人精原干细胞的体外增殖

Propagation of human spermatogonial stem cells in vitro.

作者信息

Sadri-Ardekani Hooman, Mizrak Sefika C, van Daalen Saskia K M, Korver Cindy M, Roepers-Gajadien Hermien L, Koruji Morteza, Hovingh Suzanne, de Reijke Theo M, de la Rosette Jean J M C H, van der Veen Fulco, de Rooij Dirk G, Repping Sjoerd, van Pelt Ans M M

机构信息

Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, University of Amsterdam, Amsterdam, The Netherlands.

出版信息

JAMA. 2009 Nov 18;302(19):2127-34. doi: 10.1001/jama.2009.1689.

DOI:10.1001/jama.2009.1689

PMID:19920237

Abstract

CONTEXT

Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility.

OBJECTIVE

To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation.

DESIGN, SETTING, AND PARTICIPANTS: Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation.

MAIN OUTCOME MEASURES

Propagation of spermatogonial stem cells over time.

RESULTS

Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture.

CONCLUSION

Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

摘要

背景

接受高剂量化疗的年轻男孩成年后常常面临不育问题。化疗前冷冻保存睾丸组织，后期自体移植精原干细胞理论上可恢复生育能力。

目的

从小睾丸活检组织中建立人精原干细胞的体外增殖体系，以获得足够数量的细胞用于成功移植。

设计、场所和参与者：2007年4月至2009年7月进行的研究，使用了6名因前列腺癌治疗而接受睾丸切除术的成年男性捐赠的睾丸材料。分离睾丸细胞并在添加了成分的StemPro培养基中培养；产生的生殖系干细胞簇在相同培养基中接种于包被人胎盘层粘连蛋白的培养皿上进行传代培养。通过逆转录聚合酶链反应和精原细胞标志物的免疫荧光检测精原细胞的存在。为检测培养物中功能性精原干细胞的存在，将其异种移植到免疫缺陷小鼠的睾丸中，并通过COT-1荧光原位杂交检测移植后迁移的人精原干细胞。对培养早期和后期移植的定植精原干细胞数量进行计数以确定增殖情况。

主要观察指标

精原干细胞随时间的增殖情况。

结果

睾丸细胞可培养并增殖长达15周。所有6名男性的睾丸细胞培养物中均出现了生殖系干细胞簇，且可传代培养并增殖长达28周。在整个培养期间，精原细胞标志物在RNA和蛋白质水平上的表达均得以维持。在6名男性中的4名中，即使经过长时间的体外培养，将其移植到小鼠体内也证明存在功能性精原干细胞。睾丸细胞培养物中精原干细胞数量在19天内增加了53倍，生殖系干细胞传代培养物中在64天内增加了18450倍。