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1.
Circulation ; 125(1): 87-99, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-22095829

RESUMO

BACKGROUND: Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. METHODS AND RESULTS: We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105(+)CD90(+)CD73(+)CD31(-) multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor ß, but not the smooth muscle cell marker α-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. CONCLUSIONS: Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine.


Assuntos
Extremidades/irrigação sanguínea , Isquemia/cirurgia , Células-Tronco Multipotentes/transplante , Pericitos/transplante , Células-Tronco Pluripotentes/transplante , Recuperação de Função Fisiológica/fisiologia , Animais , Células Endoteliais/transplante , Extremidades/cirurgia , Humanos , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID
2.
Pharmaceutics ; 14(6)2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35745752

RESUMO

Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127−257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800−1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted (via a GAG-analogue hydrogel) in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications.

3.
J Cell Mol Med ; 15(1): 38-51, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20041972

RESUMO

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force-frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca(2+) handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation-contraction coupling, resemble in part those of adult cardiomyocytes.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Calsequestrina/genética , Calsequestrina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/metabolismo , Imunofluorescência , Prepúcio do Pênis/citologia , Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos SCID , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Fatores de Transcrição SOXB1/genética , Retículo Sarcoplasmático/metabolismo , Teratoma/metabolismo , Teratoma/patologia
4.
Cell Rep ; 35(9): 109198, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34077720

RESUMO

Embryonic stem cell (ESC) self-renewal and cell fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell differentiation. Notably, many RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly targeted by this factor. Using cross-linking and immunoprecipitation (CLIP-seq), we find that the pluripotency-associated STAT3 and OCT4 transcription factors interact with RNA in hESCs and confirm the binding of STAT3 to the conserved NORAD long-noncoding RNA. Our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Diferenciação Celular/genética , Linhagem Celular , DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Fator de Transcrição STAT3/metabolismo
5.
Nat Biotechnol ; 25(7): 803-16, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17572666

RESUMO

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.


Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fosfatase Alcalina/metabolismo , Antígenos CD/biossíntese , Biotecnologia/métodos , Diferenciação Celular , Linhagem da Célula , Membrana Celular/metabolismo , Células Cultivadas , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Genótipo , Glicolipídeos/química , Humanos , Glicoproteínas de Membrana/biossíntese , Tetraspanina 29
6.
Stem Cells ; 26(12): 3130-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18818435

RESUMO

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca(2+)-induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP(3))-operated intracellular Ca(2+) ([Ca(2+)](i)) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP(3) pathway, on [Ca(2+)](i) transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10(-9) to 10(-7) M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10(-9) to 10(-7) M) increased the [Ca(2+)](i) transient amplitude. The involvement of 1,4,5-IP(3)-dependent intracellular Ca(2+) release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP(3) receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 microM 2-aminoethoxyphenyl borate (a 1,4,5-IP(3) receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP(3)-operated releasable Ca(2+) stores.


Assuntos
Angiotensina II/metabolismo , Cálcio/metabolismo , Células-Tronco Embrionárias/citologia , Endotelina-1/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Modelos Biológicos , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais
7.
Stem Cells ; 26(2): 440-4, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18032703

RESUMO

Embryonic stem (ES) cells represent a unique cellular model to recapitulate in vitro early steps of embryonic development and an unlimited cellular source in therapy for many diseases, as well as targets for drug discovery and toxicology screens. Although previous studies have reported epidermal differentiation of mouse and human embryonic stem (huES) cells, the heterogeneity of the resulting cell culture impairs the evaluation of differentiated cells for cell therapy. We report here the reproducible isolation of a homogenous ectodermal cell population, IT1, from human ES cells. Like primary cells, IT1 cells remain homogenous over 15 passages, expand up to 60 population doublings, and then die through senescence. Accordingly, IT1 cells display a normal karyotype and a somatic cell cycle kinetics and do not produce teratoma in nude mice. The production of K14-expressing epithelial cells driven by p63 expression strengthens the ectodermal nature of IT1 cells. Since IT1 can be isolated from different huES cell lines, it may provide a ready source of ectodermal progenitors for the development of a toxicology cell model, new-drug-screening strategies, and cell therapy transplantation.


Assuntos
Separação Celular/métodos , Ectoderma/citologia , Células-Tronco Embrionárias/citologia , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Teratoma/etiologia , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
8.
J Cell Physiol ; 216(2): 445-52, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18340642

RESUMO

The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X-inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X-inactivation during early development. To identify a human ES cell model for X-inactivation and study differences in the epigenetic state of hESC lines, we investigated X-inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X-inactivation. However, subcultures of any hESC line can vary in X-inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X-inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X-inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture.


Assuntos
Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Inativação do Cromossomo X , Animais , Cromossomos Humanos X , Mecanismo Genético de Compensação de Dose , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Camundongos , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
9.
Stem Cells ; 25(9): 2200-5, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17540853

RESUMO

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Infarto do Miocárdio/terapia , Miocárdio/citologia , Animais , Células Cultivadas , Feminino , Coração/fisiologia , Humanos , Fenótipo , Ratos , Ratos Wistar , Regeneração , Transplante Heterólogo
10.
Methods Mol Biol ; 407: 11-20, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18453245

RESUMO

Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the inner cell mass of the blastocyst. Due to their unique properties, hESCs might be used for research fields such as self-renewal, specific lineage differentiation, human developmental biology, and teratology. hESCs also have outstanding potential to serve for clinical purposes as a source for cell-based therapies. Traditionally, these cells are cultured and derived with mouse embryonic fibroblast as supportive layer, using a medium supplemented with fetal bovine serum. Future industrial and clinical implementation of hESCs will require the use of a defined medium and an animal-free culture method that will prevent their possible exposure to animal pathogens. This chapter discusses the advancements in the development of methods for the defined culture of hESCs and describes a simple method for animals serum-free and feeder layer-free culture of hESCs.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Proliferação de Células , Células Cultivadas , Fibroblastos , Humanos , Fator de Crescimento Transformador beta1/metabolismo
11.
J Electrocardiol ; 40(6 Suppl): S192-6, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17993321

RESUMO

Cardiovascular diseases are the most frequent cause of death in the industrialized world, with the main contributor being myocardial infarction. Given the high morbidity and mortality rates associated with congestive heart failure, the shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various physiological stimuli (eg, beta-adrenergic stimulation), our major long-term goal is to investigate the developmental changes in functional properties and hormonal responsiveness of human embryonic stem cells-derived cardiomyocytes (hESC-CM). Furthermore, because one of the key obstacles in advancing cardiac cell therapy is the low differentiation rate of hESC into cardiomyocytes, which reduces the clinical efficacy of cell transplantation, our second major goal is to develop efficient protocols for directing the cardiomyogenic differentiation of hESC in vitro. To accomplish the first goal, we investigated the functional properties of hESC-CM (<90 days old), respecting the contractile function and the underlying intracellular Ca(2+) handling. In addition, we performed Western blot analysis of the key Ca(2+)-handling proteins SERCA2, calsequestrin, phospholamban and the Na(+)/Ca(2+) exchanger. Our major findings were the following: (1) In contrast to the mature myocardium, hESC-CM exhibit negative force-frequency relationships and do not present postrest potentiation. (2) Ryanodine and thapsigargin do not affect the [Ca(2+)](i) transient and contraction, suggesting that, at this developmental stage, the contraction does not depend on sarcoplasmic reticulum Ca(2+) release. (3) In agreement with the finding that a voltage-dependent Ca(2+) current is present in hESC-CM and contributes to the mechanical function, verapamil completely blocks contraction. (4) Although hESC-CM express SERCA2 and Na(+)/Ca(2+) exchanger at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban are not expressed. (4) In agreement with other reports, hESC-CM are responsive to beta-adrenergic stimulation. These findings show that the mechanical function related to intracellular Ca(2+) handling of hESC-CM differs from the adult myocardium, probably because of immature sarcoplasmic reticulum capacity.


Assuntos
Potenciais de Ação/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos , Cálcio/metabolismo , Células Cultivadas , Humanos
12.
Methods Enzymol ; 420: 37-49, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17161692

RESUMO

In addition to their contribution to research fields such as early human development, self-renewal, and differentiation mechanisms, human embryonic stem cells (hESCs) may serve as a tool for drug testing and for the study of cell-based therapies. Traditionally, these cells have been cultured with mouse embryonic fibroblast (MEF) feeder layers, which allow their continuous growth in an undifferentiated state. However, for future clinical applications, hESCs should be cultured under defined conditions, preferably in a xeno-free culture system, where exposure to animal pathogens is prevented. To this end, different culture methods for hESCs, based on serum replacement and free of supportive cell layers, were developed. This chapter discusses a simple, feeder-free culture system on the basis of medium supplemented with transforming growth factor beta1 (TGFbeta1), basic fibroblast growth factor (bFGF) and fibronectin as matrix.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/normas , Células-Tronco Embrionárias/citologia , Técnicas de Cultura de Células/normas , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/farmacologia , Humanos , Fator de Crescimento Transformador beta1/farmacologia
13.
Semin Reprod Med ; 24(5): 298-303, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17123224

RESUMO

Human embryonic stem cells (hESCs) are immortal cells capable of perpetual self-renewal in culture while maintaining their undifferentiated state, high telomerase activity, normal karyotype, and specific pattern expression of embryonic surface markers and pluripotent transcription factors such as Oct-4 and Nanog. Since their first derivation in 1998, hundreds of hESC lines have been derived and characterized. Normal surplus embryos from IVF programs are the main source for the derivation of hESC lines but cell lines from poor-quality discarded embryos or embryos carrying genetic defects following preimplantation genetic diagnosis were also isolated. Such isolation is usually accomplished by either mechanical or immunosurgical removal of the trophectoderm and culture of the inner cell mass on inactivated feeder cells. In light of the future need for clinical-grade cells, the subject of defining specific culture conditions has been addressed widely. Indeed, derivation and maintenance of hESCs without feeder cells and in media free of animal products have been attained recently. This well-defined culture system may facilitate research and clinical applications, and use the remarkable potential of these exceptional cells to its fullest in both the laboratory and the clinic.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Animais , Técnicas de Cultura de Células , Humanos , Modelos Biológicos
14.
Methods Mol Biol ; 331: 43-53, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16881508

RESUMO

Since their derivation in 1998, human embryonic stem cells (hESCs) have been the center of tremendous scientific efforts in improve the existing methodologies for their isolation and maintenance to exhaust the potential use of these unique cells in cell-based therapy and developmental research. To date, there are more than 50 reported well-characterized hESC lines worldwide. hESCs are traditionally isolated from the blastocysts on mouse embryonic fibroblasts. The most used method for isolating the inner cell mass from the human blastocyst is immunosurgery. This chapter focuses on the basic methods for the derivation and maintenance of hESC lines.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Técnicas de Cocultura/métodos , Células-Tronco Pluripotentes/citologia , Animais , Linhagem Celular , Criopreservação , Feminino , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Trofoblastos/citologia
15.
Methods Mol Biol ; 331: 105-13, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16881512

RESUMO

The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes/citologia , Animais , Proteínas Sanguíneas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibronectinas/farmacologia , Humanos , Interleucina-6/farmacologia , Fator Inibidor de Leucemia , Camundongos , Células-Tronco Pluripotentes/efeitos dos fármacos , Pele/citologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1
16.
Cell Metab ; 21(3): 392-402, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25738455

RESUMO

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.


Assuntos
Acetilcoenzima A/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Glicólise/fisiologia , Histonas/metabolismo , Acetilcoenzima A/genética , Acetilação , Animais , Diferenciação Celular/genética , Linhagem Celular , Glicólise/genética , Histonas/genética , Humanos , Camundongos , Transcrição Gênica/genética , Transcrição Gênica/fisiologia
17.
Methods Mol Biol ; 997: 3-11, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23546743

RESUMO

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass (ICM) of the developing embryo. hESCs culture as cell lines in vitro and possess great potential in such research fields as developmental biology and cell-based therapy, as well as such industrial purposes as drug screening and toxicology. When ESCs were first derived by Thomson and colleagues, traditional methods of immunostaining and culturing, using primary mouse embryonic fibroblasts and medium supplemented by serum were used. Considerable efforts have since led to improved methods for isolating new lines in defined and reproducible conditions. This chapter discusses sources for embryos for ESC isolation, commonly used methods for deriving hESC lines, and a number of possible culture systems.


Assuntos
Células-Tronco Embrionárias/citologia , Animais , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Humanos
18.
Tissue Eng Part A ; 18(21-22): 2290-302, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22731654

RESUMO

Human mesenchymal stem cells (hMSCs) can be derived from various adult and fetal tissues. However, the quality of tissues for the isolation of adult and fetal hMSCs is donor dependent with a nonreproducible yield. In addition, tissue engineering and cell therapy require large-scale production of a pure population of lineage-restricted stem cells that can be easily induced to differentiate into a specific cell type. Therefore, human embryonic stem cells (hESCs) can provide an alternative, plentiful source for generation of reproducible hMSCs. We have developed efficient differentiation protocols for derivation of hMSCs from hESCs, including coculture with murine OP9 stromal cells and feeder layer-free system. Our protocols have resulted in the generation of up to 49% of hMSCs, which expressed CD105, CD90, CD29, and CD44. The hMSCs exhibited high adipogenic, chondrocytic, and osteogenic differentiation in vitro. The latter correlated with osteocalcin secretion and vascular endothelial growth factor (VEGF) production by the differentiating hMSCs. hMSC-derived osteoblasts further differentiated and formed ectopic bone in vivo, and induced the formation of blood vessels in Matrigel implants. Our protocol enables generation of a purified population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, and particularly into vasculogenesis-inducing osteoblasts, which can contribute to the development of bone repair protocols.


Assuntos
Osso e Ossos/irrigação sanguínea , Coristoma/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Separação Celular , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Técnicas de Cocultura , Colágeno/farmacologia , Combinação de Medicamentos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Cinética , Laminina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Neovascularização Fisiológica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Proteoglicanas/farmacologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Stem Cell Rev Rep ; 8(1): 137-49, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21732092

RESUMO

The regulatory pathways responsible for maintaining human embryonic stem cells (hESCs) in an undifferentiated state have yet to be elucidated. Since these pathways are thought to be governed by complex protein cues, deciphering the changes that occur in the proteomes of the ESCs during differentiation is important for understanding the expansion and differentiation processes involved. In this study, we present the first quantitative comparison of the hESC protein profile in the undifferentiated and early differentiated states. We used iTRAQ (isobaric tags for relative and absolute quantification) labeling combined with two dimensional capillary chromatography coupled with tandem mass spectrometry (µLC-MS/MS) to achieve comparative proteomics of hESCs at the undifferentiated stage, and at 6, 48, and 72 h after initiation of differentiation. In addition, two dimensional electrophoresis (2-DE) was performed on differentiating hESCs at eleven points of time during the first 72 h of differentiation. The results indicate that during the first 48 h of hESC differentiation, many processes are initiated and are later reversed, including chromatin remodeling, heterochromatin spreading, a decrease in transcription and translation, a decrease in glycolytic proteins and cytoskeleton remodeling, and a decrease in focal and cell adhesion. Only 72 h after differentiation induction did the expression of the homeobox prox1 protein increase, indicating the beginning of developmental processes.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Proteoma/metabolismo , Forma Celular , Células Cultivadas , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Cariótipo , Proteoma/genética , Proteoma/isolamento & purificação , Proteômica , Espectrometria de Massas em Tandem
20.
Eur J Hum Genet ; 20(12): 1248-55, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22713809

RESUMO

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.


Assuntos
Aneuploidia , Duplicação Cromossômica/genética , Cromossomos Humanos Par 1/genética , Mosaicismo , Células-Tronco Pluripotentes , Linhagem Celular , Hibridização Genômica Comparativa , Instabilidade Genômica/genética , Humanos , Hibridização in Situ Fluorescente
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