Long-term culture and transplantation of murine testicular germ cells.
Long-term culture and transplantation of murine testicular germ cells.
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DOI：
10.1002/j.1939-4640.2003.tb02724.x
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发表时间：
2003-09
影响因子：
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通讯作者：
Dongkee Jeong;D. Mclean;M. Griswold
中科院分区：
文献类型：
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作者：
Dongkee Jeong;D. Mclean;M. Griswold
本研究的目的是建立一个体外培养系统，以优化生殖细胞的增殖，并测量培养的生殖细胞在移植到受体后产生成熟精子的潜力。从ROSA26雄性小鼠中分离供体生殖细胞，用STO饲养细胞层在Dulbecco's最低必需培养基（DMEM）中培养，培养基中添加胎牛血清（FBS）、干细胞因子、白血病抑制因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、白细胞介素-11、l -谷氨酰胺、丙酮酸钠、2-巯基乙醇、小鼠抑癌素M和血小板源性生长因子。8-21天后，供体生殖细胞在原代培养物中形成菌落。这些培养的菌落在没有继代培养的情况下维持了4周或更长时间，在3个月的时间内增殖了8代。这些菌落具有碱性磷酸酶活性，并含有5-溴-2'-脱氧尿苷。这些菌落经生殖细胞核抗原抗体和c-kit筛选后部分呈阳性。与用DMEM和FBS培养的对照相比，用该培养基培养的生殖细胞的定植能力增强。将Rosa26供体培养的生殖细胞移植到睾丸中，通过X-gal染色和组织学筛选鉴定。在补充培养基中培养的细胞在受体小鼠的小管中定植并开始精子发生。这是一种改良的生殖细胞培养方法，可用于基因治疗和转基因动物的生产。
The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.