechanism of reprogramming whose regulation remains to be elucidated. Here, we report that miR-6126 is ubiquitously released in high abundance from both chemosensitive and chemoresistant ovarian cancer cells via exosomes. Overexpression of miR-6126 was confirmed in healthy ovarian tissue compared to ovarian cancer patient samples and correlated with better overall survival in high-grade serous ovarian cancer patients. miR-6126 acted as a tumor suppressor by directly targeting integrin β1, a key regulator of cancer cell metastasis. miR-6126 mimic treatment of cancer cells resulted in increased miR-6126 and decreased integrin β1 mRNA levels in the exosome. Functional analysis showed that treatment of endothelial cells with miR-6126 mimic significantly reduced tube formation as well as invasion and migration capacities of ovarian cancer cells in vitro. Administration of miR-6126 mimic in an orthotopic mouse model of ovarian cancer elicited a relative reduction in tumor growth, proliferating cells and microvessel density. miR-6126 inhibition promoted oncogenic behavior by leading ovarian cancer cells to release more exosomes. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression and suggest a new therapeutic approach to disrupt oncogenic phenotypes in tumors. PMID:27742688

Tumor-derived exosomes and their role in cancer progression

PubMed Central

Whiteside, Theresa L

2017-01-01

Tumor cells actively produce, release and utilize exosomes to promote tumor growth. Mechanisms through which tumor-derived exosomes subserve the tumor are under intense investigation. These exosomes are information carriers, conveying molecular and genetic messages from tumor cells to normal or other abnormal cells residing at close or distant sites. Tumor-derived exosomes are found in all body fluids. Upon the contact with target cells, they alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to angiogenesis, thrombosis, metastasis and immunosuppression. Exosomes produced by tumors carry cargos that in part mimic contents of parent cells and are of potential interest as non-invasive biomarkers of cancer. Their role in inhibiting the host antitumor responses and in mediating drug resistance is important for cancer therapy. Tumor-derived exosomes may interfere with cancer immunotherapy, but they also could serve as adjuvants and antigenic components of antitumor vaccines. Their biological roles in cancer development or progression as well as cancer therapy suggest that tumor-derived exosomes are critical components of oncogenic transformation. PMID:27117662

Tumor-Derived Exosomes and Their Role in Cancer Progression.

PubMed

Whiteside, Theresa L

2016-01-01

Tumor cells actively produce, release, and utilize exosomes to promote tumor growth. Mechanisms through which tumor-derived exosomes subserve the tumor are under intense investigation. These exosomes are information carriers, conveying molecular and genetic messages from tumor cells to normal or other abnormal cells residing at close or distant sites. Tumor-derived exosomes are found in all body fluids. Upon contact with target cells, they alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to angiogenesis, thrombosis, metastasis, and immunosuppression. Exosomes produced by tumors carry cargos that in part mimic contents of parent cells and are of potential interest as noninvasive biomarkers of cancer. Their role in inhibiting the host antitumor responses and in mediating drug resistance is important for cancer therapy. Tumor-derived exosomes may interfere with cancer immunotherapy, but they also could serve as adjuvants and antigenic components of antitumor vaccines. Their biological roles in cancer development or progression as well as cancer therapy suggest that tumor-derived exosomes are critical components of oncogenic transformation. © 2016 Elsevier Inc. All rights reserved.

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

PubMed

Biswas, Dhruba; Jiang, Peng

2016-02-06

The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

PubMed Central

Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

2016-01-01

Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Tumor-associated macrophages: implications in cancer immunotherapy.

PubMed

Petty, Amy J; Yang, Yiping

2017-03-01

Tumor-associated macrophages (TAMs), representing most of the leukocyte population in solid tumors, demonstrate great phenotypic heterogeneity and diverse functional capabilities under the influence of the local tumor microenvironment. These anti-inflammatory and protumorigenic macrophages modulate the local microenvironment to facilitate tumor growth and metastasis. In this review, we examine the origin of TAMs and the complex regulatory networks within the tumor microenvironment that facilitate the polarization of TAMs toward a protumoral phenotype. More extensively, we evaluate the mechanisms by which TAMs mediate angiogenesis, metastasis, chemotherapeutic resistance and immune evasion. Lastly, we will highlight novel interventional strategies targeting TAMs in preclinical studies and in early clinical trials that have significant potential in improving efficacy of current chemotherapeutic and/or immunotherapeutic approaches.

Structure and Electronic Properties of Nano-complex CCl4…Cr(AcacCl)3 on Evidence Derived from Vibrational Spectroscopy

NASA Astrophysics Data System (ADS)

Slabzhennikov, S. N.; Kuarton, L. A.; Ryabchenko, O. B.

In order to specify influence of intermolecular interaction on IR spectrum of interacting species, an investigation of a process CCl4 + Cr(AcacCl)3 → CCl4…Cr(AcacCl)3 has been performed by means of Hartree-Fock-Roothaan method in MIDI basis set with p- and d- polarization functions. An estimation of intermolecular interaction in geometrical parameters, electron density function both between interacting particles and inside themselves, frequencies and intensities of normal modes has been carried out. Chemical bonds with the most significant shifts of characteristics under formation of nano-complex CCl4…Cr(AcacCl)3 have been noted.

Transcriptional switch of dormant tumors to fast-growing angiogenic phenotype.

PubMed

Almog, Nava; Ma, Lili; Raychowdhury, Raktima; Schwager, Christian; Erber, Ralf; Short, Sarah; Hlatky, Lynn; Vajkoczy, Peter; Huber, Peter E; Folkman, Judah; Abdollahi, Amir

2009-02-01

Tumor dormancy has important implications for early detection and treatment of cancer. Lack of experimental models and limited clinical accessibility constitute major obstacles to the molecular characterization of dormant tumors. We have developed models in which human tumors remain dormant for a prolonged period of time (>120 days) until they switch to rapid growth and become strongly angiogenic. These angiogenic tumors retain their ability to grow fast once injected in new mice. We hypothesized that dormant tumors undergo a stable genetic reprogramming during their switch to the fast-growing phenotype. Genome-wide transcriptional analysis was done to dissect the molecular mechanisms underlying the switch of dormant breast carcinoma, glioblastoma, osteosarcoma, and liposarcoma tumors. A consensus expression signature distinguishing all four dormant versus switched fast-growing tumors was generated. In alignment with our phenotypic observation, the angiogenesis process was the most significantly affected functional gene category. The switch of dormant tumors was associated with down-regulation of angiogenesis inhibitor thrombospondin and decreased sensitivity of angiogenic tumors to angiostatin. The conversion of dormant tumors to exponentially growing tumors was also correlated with regulation and activation of pathways not hitherto linked to tumor dormancy process, such as endothelial cell-specific molecule-1, 5'-ecto-nucleotidase, tissue inhibitor of metalloproteinase-3, epidermal growth factor receptor, insulin-like growth factor receptor, and phosphatidylinositol 3-kinase signaling. Further, novel dormancy-specific biomarkers such as H2BK and Eph receptor A5 (EphA5) were discovered. EphA5 plasma levels in mice and mRNA levels in tumor specimens of glioma patients correlated with diseases stage. These data will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for development of dormancy-promoting tumor therapy

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

PubMed Central

Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

2016-01-01

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001 PMID:26920219

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

PubMed

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

(Re-)programming of subtype specific cardiomyocytes.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration

PubMed Central

Zhang, Lijuan; Du, Jianhai; Justus, Sally; Hsu, Chun-Wei; Bonet-Ponce, Luis; Wu, Wen-Hsuan; Tsai, Yi-Ting; Wu, Wei-Pu; Jia, Yading; Duong, Jimmy K.; Mahajan, Vinit B.; Lin, Chyuan-Sheng; Wang, Shuang; Hurley, James B.

2016-01-01

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy–mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders. PMID:27841758

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

PubMed

Ge, Haitao; Mu, Luyan; Jin, Linchun; Yang, Changlin; Chang, Yifan Emily; Long, Yu; DeLeon, Gabriel; Deleyrolle, Loic; Mitchell, Duane A; Kubilis, Paul S; Lu, Dunyue; Qi, Jiping; Gu, Yunhe; Lin, Zhiguo; Huang, Jianping

2017-10-01

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM. © 2017 UICC.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

PubMed

Cieślar-Pobuda, Artur; Knoflach, Viktoria; Ringh, Mikael V; Stark, Joachim; Likus, Wirginia; Siemianowicz, Krzysztof; Ghavami, Saeid; Hudecki, Andrzej; Green, Jason L; Łos, Marek J

2017-07-01

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Composite carbohydrate interpenetrating polyelectrolyte nano-complexes (IPNC) as a controlled oral delivery system of citalopram HCl for pediatric use: in-vitro/in-vivo evaluation and histopathological examination.

PubMed

Kamel, Rabab; Abbas, Haidy; El-Naa, Mona

2018-06-01

Citalopram HCl (CH) is one of the few drugs which can be used safely in childhood psychiatric disorders. This study was focused on the preparation of interpenetrating polyelectrolytes nano-complexes (IPNC) to transform the hydrophilic carbohydrate polymers into an insoluble form. The IPNCs were loaded with CH to sustain its effect. The IPNC2 (composed of chitosan:pectin in a 3:1 ratio) showed the most extended drug release pattern (P < 0.05) and followed a Higuchi-order kinetics model. It was characterized using SEM, X-rays diffractometry, and FTIR. In-vivo studies were performed using immature rats with induced depression, and were based on the investigation of behavioral, biochemical, and histopathological changes at different time intervals up to 24 h. Rats treated with IPNC2 showed a significant more rapid onset of action and more extended effect in the behavioral tests, in addition to a significantly higher serotonin brain level up to 24 h, compared to rats treated with the market product (P < 0.05). The histopathological examination showed a profound amelioration of the cerebral cortex features of the depressed rats after IPNC2 administration. This study proves the higher efficacy and more extended effect of the new polyelectrolytes nano-complexes compared to the market product.

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

PubMed Central

Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

2014-01-01

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Exploring the Mechanisms of Differentiation, Dedifferentiation, Reprogramming and Transdifferentiation

PubMed Central

Xu, Li; Zhang, Kun; Wang, Jin

2014-01-01

We explored the underlying mechanisms of differentiation, dedifferentiation, reprogramming and transdifferentiation (cell type switchings) from landscape and flux perspectives. Lineage reprogramming is a new regenerative method to convert a matured cell into another cell including direct transdifferentiation without undergoing a pluripotent cell state and indirect transdifferentiation with an initial dedifferentiation-reversion (reprogramming) to a pluripotent cell state. Each cell type is quantified by a distinct valley on the potential landscape with higher probability. We investigated three driving forces for cell fate decision making: stochastic fluctuations, gene regulation and induction, which can lead to cell type switchings. We showed that under the driving forces the direct transdifferentiation process proceeds from a differentiated cell valley to another differentiated cell valley through either a distinct stable intermediate state or a certain series of unstable indeterminate states. The dedifferentiation process proceeds through a pluripotent cell state. Barrier height and the corresponding escape time from the valley on the landscape can be used to quantify the stability and efficiency of cell type switchings. We also uncovered the mechanisms of the underlying processes by quantifying the dominant biological paths of cell type switchings on the potential landscape. The dynamics of cell type switchings are determined by both landscape gradient and flux. The flux can lead to the deviations of the dominant biological paths for cell type switchings from the naively expected landscape gradient path. As a result, the corresponding dominant paths of cell type switchings are irreversible. We also classified the mechanisms of cell fate development from our landscape theory: super-critical pitchfork bifurcation, sub-critical pitchfork bifurcation, sub-critical pitchfork with two saddle-node bifurcation, and saddle-node bifurcation. Our model showed good

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and

Genetics Home Reference: tumor necrosis factor receptor-associated periodic syndrome

MedlinePlus

... Email Facebook Twitter Home Health Conditions TRAPS Tumor necrosis factor receptor-associated periodic syndrome Printable PDF Open ... to view the expand/collapse boxes. Description Tumor necrosis factor receptor-associated periodic syndrome (commonly known as ...

Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

PubMed Central

Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

2015-01-01

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

Reprogramming cellular functions with engineered membrane proteins.

PubMed

Arber, Caroline; Young, Melvin; Barth, Patrick

2017-10-01

Taking inspiration from Nature, synthetic biology utilizes and modifies biological components to expand the range of biological functions for engineering new practical devices and therapeutics. While early breakthroughs mainly concerned the design of gene circuits, recent efforts have focused on engineering signaling pathways to reprogram cellular functions. Since signal transduction across cell membranes initiates and controls intracellular signaling, membrane receptors have been targeted by diverse protein engineering approaches despite limited mechanistic understanding of their function. The modular architecture of several receptor families has enabled the empirical construction of chimeric receptors combining domains from distinct native receptors which have found successful immunotherapeutic applications. Meanwhile, progress in membrane protein structure determination, computational modeling and rational design promise to foster the engineering of a broader range of membrane receptor functions. Marrying empirical and rational membrane protein engineering approaches should enable the reprogramming of cells with widely diverse fine-tuned functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer.

PubMed

Tagalakis, Aristides D; McAnulty, Robin J; Devaney, James; Bottoms, Stephen E; Wong, John B; Elbs, Martin; Writer, Michele J; Hailes, Helen C; Tabor, Alethea B; O'Callaghan, Christopher; Jaffe, Adam; Hart, Stephen L

2008-05-01

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

PubMed Central

Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

2009-01-01

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

Conditionally reprogrammed cells (CRC) methodology does not allow the in vitro expansion of patient-derived primary and metastatic lung cancer cells.

PubMed

Sette, Giovanni; Salvati, Valentina; Giordani, Ilenia; Pilozzi, Emanuela; Quacquarini, Denise; Duranti, Enrico; De Nicola, Francesca; Pallocca, Matteo; Fanciulli, Maurizio; Falchi, Mario; Pallini, Roberto; De Maria, Ruggero; Eramo, Adriana

2018-07-01

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells. © 2018 UICC.

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress

PubMed Central

2013-01-01

redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific. PMID:24359630

Identification of secretaglobin Scgb2a1 as a target for developmental reprogramming by BPA in the rat prostate.

PubMed

Wong, Rebecca Lee Yean; Wang, Quan; Treviño, Lindsey S; Bosland, Maarten C; Chen, Jing; Medvedovic, Mario; Prins, Gail S; Kannan, Kurunthachalam; Ho, Shuk-Mei; Walker, Cheryl Lyn

2015-01-01

Secretoglobins are a superfamily of secreted proteins thought to participate in inflammation, tissue repair, and tumorigenesis. Secretoglobin family 2A member 1 (Scgb2a1) is a component of prostatein, a major androgen-binding protein secreted by the rat prostate. Using a rat model for developmental reprogramming of susceptibility to prostate carcinogenesis, we identified, by RNA-seq, that Scgb2a1 is significantly upregulated (>100-fold) in the prostate of adult rats neonatally exposed to bisphenol A (BPA), with increased gene expression confirmed by quantitative RT-PCR and chromatin immunoprecipitation for histone H3 lysine 9 acetylation. Bisulfite analysis of both CpG islands located within 10 kb of the Scgb2a1 promoter identified significant hypomethylation of the CpG island upstream of the transcription start site of this gene in the reprogrammed prostate. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development, with potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding.

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Filaggrin gene polymorphism associated with Epstein-Barr virus-associated tumors in China.

PubMed

Yang, Yang; Liu, Wen; Zhao, Zhenzhen; Zhang, Yan; Xiao, Hua; Luo, Bing

2017-08-01

Mutations of filaggrin gene (FLG) have been identified as the cause of ichthyosis vulgaris, while recently FLG mutations were found to be associated with gastric cancer. This study aimed to investigate the association of filaggrin polymorphism with Epstein-Barr virus-associated tumors in China. A total of 200 patients with three types of tumors and 117 normal control samples were genotyped at three common FLG mutation loci (rs3126085, K4671X, R501X) by using Sequenom MassARRAY technique. The χ 2 test was used to evaluate the relationship between the mutation and the three kinds of tumors. A two-sided P value of <0.05 was considered statistically significant. The results showed that two single-nucleotide polymorphism (SNP) loci (rs3126085, K4671X) were significantly associated with nasopharyngeal carcinoma in genetic model. In addition, the two SNPs K4671X and rs3126085 were related to Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC), respectively. Furthermore, allele distributions in EBVaGC and EBVnGC were verified to be different in both SNP loci.

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

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

Zhai, Yingying; Chen, Xi; Yu, Dehai

2015-09-10

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phasemore » blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. - Highlights: • Histone deacetylase inhibitor valproic acid enhances iPSC induction. • Valproic acid suppresses reprogramming-induced senescence stress. • Valproic acid downregulates the p16/p21 pathway in reprogramming. • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.« less

Decoy receptor 3 enhances tumor progression via induction of tumor-associated macrophages.

PubMed

Tai, Shyh-Kuan; Chang, Hsin-Chuan; Lan, Keng-Li; Lee, Chun-Ting; Yang, Chih-Ya; Chen, Nien-Jung; Chou, Teh-Ying; Tarng, Der-Cherng; Hsieh, Shie-Liang

2012-03-01

Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

PubMed Central

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells. PMID:28796841

Single cell qPCR reveals that additional HAND2 and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes.

PubMed

Bektik, Emre; Dennis, Adrienne; Prasanna, Prateek; Madabhushi, Anant; Fu, Ji-Dong

2017-01-01

The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional HAND2 or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more HAND2 expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, HAND2 and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

PubMed

Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

Blood pressure reprogramming adapter assists signal recording

NASA Technical Reports Server (NTRS)

Vick, H. A.

1967-01-01

Blood pressure reprogramming adapter separates the two components of a blood pressure signal, a dc pressure signal and an ac Korotkoff sounds signal, so that the Korotkoff sounds are recorded on one channel as received while the dc pressure signal is converted to FM and recorded on a second channel.

Dendritic cell reprogramming by endogenously produced lactic acid.

PubMed

Nasi, Aikaterini; Fekete, Tünde; Krishnamurthy, Akilan; Snowden, Stuart; Rajnavölgyi, Eva; Catrina, Anca I; Wheelock, Craig E; Vivar, Nancy; Rethi, Bence

2013-09-15

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration-dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration-dependent pathway was independent of peroxisome proliferator-activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid-mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.

Retinoic Acid Inducible Gene 1 Protein (RIG1)-like Receptor Pathway is Required for Efficient Nuclear Reprogramming

PubMed Central

Sayed, Nazish; Ospino, Frank; Himmati, Farhan; Lee, Jieun; Chanda, Palas; Mocarski, Edward S.; Cooke, John P.

2017-01-01

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. PMID:28276156

RAGE expression in tumor-associated macrophages promotes angiogenesis in glioma.

PubMed

Chen, Xuebo; Zhang, Leying; Zhang, Ian Y; Liang, Junling; Wang, Huaqing; Ouyang, Mao; Wu, Shihua; da Fonseca, Anna Carolina Carvalho; Weng, Lihong; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Natarajan, Rama; Badie, Behnam

2014-12-15

Interaction of RAGE (the receptor for advanced glycation endproducts) with its ligands can promote tumor progression, invasion, and angiogenesis. Although blocking RAGE signaling has been proposed as a potential anticancer strategy, functional contributions of RAGE expression in the tumor microenvironment (TME) have not been investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth of gliomas. In both invasive and noninvasive glioma models, animal survival was prolonged in RAGE knockout (Ager(-/-)) mice. However, the improvement in survival in Ager(-/-) mice was not due to changes in tumor growth rate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TME abrogated angiogenesis by downregulating the expression of proangiogenic factors, which prevented normal vessel formation, thereby generating a leaky vasculature. These alterations were most prominent in noninvasive gliomas, in which the expression of VEGF and proinflammatory cytokines were also lower in tumor-associated macrophages (TAM) in Ager(-/-) mice. Interestingly, reconstitution of Ager(-/-) TAM with wild-type microglia or macrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associated microglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis. ©2014 American Association for Cancer Research.

Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake.

PubMed

Flavahan, William A; Wu, Qiulian; Hitomi, Masahiro; Rahim, Nasiha; Kim, Youngmi; Sloan, Andrew E; Weil, Robert J; Nakano, Ichiro; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Li, Meizhang; Lathia, Justin D; Rich, Jeremy N; Hjelmeland, Anita B

2013-10-01

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) owing to preferential BTIC survival and to adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3, and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, tumor initiating cells may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may maintain the tumor hierarchy and portend poor prognosis.

PHACES syndrome associated with carcinoid endobronchial tumor.

PubMed

Mama, Nadia; H'mida, Dorra; Lahmar, Imen; Yacoubi, Mohamed Tahar; Tlili-Graiess, Kalthoum

2014-05-01

PHACES syndrome consists of the constellation of manifestations including posterior fossa anomalies of the brain (most commonly Dandy-Walker malformations), hemangiomas of the face and scalp, arterial abnormalities, cardiac defects, eye anomalies and sternal defects. We present a case with a possible PHACES syndrome including sternal cleft and supraumbilical raphé, precordial skin tag, persistent left superior vena cava and subtle narrowing of the aorta with an endobronchial carcinoid tumor. All these anomalies were discovered on chest multi-detector CT. This is a unique case of PHACES syndrome associated with carcinoid tumor. Review of the literature revealed 3 cases of PHACES syndrome with glial tumor. The authors tried to find the relationship between PHACES syndrome and carcinoid tumors or gliomas, which all derive from the neural crest cells.

Direct Cardiac Reprogramming as a Novel Therapeutic Strategy for Treatment of Myocardial Infarction.

PubMed

Ma, Hong; Wang, Li; Liu, Jiandong; Qian, Li

2017-01-01

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a novel therapy for the treatment of heart failure, a common and morbid disease that is usually caused by irreversible loss of functional cardiomyocytes (CMs). Recently, we and others showed that in a murine model of acute myocardial infarction, delivery of three transcription factors, Gata4, Mef2c, and Tbx5 converted endogenous cardiac fibroblasts into functional iCMs. These iCMs integrated electrically and mechanically with surrounding myocardium, resulting in a reduction in scar size and an improvement in heart function. Our findings suggest that iCM reprogramming may be a means of regenerating functional CMs in vivo for patients with heart disease. However, because relatively little is known about the factors that regulate iCM reprogramming, the applicability of iCM reprogramming is currently limited to the experimental settings in which it has been attempted. Specific hurdles include the relatively low conversion rate of iCMs and the need for reprogramming to occur in the context of acute injury. Therefore, before this treatment can become a viable therapy for human heart disease, the optimal condition for efficient iCM generation must be determined. Here, we provide a detailed protocol for both in vitro and in vivo iCM generation that has been optimized so far in our lab. We hope that this protocol will lay a foundation for future further improvement of iCM generation and provide a platform for mechanistic studies.

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

PubMed

Hannemann, Sebastian; Galán, Jorge E

2017-07-01

Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

End of inevitability: programming and reprogramming.

PubMed

Turksen, Kursad

2013-08-01

Stem cell commitment and differentiation leading to functional cell types and organs has generally been considered unidirectional and deterministic. Starting first with a landmark study 50 years ago, and now with more recent observations, this paradigm has been challenged, necessitating a rethink of what constitutes both programming and reprogramming processes, and how we can use this new understanding for new approaches to drug discovery and regenerative medicine.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

PubMed

Olariu, Victor; Manesso, Erica; Peterson, Carsten

2017-06-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths

PubMed Central

Olariu, Victor; Manesso, Erica

2017-01-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis–Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming. PMID:28680655

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

PubMed

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-07-17

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. © 2014 The Authors.

MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

PubMed Central

Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2014-01-01

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

PubMed

Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi

2017-11-01

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.

Direct Reprogramming of Fibroblasts via a Chemically Induced XEN-like State.

PubMed

Li, Xiang; Liu, Defang; Ma, Yantao; Du, Xiaomin; Jing, Junzhan; Wang, Lipeng; Xie, Bingqing; Sun, Da; Sun, Shaoqiang; Jin, Xueqin; Zhang, Xu; Zhao, Ting; Guan, Jingyang; Yi, Zexuan; Lai, Weifeng; Zheng, Ping; Huang, Zhuo; Chang, Yanzhong; Chai, Zhen; Xu, Jun; Deng, Hongkui

2017-08-03

Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state. Copyright © 2017 Elsevier Inc. All rights reserved.

X Chromosome of female cells shows dynamic changes in status during human somatic cell reprogramming.

PubMed

Kim, Kun-Yong; Hysolli, Eriona; Tanaka, Yoshiaki; Wang, Brandon; Jung, Yong-Wook; Pan, Xinghua; Weissman, Sherman Morton; Park, In-Hyun

2014-06-03

Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.

The therapeutic potential of cell identity reprogramming for the treatment of aging-related neurodegenerative disorders.

PubMed

Smith, Derek K; He, Miao; Zhang, Chun-Li; Zheng, Jialin C

2017-10-01

Neural cell identity reprogramming strategies aim to treat age-related neurodegenerative disorders with newly induced neurons that regenerate neural architecture and functional circuits in vivo. The isolation and neural differentiation of pluripotent embryonic stem cells provided the first in vitro models of human neurodegenerative disease. Investigation into the molecular mechanisms underlying stem cell pluripotency revealed that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs) and these cells could be used to model Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, and Parkinson disease. Additional neural precursor and direct transdifferentiation strategies further enabled the induction of diverse neural linages and neuron subtypes both in vitro and in vivo. In this review, we highlight neural induction strategies that utilize stem cells, iPSCs, and lineage reprogramming to model or treat age-related neurodegenerative diseases, as well as, the clinical challenges related to neural transplantation and in vivo reprogramming strategies. Copyright © 2016. Published by Elsevier Ltd.

The therapeutic potential of cell identity reprogramming for the treatment of aging-related neurodegenerative disorders

PubMed Central

Smith, Derek K.; He, Miao; Zhang, Chun-Li; Zheng, Jialin C.

2018-01-01

Neural cell identity reprogramming strategies aim to treat age-related neurodegenerative disorders with newly induced neurons that regenerate neural architecture and functional circuits in vivo. The isolation and neural differentiation of pluripotent embryonic stem cells provided the first in vitro models of human neurodegenerative disease. Investigation into the molecular mechanisms underlying stem cell pluripotency revealed that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs) and these cells could be used to model Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, and Parkinson disease. Additional neural precursor and direct transdifferentiation strategies further enabled the induction of diverse neural linages and neuron subtypes both in vitro and in vivo. In this review, we highlight neural induction strategies that utilize stem cells, iPSCs, and lineage reprogramming to model or treat age-related neurodegenerative diseases, as well as, the clinical challenges related to neural transplantation and in vivo reprogramming strategies. PMID:26844759

Chemical compound-based direct reprogramming for future clinical applications

PubMed Central

Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

Targeting Unique Metabolic Properties of Breast Tumor Initiating Cells

PubMed Central

Feng, Weiguo; Gentles, Andrew; Nair, Ramesh V.; Huang, Min; Lin, Yuan; Lee, Cleo Y.; Cai, Shang; Scheeren, Ferenc A.; Kuo, Angera H.; Diehn, Maximilian

2014-01-01

Normal stem cells from a variety of tissues display unique metabolic properties compared to their more differentiated progeny. However, relatively little is known about heterogeneity of metabolic properties cancer stem cells, also called tumor initiating cells (TICs). In this study we show that, analogous to some normal stem cells, breast TICs have distinct metabolic properties compared to non-tumorigenic cancer cells (NTCs). Transcriptome profiling using RNA-Seq revealed TICs under-express genes involved in mitochondrial biology and mitochondrial oxidative phosphorylation and metabolic analyses revealed TICs preferentially perform glycolysis over oxidative phosphorylation compared to NTCs. Mechanistic analyses demonstrated that decreased expression and activity of pyruvate dehydrogenase (Pdh), a key regulator of oxidative phosphorylation, play a critical role in promoting the pro-glycolytic phenotype of TICs. Metabolic reprogramming via forced activation of Pdh preferentially eliminates TICs both in vitro and in vivo. Our findings reveal unique metabolic properties of TICs and demonstrate that metabolic reprogramming represents a promising strategy for targeting these cells. PMID:24497069

Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

PubMed

Perera, P I P; Ordoñez, C A; Dedicova, B; Ortega, P E M

2014-05-21

Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion

PubMed Central

Nguyen, Van T. M.; Barozzi, Iros; Faronato, Monica; Lombardo, Ylenia; Steel, Jennifer H.; Patel, Naina; Darbre, Philippa; Castellano, Leandro; Győrffy, Balázs; Woodley, Laura; Meira, Alba; Patten, Darren K.; Vircillo, Valentina; Periyasamy, Manikandan; Ali, Simak; Frige, Gianmaria; Minucci, Saverio; Coombes, R. Charles; Magnani, Luca

2015-01-01

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients. PMID:26610607

In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells

PubMed Central

Li, Hedong; Chen, Gong

2017-01-01

Neuroregeneration in the central nervous system (CNS) has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart and liver, and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient’s own internal cells for tissue repair. PMID:27537482

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

PubMed Central

Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

2016-01-01

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

Metabolic reprogramming: a cancer hallmark even warburg did not anticipate.

PubMed

Ward, Patrick S; Thompson, Craig B

2012-03-20

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

MicroRNA in Metabolic Re-Programming and Their Role in Tumorigenesis

PubMed Central

Tomasetti, Marco; Amati, Monica; Santarelli, Lory; Neuzil, Jiri

2016-01-01

The process of metabolic re-programing is linked to the activation of oncogenes and/or suppression of tumour suppressor genes, which are regulated by microRNAs (miRNAs). The interplay between oncogenic transformation-driven metabolic re-programming and modulation of aberrant miRNAs further established their critical role in the initiation, promotion and progression of cancer by creating a tumorigenesis-prone microenvironment, thus orchestrating processes of evasion to apoptosis, angiogenesis and invasion/migration, as well metastasis. Given the involvement of miRNAs in tumour development and their global deregulation, they may be perceived as biomarkers in cancer of therapeutic relevance. PMID:27213336

Staircase polymetalsilicon nanocomplexes - Polymetalphenyl siloxanes: Structure and properties

NASA Astrophysics Data System (ADS)

Shapkin, N. P.; Balanov, M. I.; Razov, V. I.; Gardionov, S. V.; Mayorov, V. Yu; Tokar, E. A.; Papynov, E. K.; Korochentsev, V. V.; Leont'ev, L. B.; Slobodyuk, A. B.; Modin, E. B.

2018-03-01

Polyphenyl siloxanes containing chromium, iron, and aluminum in the backbone chain have been synthesized. The structure of the obtained staircase nano-metal complexes has been studied by the methods of XRD analysis and IR, 29Si and 27Al NMR, and XPS spectroscopy and scanning electron microscopy. Physical-chemical characteristics of these compounds have been investigated by the positron annihilation spectroscopy (PAS) and low-temperature nitrogen adsorption. The data of X-ray diffraction analysis (XRD) enabled us to calculate the size and volume of coherent scattering regions (CSR) and the cross-section area of the polymer chains. By means of the PAS method, the specific volumes of positron (Ve+) and positronium (Vps) "traps" have been calculated. The data of 29Si NMR spectroscopy have shown the presence of T2 and T3 fragments in the structure. As was shown on the basis of the data of 27Al NMR and XPS spectroscopy, tetrahedral (66%) and octahedral surroundings of the metal atom were realized in the backbone chain. The obtained data were used to describe a spatial layered structure of phenyl siloxanes containing trivalent metals. The electron microscopy of nanocomplexes revealed the presence of spherical particles, whose size changes in cases of chromium, iron, and aluminum. Using the data of low-temperature nitrogen adsorption, it was assumed that the specific surface area was filled with a layer of compacted spherical particles, whereas the layer thickness was determined, in its turn, by the specific polarizing potential (SPP) calculated as a ratio of the polarizing potential (PP) to the volume of voids between coherent scattering regions. Similar dependence is observed between the layer thickness and the specific polarizing potential calculated as a ratio of the polarizing potential to the positronium "trap" volume. A direct dependence between the thickness of the spherical particles layer and the specific polarizing potential has been demonstrated. The assumption

Magnetic Resonance Imaging of Tumor Associated Macrophages: Clinical Translation.

PubMed

Aghighi, Maryam; Theruvath, Ashok Joseph; Pareek, Anuj; Pisani, Laura; Alford, Raphael; Muehe, Anne Monika; Sethi, Tarsheen K; Holdsworth, Samantha J; Hazard, Florette K; Gratzinger, Dita; Luna-Fineman, Sandra; Advani, Ranjana H; Spunt, Sheri L; Daldrup-Link, Heike E

2018-05-15

Tumor associated macrophages (TAM) in malignant tumors have been linked to tumor aggressiveness and represent a new target for cancer immunotherapy. As new TAM-targeted immunotherapies are entering clinical trials, it is important to detect and quantify TAM with non-invasive imaging techniques. The purpose of this study was to determine if ferumoxytol-enhanced MRI can detect TAM in lymphomas and bone sarcomas of pediatric patients and young adults. In a first-in-patient , IRB-approved prospective clinical trial, 25 pediatric and young adult patients with lymphoma or bone sarcoma underwent ferumoxytol-enhanced MRI. To confirm ferumoxytol enhancement, five pilot patients (2 lymphoma, 3 bone sarcoma) underwent pre- and post-contrast MRI. Subsequently, 20 patients (10 lymphoma, 10 bone sarcoma) underwent ferumoxytol-enhanced MRI 24-48 hours after intravenous injection, followed by tumor biopsy/resection and macrophage staining. To determine if ferumoxytol-MRI can differentiate tumors with different TAM content, we compared T2* relaxation times of lymphomas and bone sarcomas. Tumor T2* values of 20 patients were correlated with CD68+ and CD163+ TAM quantities on histopathology. Significant ferumoxytol tumor enhancement was noted on post-contrast scans compared to pre-contrast scans ( P = 0.036). Bone sarcomas and lymphomas demonstrated significantly different MRI enhancement and TAM density ( P < 0.05). Within each tumor group, T2* signal enhancement on MR images correlated significantly with the density of CD68+ and CD163+ TAM ( P < 0.05). Ferumoxytol-enhanced MRI is immediately clinically applicable and could be used to stratify patients with TAM-rich tumors to immune-targeted therapies and to monitor tumor response to these therapies. Copyright ©2018, American Association for Cancer Research.

Cancer associated fibroblasts promote tumor growth and metastasis by modulating the tumor immune microenvironment in a 4T1 murine breast cancer model.

PubMed

Liao, Debbie; Luo, Yunping; Markowitz, Dorothy; Xiang, Rong; Reisfeld, Ralph A

2009-11-23

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer. We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8(+) T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression. Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.

Brain Tumor Initiating Cells Adapt to Restricted Nutrition through Preferential Glucose Uptake

PubMed Central

Flavahan, William A.; Wu, Qiulian; Hitomi, Masahiro; Rahim, Nasiha; Kim, Youngmi; Sloan, Andrew E.; Weil, Robert J.; Nakano, Ichiro; Sarkaria, Jann N.; Stringer, Brett W.; Day, Bryan W.; Li, Meizhang; Lathia, Justin D.; Rich, Jeremy N.; Hjelmeland, Anita B.

2013-01-01

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) due to preferential BTIC survival and adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3 and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, TICs may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may instruct the tumor hierarchy and portend poor prognosis. PMID:23995067

NANOG priming before full reprogramming may generate germ cell tumours.

PubMed

Grad, I; Hibaoui, Y; Jaconi, M; Chicha, L; Bergström-Tengzelius, R; Sailani, M R; Pelte, M F; Dahoun, S; Mitsiadis, T A; Töhönen, V; Bouillaguet, S; Antonarakis, S E; Kere, J; Zucchelli, M; Hovatta, O; Feki, A

2011-11-09

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Dexamethasone Alleviates Tumor-Associated Brain Damage and Angiogenesis

PubMed Central

Fan, Zheng; Sehm, Tina; Rauh, Manfred; Buchfelder, Michael

2014-01-01

Children and adults with the most aggressive form of brain cancer, malignant gliomas or glioblastoma, often develop cerebral edema as a life-threatening complication. This complication is routinely treated with dexamethasone (DEXA), a steroidal anti-inflammatory drug with pleiotropic action profile. Here we show that dexamethasone reduces murine and rodent glioma tumor growth in a concentration-dependent manner. Low concentrations of DEXA are already capable of inhibiting glioma cell proliferation and at higher levels induce cell death. Further, the expression of the glutamate antiporter xCT (system Xc −; SLC7a11) and VEGFA is up-regulated after DEXA treatment indicating early cellular stress responses. However, in human gliomas DEXA exerts differential cytotoxic effects, with some human glioma cells (U251, T98G) resistant to DEXA, a finding corroborated by clinical data of dexamethasone non-responders. Moreover, DEXA-resistant gliomas did not show any xCT alterations, indicating that these gene expressions are associated with DEXA-induced cellular stress. Hence, siRNA-mediated xCT knockdown in glioma cells increased the susceptibility to DEXA. Interestingly, cell viability of primary human astrocytes and primary rodent neurons is not affected by DEXA. We further tested the pharmacological effects of DEXA on brain tissue and showed that DEXA reduces tumor-induced disturbances of the microenvironment such as neuronal cell death and tumor-induced angiogenesis. In conclusion, we demonstrate that DEXA inhibits glioma cell growth in a concentration and species-dependent manner. Further, DEXA executes neuroprotective effects in brains and reduces tumor-induced angiogenesis. Thus, our investigations reveal that DEXA acts pleiotropically and impacts tumor growth, tumor vasculature and tumor-associated brain damage. PMID:24714627

RAGE Expression in Tumor-associated Macrophages Promotes Angiogenesis in Glioma

PubMed Central

Zhang, Ian Y.; Liang, Junling; Wang, Huaqing; Ouyang, Mao; Wu, Shihua; da Fonseca, Anna Carolina Carvalho; Weng, Lihong; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Natarajan, Rama; Badie, Behnam

2014-01-01

Interaction of RAGE with its ligands can promote tumor progression, invasion and angiogenesis. Although blocking RAGE signaling has been proposed as a potential anti-cancer strategy, functional contributions of RAGE expression in the tumor microenvironment (TME) has not been investigated in detail. Here, we evaluated the effect of genetic depletion of RAGE in TME on the growth of gliomas. In both invasive and non-invasive glioma models, animal survival was prolonged in RAGE knockout (Ager−/−) mice. However, the improvement in survival in Ager−/− mice was not due to changes in tumor growth rate but rather to a reduction in tumor-associated inflammation. Furthermore, RAGE ablation in the TME abrogated angiogenesis by downregulating the expression of pro-angiogenic factors which prevented normal vessel formation, thereby generating a leaky vasculature. These alterations were most prominent in non-invasive gliomas, where the expression of VEGF and pro-inflammatory cytokines were also lower in tumor-associated macrophages (TAM) in Ager−/− mice. Interestingly, reconstitution of Ager−/− TAM with wild-type microglia or macrophages normalized tumor vascularity. Our results establish that RAGE signaling in glioma-associated microglia and TAM drives angiogenesis, underscoring the complex role of RAGE and its ligands in gliomagenesis. PMID:25326491

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease.

PubMed

Cantone, Irene; Fisher, Amanda G

2017-11-05

X-chromosome inactivation (XCI) is an exemplar of epigenetic regulation that is set up as pluripotent cells differentiate. Once established, XCI is stably propagated, but can be reversed in vivo or by pluripotent reprogramming in vitro Although reprogramming provides a useful model for inactive X (Xi) reactivation in mouse, the relative instability and heterogeneity of human embryonic stem (ES) cells and induced pluripotent stem cells hampers comparable progress in human. Here we review studies aimed at reactivating the human Xi using different reprogramming strategies. We outline our recent results using mouse ES cells to reprogramme female human fibroblasts by cell-cell fusion. We show that pluripotent reprogramming induces widespread and rapid chromatin remodelling in which the human Xi loses XIST and H3K27m3 enrichment and selected Xi genes become reactivated, ahead of mitotic division. Using RNA sequencing to map the extent of human Xi reactivation, and chromatin-modifying drugs to potentiate reactivation, we outline how this approach could be used to better design strategies to re-express human X-linked loci. As cell fusion induces the expression of human pluripotency genes that represent both the 'primed' and 'naive' states, this approach may also offer a fresh opportunity to segregate human pluripotent states with distinct Xi expression profiles, using single-cell-based approaches.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'. © 2017 The Author(s).

Nanomedicine Strategies to Target Tumor-Associated Macrophages

PubMed Central

Binnemars-Postma, Karin; Storm, Gert; Prakash, Jai

2017-01-01

In recent years, the influence of the tumor microenvironment (TME) on cancer progression has been better understood. Macrophages, one of the most important cell types in the TME, exist in different subtypes, each of which has a different function. While classically activated M1 macrophages are involved in inflammatory and malignant processes, activated M2 macrophages are more involved in the wound-healing processes occurring in tumors. Tumor-associated macrophages (TAM) display M2 macrophage characteristics and support tumor growth and metastasis by matrix remodeling, neo-angiogenesis, and suppressing local immunity. Due to their detrimental role in tumor growth and metastasis, selective targeting of TAM for the treatment of cancer may prove to be beneficial in the treatment of cancer. Due to the plastic nature of macrophages, their activities may be altered to inhibit tumor growth. In this review, we will discuss the therapeutic options for the modulation and targeting of TAM. Different therapeutic strategies to deplete, inhibit recruitment of, or re-educate TAM will be discussed. Current strategies for the targeting of TAM using nanomedicine are reviewed. Passive targeting using different nanoparticle systems is described. Since TAM display a number of upregulated surface proteins compared to non-TAM, specific targeting using targeting ligands coupled to nanoparticles is discussed in detail. PMID:28471401

Induced Pluripotency and Epigenetic Reprogramming

PubMed Central

Hochedlinger, Konrad; Jaenisch, Rudolf

2015-01-01

SUMMARY Induced pluripotency defines the process by which somatic cells are converted into induced pluripotent stem cells (iPSCs) upon overexpression of a small set of transcription factors. In this article, we put transcription factor–induced pluripotency into a historical context, review current methods to generate iPSCs, and discuss mechanistic insights that have been gained into the process of reprogramming. In addition, we focus on potential therapeutic applications of induced pluripotency and emerging technologies to efficiently engineer the genomes of human pluripotent cells for scientific and therapeutic purposes. PMID:26626939

Targeting of Pancreatic Cancer with Magneto-Fluorescent Theranostic Gold Nanoshells

PubMed Central

Chen, Wenxue; Ayala-Orozco, Ciceron; Biswal, Nrusingh C.; Perez-Torres, Carlos; Bartels, Marc; Bardhan, Rizia; Stinnet, Gary; Liu, Xian-De; Ji, Baoan; Deorukhkar, Amit; Brown, Lisa V.; Guha, Sushovan; Pautler, Robia G.; Krishnan, Sunil; Halas, Naomi J; Joshi, Amit

2014-01-01

Aim We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. Materials and Methods Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the NIR dye ICG, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. Results AntiNGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2 weighted MR imaging with higher tumor contrast than can be obtained using long-circulating but non-targeted PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. Conclusions Theranostic gold nanoshells with embedded NIR and MR contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy. PMID:24063415

Targeting pancreatic cancer with magneto-fluorescent theranostic gold nanoshells.

PubMed

Chen, Wenxue; Ayala-Orozco, Ciceron; Biswal, Nrusingh C; Perez-Torres, Carlos; Bartels, Marc; Bardhan, Rizia; Stinnet, Gary; Liu, Xian-De; Ji, Baoan; Deorukhkar, Amit; Brown, Lisa V; Guha, Sushovan; Pautler, Robia G; Krishnan, Sunil; Halas, Naomi J; Joshi, Amit

2014-01-01

We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.

Colon tumor mutations and epigenetic changes associated with genetic polymorphism: Insight into disease pathways

PubMed Central

Slattery, Martha L.; Wolff, Roger K.; Curtin, Karen; Fitzpatrick, Frank; Herrick, Jennifer; Potter, John D.; Caan, Bette J.; Samowitz, Wade S.

2010-01-01

Variation in genes associated with serum levels of proteins may be useful for examining specific disease pathways. Using data from a large study of colon cancer, we examine genetic variants in insulin, inflammation, estrogen, metabolizing enzymes, and energy homeostasis genes to explore associations with microsatellite instability (MSI), CpG Island methylator phenotype (CIMP), mutations of p53 in exons 5 through 8, and mutations in codons 12 and 13 of Ki-ras. Insulin-related genes were associated with CIMP positive and MSI tumors, with the strongest associations among aspirin users. The Fok1 Vitamin D Receptor (VDR) polymorphism was associated with CIMP positive/Ki-ras mutated tumors; the Poly A and CDX2 VDR polymorphisms were associated only with Ki-ras mutated tumors. NAT2 was associated with CIMP positive/Ki-ras mutated tumors but not with MSI tumors. The TCF7L2 rs7903146 polymorphism was associated with p53 mutated tumors. Most associations varied by recent aspirin/NSAID use: IL6 rs1800796 and rs1800795 polymorphisms were associated inversely with tumor mutations in the presence of aspirin/NSAIDs; POMC significantly reduced risk of Ki-ras- mutated tumors when aspirin/NSAIDs were not used; the TCF7L2 rs7903146 was associated with reduced risk of Ki-ras-mutated tumors in the presence of aspirin and increased risk in the absence of aspirin. These data, although exploratory, identify specific tumor subsets that may be associated with specific exposures/polymorphism combinations. The important modifying effects of aspirin/NSAIDs on associations with genetic polymorphisms reinforce the underlying role of inflammation in the etiology of colon cancer. PMID:18992263

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor*

PubMed Central

Dai, Xiangpeng; Hao, Jie; Hou, Xiao-jun; Hai, Tang; Fan, Yong; Yu, Yang; Jouneau, Alice; Wang, Liu; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming. PMID:20566633

[Advances in nanoparticle-targeting tumor associated macrophages for cancer imaging and therapy].

PubMed

Fengliang, Guo; Guping, Tang; Qinglian, H U

2017-03-25

Tumor tissues are composed of tumor cells and complicate microenvironment. Tumor associated macrophages (TAMs) as an important component in tumor microenvironment, play fundamental roles in tumor progression, metastasis and microenvironment regulation. Recently, studies have found that nanotechnology, as an emerging platform, provides unique potential for cancer imaging and therapy. With the nanotechnology, TAMs imaging presents direct evidence for cancer development, progression, and the effectiveness of cancer treatments; it also can regulate the immunosuppression of tumor microenvironment and improve therapeutic efficiency through TAMs targeted killing or phenotypic transformation. In this article, we illustrate the function of TAMs and review the latest development in nano-carriers and their applications in tumor associated macrophage targeting cancer imaging and therapy.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

PubMed

Dormiani, Kianoush; Mir Mohammad Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Forouzanfar, Mahboobeh; Baharvand, Hossein; Ghaedi, Kamran; Nasr-Esfahani, Mohammad Hossein

2017-01-01

Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

AMP-Activated Protein Kinase as a Reprogramming Strategy for Hypertension and Kidney Disease of Developmental Origin.

PubMed

Tain, You-Lin; Hsu, Chien-Ning

2018-06-12

Suboptimal early-life conditions affect the developing kidney, resulting in long-term programming effects, namely renal programming. Adverse renal programming increases the risk for developing hypertension and kidney disease in adulthood. Conversely, reprogramming is a strategy aimed at reversing the programming processes in early life. AMP-activated protein kinase (AMPK) plays a key role in normal renal physiology and the pathogenesis of hypertension and kidney disease. This review discusses the regulation of AMPK in the kidney and provides hypothetical mechanisms linking AMPK to renal programming. This will be followed by studies targeting AMPK activators like metformin, resveratrol, thiazolidinediones, and polyphenols as reprogramming strategies to prevent hypertension and kidney disease. Further studies that broaden our understanding of AMPK isoform- and tissue-specific effects on renal programming are needed to ultimately develop reprogramming strategies. Despite the fact that animal models have provided interesting results with regard to reprogramming strategies targeting AMPK signaling to protect against hypertension and kidney disease with developmental origins, these results await further clinical translation.

Factors associated with tumor size of hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Siregar, G. A.; Buulolo, B. A.

2018-03-01

Determining the association of age and laboratory parameters with tumor size of hepatocellular carcinoma (HCC). The study was at Adam Malik Hospital Medan from June- December 2016. 100 HCC patients were enrolled; those with excluding liver metastatic. Baseline characteristics of gender, age, obtaining etiology of HCC. Liver function tests, viral marker, and INR were done. Based on tumor size from abdomen CT, patients were three groups: tumor size below 3 cm, 3-5 cm, and above 5 cm size. Patients were also divided based on Child-Pugh class. Correlation of age and laboratory results with tumor size of HCC patients were analyzed. Age have negative correlation with tumor size in HCC patients (r=-0.297, p=0.032) while AFP have positive correlation with tumor size (r0.446, p=<0.001). Total bilirubin, AST, and ALT have negative correlation but non-significant (r=-0.045, -0.078, - 0.126 respectively). Albumin and INR have positive correlation but non-significant (r=0.021, 0.112 respectively). Our study suggests that older age correlates with smaller tumor size, while AFP level has a significant correlation with tumor size in HCC patients. AFP level may be a useful marker for determining the prognosis of HCC patients.

Cancer: A Problem of Developmental Biology; Scientific Evidence for Reprogramming and Differentiation Therapy.

PubMed

Sell, Stewart; Nicolini, Andrea; Ferrari, Paola; Biava, Pier M

2016-01-01

Current medical literature acknowledges that embryonic micro-environment is able to suppress tumor development. Administering carcinogenic substances during organogenesis in fact leads to embryonic malformations, but not to offspring tumor growth. Once organogenesis has ended, administration of carcinogenic substances causes a rise in offspring tumor development. These data indicate that cancer can be considered a deviation in normal development, which can be regulated by factors of the embryonic microenvironment. Furthermore, it has been demonstrated that teratoma differentiates into normal tissues once it is implanted in the embryo. Recently, it has been shown that implanting a melanoma in Zebrafish embryo did not result in a tumor development; however, it did in the adult specimen. This demonstrates that cancer cells can differentiate into normal tissues when implanted in the embryo. In addition, it was demonstrated that other tumors can revert into a normal phenotype and/or differentiate into normal tissue when implanted in the embryo. These studies led some authors to define cancer as a problem of developmental biology and to predict the present concept of "cancer stem cells theory". In this review, we record the most important researches about the reprogramming and differentiation treatments of cancer cells to better clarify how the substances taken from developing embryo or other biological substances can induce differentiation of malignant cells. Lastly, a model of cancer has been proposed here, conceived by one of us, which is consistent with the reality, as demonstrated by a great number of researches. This model integrates the theory of the "maturation arrest" of cancer cells as conceived by B. Pierce with the theory which describes cancer as a process of deterministic chaos determined by genetic and/or epigenetic alterations in differentiated cells, which leads a normal cell to become cancerous. All the researches here described demonstrated that cancer

A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

PubMed Central

Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

2015-01-01

SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

Prenatal Choline Supplementation Diminishes Early-Life Iron Deficiency-Induced Reprogramming of Molecular Networks Associated with Behavioral Abnormalities in the Adult Rat Hippocampus.

PubMed

Tran, Phu V; Kennedy, Bruce C; Pisansky, Marc T; Won, Kyoung-Jae; Gewirtz, Jonathan C; Simmons, Rebecca A; Georgieff, Michael K

2016-03-01

Early-life iron deficiency is a common nutrient deficiency worldwide. Maternal iron deficiency increases the risk of schizophrenia and autism in the offspring. Postnatal iron deficiency in young children results in cognitive and socioemotional abnormalities in adulthood despite iron treatment. The rat model of diet-induced fetal-neonatal iron deficiency recapitulates the observed neurobehavioral deficits. We sought to establish molecular underpinnings for the persistent psychopathologic effects of early-life iron deficiency by determining whether it permanently reprograms the hippocampal transcriptome. We also assessed the effects of maternal dietary choline supplementation on the offspring's hippocampal transcriptome to identify pathways through which choline mitigates the emergence of long-term cognitive deficits. Male rat pups were made iron deficient (ID) by providing pregnant and nursing dams an ID diet (4 g Fe/kg) from gestational day (G) 2 through postnatal day (PND) 7 and an iron-sufficient (IS) diet (200 g Fe/kg) thereafter. Control pups were provided IS diet throughout. Choline (5 g/kg) was given to half the pregnant dams in each group from G11 to G18. PND65 hippocampal transcriptomes were assayed by next generation sequencing (NGS) and analyzed with the use of knowledge-based Ingenuity Pathway Analysis. Real-time polymerase chain reaction was performed to validate a subset of altered genes. Formerly ID rats had altered hippocampal expression of 619 from >10,000 gene loci sequenced by NGS, many of which map onto molecular networks implicated in psychological disorders, including anxiety, autism, and schizophrenia. There were significant interactions between iron status and prenatal choline treatment in influencing gene expression. Choline supplementation reduced the effects of iron deficiency, including those on gene networks associated with autism and schizophrenia. Fetal-neonatal iron deficiency reprograms molecular networks associated with the

Prognostic significance of monocarboxylate transporter expression in oral cavity tumors

PubMed Central

Simões-Sousa, Susana; Granja, Sara; Pinheiro, Céline; Fernandes, Daniela; Longatto-Filho, Adhemar; Laus, Ana Carolina; Alves, Cira Danielle Casado; Suárez-Peñaranda, J. M.; Pérez-Sayáns, Mario; Lopes Carvalho, Andre; Schmitt, Fernando C.; García-García, Abel; Baltazar, Fatima

2016-01-01

ABSTRACT Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer. The majority of patients present advanced stage disease and has poor survival. Therefore, it is imperative to search for new biomarkers and new alternative and effective treatment options. Most cancer cells rely on aerobic glycolysis to generate energy and metabolic intermediates. This phenotype is a hallmark of cancer, characterized by an increase in glucose consumption and production of high amounts of lactate. Consequently, cancer cells need to up-regulate many proteins and enzymes related with the glycolytic metabolism. Thus, the aim of this study was to characterize metabolic phenotype of oral cavity cancers (OCC) by assessing the expression pattern of monocarboxylate transporters (MCTs) 1, 2 and 4 and other proteins related with the glycolytic phenotype. Material and Methods: We evaluated the immunohistochemical expression of MCT1, MCT4, CD147, GLUT1 and CAIX in 135 human samples of OCC and investigated the correlation with clinicopathological parameters and the possible association with prognosis. Results: We observed that all proteins analyzed presented significantly higher plasma membrane expression in neoplastic compared to non-neoplastic samples. MCT4 was significantly associated with T-stage and advanced tumoral stage, while CD147 was significantly correlated with histologic differentiation. Interestingly, tumors expressing both MCT1 and MCT4 but negative for MCT2 were associated with shorter overall survival. Conclusion: Overexpression of MCT1/4, CD147, GLUT1 and CAIX, supports previous findings of metabolic reprograming in OCC, warranting future studies to explore the hyper-glycolytic phenotype of these tumors. Importantly, MCT expression revealed to have a prognostic value in OCC survival. PMID:27232157

Tumor cell-associated immune checkpoint molecules - Drivers of malignancy and stemness.

PubMed

Marcucci, Fabrizio; Rumio, Cristiano; Corti, Angelo

2017-12-01

Inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor cells in different tumor types. It was thought that the main function of tumor cell-associated immune checkpoint molecules would be the modulation (down- or upregulation) of antitumor immune responses. In recent years, however, it has become clear that the expression of immune checkpoint molecules on tumor cells has important consequences on the biology of the tumor cells themselves. In particular, a causal relationship between the expression of these molecules and the acquisition of malignant traits has been demonstrated. Thus, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells, the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize. Herein, we review this evidence, with a main focus on PD-L1, the most intensively investigated tumor cell-associated immune checkpoint molecule and for which most information is available. Then, we discuss more concisely other tumor cell-associated immune checkpoint molecules that have also been shown to induce the acquisition of malignant traits, such as PD-1, B7-H3, B7-H4, Tim-3, CD70, CD28, CD137, CD40 and CD47. Open questions in this field as well as some therapeutic approaches that can be derived from this knowledge, are also addressed. Copyright © 2017 Elsevier B.V. All rights reserved.

pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis

PubMed Central

Choi, Joung-Woo; Jung, Soo-Jung; Kasala, Dayananda; Hwang, June Kyu; Hu, Jun; Bae, You Han; Yun, Chae-Ok

2015-01-01

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH 6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH 6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH 7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX. PMID:25575865

In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes

PubMed Central

Li, Weida; Nakanishi, Mio; Zumsteg, Adrian; Shear, Matthew; Wright, Christopher; Melton, Douglas A; Zhou, Qiao

2014-01-01

Direct lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin+ β-cells, glucagon+ α-cells, and somatostatin+ δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to β-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and β-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001 PMID:24714494

Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

PubMed Central

Li, Chunhe; Wang, Jin

2013-01-01

Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

PubMed

Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

2017-09-01

Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Association between tumor-stroma ratio and prognosis in solid tumor patients: a systematic review and meta-analysis.

PubMed

Wu, Jiayuan; Liang, Caixia; Chen, Manyu; Su, Wenmei

2016-10-18

Tumor-related stroma plays an active role in tumor invasion and metastasis. The tumor-stroma ratio (TSR) in the pathologic specimen has drawn increasing attention from the field of predicting tumor prognosis. However, the prognostic value of TSR in solid tumors necessitates further elucidation. We conducted a meta-analysis on 14 studies with 4238 patients through a comprehensive electronic search on databases updated on May 2016 to explore the relationship between TSR and prognosis of solid tumors. The overall hazard ratio showed that rich stroma in tumor tissue was associated with poor overall survival (OS) (14 studies, 4238 patients) and disease-free survival (DFS) (9 studies, 2235 patients) of patients with solid tumors. The effect of low TSR on poor OS was observed among various cancer types, but not in the early stage of cervical caner. A significant relationship between low TSR and poor OS was also observed in the subgroup analyses based on study region, blinding status, and Newcastle-Ottawa Scale (NOS) score. Subgroup analyses indicated that cancer type, clinical stage, study region, blinding status, and NOS score did not affect the prognostic value of TSR for DFS. Moreover, low TSR was significantly correlated with the serious clinical stage, advanced depth of invasion, and positive lymph node metastasis. These findings indicate that a high proportion of stroma in cancer tissue is associated with poor clinical outcomes in cancer patients, and TSR may serve as an independent prognostic factor for solid tumors.

Tumor microvessel density–associated mast cells in canine nodal lymphoma

PubMed Central

Mann, Elizabeth; Whittington, Lisa

2014-01-01

Objective: Mast cells are associated in angiogenesis in various human and animal neoplasms. However, association of mast cells with tumor microvessel density in canine lymphoma was not previously documented. The objective of the study is to determine if mast cells are increased in canine nodal lymphomas and to evaluate their correlation with tumor microvessel density and grading of lymphomas. Methods: Nodal lymphomas from 33 dogs were studied and compared with nonneoplastic lymph nodes from 6 dogs as control. Mast cell count was made on Toluidine blue stained sections. Immunohistochemistry using antibody against Factor VIII was employed to visualize and determine microvessel density. Results: The mast cell count in lymphoma (2.95 ± 2.4) was significantly higher (p < 0.05) than that in the control (0.83 ± 0.3) and was positively correlated with tumor microvessel density (r = 0.44, p = 0.009). Significant difference was not observed in mast cell count and tumor microvessel density among different gradings of lymphomas. Conclusions: Mast cells are associated with tumor microvessel density in canine nodal lymphoma with no significant difference among gradings of lymphomas. Mast cells may play an important role in development of canine nodal lymphomas. Further detailed investigation on the role of mast cells as important part of tumor microenvironment in canine nodal lymphomas is recommended. PMID:26770752

Coordination of m6A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming

PubMed Central

Aguilo, Francesca; Zhang, Fan; Sancho, Ana; Fidalgo, Miguel; Di Cecilia, Serena; Vashisht, Ajay; Lee, Dung-Fang; Chen, Chih-Hung; Rengasamy, Madhumitha; Andino, Blanca; Jahouh, Farid; Roman, Angel; Krig, Sheryl R.; Wang, Rong; Zhang, Weijia; Wohlschlegel, James A.; Wang, Jianlong; Walsh, Martin J.

2015-01-01

SUMMARY Epigenetic and epitranscriptomic networks have important functions in maintaining pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However the mechanisms integrating the actions of these distinct networks are only partially understood. Here, we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates transcription of key pluripotency genes, and modulates N6-methyladenosine (m6A) deposition on their transcripts by sequestering the enzyme m6A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m6A RNA levels, and enhances m6A modification of Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3-associated, and is enriched at promoters of m6A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m6A RNA modification to insure ESC identity. PMID:26526723

Near-infrared-absorbing gold nanopopcorns with iron oxide cluster core for magnetically amplified photothermal and photodynamic cancer therapy.

PubMed

Bhana, Saheel; Lin, Gan; Wang, Lijia; Starring, Hunter; Mishra, Sanjay R; Liu, Gang; Huang, Xiaohua

2015-06-03

We present the synthesis and application of a new type of dual magnetic and plasmonic nanostructures for magnetic-field-guided drug delivery and combined photothermal and photodynamic cancer therapy. Near-infrared-absorbing gold nanopopcorns containing a self-assembled iron oxide cluster core were prepared via a seed-mediated growth method. The hybrid nanostructures are superparamagnetic and show great photothermal conversion efficiency (η=61%) under near-infrared irradiation. Compact and stable nanocomplexes for photothermal-photodynamic therapy were formed by coating the nanoparticles with near-infrared-absorbing photosensitizer silicon 2,3-naphthalocyannie dihydroxide and stabilization with poly(ethylene glycol) linked with 11-mercaptoundecanoic acid. The nanocomplex showed enhanced release and cellular uptake of the photosensitizer with the use of a gradient magnetic field. In vitro studies using two different cell lines showed that the dual mode photothermal and photodynamic therapy with the assistance of magnetic-field-guided drug delivery dramatically improved the therapeutic efficacy of cancer cells as compared to the combination treatment without using a magnetic field and the two treatments alone. The "three-in-one" nanocomplex has the potential to carry therapeutic agents deep into a tumor through magnetic manipulation and to completely eradicate tumors by subsequent photothermal and photodynamic therapies without systemic toxicity.

Tumor Expression of CD200 Inhibits IL-10 Production by Tumor-Associated Myeloid Cells and Prevents Tumor Immune Evasion of CTL Therapy

PubMed Central

Wang, Lixin; Liu, Jin-Qing; Talebian, Fatemeh; El-Omrani, Hani Y.; Khattabi, Mazin; Yu, Li; Bai, Xue-Feng

2010-01-01

CD200 is a cell-surface glycoprotein that functions through interaction with the CD200 receptor (CD200R) on myeloid lineage cells to regulate myeloid cell functions. Expression of CD200 has been implicated in multiple types of human cancer, however the impact of tumor expression of CD200 on tumor immunity remains poorly understood. To evaluate this issue, we generated CD200-positive mouse plasmacytoma J558 and mastocytoma P815 cells. We found that established CD200-positive tumors were often completely rejected by adoptively transferred CTL without tumor recurrence; in contrast, CD200-negative tumors were initially rejected by adoptively transferred CTL but the majority of tumors recurred. Tumor expression of CD200 significantly inhibited suppressive activity and IL-10 production by tumor-associated myeloid cells (TAMC), and as a result, more CTL accumulated in the tumor and exhibited a greater capacity to produce IFN-γ in CD200-positive tumors than in CD200-negative tumors. Neutralization of IL-10 significantly inhibited the suppressor activity of TAMC, and IL-10-deficiency allowed TAMC to kill cancer cells and their antigenic variants, which prevented tumor recurrence during CTL therapy. Thus, tumor expression of CD200 prevents tumor recurrence via inhibiting IL-10 production by TAMC. PMID:20662098

Tetramer formation of tumor suppressor protein p53: Structure, function, and applications.

PubMed

Kamada, Rui; Toguchi, Yu; Nomura, Takao; Imagawa, Toshiaki; Sakaguchi, Kazuyasu

2016-11-04

Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal, and reprogramming of differentiated cells into stem cells, immune system, and metastasis. More than 50% of human tumors have TP53 gene mutations, and most of them are missense mutations that presumably reduce tumor suppressor activity of p53. This review focuses on the role of the tetramerization (oligomerization), which is modulated by the protein concentration of p53, posttranslational modifications, and/or interactions with its binding proteins, in regulating the tumor suppressor function of p53. Functional control of p53 by stabilizing or inhibiting oligomer formation and its bio-applications are also discussed. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 598-612, 2016. © 2015 Wiley Periodicals, Inc.

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.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

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.

Fast-ball sports experts depend on an inhibitory strategy to reprogram their movement timing.

PubMed

Nakamoto, Hiroki; Ikudome, Sachi; Yotani, Kengo; Maruyama, Atsuo; Mori, Shiro

2013-07-01

The purpose of our study was to clarify whether an inhibitory strategy is used for reprogramming of movement timing by experts in fast-ball sports when they correct their movement timing due to unexpected environmental changes. We evaluated the influence of disruption of inhibitory function of the right inferior frontal gyrus (rIFG) on reprogramming of movement timing of experts and non-experts in fast-ball sports. The task was to manually press a button to coincide with the arrival of a moving target. The target moved at a constant velocity, and its velocity was suddenly either increased or decreased in some trials. The task was performed either with or without transcranial magnetic stimulation (TMS), which was delivered to the region of the rIFG. Under velocity change conditions without TMS, the experts showed significantly smaller timing errors and a higher rate of reprogramming of movement timing than the non-experts. Moreover, TMS application during the task significantly diminished the expert group's performance, but not the control group, particularly in the condition where the target velocity decreases. These results suggest that experts use an inhibitory strategy for reprogramming of movement timing. In addition, the rIFG inhibitory function contributes to the superior movement correction of experts in fast-ball sports.

Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation

PubMed Central

Rodgers, Ali B.; Morgan, Christopher P.; Bronson, Stefanie L.; Revello, Sonia; Bale, Tracy L.

2013-01-01

Neuropsychiatric disease frequently presents with an underlying hypo- or hyper- reactivity of the HPA stress axis, suggesting an exceptional vulnerability of this circuitry to external perturbations. Parental lifetime exposures to environmental challenges are associated with increased offspring neuropsychiatric disease risk, and likely contribute to stress dysregulation. While maternal influences have been extensively examined, much less is known regarding the specific role of paternal factors. To investigate the potential mechanisms by which paternal stress may contribute to offspring hypothalamic-pituitary-adrenal (HPA) axis dysregulation, we exposed mice to six weeks of chronic stress prior to breeding. As epidemiological studies support variation in paternal germ cell susceptibility to reprogramming across the lifespan, male stress exposure occurred either throughout puberty or in adulthood. Remarkably, offspring of sires from both paternal stress groups displayed significantly reduced HPA axis stress responsivity. Gene set enrichment analyses in offspring stress regulating brain regions, the paraventricular nucleus (PVN) and the bed nucleus of stria terminalis (BNST), revealed global pattern changes in transcription suggestive of epigenetic reprogramming and consistent with altered offspring stress responsivity, including increased expression of glucocorticoid-responsive genes in the PVN. In examining potential epigenetic mechanisms of germ cell transmission, we found robust changes in sperm miRNA (miR) content, where nine specific miRs were significantly increased in both paternal stress groups. Overall, these results demonstrate that paternal experience across the lifespan can induce germ cell epigenetic reprogramming and impact offspring HPA stress axis regulation, and may therefore offer novel insight into factors influencing neuropsychiatric disease risk. PMID:23699511

Heterogeneous assembled nanocomplexes for ratiometric detection of highly reactive oxygen species in vitro and in vivo.

PubMed

Ju, Enguo; Liu, Zhen; Du, Yingda; Tao, Yu; Ren, Jinsong; Qu, Xiaogang

2014-06-24

Probes for detecting highly reactive oxygen species (hROS) are critical to both understanding the etiology of the disease and optimizing therapeutic interventions. However, problems such as low stability due to autoxidation and photobleaching and unsuitability for biological application in vitro and in vivo, as well as the high cost and complex procedure in synthesis and modification, largely limit their application. In this work, binary heterogeneous nanocomplexes (termed as C-dots-AuNC) constructed from gold clusters and carbon dots were reported. The fabrication takes full advantages of the inherent active groups on the surface of the nanoparticles to avoid tedious modification and chemical synthetic processes. Additionally, the assembly endowed C-dots-AuNC with improved performance such as the fluorescence enhancement of AuNCs and stability of C-dots to hROS. Moreover, the dual-emission property allows sensitive imaging and monitoring of the hROS signaling in living cells with high contrast. Importantly, with high physiological stability and excellent biocompatibility, C-dots-AuNC allows for the detection of hROS in the model of local ear inflammation.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression.

PubMed

Umei, Tomohiko C; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-08-19

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

PubMed Central

Umei, Tomohiko C.; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Kurotsu, Shota; Tamura, Fumiya; Osakabe, Rina; Tani, Hidenori; Nara, Kaori; Miyoshi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

2017-01-01

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming. PMID:28825623

Cell Fate Reprogramming by Control of Intracellular Network Dynamics

PubMed Central

Zañudo, Jorge G. T.; Albert, Réka

2015-01-01

Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell’s fate, such as disease therapeutics and stem cell reprogramming. Here we develop a novel network control framework that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our approach drives any initial state to the target state with 100% effectiveness and needs to be applied only transiently for the network to reach and stay in the desired state. We illustrate our method’s potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of helper T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. PMID:25849586

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

PubMed Central

Malyshev, Igor; Malyshev, Yuri

2015-01-01

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

PubMed

Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S; Benigni, Ariela

2015-04-14

The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American

Regenerating the human heart: direct reprogramming strategies and their current limitations.

PubMed

Ghiroldi, Andrea; Piccoli, Marco; Ciconte, Giuseppe; Pappone, Carlo; Anastasia, Luigi

2017-10-27

Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

Simulating Heterogeneous Tumor Cell Populations

PubMed Central

Bar-Sagi, Dafna; Mishra, Bud

2016-01-01

Certain tumor phenomena, like metabolic heterogeneity and local stable regions of chronic hypoxia, signify a tumor’s resistance to therapy. Although recent research has shed light on the intracellular mechanisms of cancer metabolic reprogramming, little is known about how tumors become metabolically heterogeneous or chronically hypoxic, namely the initial conditions and spatiotemporal dynamics that drive these cell population conditions. To study these aspects, we developed a minimal, spatially-resolved simulation framework for modeling tissue-scale mixed populations of cells based on diffusible particles the cells consume and release, the concentrations of which determine their behavior in arbitrarily complex ways, and on stochastic reproduction. We simulate cell populations that self-sort to facilitate metabolic symbiosis, that grow according to tumor-stroma signaling patterns, and that give rise to stable local regions of chronic hypoxia near blood vessels. We raise two novel questions in the context of these results: (1) How will two metabolically symbiotic cell subpopulations self-sort in the presence of glucose, oxygen, and lactate gradients? We observe a robust pattern of alternating striations. (2) What is the proper time scale to observe stable local regions of chronic hypoxia? We observe the stability is a function of the balance of three factors related to O2—diffusion rate, local vessel release rate, and viable and hypoxic tumor cell consumption rate. We anticipate our simulation framework will help researchers design better experiments and generate novel hypotheses to better understand dynamic, emergent whole-tumor behavior. PMID:28030620

Carcinogenesis associated with parasites other than Schistosoma, Opisthorchis and Clonorchis: A systematic review.

PubMed

Machicado, Claudia; Marcos, Luis A

2016-06-15

Only three helminths (Schistosoma haematobium, Opisthorchis viverrini and Clonorchis sinensis) are directly associated with carcinogenesis in humans whereas the role of other parasites in cancer remains unclear. This study aimed to perform a systematic review to identify recent insights in the role of other parasite infections in carcinogenesis. We conducted systematic searches of MEDLINE and EMBASE on July 2015. Our primary outcome was the association between parasitic infections and carcinogenesis. Out of 1,266 studies, 19 were selected for detailed evaluation (eight for helminths and 11 for protozoa). The mechanisms of helminth-induced cancer included chronic inflammation, sustained proliferation, modulation of the host immune system, reprogramming of glucose metabolism and redox signaling, induction of genomic instability and destabilization of suppressor tumor proteins, stimulation of angiogenesis, resisting cell death, and activation of invasion and metastasis. In addition to the current knowledge, the following parasites were found in cancers or tumors: Echinococcus, Strongyloides, Fasciola, Heterakis, Platynosomum and Trichuris. Additional parasites were found in this systematic review that could potentially be associated with cancers or tumors but further evidence is needed to elaborate a cause-effect relationship. © 2016 UICC.

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

Antibody induction directed against the tumor-associated MUC4 glycoprotein.

PubMed

Cai, Hui; Palitzsch, Björn; Hartmann, Sebastian; Stergiou, Natascha; Kunz, Horst; Schmitt, Edgar; Westerlind, Ulrika

2015-04-13

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor-associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem-repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4-based vaccines induced very strong antigen-specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aggressive rat prostate tumors reprogram the benign parts of the prostate and regional lymph nodes prior to metastasis

PubMed Central

Thysell, Elin; Halin Bergström, Sofia; Bergh, Anders

2017-01-01

In order to grow and spread tumors need to interact with adjacent tissues. We therefore hypothesized that small but aggressive prostate cancers influence the rest of the prostate and regional lymph nodes differently than tumors that are more indolent. Poorly metastatic (Dunning AT1) or highly metastatic (Dunning MLL) rat prostate tumor cells were injected into the ventral prostate lobe of immunocompetent rats. After 10 days—when the tumors occupied about 30% of the prostate lobe and lymph node metastases were undetectable—the global gene expression in tumors, benign parts of the prostate, and regional iliac lymph nodes were examined to define tumor-induced changes related to preparation for future metastasis. The tumors induced profound effects on the gene expression profiles in the benign parts of the prostate and these were strikingly different in the two tumor models. Gene ontology enrichment analysis suggested that tumors with high metastatic capacity were more successful than less metastatic tumors in inducing tumor-promoting changes and suppressing anti-tumor immune responses in the entire prostate. Some of these differences such as altered angiogenesis, nerve density, accumulation of T-cells and macrophages were verified by immunohistochemistry. Gene expression alterations in the regional lymph nodes suggested decreased quantity and activation of immune cells in MLL-lymph nodes that were also verified by immunostaining. In summary, even when small highly metastatic prostate tumors can affect the entire tumor-bearing organ and pre-metastatic lymph nodes differently than less metastatic tumors. When the kinetics of these extratumoral influences (by us named TINT = tumor instructed normal tissue) are more precisely defined they could potentially be used as markers of disease aggressiveness and become therapeutic targets. PMID:28472073

Prenatal Choline Supplementation Diminishes Early-Life Iron Deficiency–Induced Reprogramming of Molecular Networks Associated with Behavioral Abnormalities in the Adult Rat Hippocampus123

PubMed Central

Tran, Phu V; Kennedy, Bruce C; Pisansky, Marc T; Won, Kyoung-Jae; Gewirtz, Jonathan C; Simmons, Rebecca A; Georgieff, Michael K

2016-01-01

Background: Early-life iron deficiency is a common nutrient deficiency worldwide. Maternal iron deficiency increases the risk of schizophrenia and autism in the offspring. Postnatal iron deficiency in young children results in cognitive and socioemotional abnormalities in adulthood despite iron treatment. The rat model of diet-induced fetal-neonatal iron deficiency recapitulates the observed neurobehavioral deficits. Objectives: We sought to establish molecular underpinnings for the persistent psychopathologic effects of early-life iron deficiency by determining whether it permanently reprograms the hippocampal transcriptome. We also assessed the effects of maternal dietary choline supplementation on the offspring’s hippocampal transcriptome to identify pathways through which choline mitigates the emergence of long-term cognitive deficits. Methods: Male rat pups were made iron deficient (ID) by providing pregnant and nursing dams an ID diet (4 g Fe/kg) from gestational day (G) 2 through postnatal day (PND) 7 and an iron-sufficient (IS) diet (200 g Fe/kg) thereafter. Control pups were provided IS diet throughout. Choline (5 g/kg) was given to half the pregnant dams in each group from G11 to G18. PND65 hippocampal transcriptomes were assayed by next generation sequencing (NGS) and analyzed with the use of knowledge-based Ingenuity Pathway Analysis. Real-time polymerase chain reaction was performed to validate a subset of altered genes. Results: Formerly ID rats had altered hippocampal expression of 619 from >10,000 gene loci sequenced by NGS, many of which map onto molecular networks implicated in psychological disorders, including anxiety, autism, and schizophrenia. There were significant interactions between iron status and prenatal choline treatment in influencing gene expression. Choline supplementation reduced the effects of iron deficiency, including those on gene networks associated with autism and schizophrenia. Conclusions: Fetal-neonatal iron deficiency

Cancer-promoting tumor-associated macrophages: new vistas and open questions.

PubMed

Mantovani, Alberto; Germano, Giovanni; Marchesi, Federica; Locatelli, Marco; Biswas, Subhra K

2011-09-01

Tumor-associated macrophages (TAMs) are key components of the tumor macroenvironment. Cancer- and host cell-derived signals generally drive the functions of TAMs towards an M2-like polarized, tumor-propelling mode; however, when appropriately re-educated. TAMs also have the potential to elicit tumor destructive reactions. Here, we discuss recent advances regarding the immunobiology of TAMs and highlight open questions including the mechanisms of their accumulation (recruitment versus proliferation), their diversity and how to best therapeutically target these cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

PubMed

Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

2013-01-01

Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This

Lmo2 expression defines tumor cell identity during T-cell leukemogenesis.

PubMed

García-Ramírez, Idoia; Bhatia, Sanil; Rodríguez-Hernández, Guillermo; González-Herrero, Inés; Walter, Carolin; González de Tena-Dávila, Sara; Parvin, Salma; Haas, Oskar; Woessmann, Wilhelm; Stanulla, Martin; Schrappe, Martin; Dugas, Martin; Natkunam, Yasodha; Orfao, Alberto; Domínguez, Verónica; Pintado, Belén; Blanco, Oscar; Alonso-López, Diego; De Las Rivas, Javier; Martín-Lorenzo, Alberto; Jiménez, Rafael; García Criado, Francisco Javier; García Cenador, María Begoña; Lossos, Izidore S; Vicente-Dueñas, Carolina; Borkhardt, Arndt; Hauer, Julia; Sánchez-García, Isidro

2018-06-07

The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel.

PubMed

Sato, Kei A; Hachiya, Tsuyoshi; Iwaya, Takeshi; Kume, Kohei; Matsuo, Teppei; Kawasaki, Keisuke; Abiko, Yukito; Akasaka, Risaburo; Matsumoto, Takayuki; Otsuka, Koki; Nishizuka, Satoshi S

2016-01-01

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

Shifting behaviour: epigenetic reprogramming in eusocial insects.

PubMed

Patalano, Solenn; Hore, Timothy A; Reik, Wolf; Sumner, Seirian

2012-06-01

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity. Copyright © 2012 Elsevier Ltd. All rights reserved.

CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways.

PubMed

Li, Jibin; Huang, Qichao; Long, Xiaoyu; Zhang, Jing; Huang, Xiaojun; Aa, Jiye; Yang, Hushan; Chen, Zhinan; Xing, Jinliang

2015-12-01

CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive. We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms. Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells. Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

PubMed

Ebrahimi, Behnam

2016-01-01

Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Developmental Programming of Renal Function and Re-Programming Approaches.

PubMed

Nüsken, Eva; Dötsch, Jörg; Weber, Lutz T; Nüsken, Kai-Dietrich

2018-01-01

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin-angiotensin-aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early

Associations of breast cancer risk factors with tumor subtypes: a pooled analysis from the Breast Cancer Association Consortium studies.

PubMed

Yang, Xiaohong R; Chang-Claude, Jenny; Goode, Ellen L; Couch, Fergus J; Nevanlinna, Heli; Milne, Roger L; Gaudet, Mia; Schmidt, Marjanka K; Broeks, Annegien; Cox, Angela; Fasching, Peter A; Hein, Rebecca; Spurdle, Amanda B; Blows, Fiona; Driver, Kristy; Flesch-Janys, Dieter; Heinz, Judith; Sinn, Peter; Vrieling, Alina; Heikkinen, Tuomas; Aittomäki, Kristiina; Heikkilä, Päivi; Blomqvist, Carl; Lissowska, Jolanta; Peplonska, Beata; Chanock, Stephen; Figueroa, Jonine; Brinton, Louise; Hall, Per; Czene, Kamila; Humphreys, Keith; Darabi, Hatef; Liu, Jianjun; Van 't Veer, Laura J; van Leeuwen, Flora E; Andrulis, Irene L; Glendon, Gord; Knight, Julia A; Mulligan, Anna Marie; O'Malley, Frances P; Weerasooriya, Nayana; John, Esther M; Beckmann, Matthias W; Hartmann, Arndt; Weihbrecht, Sebastian B; Wachter, David L; Jud, Sebastian M; Loehberg, Christian R; Baglietto, Laura; English, Dallas R; Giles, Graham G; McLean, Catriona A; Severi, Gianluca; Lambrechts, Diether; Vandorpe, Thijs; Weltens, Caroline; Paridaens, Robert; Smeets, Ann; Neven, Patrick; Wildiers, Hans; Wang, Xianshu; Olson, Janet E; Cafourek, Victoria; Fredericksen, Zachary; Kosel, Matthew; Vachon, Celine; Cramp, Helen E; Connley, Daniel; Cross, Simon S; Balasubramanian, Sabapathy P; Reed, Malcolm W R; Dörk, Thilo; Bremer, Michael; Meyer, Andreas; Karstens, Johann H; Ay, Aysun; Park-Simon, Tjoung-Won; Hillemanns, Peter; Arias Pérez, Jose Ignacio; Menéndez Rodríguez, Primitiva; Zamora, Pilar; Benítez, Javier; Ko, Yon-Dschun; Fischer, Hans-Peter; Hamann, Ute; Pesch, Beate; Brüning, Thomas; Justenhoven, Christina; Brauch, Hiltrud; Eccles, Diana M; Tapper, William J; Gerty, Sue M; Sawyer, Elinor J; Tomlinson, Ian P; Jones, Angela; Kerin, Michael; Miller, Nicola; McInerney, Niall; Anton-Culver, Hoda; Ziogas, Argyrios; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Yang, Show-Lin; Yu, Jyh-Cherng; Chen, Shou-Tung; Hsu, Giu-Cheng; Haiman, Christopher A; Henderson, Brian E; Le Marchand, Loic; Kolonel, Laurence N; Lindblom, Annika; Margolin, Sara; Jakubowska, Anna; Lubiński, Jan; Huzarski, Tomasz; Byrski, Tomasz; Górski, Bohdan; Gronwald, Jacek; Hooning, Maartje J; Hollestelle, Antoinette; van den Ouweland, Ans M W; Jager, Agnes; Kriege, Mieke; Tilanus-Linthorst, Madeleine M A; Collée, Margriet; Wang-Gohrke, Shan; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Mononen, Kari; Grip, Mervi; Hirvikoski, Pasi; Winqvist, Robert; Mannermaa, Arto; Kosma, Veli-Matti; Kauppinen, Jaana; Kataja, Vesa; Auvinen, Päivi; Soini, Ylermi; Sironen, Reijo; Bojesen, Stig E; Ørsted, David Dynnes; Kaur-Knudsen, Diljit; Flyger, Henrik; Nordestgaard, Børge G; Holland, Helene; Chenevix-Trench, Georgia; Manoukian, Siranoush; Barile, Monica; Radice, Paolo; Hankinson, Susan E; Hunter, David J; Tamimi, Rulla; Sangrajrang, Suleeporn; Brennan, Paul; McKay, James; Odefrey, Fabrice; Gaborieau, Valerie; Devilee, Peter; Huijts, P E A; Tollenaar, R A E M; Seynaeve, C; Dite, Gillian S; Apicella, Carmel; Hopper, John L; Hammet, Fleur; Tsimiklis, Helen; Smith, Letitia D; Southey, Melissa C; Humphreys, Manjeet K; Easton, Douglas; Pharoah, Paul; Sherman, Mark E; Garcia-Closas, Montserrat

2011-02-02

Previous studies have suggested that breast cancer risk factors are associated with estrogen receptor (ER) and progesterone receptor (PR) expression status of the tumors. We pooled tumor marker and epidemiological risk factor data from 35,568 invasive breast cancer case patients from 34 studies participating in the Breast Cancer Association Consortium. Logistic regression models were used in case-case analyses to estimate associations between epidemiological risk factors and tumor subtypes, and case-control analyses to estimate associations between epidemiological risk factors and the risk of developing specific tumor subtypes in 12 population-based studies. All statistical tests were two-sided. In case-case analyses, of the epidemiological risk factors examined, early age at menarche (≤12 years) was less frequent in case patients with PR(-) than PR(+) tumors (P = .001). Nulliparity (P = 3 × 10(-6)) and increasing age at first birth (P = 2 × 10(-9)) were less frequent in ER(-) than in ER(+) tumors. Obesity (body mass index [BMI] ≥ 30 kg/m(2)) in younger women (≤50 years) was more frequent in ER(-)/PR(-) than in ER(+)/PR(+) tumors (P = 1 × 10(-7)), whereas obesity in older women (>50 years) was less frequent in PR(-) than in PR(+) tumors (P = 6 × 10(-4)). The triple-negative (ER(-)/PR(-)/HER2(-)) or core basal phenotype (CBP; triple-negative and cytokeratins [CK]5/6(+) and/or epidermal growth factor receptor [EGFR](+)) accounted for much of the heterogeneity in parity-related variables and BMI in younger women. Case-control analyses showed that nulliparity, increasing age at first birth, and obesity in younger women showed the expected associations with the risk of ER(+) or PR(+) tumors but not triple-negative (nulliparity vs parity, odds ratio [OR] = 0.94, 95% confidence interval [CI] = 0.75 to 1.19, P = .61; 5-year increase in age at first full-term birth, OR = 0.95, 95% CI = 0.86 to 1.05, P = .34; obesity in younger women, OR = 1.36, 95% CI = 0.95 to 1

Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System.

PubMed

Ma, Yingcong; Sha, Meng; Cheng, Shixuan; Yao, Wang; Li, Zhongjun; Qi, Xian-Rong

2018-06-14

The CD44 protein, as a predominant receptor for hyaluronan (HA), is highly expressed on the surface of multiple tumor cells. HA, as a targeting molecule for a CD44-contained delivery system, increases intracellular drug concentration in tumor tissue. However, due to the weak binding ability of hyaluronan oligosaccharide to CD44, targeting for tumor drug delivery has been restricted. In this study, we first use a HA tetrasaccharide cluster as the target ligand to enhance the binding ability to CD44. A polyamidoamine (PAMAM) dendrimer was modified by a HA tetrasaccharide cluster as a nonviral vector for small interfering RNA (siRNA) delivery. The dendrimer/siRNA nanocomplexes increased the cellular uptake capacity of siRNA through the CD44 receptor-mediated endocytosis pathway, allowing the siRNA to successfully escape the endosome/lysosome. Compared with the control group, nanocomplexes effectively reduced the expression of GFP protein and mRNA in MDA-MB-231-GFP cells. This delivery system provides a foundation to increase the clinical applications of PAMAM nanomaterials.

Recoverin-associated retinopathy secondary to Warthin tumor of parotid gland.

PubMed

Machida, Shigeki; Ohguro, Hiroshi; Ishida, Kazunori; Suzuki, Masamichi; Kawagishi, Kazuaki

2014-10-01

To present a case of photoreceptor degeneration associated with a benign Warthin tumor of the parotid gland. A 57-year-old man visited our clinic complaining of blurred vision in both eyes. His best-corrected visual acuity was 0.07 in the right and 0.04 in the left eyes. All components of the full-field electroretinograms (ERGs) were reduced in both eyes. The focal macular ERGs were extinguished in both eyes, which was consistent with the deterioration of the outer retina in optical coherence tomographic images. Positron emission tomography showed (18)F-fluorodeoxy glucose accumulation in the left parotid gland. Parotidectomy was performed, and the histopathology of the specimen had features compatible with a Warthin tumor without malignancy. Western blot analysis of the patient's sera detected an antibody against recoverin. In addition, the tumor tissue had an aberrant expression of recoverin. The findings in this case indicate that recoverin-associated retinopathy can develop secondary to a benign Warthin tumor.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

PubMed

Hysolli, Eriona; Tanaka, Yoshiaki; Su, Juan; Kim, Kun-Yong; Zhong, Tianyu; Janknecht, Ralf; Zhou, Xiao-Ling; Geng, Lin; Qiu, Caihong; Pan, Xinghua; Jung, Yong-Wook; Cheng, Jijun; Lu, Jun; Zhong, Mei; Weissman, Sherman M; Park, In-Hyun

2016-07-12

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

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

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

DOE PAGES

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; ...

2015-05-07

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

Tumor-associated myeloid cells as guiding forces of cancer cell stemness.

PubMed

Sica, Antonio; Porta, Chiara; Amadori, Alberto; Pastò, Anna

2017-08-01

Due to their ability to differentiate into various cell types and to support tissue regeneration, stem cells simultaneously became the holy grail of regenerative medicine and the evil obstacle in cancer therapy. Several studies have investigated niche-related conditions that favor stemness properties and increasingly emphasized their association with an inflammatory environment. Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are major orchestrators of cancer-related inflammation, able to dynamically express different polarized inflammatory programs that promote tumor outgrowth, including tumor angiogenesis, immunosuppression, tissue remodeling and metastasis formation. In addition, these myeloid populations support cancer cell stemness, favoring tumor maintenance and progression, as well as resistance to anticancer treatments. Here, we discuss inflammatory circuits and molecules expressed by TAMs and MDSCs as guiding forces of cancer cell stemness.

Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma

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

Qin, Tian; Wang, Chenlong; Chen, Xuewei

Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50 mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing themore » coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy. - Highlights: • Dopamine induces tumor growth inhibition and vascular normalization in rat C6 glioma. • Dopamine switches macrophage phenotype from M2 to M1. • Dopamine-induced vascular normalization is mediated by macrophage polarization. • Dopamine is a promising agent targeting the microvasculature in

Testis tumor associated to microlithiasis.

PubMed

Jesus, Lisieux Eyer de; Maciel, Felipe; Monnerat, Andrea Lima C; Fernandes, Marcia Antunes; Dekermache, Samuel

2013-12-01

To discuss the relationship between testicular microlithiasis and testis tumors in children and to consider the chances of testis preserving surgery in specific cases. Pre-adolescent presenting testicular microlithiasis and a larger left testis, corresponding to a cystic testicular tumor. The tumor was excised, with ipsilateral testis preservation. Histology diagnosed a testis dermoid tumor. The relationship between testis tumors and testicular microlithiasis is ill defined in children. Pediatric urologists need to develop specific follow-up protocols for pre-pubertal children.

Exploring the molecular aspects associated with testicular germ cell tumors: a review

PubMed Central

Facchini, Gaetano; Rossetti, Sabrina; Cavaliere, Carla; D’Aniello, Carmine; Di Franco, Rossella; Iovane, Gelsomina; Grimaldi, Giovanni; Piscitelli, Raffaele; Muto, Paolo; Botti, Gerardo; Perdonà, Sisto; Veneziani, Bianca Maria; Berretta, Massimiliano; Montanari, Micaela

2018-01-01

Testicular germ cell tumors (TGCTs) represent the most common solid tumors affecting young men. They constitute a distinct entity because of their embryonic origin and their unique biological behavior. Recent preclinical data regarding biological signaling machinery as well as genetic and epigenetic mechanisms associated with molecular patterns of tumors have contribute to explain the pathogenesis and the differentiation of TGCTs and to understand the mechanisms responsible for the development of resistance to treatment. In this review, we discuss the main genetic and epigenetic events associated with TGCTs development in order to better define their role in the pathogenesis of these tumors and in cisplatin-acquired resistance. PMID:29416701

Fibrinolysis in Tumor Associated Angiogenesis

DTIC Science & Technology

2005-07-01

tPA expression in mammary vessel assays and in animals with use of retroviral vectors to deliver antisense RNA . We still intend to use that strategy...pads of nude mice. RNA obtained from tumor- and mam- mary fat pad-associated endothelial cells was used to synthesize cDNA and cDNA libraries, which... RNA (mRNA) for tPA and MT1-MMP, with some upregulation of uPA expression. BODY 1. Confirm differential expression of tPA and MT1-MIP with GAPDH control

Transcriptional Modulation of Tumor-Associated Macrophages to Facilitate Prostate Cancer Immunotherapy

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0334 TITLE : TRANSCRIPTIONAL MODULATION OF TUMOR-ASSOCIATED MACROPHAGES TO FACILITATE PROSTATE CANCER IMMUNOTHERAPY...OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT...Annual 3. DATES COVERED (From - To) 1SEP2016 - 31AUG2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER TRANSCRIPTIONAL MODULATION OF TUMOR-ASSOCIATED

Testis tumor associated to microlithiasis

PubMed Central

de Jesus, Lisieux Eyer; Maciel, Felipe; Monnerat, Andrea Lima C.; Fernandes, Marcia Antunes; Dekermache, Samuel

2013-01-01

OBJECTIVE: To discuss the relationship between testicular microlithiasis and testis tumors in children and to consider the chances of testis preserving surgery in specific cases. CASE DESCRIPTION: Pre-adolescent presenting testicular microlithiasis and a larger left testis, corresponding to a cystic testicular tumor. The tumor was excised, with ipsilateral testis preservation. Histology diagnosed a testis dermoid tumor. COMMENTS: The relationship between testis tumors and testicular microlithiasis is ill defined in children. Pediatric urologists need to develop specific follow-up protocols for pre-pubertal children. PMID:24473964

PD-1 expression by tumor-associated macrophages inhibits phagocytosis and tumor immunity

PubMed Central

Gordon, Sydney R.; Maute, Roy L.; Dulken, Ben W.; Hutter, Gregor; George, Benson M.; McCracken, Melissa N.; Gupta, Rohit; Tsai, Jonathan M.; Sinha, Rahul; Corey, Daniel; Ring, Aaron M.; Connolly, Andrew J.; Weissman, Irving L.

2017-01-01

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells to induce immune tolerance.1,2 Tumor cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating escape from the immune system.3,4 Monoclonal antibodies blocking PD-1/PD-L1 have shown remarkable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small cell lung cancer, and Hodgkin’s lymphoma.5–9 Although it is well-established that PD-1/PD-L1 blockade activates T cells, little is known about the role that this pathway may have on tumor-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models, and with increasing disease stage in primary human cancers. TAM PD-1 expression negatively correlates with phagocytic potency against tumor cells, and blockade of PD-1/PD-L1 in vivo increases macrophage phagocytosis, reduces tumor growth, and lengthens survival in mouse models of cancer in a macrophage-dependent fashion. Our results suggest that PD-1/PD-L1 therapies may also function through a direct effect on macrophages, with significant implications for treatment with these agents. PMID:28514441

Associations of Breast Cancer Risk Factors With Tumor Subtypes: A Pooled Analysis From the Breast Cancer Association Consortium Studies

PubMed Central

Chang-Claude, Jenny; Goode, Ellen L.; Couch, Fergus J.; Nevanlinna, Heli; Milne, Roger L.; Gaudet, Mia; Schmidt, Marjanka K.; Broeks, Annegien; Cox, Angela; Fasching, Peter A.; Hein, Rebecca; Spurdle, Amanda B.; Blows, Fiona; Driver, Kristy; Flesch-Janys, Dieter; Heinz, Judith; Sinn, Peter; Vrieling, Alina; Heikkinen, Tuomas; Aittomäki, Kristiina; Heikkilä, Päivi; Blomqvist, Carl; Lissowska, Jolanta; Peplonska, Beata; Chanock, Stephen; Figueroa, Jonine; Brinton, Louise; Hall, Per; Czene, Kamila; Humphreys, Keith; Darabi, Hatef; Liu, Jianjun; Van ‘t Veer, Laura J.; van Leeuwen, Flora E.; Andrulis, Irene L.; Glendon, Gord; Knight, Julia A.; Mulligan, Anna Marie; O’Malley, Frances P.; Weerasooriya, Nayana; John, Esther M.; Beckmann, Matthias W.; Hartmann, Arndt; Weihbrecht, Sebastian B.; Wachter, David L.; Jud, Sebastian M.; Loehberg, Christian R.; Baglietto, Laura; English, Dallas R.; Giles, Graham G.; McLean, Catriona A.; Severi, Gianluca; Lambrechts, Diether; Vandorpe, Thijs; Weltens, Caroline; Paridaens, Robert; Smeets, Ann; Neven, Patrick; Wildiers, Hans; Wang, Xianshu; Olson, Janet E.; Cafourek, Victoria; Fredericksen, Zachary; Kosel, Matthew; Vachon, Celine; Cramp, Helen E.; Connley, Daniel; Cross, Simon S.; Balasubramanian, Sabapathy P.; Reed, Malcolm W. R.; Dörk, Thilo; Bremer, Michael; Meyer, Andreas; Karstens, Johann H.; Ay, Aysun; Park-Simon, Tjoung-Won; Hillemanns, Peter; Arias Pérez, Jose Ignacio; Rodríguez, Primitiva Menéndez; Zamora, Pilar; Benítez, Javier; Ko, Yon-Dschun; Fischer, Hans-Peter; Hamann, Ute; Pesch, Beate; Brüning, Thomas; Justenhoven, Christina; Brauch, Hiltrud; Eccles, Diana M.; Tapper, William J.; Gerty, Sue M.; Sawyer, Elinor J.; Tomlinson, Ian P.; Jones, Angela; Kerin, Michael; Miller, Nicola; McInerney, Niall; Anton-Culver, Hoda; Ziogas, Argyrios; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Yu, Jyh-Cherng; Chen, Shou-Tung; Hsu, Giu-Cheng; Haiman, Christopher A.; Henderson, Brian E.; Le Marchand, Loic; Kolonel, Laurence N.; Lindblom, Annika; Margolin, Sara; Jakubowska, Anna; Lubiński, Jan; Huzarski, Tomasz; Byrski, Tomasz; Górski, Bohdan; Gronwald, Jacek; Hooning, Maartje J.; Hollestelle, Antoinette; van den Ouweland, Ans M. W.; Jager, Agnes; Kriege, Mieke; Tilanus-Linthorst, Madeleine M. A.; Collée, Margriet; Wang-Gohrke, Shan; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Mononen, Kari; Grip, Mervi; Hirvikoski, Pasi; Winqvist, Robert; Mannermaa, Arto; Kosma, Veli-Matti; Kauppinen, Jaana; Kataja, Vesa; Auvinen, Päivi; Soini, Ylermi; Sironen, Reijo; Bojesen, Stig E.; Dynnes Ørsted, David; Kaur-Knudsen, Diljit; Flyger, Henrik; Nordestgaard, Børge G.; Holland, Helene; Chenevix-Trench, Georgia; Manoukian, Siranoush; Barile, Monica; Radice, Paolo; Hankinson, Susan E.; Hunter, David J.; Tamimi, Rulla; Sangrajrang, Suleeporn; Brennan, Paul; McKay, James; Odefrey, Fabrice; Gaborieau, Valerie; Devilee, Peter; Huijts, P.E.A.; Tollenaar, RAEM.; Seynaeve, C.; Dite, Gillian S.; Apicella, Carmel; Hopper, John L.; Hammet, Fleur; Tsimiklis, Helen; Smith, Letitia D.; Southey, Melissa C.; Humphreys, Manjeet K.; Easton, Douglas; Pharoah, Paul; Sherman, Mark E.; Garcia-Closas, Montserrat

2011-01-01

Background Previous studies have suggested that breast cancer risk factors are associated with estrogen receptor (ER) and progesterone receptor (PR) expression status of the tumors. Methods We pooled tumor marker and epidemiological risk factor data from 35 568 invasive breast cancer case patients from 34 studies participating in the Breast Cancer Association Consortium. Logistic regression models were used in case–case analyses to estimate associations between epidemiological risk factors and tumor subtypes, and case–control analyses to estimate associations between epidemiological risk factors and the risk of developing specific tumor subtypes in 12 population-based studies. All statistical tests were two-sided. Results In case–case analyses, of the epidemiological risk factors examined, early age at menarche (≤12 years) was less frequent in case patients with PR− than PR+ tumors (P = .001). Nulliparity (P = 3 × 10−6) and increasing age at first birth (P = 2 × 10−9) were less frequent in ER− than in ER+ tumors. Obesity (body mass index [BMI] ≥ 30 kg/m2) in younger women (≤50 years) was more frequent in ER−/PR− than in ER+/PR+ tumors (P = 1 × 10−7), whereas obesity in older women (>50 years) was less frequent in PR− than in PR+ tumors (P = 6 × 10−4). The triple-negative (ER−/PR−/HER2−) or core basal phenotype (CBP; triple-negative and cytokeratins [CK]5/6+ and/or epidermal growth factor receptor [EGFR]+) accounted for much of the heterogeneity in parity-related variables and BMI in younger women. Case–control analyses showed that nulliparity, increasing age at first birth, and obesity in younger women showed the expected associations with the risk of ER+ or PR+ tumors but not triple-negative (nulliparity vs parity, odds ratio [OR] = 0.94, 95% confidence interval [CI] = 0.75 to 1.19, P = .61; 5-year increase in age at first full-term birth, OR = 0.95, 95% CI = 0.86 to 1.05, P = .34; obesity in younger women, OR = 1.36, 95

Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

PubMed

Ho, Lin; Hsu, Shan-Hui

2018-04-01

3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be

Self-assembly of an upconverting nanocomplex and its application to turn-on detection of metalloproteinase-9 in living cells

NASA Astrophysics Data System (ADS)

Nguyen, Phuong-Diem; Thanh Cong, Vu; Baek, Changyoon; Min, Junhong

2016-10-01

Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml-1. Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.

Loss of 11q and 16q in Wilms tumors is associated with anaplasia, tumor recurrence, and poor prognosis.

PubMed

Wittmann, Stefanie; Zirn, Birgit; Alkassar, Muhannad; Ambros, Peter; Graf, Norbert; Gessler, Manfred

2007-02-01

Allele loss of chromosome arms 11q and 16q in Wilms tumors has been associated with different clinical parameters in prior studies. To substantiate these findings in a large collection of tumors treated according to the GPOH/SIOP protocol and to narrow down critical regions, we performed loss of heterozygosity (LOH) analyses of chromosome arms 11q and 16q on 225 Wilms tumors. On chromosome arm 11q an overall rate of allele loss of 19.6% (44 of 225 tumors) was found using eleven markers that were almost evenly distributed along the long arm. Chromosome arm 16q was analyzed with six markers selected from gene-rich regions that identified an LOH rate of 18.4% (41/223). Evaluation of LOH with respect to clinical data revealed significant associations of LOH 11q with histology: LOH 11q was 3-4 times more frequent in mixed type and diffuse anaplastic tumors. In contrast, epithelial as well as stromal type tumors never exhibited allele loss on 11q. Furthermore, a significant correlation with tumor recurrence and death was detected, but only for tumors that lost the entire long arm of chromosome 11. Similarly, LOH 16q was correlated with higher risks of later relapse, especially in tumors with complete loss of the long arm. Hence, analyses of LOH on 11q and 16q appear to be helpful to identify tumors with a higher risk of relapse and adverse outcome, which need adjusted therapeutic approaches. Copyright 2006 Wiley-Liss, Inc.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

PubMed

Arczewska, Katarzyna D; Tomazella, Gisele G; Lindvall, Jessica M; Kassahun, Henok; Maglioni, Silvia; Torgovnick, Alessandro; Henriksson, Johan; Matilainen, Olli; Marquis, Bryce J; Nelson, Bryant C; Jaruga, Pawel; Babaie, Eshrat; Holmberg, Carina I; Bürglin, Thomas R; Ventura, Natascia; Thiede, Bernd; Nilsen, Hilde

2013-05-01

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

PubMed Central

Herrmann, Andreas; Priceman, Saul J.; Kujawski, Maciej; Xin, Hong; Cherryholmes, Gregory A.; Zhang, Wang; Zhang, Chunyan; Lahtz, Christoph; Kowolik, Claudia; Forman, Steve J.; Kortylewski, Marcin; Yu, Hua

2014-01-01

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte–associated antigen 4 (CTLA4apt) allows gene silencing in exhausted CD8+ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8+ T cells in the tumor milieu; therefore, CTLA4apt fused to a STAT3-targeting siRNA (CTLA4apt–STAT3 siRNA) resulted in internalization into tumor-associated CD8+ T cells and silencing of STAT3, which activated tumor antigen–specific T cells in murine models. Both local and systemic administration of CTLA4apt–STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4apt–STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4apt–STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4apt-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis. PMID:24892807

Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

Inhibition of Lysyl Oxidases Impairs Migration and Angiogenic Properties of Tumor-Associated Pericytes.

PubMed

Ribeiro, Aline Lopes; Kaid, Carolini; Silva, Patrícia B G; Cortez, Beatriz A; Okamoto, Oswaldo Keith

2017-01-01

Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFR β . Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β -aminopropionitrile ( β APN) significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with β APN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents.

Inhibition of Lysyl Oxidases Impairs Migration and Angiogenic Properties of Tumor-Associated Pericytes

PubMed Central

Kaid, Carolini; Silva, Patrícia B. G.; Cortez, Beatriz A.

2017-01-01

Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFRβ. Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β-aminopropionitrile (βAPN) significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with βAPN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents. PMID:28553358

NGF reprograms metastatic melanoma to a bipotent glial-melanocyte neural crest-like precursor

PubMed Central

Kasemeier-Kulesa, Jennifer C.; Romine, Morgan H.; Morrison, Jason A.; Bailey, Caleb M.; Welch, Danny R.

2018-01-01

ABSTRACT Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type. PMID:29175861

Tumor-associated macrophages drive spheroid formation during early transcoelomic metastasis of ovarian cancer

PubMed Central

Yin, Mingzhu; Li, Xia; Tan, Shu; Zhou, Huanjiao Jenny; Ji, Weidong; Bellone, Stefania; Xu, Xiaocao; Zhang, Haifeng; Santin, Alessandro D.; Lou, Ge

2016-01-01

Tumor-associated macrophages (TAMs) can influence ovarian cancer growth, migration, and metastasis, but the detailed mechanisms underlying ovarian cancer metastasis remain unclear. Here, we have shown a strong correlation between TAM-associated spheroids and the clinical pathology of ovarian cancer. Further, we have determined that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in an established mouse model for epithelial ovarian cancer. M2 macrophage–like TAMs were localized in the center of spheroids and secreted EGF, which upregulated αMβ2 integrin on TAMs and ICAM-1 on tumor cells to promote association between tumor cells and TAM. Moreover, EGF secreted by TAMs activated EGFR on tumor cells, which in turn upregulated VEGF/VEGFR signaling in surrounding tumor cells to support tumor cell proliferation and migration. Pharmacological blockade of EGFR or antibody neutralization of ICAM-1 in TAMs blunted spheroid formation and ovarian cancer progression in mouse models. These findings suggest that EGF secreted from TAMs plays a critical role in promoting early transcoelomic metastasis of ovarian cancer. As transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our findings uncover a mechanism for TAM-mediated spheroid formation and provide a potential target for the treatment of ovarian cancer and other transcoelomic metastatic cancers. PMID:27721235

Tumor-associated macrophages and stromal TNF-α regulate collagen structure in a breast tumor model as visualized by second harmonic generation

NASA Astrophysics Data System (ADS)

Burke, Ryan M.; Madden, Kelley S.; Perry, Seth W.; Zettel, Martha L.; Brown, Edward B.

2013-08-01

Collagen fibers can be imaged with second harmonic generation (SHG) and are associated with efficient tumor cell locomotion. Preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype, and changes in SHG emission properties accompany changes in metastatic outcome. We therefore attempted to elucidate the cellular and molecular machinery that influences SHG in order to understand how the microstructure of tumor collagen fibers is regulated. By quantifying SHG and immunofluorescence (IF) from tumors grown in mice with and without stromal tumor necrosis factor (TNF)-α and in the presence or absence of tumor-associated macrophages (TAMs), we determined that depletion of TAMs alters tumor collagen fibrillar microstructure as quantified by SHG and IF. Furthermore, we determined that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar microstructure and that subsequent depletion of TAMs has no further effect. In each case, metastatic burden correlated with optical readouts of collagen microstructure. Our results implicate TAMs and stromal TNF-α as regulators of breast tumor collagen microstructure and suggest that this regulation plays a role in tumor metastasis. Furthermore, these results indicate that quantification of SHG represents a useful strategy for evaluating the cells and molecular pathways responsible for manipulating fibrillar collagen in breast tumor models.

Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

PubMed

Burns, David; Blau, Helen M

2014-07-01

Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Epigenetic regulation of cell fate reprogramming in aging and disease: A predictive computational model.

PubMed

Folguera-Blasco, Núria; Cuyàs, Elisabet; Menéndez, Javier A; Alarcón, Tomás

2018-03-01

Understanding the control of epigenetic regulation is key to explain and modify the aging process. Because histone-modifying enzymes are sensitive to shifts in availability of cofactors (e.g. metabolites), cellular epigenetic states may be tied to changing conditions associated with cofactor variability. The aim of this study is to analyse the relationships between cofactor fluctuations, epigenetic landscapes, and cell state transitions. Using Approximate Bayesian Computation, we generate an ensemble of epigenetic regulation (ER) systems whose heterogeneity reflects variability in cofactor pools used by histone modifiers. The heterogeneity of epigenetic metabolites, which operates as regulator of the kinetic parameters promoting/preventing histone modifications, stochastically drives phenotypic variability. The ensemble of ER configurations reveals the occurrence of distinct epi-states within the ensemble. Whereas resilient states maintain large epigenetic barriers refractory to reprogramming cellular identity, plastic states lower these barriers, and increase the sensitivity to reprogramming. Moreover, fine-tuning of cofactor levels redirects plastic epigenetic states to re-enter epigenetic resilience, and vice versa. Our ensemble model agrees with a model of metabolism-responsive loss of epigenetic resilience as a cellular aging mechanism. Our findings support the notion that cellular aging, and its reversal, might result from stochastic translation of metabolic inputs into resilient/plastic cell states via ER systems.

Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

PubMed Central

Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

2012-01-01

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self

Malignant tumors associated with ovarian mature teratoma: A single institution experience.

PubMed

Trabzonlu, Levent; Durmaz, Guray; Vural, Cigdem; Muezzinoglu, Bahar; Corakci, Aydin

2017-05-01

The aims of this study are to present demographical features of cases diagnosed with malignant tumor associated with ovarian mature teratoma and to analyze histopathological features and clinical follow up of these tumors. Single-institution retrospective charts were reviewed to identify all cases of ovarian mature teratoma diagnosed from 1998 to 2015. Clinicopathological parameters that were analyzed include age, tumor size, tumor stage, histological type, laterality, IOC diagnosis and whether or not patient has received adjuvant chemotherapy. A total of 218 ovarian mature teratoma cases were identified during the study period. Of the 218 ovarian mature teratoma specimens, eight (3.7%) exhibited malignant tumors. The average age for cases of malignancy associated with ovarian mature teratoma was 44.6 years. The average size of tumors was 10.36cm. On final pathology, histological types of tumors were as follows: two cases each of squamous cell carcinoma and papillary thyroid carcinoma; one case each of mucinous adenocarcinoma, metastatic adenocarcinoma, sebaceous carcinoma and oligodendroglioma. Only one patient with Stage IIB tumor died of disease. One patient was alive with metastatic disease two months after initial diagnosis. Mean and median follow-up times were 64.1 and 49 months, respectively. An ovarian mass that has characteristics of a teratoma in a postmenopausal patient should alert for malignancy -regardless of tumor size. IOC is a valuable tool for the detection of malignancy and should be requested to determine the modality of surgical approach. Copyright © 2017 Elsevier GmbH. All rights reserved.

THE HISTONE DEACETYLASE SIRT6 IS A NOVEL TUMOR SUPPRESSOR THAT CONTROLS CANCER METABOLISM

PubMed Central

Sebastián, Carlos; Zwaans, Bernardette M. M.; Silberman, Dafne M.; Gymrek, Melissa; Goren, Alon; Zhong, Lei; Ram, Oren; Truelove, Jessica; Guimaraes, Alexander R.; Toiber, Debra; Cosentino, Claudia; Greenson, Joel K.; Mac Donald, Alasdair I; McGlynn, Liane; Maxwell, Fraser; Edwards, Joanne; Giacosa, Sofia; Guccione, Ernesto; Weissleder, Ralph; Bernstein, Bradley E.; Regev, Aviv; Shiels, Paul G.; Lombard, David B.; Mostoslavsky, Raul

2012-01-01

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a novel tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, while transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. Using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size and aggressiveness of tumors. SIRT6 also functions as a novel regulator of ribosome metabolism by co-repressing MYC transcriptional activity. Lastly, SIRT6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism. PMID:23217706

Authentication in Reprogramming of Sensor Networks for Mote Class Adversaries

DTIC Science & Technology

2006-01-01

based approach. In this paper, we propose a symmetric key-based protocol for authenticating the reprogramming process. Our protocol is based on the ... secret instantiation algorithm, which requires only O(log n) keys to be maintained at each sensor. We integrate this algorithm with the existing

Mixed endocrine gastric tumors associated with hypergastrinemia of antral origin.

PubMed Central

Larsson, L. I.; Rehfeld, J. F.; Stockbrügger, R.; Blohme, G.; Schöön, I. M.; Lundqvist, G.; Kindblom, L. G.; Säve-Söderberg, J.; Grimelius, L.; Olbe, L.

1978-01-01

A patient with atrophic gastritis and excessively raised serum gastrin concentrations (4000 to 5000 pg/ml) was found to have multiple polypous tumors of the gastric corpus mucosa. Following gastrectomy, serum gastrin concentrations decreased to undetectable levels. The tumors consisted of a mixed population of endocrine cells. The majority of tumor cells were of the ECL type, but, in addition, enterochromaffin cells of various subtypes as well as agranular cells were found. The tumors were locally invasive and invaded the walls of submucosal blood vessels. The surrounding mucosa showed a severe atrophic gastritis with intestinalization and contained numerous goblet cells, enterochromaffin cells, and cholecystokinin cells. Cholecystokinin cells do not occur in the normal oxyntic mucosa. Hence, the observation of this cell type in intestinalized gastric epithelium suggests that "intestinalization also is associated with changes in endocrine cell populations. Gastrin has been shown to affect the function of the ECL cells. Indications for a trophic action of gastrin on these cells have been obtained. It is discussed whether greatly raised serum gastrin levels in patients with atrophic gastritis may be associated with increased risks for the development of certain types of gastric tumors. Images Figure 4 Figure 5 Figure 6 Figure 7 Figure 1 Figure 2 Figure 3 PMID:696807