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

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.

Gametes from stem cells: Status and applications in animal reproduction

“…Similar to PGCs, VSELs have the ability to differentiate into the three germ layers in vitro (Havens et al, ). Although their potential is still to be studied in livestock species, some evidence from mouse and human studies supports them as candidates for gamete generation with capacity to undergo oocyte‐specific (Sriraman, Bhartiya, Anand, & Bhutda, ) and sperm‐specific (Anand, Patel, & Bhartiya, ) differentiation.…”

Making gametes from pluripotent stem cells: embryonic stem cells or very small embryonic-like stem cells?

“…The SSCs in turn divide rapidly by symmetric cell divisions followed by clonal expansion to form cell aggregates ( Figure 1). We had earlier shown that the testicular cells isolated from chemoablated testis on culture spontaneously differentiate into sperm (12). The Sertoli cells attach to the culture surface and provide a somatic support and act as a source of growth factors and cytokines required for stem cells differentiation.…”

“…A critique to the study (12) was that all testicular cells that survived chemotherapy were used for in vitro culture. It could possibly be the few surviving SSCs that may have given rise to sperm in vitro rather than the VSELs.…”

“…VSELs and SSCs are not two distinct stem cell populations, rather VSELs are the stem cells and SSCs are the progenitors (26). Indeed VSELs are the 'true' and most primitive stem cells in the testis which give rise to SSCs by asymmetric cell division (12,22). But criticism is always good and to further satisfy the reviewers, we have now studied differentiation of mouse bone marrow derived VSELs (no chance of contaminating SSCs) on a testicular niche and observed they successfully give rise to SSCs and We were intrigued to observe that PGCs were recently defined 'unipotent' in Nature journal (27) on the assumption that they only give rise to gametes in the body compared to totipotent or pluripotent early stage embryo/ICM which can form the whole body.…”

National multicentric M13 Stem Cell Trial reports negative outcome - Need to look at VSELs as an alternative to bone marrow MNCs for cardiac regeneration

“…More clarity is needed to define stem cells and distinguish them from the progenitors in the body organs16. VSELs survive oncotherapy in mammalian testis17, ovary18 and also in the BM19 and could possibly result in spontaneous pregnancies. Regenerative potential of VSELs has been reported in mice for lung epithelium20; cardiac tissue2122; neurons23; pancreas2425; liver26; and in humans for critical limb ischaemia27; cardiac tissue2829; and diabetes30.…”