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
Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation ofpig spermatogonial stem cells （pSSCs） has not been tested. The aim of this...Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation ofpig spermatogonial stem cells （pSSCs） has not been tested. The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium, pSSCs were cryopreserved with freezing media containing different concentrations of sucrose （70, 140, 210, and 280 mmol L^-1） and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue （TB） staining, SYBR-14/propidium iodide （PI） dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^-1 sucrose. Moreover, the 210 mmol L^-1 sucrose group yielded the highest survival rate among all the groups （P〈0.05）. The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR （qRT-PCR） indicated that the mRNA levels of three apoptosis-promoting genes （BAX, APAF1 and CASPASE9） were significantly higher in thawed cells than in cells before freezing （P〈0.05）. Moreover, the mRNA level of one anti-apoptotic gene （XIAP） was significantly lower in thawed cells than in cells before freezing （P〈0.05）. When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups （P〈0.05）. Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L-1 sucrose group. Both qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^-1, which possibly assisted pSSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs.展开更多
Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of...Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of cell surface markers and transcription factors.However,it has been challenging to define micro-heterogeneity within“stem cell”populations,each of which stands at a different level of differentiation.As stem cells become defined at a single-cell level,their differentiation path becomes clearly defined.Here,this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches,with an emphasis on practical considerations in stem cell biology.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.
基金supported by the China Postdoctoral Science Foundation(2014M560809)the Shaanxi Province Postdoctoral Science Foundation,China+1 种基金the Fundamental Research Funds for the Central Universities,China(NWSUAF,2452015145)the National Basic Research Program of China(2014CB943100)
文摘Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation ofpig spermatogonial stem cells （pSSCs） has not been tested. The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium, pSSCs were cryopreserved with freezing media containing different concentrations of sucrose （70, 140, 210, and 280 mmol L^-1） and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue （TB） staining, SYBR-14/propidium iodide （PI） dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^-1 sucrose. Moreover, the 210 mmol L^-1 sucrose group yielded the highest survival rate among all the groups （P〈0.05）. The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR （qRT-PCR） indicated that the mRNA levels of three apoptosis-promoting genes （BAX, APAF1 and CASPASE9） were significantly higher in thawed cells than in cells before freezing （P〈0.05）. Moreover, the mRNA level of one anti-apoptotic gene （XIAP） was significantly lower in thawed cells than in cells before freezing （P〈0.05）. When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated groups （P〈0.05）. Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L-1 sucrose group. Both qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^-1, which possibly assisted pSSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs.
基金This research was supported by National Institute of Health Grants R01DE026666 and R01DE030630(to NO)and R01DE029181(to WO).
文摘Single-cell sequencing technologies have rapidly progressed in recent years,and been applied to characterize stem cells in a number of organs.Somatic(postnatal)stem cells are generally identified using combinations of cell surface markers and transcription factors.However,it has been challenging to define micro-heterogeneity within“stem cell”populations,each of which stands at a different level of differentiation.As stem cells become defined at a single-cell level,their differentiation path becomes clearly defined.Here,this viewpoint discusses the potential synergy of single-cell sequencing analyses with in vivo lineage-tracing approaches,with an emphasis on practical considerations in stem cell biology.
