Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the ...Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.展开更多
Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at ...Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.展开更多
Human induced pluripotent stem （hiPS） cells are considered a potential source for the generation of insulin-producing pancreatic β-ceUs because of their differentiation capacity. In this study, we have developed a ...Human induced pluripotent stem （hiPS） cells are considered a potential source for the generation of insulin-producing pancreatic β-ceUs because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently dif- ferentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 （PDX1）-positive pancreatic progenitors and then into neurogenin 3 （NGN3）-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-l-methylxanthine （IBMX）, exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic β-cell markers. Most notably, the differentiated cells contained en- dogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic β-ceUs under xeno-free conditions and highlight their poten- tial to treat patients with type I diabetes.展开更多
While human induced pluripotent stem cells(hiPSCs)have promising applications in regenerative medicine,most of the hiPSC lines available today are not suitable for clinical applications due to contamination with nonhu...While human induced pluripotent stem cells(hiPSCs)have promising applications in regenerative medicine,most of the hiPSC lines available today are not suitable for clinical applications due to contamination with nonhuman materials,such as sialic acid,and potential pathogens from animal-product-containing cell culture systems.Although several xeno-free cell culture systems have been established recently,their use of human fibroblasts as feeders reduces the clinical potential of hiPSCs due to batch-to-batch variation in the feeders and time-consuming preparation processes.In this study,we have developed a xeno-free and feeder-cell-free human embryonic stem cell(hESC)/hiPSC culture system using human plasma and human placenta extracts.The system maintains the self-renewing capacity and pluripotency of hESCs for more than 40 passages.Human iPSCs were also derived from human dermal fibroblasts using this culture system by overexpressing three transcription factors—Oct4,Sox2 and Nanog.The culture system developed here is inexpensive and suitable for large scale production.展开更多
文摘Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.
文摘Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.
文摘Human induced pluripotent stem （hiPS） cells are considered a potential source for the generation of insulin-producing pancreatic β-ceUs because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently dif- ferentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 （PDX1）-positive pancreatic progenitors and then into neurogenin 3 （NGN3）-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-l-methylxanthine （IBMX）, exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic β-cell markers. Most notably, the differentiated cells contained en- dogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic β-ceUs under xeno-free conditions and highlight their poten- tial to treat patients with type I diabetes.
基金by the National High Technology Research and Development Program(863 ProgramGrant No.2006AA02A106)+3 种基金the National Basic Research Program(973 ProgramGrant Nos.2006CB943901,2010CB945024,and 2011CB965002)the Knowledge Innovation Program of the Chinese Academy of Sciences(KSCX2-YW-R-50)the National Foundation of Science and Technology(No.30640005).
文摘While human induced pluripotent stem cells(hiPSCs)have promising applications in regenerative medicine,most of the hiPSC lines available today are not suitable for clinical applications due to contamination with nonhuman materials,such as sialic acid,and potential pathogens from animal-product-containing cell culture systems.Although several xeno-free cell culture systems have been established recently,their use of human fibroblasts as feeders reduces the clinical potential of hiPSCs due to batch-to-batch variation in the feeders and time-consuming preparation processes.In this study,we have developed a xeno-free and feeder-cell-free human embryonic stem cell(hESC)/hiPSC culture system using human plasma and human placenta extracts.The system maintains the self-renewing capacity and pluripotency of hESCs for more than 40 passages.Human iPSCs were also derived from human dermal fibroblasts using this culture system by overexpressing three transcription factors—Oct4,Sox2 and Nanog.The culture system developed here is inexpensive and suitable for large scale production.
