Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro

Reproductive Biology and Endocrinology

Deepa Bhartiya¹  Hiren Patel¹  Sandhya Anand¹ 

[1] Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai 400 012, India

关键词: PGCs;    ES cells;    Sperm;    SSCs;    VSELs;    Testis;   

Others  :  1175057
DOI  :  10.1186/s12958-015-0031-2

 received in 2015-02-20, accepted in 2015-04-14,  发布年份 2015

【 摘 要 】

Background

Extensive research is ongoing to empower cancer survivors to have biological parenthood. For this, sperm are cryopreserved prior to therapy and in younger children testicular biopsies are cryopreserved with a hope to mature the germ cells into sperm later on for assisted reproduction. In addition, lot of hope was bestowed on pluripotent embryonic and induced pluripotent stem cells to differentiate into sperm and oocytes. However, obtaining functional gametes from pluripotent stem cells still remains a distant dream and major bottle-neck appears to be their inefficient differentiation into primordial germ cells (PGCs). There exists yet another population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) in adult body organs including gonads. We have earlier reported that busulphan (25 mg/Kg) treatment to 4 weeks old mice destroys actively dividing cells and sperm but VSELs survive and differentiate into sperm when a healthy niche is provided in vivo.

Methods

Mouse testicular VSELs that survived busulphan treatment were cultured for 3 weeks. A mix of surviving cells in seminiferous tubules (VSELs, possibly few spermatogonial stem cells and Sertoli cells) were cultured using Sertoli cells conditioned medium containing fetal bovine serum, follicle stimulating hormone and with no additional growth factors.

Results

Stem cells underwent proliferation and clonal expansion in culture and spontaneously differentiated into sperm whereas Sertoli cells attached and provided a somatic support. Transcripts specific for various stages of spermatogenesis were up-regulated by qRT-PCR studies on day 7 suggesting VSELs (Sca1) and SSCs (Gfra) proliferate (Pcna), undergo spermatogenesis (spermatocyte specific marker prohibitin), meiosis (Scp3) and differentiate into sperm (post-meiotic marker protamine).

Conclusions

Process of spermatogenesis and spermiogenesis was replicated in vitro starting with testicular cells that survived busulphan treatment. We have earlier reported similar ability of ovarian VSELs enriched in the ovary surface epithelial cells to form oocyte-like structures in vitro. This striking potential of spontaneous differentiation of primitive testicular cells including VSELs that survive chemotherapy is being described for the first time in the present study.

【 授权许可】

   
2015 Anand et al.; licensee BioMed Central.

【 图 表 】

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