RESUMO
BACKGROUND: Human embryonic stem cells (hESCs) can differentiate into any human cell type, including CNS cells, and thus have high potential in regenerative medicine. Several protocols exist for neuronal differentiation of hESCs, which do not necessarily work for all hESC lines. MATERIALS & METHODS: We tested the differentiation capacity of four similarly derived and cultured hESC lines (HS181, HS360, HS362 and HS401) in suspension culture in relatively simple neural differentiation medium for up to 20 weeks. RESULTS: All the hESC lines differentiated into neuronal cells, but in a line-dependent manner. Using our method, the HS181- and HS360-derived neurospheres differentiated in vitro into pure neuronal cell populations within 6 weeks, whereas HS362 and HS401 reached their peak of differentiation in 12 weeks, but never produced pure neuronal cell populations using the present method. The withdrawal of FGF from suspension culture increased the in vitro differentiation potential. The hESC-derived neurospheres formed functional neuronal networks when replated on a microelectrode array and responded as expected to pharmacologic modulation. CONCLUSION: Simple neurosphere culture is a suitable method for producing hESC-derived neuronal cells that can form functional neuronal networks from a number of hESC lines. The variation in the differentiation potential of hESC lines into neuronal cells must be carefully considered by those comparing various differentiation methods and designing transplantation therapies for neuronal disorders.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Técnicas de Cultura de Células , Linhagem da Célula , Meios de Cultura/química , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa/métodos , Fatores de TempoRESUMO
BACKGROUND: The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed. CONCLUSION/SIGNIFICANCE: Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.
Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Células-Tronco/citologia , Adipócitos/citologia , Diferenciação Celular , Meios de Cultura , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , MétodosRESUMO
BACKGROUND: Glutamate metabolism is associated with myocardial ischemia-reperfusion, but it is not clear whether glutamate reveals ongoing ischemia (OI). We evaluated whether microdialysis would detect OI induced by coronary artery ligation in a rat cardiac transplantation model. MATERIAL AND METHODS: A total of 24 Fischer 344 rats underwent syngeneic heterotopic cardiac transplantation. Of these, 16 rats underwent ligation of the left anterior coronary artery (LAD) of the heart to induce ongoing ischemia (OI), of which eight grafts received intra-aortally Gabapentin (12 mg/graft), a glutamate-release inhibitor and eight grafts with transplantation only served as the control. With a microdialysis catheter samples for glucose, lactate, pyruvate, glutamate, and glycerol were analysed spectrophotometrically. Histology and aquaporin 7 evaluations were performed after graft harvesting. RESULTS: Glutamate was elevated after 15 min of reperfusion in OI as compared with Control (14.31 +/- 5.03 microM vs 6.75 +/- 2.21 microM, p = 0.05), respectively. Glycerol remained high in OI (61.89 +/- 46.13 microM to 15.84 +/- 0.85 microM, p = ns) and low in Control (12.33 +/- 3.36 microM to 5.52 +/- 0.25 microM, p = ns). Gabapentin decreased glutamate release from 7.32 +/- 1.57 microM to 2.71 +/- 0.64 microM, (p < 0.05) and resulted in decrease of glycerol levels from 24.64 +/- 4.03 microM to 10.43 +/- 2.49 microM, (p < 0.05) in OI. The expression of aquaporin 7 and histology confirmed OI. CONCLUSIONS: We suggest that glutamate release may be used as an early indicator of OI after cardiac arrest.
Assuntos
Ácido Glutâmico/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Animais , Aquaporinas/genética , Aquaporinas/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Glicerol/metabolismo , Ácido Láctico/metabolismo , Microdiálise , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Piruvatos/metabolismo , Curva ROC , Ratos , Ratos Endogâmicos F344RESUMO
An optimal surface for culturing human embryonic stem cell (hESC)-derived neuronal cells is of high interest. In this study, a specific antibody to a neural cell adhesion molecule (NCAM) was immobilised on a solid surface of polystyrene and used as a selective matrix for culturing of hESC-derived neuronal cells. Thereafter, hESC-derived neurospheres were seeded on the matrix. The neurospheres did not attach to the NCAM antibody containing matrix whereas individual neuronal cells did. The neuronal cell attachment was depended on the NCAM antibody concentration. The neuronal cells were viable on the NCAM antibody containing matrix during an 8 day follow-up and exhibited typical bipolar morphology of immature neurons. Specific binding of the NCAM antigen to an immunoglobulin-polymer coated surface was verified by surface plasmon resonance (SPR) measurements. This study is to our knowledge the first demonstrating the use of an antibody layer as a selective surface for hESC-derived neuronal cells.
Assuntos
Anticorpos/imunologia , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Moléculas de Adesão de Célula Nervosa/imunologia , Neurônios/citologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Diferenciação Celular/fisiologia , Forma Celular/fisiologia , Células Cultivadas , Meios de Cultura , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Humanos , Imunoglobulinas/imunologia , Neurogênese/fisiologia , Neurônios/metabolismo , Poliestirenos/química , Ligação Proteica/imunologia , Ressonância de Plasmônio de Superfície/métodosRESUMO
The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Técnicas Biossensoriais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Estimulação Elétrica/métodos , Células-Tronco Embrionárias/citologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , GABAérgicos/farmacologia , Humanos , Microeletrodos , Neurônios/citologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
Cortical stem cell transplantation may help replace lost brain cells after stroke and improve the functional outcome. In this study, we transplanted human embryonic stem cell (hESC)-derived neural precursor cells (hNPCs) or vehicle into the cortex of rats after permanent distal middle cerebral artery occlusion (dMCAO) or sham-operation, and followed functional recovery in the cylinder and staircase tests. The hNPCs were examined prior to transplantation, and they expressed neuroectodermal markers but not markers for undifferentiated hESCs or non-neural cells. The rats were housed in either enriched environment or standard cages to examine the effects of additive rehabilitative therapy. In the behavioral tests dMCAO groups showed significant impairments compared with sham group before transplantation. Vehicle groups remained significantly impaired in the cylinder test 1 and 2 months after vehicle injection, whereas hNPC transplanted groups did not differ from the sham group. Rehabilitation or hNPC transplantation had no effect on reaching ability measured in the staircase test, and no differences were found in the cortical infarct volumes. After 2 months we measured cell survival and differentiation in vivo using stereology and confocal microscopy. Housing had no effect on cell survival or differentiation. The majority of the transplanted hNPCs were positive for the neural precursor marker nestin. A portion of transplanted cells expressed neuronal markers 2 months after transplantation, whereas only a few cells co-localized with astroglial or oligodendrocyte markers. In conclusion, hESC-derived neural precursor transplants provided some improvement in sensorimotor function after dMCAO, but did not restore more complicated sensorimotor functions.
Assuntos
Córtex Cerebral/cirurgia , Células-Tronco Embrionárias/transplante , Sobrevivência de Enxerto/fisiologia , Recuperação de Função Fisiológica/fisiologia , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/cirurgia , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Ambiente Controlado , Humanos , Masculino , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Paresia/etiologia , Paresia/fisiopatologia , Paresia/cirurgia , Ratos , Ratos Wistar , Células-Tronco/citologia , Células-Tronco/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Resultado do TratamentoRESUMO
The regenerative potential of stem cells from various sources has been under intense investigation in the experimental models of cerebral ischemia. To end up with a restorative therapeutic treatment, it is crucial to get the cell transplants to the site of injury. Here, we evaluated the feasibility of small animal SPECT/CT in assessing the definite accumulation of (111)In-oxine-labeled human embryonic stem (ES) cell-derived neural progenitors and rat hippocampal progenitors after intravenous or intra-arterial administration (femoral vein vs. common carotid artery) in middle cerebral artery occlusion (MCAO) and sham-operated rats. Cell detection was carried out immediately and 24h after the infusion using a SPECT/CT device. The results showed that after intravenous injections both cell types accumulated primarily into internal organs, instead of brain. In contrast, after intra-arterial injection, a weak signal was detected in the ischemic hemisphere. Additional studies showed that the detection sensitivity of SPECT/CT device was approximately 1000 (111)In-oxine-labeled cells and labeling did not affect the cell viability. In conclusion, a small animal SPECT is powerful technique to study the whole body biodistribution of cell-based therapies. Our data showed that intravenous administration is not an optimal route to deliver neural progenitor cell-containing transplants into the brain after MCAO in rats.