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Searching for culture condition optimal for proliferation of spermatogonial stem cells

寻找最适合精原干细胞增殖的培养条件

基本信息

批准号：
15591702
负责人：
OGAWA Takehiko
金额：
$ 1.34万
依托单位：
YOKOHAMA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591702/
关键词：
spermatogenesis stem cell cell culture

项目摘要

Culturing of Spermatogonial Stem CellsIn April, 2003, it was reported that male germline stem cells, collected from neonatal mouse testes (strains of DBA/2 and ICR), were cultured for long period on feeder layer of mouse embryonic fibroblasts with 4 growth factors (EGF, bFGF, LIF, GDNF). We immediately repeated and confirmed results of the report and progressed it one step beyond by establishing germline stem cells (GS cells) from not only neonates but also from adult mice. The GS cells expanded exponentially in vitro. They produce sperm when transplanted recipient mouse testes. They did not produce tumor nor showed abnormal proliferation in the body of recipient mice. The GS cells was re-derived from recipient mouse testes, which showed that GS cells in vitro and spermatogonial stem cells in the testis were identical or mutually convertible.Fertilization of recipient mice with spermatogonial transplantationIn order to make recipient mice fertile after spermatogonial transplantation, immature mice (10-15 days old) were treated with irradiation before transplantation. The aim of irradiation was to make empty niche in the testis by removing endogenous germ cells for efficient colonization of donor stem cells. The dose of irradiation turned out to be optimal at around 12Gy for that purpose. One to Three days after the irradiation, the recipient mice received spermatogonial transplantation. Two months after the transplantation onward, the recipient mice were mated with females to test their fertility. It was confirmed that mice became fertile fathered pups of donor germ cell origin at high rate (about 70%). When we used GS cells, the fertility rate of recipient mice became higher, around 80%. The important factors for making recipient fertile with donor germ cells therefore include age of recipient, stem cell concentration of donor cell suspension.

精原干细胞的培养2003年4月，报道了从新生小鼠睾丸(DBA/2株和ICR株)采集的雄性生殖系干细胞，在含4种生长因子(EGF、bFGF、LIF、GDNF)的小鼠胚胎成纤维细胞饲养层上进行长期培养。我们立即重复和确认了报告的结果，并通过建立不仅来自新生儿的生殖系干细胞(GS细胞)，也从成年小鼠建立了生殖系干细胞(GS细胞)，使其更上一层楼。GS细胞在体外呈指数扩增。它们在移植受体小鼠的睾丸时会产生精子。它们在受体小鼠体内既没有产生肿瘤，也没有表现出异常增殖。从受体小鼠睾丸中分离出GS细胞，表明体外培养的GS细胞与睾丸内的精原干细胞是相同或可相互转化的。受体小鼠精原细胞移植的受精作用为了使受体小鼠在精原移植后生育，在移植前对未成熟小鼠(10-15天)进行照射。照射的目的是通过去除内源性生殖细胞在睾丸中留下空巢，以便有效地定植供体干细胞。为达到这一目的，照射剂量在12Gy时是最佳的。照射后1~3天，受体小鼠接受精原移植。移植两个月后，接受移植的小鼠与雌性交配，以测试它们的生育能力。已证实小鼠成为供体生殖细胞来源的可育父亲幼鼠的比率很高(约70%)。当我们使用GS细胞时，受体小鼠的受精率变得更高，约为80%。因此，受者年龄、供体细胞悬液中干细胞浓度是影响供体生殖细胞受精率的重要因素。