Abstract
BackgroundExtensive 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.MethodsMouse 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.ResultsStem 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).ConclusionsProcess 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0031-2) contains supplementary material, which is available to authorized users.
Highlights
Extensive research is ongoing to empower cancer survivors to have biological parenthood
We obtained similar results initial presence of few spermatogonial cells which may have survived busulphan treatment cannot be ruled out in our generic cultures. This was anyways not an issue of great concern to us as we have earlier reported that very small embryonic-like stem cells (VSELs) are the quiescent stem cells which undergo asymmetric cell division to give rise to the spermatogonial stem cells (SSCs) which further undergo symmetric cell divisions, proliferate rapidly, differentiate, undergo meiosis to produce sperm [16]
Present study demonstrates for the first time that testicular cells which survive busulphan treatment in adult mice testis undergo spontaneous proliferation, clonal expansion, differentiation and meiosis resulting in sperm formation during in vitro culture for 3 weeks
Summary
Extensive research is ongoing to empower cancer survivors to have biological parenthood. Lot of hope was bestowed on pluripotent embryonic and induced pluripotent stem cells to differentiate into sperm and oocytes. 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. Pluripotent stem cells (PSCs) including embryonic stem (ES) and induced pluripotent stem (iPS) cells have been studied for differentiation into gametes but the goal to achieve normal gametes to treat infertility still remains a distant dream [1]. Eguizabal et al [10] achieved complete meiosis using human iPS cells, but the efficiency remains relatively low
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