Lo Kirk C, Brugh Victor M, Parker Michele, Lamb Dolores J
Department of Urology, Mount Sinai Hospital, University of Toronto, Ontario, Canada, M5G 1X5.
Biol Reprod. 2005 Mar;72(3):767-71. doi: 10.1095/biolreprod.104.033464. Epub 2004 Dec 1.
Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho(low)) or high (Rho(hi)) fluorescent intensity. Sterile recipients, W/W(v) mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho(low), Rho(hi), and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho(low) cells showed a 17- and 20-fold enrichment over those of Rho(hi) and nonsorted cells, respectively.
干细胞在组织替代方面具有巨大的治疗潜力。为了进一步研究干细胞，必须将它们分离出来。目前已有用于富集和研究造血干细胞的技术，但到目前为止，尚未描述精原干细胞的纯化技术。造血干细胞的富集技术包括使用结合了Hoechst 33342和罗丹明123（Rho）染料的荧光激活细胞分选分析。在我们实验室中，使用Hoechst染料分离精原干细胞并不成功，我们的结果与其他实验室的结果相矛盾。利用Rho染料的差异染色，我们报告了一种富集小鼠精原干细胞的新方法。从隐睾ROSA26雄性小鼠中采集睾丸细胞。然后用Hoechst和Rho染料对这些细胞群体进行染色，使其能够通过流式细胞术分选到Hoechst低强度细胞的侧群（SP）以及低（Rho(low)）或高（Rho(hi)）荧光强度的群体中。将具有内在生殖细胞缺陷的无菌受体W/W(v)小鼠移植Hoechst SP细胞、Rho(low)、Rho(hi)和未分选的供体细胞。未从Hoechst SP细胞中获得精原干细胞集落。移植的Rho(low)细胞产生的精原干细胞集落数量分别比Rho(hi)和未分选细胞的集落数量富集了17倍和20倍。
