Isolation, culture and primary identification of human primordial germ cells

LENG Shao-hua; LI Cai-xia; LIANG Zhi-qing; WANG Feng-ying

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Isolation, culture and primary identification of human primordial germ cells

更新时间：2025-01-15

- Isolation, culture and primary identification of human primordial germ cells
- Journal of Army Medical University Vol. 31, Issue 8, Pages: 679-683(2009)
- 作者机构：
  
  第三军医大学西南医院妇产科
- 作者简介：
- 基金信息：
- DOI：
  CLC： R321.1;R329-33
- Published：2009，
  
  Published Online：30 April 2009，
- 稿件说明：
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LENG SHAO-HUA, LI CAI-XIA, LIANG ZHI-QING, et al. Isolation, culture and primary identification of human primordial germ cells. [J]. Journal of army medical university, 2009, 31(8): 679-683.
DOI：

LENG SHAO-HUA, LI CAI-XIA, LIANG ZHI-QING, et al. Isolation, culture and primary identification of human primordial germ cells. [J]. Journal of army medical university, 2009, 31(8): 679-683. DOI：

摘要

目的探讨体外分离培养人原始生殖细胞的条件和方法。方法分离、培养2～3个月人胚胎的成纤维细胞，经

Co照射后作为饲养层；从5～9周人胚胎的生殖嵴和肠背系膜组织中分离原始生殖细胞，培养在含有bFGF、LIF和Forskolin的人胚胎成纤维细胞饲养层上；同时采用免疫细胞化学和RT-PCR方法检测人原始生殖细胞阶段特异性胚胎抗原SSEA-1、SSEA-4以及特异性基因Oct-4、Ifitm-3、stella、Mvh、DAZL的表达，并进行类胚体培养。结果分离的人原始生殖细胞在饲养层上可以增殖形成典型的鸟巢状集落; 细胞染色体均为正常的二倍体核型; 阶段特异性胚胎抗原SSEA-1、SSEA-4表达阳性，同时检测到生殖干细胞特异性基因Oct-4、Ifitm-3、stella、Mvh、DAZL表达。人原始生殖细胞在悬滴培养时能够形成类胚体，表明其具有多项分化潜能。结论用人胚胎成纤维细胞作为饲养层进行体外培养人胚胎生殖嵴和肠背系膜组织

分离的原始生殖细胞形成EG细胞集落，初步鉴定为人胚胎生殖细胞。

Abstract

Objective To explore how to isolate and culture human primordial germ cells (PGCs). Methods Human embryonic fibroblasts (HEFs) isolated from embryos at 2 to 3 months of age received a radiation of

Co γ-ray and then served as feeder layer cells. Cells collected from trypsinized human embryonic gonadal ridges and dorsal mesenteries (5 to 9 weeks post-fertilization) were cultured on HEF feeder layers in the medium with recombinant human leukemia inhibitory factor (LIF)

recombinant human basic fibroblast growth factor (bFGF) and forskolin. Histochemical and immunocytochemical assays

were used to identify the stage specific embryonic antigen-1and 4 (SSEA-1/4) of the cultured cells. Reverse transcription polymerase chain reaction (RT-PCR) was used to identify the expressions of specific genes

including Oct-4

Ifitm-3

stella

Mvh and DAZL. Embryonic bodies were cultured as while. Results The collected cells were growing on the feeder layer and formed a typical nestlike multicellular colonies. The cells showed diploid chromosome in karyotype analysis and expressed SSEA-1/4 and specific genes

Oct-4

Ifitm-3

stella

Mvh and DAZL

indicating that the cells were PGCs. When cultured in hanging drop

the PGCs developed into embryonic bodies

showing multipotential ability. Conclusion Human PGCs can be isolated from genital ridges and dorsal mesenteries of embryos and cultured

in vitro

on HEF feeder layer. The formation of the embryonic stem cell colony can be observed

and the cells cultured by this method is confirmed to be embryonic germ cells.

关键词

人原始生殖细胞细胞培养胚胎生殖细胞

Keywords

human primordial germ cellscell cultureembryonic germ cells

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