人精原干细胞的体外培养及其功能鉴定

目的:通过应用多种培养条件下的生殖细胞共培养体系,寻求人精原干细胞(SSC)长期体外生存和扩增的优化途径。方法:应用磁式分选所获得的α6+Thy-1+c-k it-细胞,分别以STO、MEF、Sertoli细胞为饲养层,于DMEM/F12、DMEM或DMEM-SF培养液条件下短期培养,然后选择胎儿Sertoli细胞作为共培养体系,在32℃、5%CO2、DMEM/F12(含10%胎牛血清)培养条件下,分别观察成人和胎儿生殖细胞的长期培养结果。并应用免疫组化技术和SSC移植技术对培养细胞进行初步生物学特性和功能鉴定。结果:在短期培养中,α6+Thy-1+c-k it-细胞在存在饲养层时,可在DMEM和DMEM/F12培养液中稳定生存,1周内存活率可达90%。长期培养条件下,α6+Thy-1+c-k it-细胞逐渐与Sertoli细胞形成紧密的贴附或镶嵌关系,呈单个、成对、短链状或小团簇状;胎儿SSC与成人类似,部分以圆球形散在分布于Sertoli细胞表面,并常可观察到明显的成对或短链状细胞,部分呈有突起的长梭形扁平细胞,与Sertoli细胞相互形成紧密连接;经反复传代3个月,仍可观察到稳定存在的圆球形SSC贴附于Ser-toli细胞表面。由PKH26标记的α6+Thy-1+c-k it-细胞在移植后2个月可迁移至裸鼠生精小管基膜,表现为单个或成对的细胞。生精小管内荧光细胞计数显示α6+Thy-1+c-k it-细胞在体外培养2、4周后仍然保留54.9%和9.2%的SSC。结论:该培养系统的进一步优化将有利于体外产生大量SSC,并有助于进一步深入了解SSC的生物学特性和男性生精功能障碍的治疗。

Long-term Survival of Human Spermatogonial Stem Cells in vitro and Its Functional Identification

OBJECTIVE: The culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems. METHODS: The alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique. RESULTS: The alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively. CONCLUSION: Human SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.