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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和ICR品系）收集的雄性生殖干细胞在含有4种生长因子（EGF、bFGF、LIF、GDNF）的小鼠胚胎成纤维细胞饲养层上长期培养。我们立即重复并确认了报告的结果，并通过建立不仅来自新生儿而且来自成年小鼠的生殖系干细胞（GS细胞）进一步推进了该研究。GS细胞在体外呈指数增长。它们在移植受体小鼠睾丸时产生精子。它们在受体小鼠体内不产生肿瘤，也不显示异常增殖。从受体小鼠睾丸中再衍生出GS细胞，表明体外培养的GS细胞与睾丸中的精原干细胞是相同的或相互转化的。精原移植受体小鼠的受精为了使精原移植后的受体小鼠具有生育能力，在移植前对未成熟小鼠（10-15日龄）进行辐射处理。照射的目的是通过去除内源性生殖细胞在睾丸中形成空巢，以有效地定植供体干细胞。辐射剂量在12 Gy左右是最佳的。照射后1 ~ 3d，受体小鼠接受精原细胞移植。移植后两个月，受体小鼠与雌性小鼠交配以测试其生育能力。证实小鼠以高比率（约70%）成为供体生殖细胞来源的具有生育力的父代幼仔。当我们使用GS细胞时，受体小鼠的生育率变得更高，约为80%。因此，使受体能够用供体生殖细胞生育的重要因素包括受体的年龄、供体细胞悬液的干细胞浓度。