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1.
Nature ; 471(7336): 63-7, 2011 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-21368825

RESUMO

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.


Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutagênese/genética , Mutação Puntual/genética , Células Cultivadas , Análise Mutacional de DNA , Epistasia Genética/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Fases de Leitura Aberta/genética
2.
J Biol Chem ; 290(5): 3121-36, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25488666

RESUMO

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation.


Assuntos
Doença de Depósito de Glicogênio Tipo II/metabolismo , Doença de Depósito de Glicogênio Tipo II/patologia , Complexo de Golgi/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/patologia , Western Blotting , Células Cultivadas , Genótipo , Glicosilação , Humanos , Imuno-Histoquímica
3.
Nature ; 457(7227): 277-80, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19098894

RESUMO

Spinal muscular atrophy is one of the most common inherited forms of neurological disease leading to infant mortality. Patients have selective loss of lower motor neurons resulting in muscle weakness, paralysis and often death. Although patient fibroblasts have been used extensively to study spinal muscular atrophy, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem cells from skin fibroblast samples taken from a child with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show that human induced pluripotent stem cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.


Assuntos
Reprogramação Celular , Fibroblastos/citologia , Modelos Biológicos , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Separação Celular , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Criança , Feminino , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Pele/citologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
4.
Circ Res ; 111(9): 1125-36, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22912385

RESUMO

RATIONALE: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. OBJECTIVE: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. METHODS AND RESULTS: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. CONCLUSIONS: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Colágeno , Matriz Extracelular/fisiologia , Laminina , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Proteoglicanas , Ativinas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Combinação de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
5.
Cell Prolif ; 57(5): e13588, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38124457

RESUMO

'Requirements for Human Natural Killer Cells' is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.


Assuntos
Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/citologia , Humanos , China , Controle de Qualidade
6.
Cell Prolif ; 57(4): e13563, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37881164

RESUMO

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.


Assuntos
Neurônios Dopaminérgicos , Mesencéfalo , Humanos , China , Neurônios Dopaminérgicos/metabolismo
7.
Blood ; 118(7): 1797-800, 2011 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-21708888

RESUMO

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.


Assuntos
Linfócitos B/citologia , Linfócitos B/virologia , Herpesvirus Humano 4/isolamento & purificação , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/virologia , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr/genética , Vetores Genéticos/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , Transfecção
8.
Blood ; 117(14): e109-19, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-21296996

RESUMO

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.


Assuntos
Células da Medula Óssea/fisiologia , Neoplasias da Medula Óssea/patologia , Reprogramação Celular/fisiologia , Sangue Fetal/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Leucócitos Mononucleares/fisiologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Técnicas de Cultura de Células/métodos , Desdiferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/genética , Técnicas de Cocultura/métodos , Eficiência , Sangue Fetal/metabolismo , Sangue Fetal/fisiologia , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Camundongos , Análise em Microsséries , Transgenes/fisiologia
9.
Stem Cells ; 30(3): 461-70, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22213079

RESUMO

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Proteína Quinase C/fisiologia , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Regulação para Baixo , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
10.
Proc Natl Acad Sci U S A ; 107(9): 4335-40, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20160098

RESUMO

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.


Assuntos
Diferenciação Celular , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Transdução de Sinais , Transgenes
11.
Food Chem Toxicol ; 176: 113807, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37121429

RESUMO

Cadmium (Cd), commonly found in diet and drinking water, is known to be harmful to the human liver. Nevertheless, the effects and mechanisms of gestational Cd exposure on fetal liver development remain unclear. Here, we reported that gestational Cd (150 mg/L) exposure obviously downregulated the expression of critical proteins including PCNA, Ki67 and VEGF-A in proliferation and angiogenesis in fetal livers, and lowered the estradiol concentration in fetal livers and placentae. Maternal estradiol supplement alleviated aforesaid impairments in fetal livers. Our data showed that the levels of pivotal estrogen synthases, such as CYP17A1 and 17ß-HSD, was markedly decreased in Cd-stimulated placentae but not fetal livers. Ground on ovariectomy (OVX), we found that maternal ovarian-derived estradiol had no major effects on Cd-impaired development in fetal liver. In addition, Cd exposure activated placental PERK signaling, and inhibited PERK activity could up-regulated the expressions of CYP17A1 and 17ß-HSD in placental trophoblasts. Collectively, gestational Cd exposure inhibited placenta-derived estrogen synthesis via activating PERK signaling, and therefore impaired fetal liver development. This study suggests a protective role for placenta-derived estradiol in fetal liver dysplasia shaped by toxicants, and provides a theoretical basis for toxicants to impede fetal liver development by disrupting the placenta-fetal-liver axis.


Assuntos
Cádmio , Trofoblastos , Gravidez , Feminino , Humanos , Cádmio/toxicidade , Cádmio/metabolismo , Trofoblastos/metabolismo , Placenta/metabolismo , Fígado/metabolismo , Estradiol , Estrogênios
12.
Cell Prolif ; 55(4): e13182, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35083805

RESUMO

'Requirements for Human-Induced Pluripotent Stem Cells' is the first set of guidelines on human-induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human-induced pluripotent stem cells, which apply to the production and quality control of human-induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human-induced pluripotent stem cells for applications.


Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , China , Humanos
13.
Cell Prolif ; 55(4): e13153, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34773310

RESUMO

'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.


Assuntos
Neurônios , Pigmentos da Retina , China , Células Epiteliais , Humanos
14.
Cell Prolif ; 55(4): e13147, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34936148

RESUMO

'Requirements for Primary Human Hepatocyte' is the first set of guidelines on Primary Human Hepatocyte in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for Primary Human Hepatocyte, which is applicable to the quality control for Primary Human Hepatocyte. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of Primary Human Hepatocyte for applications.


Assuntos
Hepatócitos , China , Humanos
15.
Cell Prolif ; 55(4): e13152, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34936155

RESUMO

'Requirements for human haematopoietic stem/progenitor cells' is the first set of guidelines on human haematopoietic stem/progenitor cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, inspection methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements for human haematopoietic stem/progenitor cells, which is applicable to the quality control for human haematopoietic stem/progenitor cells. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human haematopoietic stem/progenitor cells for applications.


Assuntos
Células-Tronco Hematopoéticas , China , Humanos
16.
Cell Prolif ; 55(4): e13141, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34936710

RESUMO

Mesenchymal stem cells (MSCs) have attracted great interest for cell therapy and tissue regeneration due to their self-renewal capacity, multipotency and potent immunomodulatory effects on immune cells. However, heterogeneity of MSCs has become a prominent obstacle to limit their translation into practice, as cells from different tissue sources or each individual have great differences in their transcriptomic signatures, differentiation potential and biological functions. Therefore, there is an urgent need for consensus standard for the quality control and technical specifications of MSCs. 'Human Mesenchymal Stem Cells' is the latest set of guidelines on hMSC in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hMSC, which is applicable to the quality control for hMSC. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hMSC for clinical development and therapeutic applications.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , China , Humanos , Imunomodulação
17.
Cell Prolif ; 55(4): e13150, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34708452

RESUMO

'Requirements for human cardiomyocytes', jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human cardiomyocytes in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packing requirements, storage requirements, transportation requirements and waste disposal requirements for human cardiomyocytes, which is designed to normalize and standardize human cardiomyocyte research and production. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that the publication of this guideline will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human cardiomyocytes for applications.


Assuntos
Miócitos Cardíacos , China , Humanos
18.
Circ Res ; 104(4): e30-41, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19213953

RESUMO

Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated. The aim of this study was to characterize the cardiac differentiation potential of human iPS cells generated using OCT4, SOX2, NANOG, and LIN28 transgenes compared to human embryonic stem (ES) cells. The iPS and ES cells were differentiated using the embryoid body (EB) method. The time course of developing contracting EBs was comparable for the iPS and ES cell lines, although the absolute percentages of contracting EBs differed. RT-PCR analyses of iPS and ES cell-derived cardiomyocytes demonstrated similar cardiac gene expression patterns. The pluripotency genes OCT4 and NANOG were downregulated with cardiac differentiation, but the downregulation was blunted in the iPS cell lines because of residual transgene expression. Proliferation of iPS and ES cell-derived cardiomyocytes based on 5-bromodeoxyuridine labeling was similar, and immunocytochemistry of isolated cardiomyocytes revealed indistinguishable sarcomeric organizations. Electrophysiology studies indicated that iPS cells have a capacity like ES cells for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics. Both iPS and ES cell-derived cardiomyocytes exhibited responsiveness to beta-adrenergic stimulation manifest by an increase in spontaneous rate and a decrease in action potential duration. We conclude that human iPS cells can differentiate into functional cardiomyocytes, and thus iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Sarcômeros/metabolismo , Fatores de Tempo , Transdução Genética
19.
Stem Cells ; 27(3): 559-67, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19259936

RESUMO

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.


Assuntos
Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Sistema Hematopoético/citologia , Sistema Hematopoético/metabolismo , Células-Tronco Pluripotentes/citologia , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Leucossialina/metabolismo , Células-Tronco Pluripotentes/metabolismo
20.
J Phys Chem A ; 114(25): 6795-802, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20518517

RESUMO

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) measurements were carried out on the 1-propanol-water (abbreviated as 1PA-W) mixtures over the entire 1-propanol molar fraction range at 298 K. The two bands at approximately 1053 and approximately 1068 cm(-1), assigned to the vibrational modes of the gauche (v(C-C-C-O-G)) and the trans (v(C-C-C-O-T)) conformational isomers, respectively, which both include C-O and C-C stretching motions, were used to monitor the structural changes of the mixtures. When the water to 1-propanol molar ratio (WPR) is smaller than 0.2, the absorbance ratio of the two bands (A(vC-C-C-O-G)/A(vC-C-C-O-T)) remains constant at 1.42, characteristic of the existence of the 1-propanol aggregate chains, hydrogen-bonded by the O-H groups of 1-propanol in gauche conformations. When increasing the WPR from 0.2 to 20, there is an abrupt decrease in the absorbance ratio (A(vC-C-C-O-G)/A(vC-C-C-O-T)) from 1.42 to 1.01, corresponding to penetration of water molecules into the gauche-aggregate chains. The penetrated water molecules disrupt the 1PA chains and transform these gauche-aggregate 1PA chains to trans-aggregate chains, which are 1PA dimers of trans-conformation. The structural change induces complicated spectroscopic changes, including the red shifts of the series of bands 1016, 1053, and 1098 cm(-1) and blue shifts of the bands 2877, 2937, and 2961 cm(-1). With further increase of WPR up to 100, the absorbance ratio of A(vC-C-C-O-G)/A(vC-C-C-O-T) increases from 0.98 to 1.07, indicating a transformation of partial 1PA dimers to single molecules with gauche-conformation in the water hydrogen-bonding network. Together with results from quantum calculations at the B3L YP/6-31G (d, p) level, and two-dimensional infrared correlation and excess spectroscopy analysis, the structural evolution of water and 1PA molecules in 1PA-W mixtures has been inferred.


Assuntos
1-Propanol/química , Conformação Molecular , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Água/química , Ligação de Hidrogênio , Modelos Moleculares , Teoria Quântica , Solventes/química , Temperatura , Vibração
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