[人精原干细胞体外长期存活及其功能鉴定]

[Long-term survival of human spermatogonial stem cells in vitro and its functional identification].

作者信息

Li Yan-feng, Guo Ying-lu, Li Xiao-hong, Jin Feng-shuo, Sun Zhong-yi

机构信息

Department of Urology, Institute of Field Surgery, Daping Hospital, the Third Military Medical University, Chongqing 400042, China.

出版信息

Zhonghua Nan Ke Xue. 2005 Dec;11(12):886-90, 894.

PMID:16398355

Abstract

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.

摘要

目的

人类精原干细胞（SSC）的培养尚未得到详细研究。在此，我们尝试通过使用几种不同的共培养系统来探索人类SSC的优化培养方法。

方法

通过免疫磁珠分选技术获得的α6⁺Thy-1⁺c-kit⁻细胞在不同的共培养系统中培养。通过显微镜或免疫细胞化学分析对其形态、生物学特性和存活率进行深入观察。通过生殖细胞移植技术评估人类SSC在培养期间的长期存活率。

结果

α6⁺Thy-1⁺c-kit⁻细胞在添加胎牛血清（FBS）的饲养层上的DMEM和DMEM/F12培养基中能够稳定存活。1周内的存活率超过90%。长期培养显示细胞逐渐以分散的单细胞或聚集团块的方式附着在支持细胞表面。部分SSC与支持细胞的附着变得更加紧密或嵌入支持细胞之间。它们能够在体外存活甚至增殖超过3个月。生殖细胞移植研究表明，一些用PKH26标记的α6⁺Thy-1⁺c-kit⁻细胞可驻留在裸鼠生精小管的基底膜上，移植后2个月表现为单个或成对细胞。通过计算生精小管中的荧光细胞对培养细胞进行功能评估，结果显示α6⁺Thy-1⁺c-kit⁻细胞中的SSC在培养2周和4周后分别仍保留54.9%和9.2%。