Xenotransplantation assessment: morphometric study of human spermatogonial stem cells in recipient mouse testes

Abstract

The purpose of this study was (i) To establish in vitro propagation of human spermatogonial stem cells (hSSCs) from small testicular biopsies to obtain a high number of cells; (ii) to evaluate the presence of functional hSSCs in culture system by RT-PCR using DAZL, α6-Integrin, β1-Integrin genes; and (iii) to evaluate the effects of cell concentration on successful xenotransplantation of hSSCs in mice testis. Donor hSSCs were obtained from men with maturation arrest of spermatogenesis duration 1 year ago. These cells were propagated in DMEM containing 1 ng ml(-1) bFGF (basic fibroblast grow factor) and 1500 U ml LIF (leucaemia inhibitory factor) for 5 weeks. Different concentrations of hSSCs transplanted into seminiferous tubules of busulfan-treated immunodeficient mice and analysed up to 8 weeks after transplantation. The results showed that expression of DAZL and α6-Integrin mRNA was increased as well as the colony formation of SSCs in vtro culture during 5 weeks. Proliferation occurred about 4 weeks after transplantation, but meiotic differentiation was not observed in recipient testis after 8 weeks. The difference in donor cells concentration had effect on homing spermatogenesis in recipient testis. Homologous transplantation of proliferated SSCs to seminiferous tubules of that patient individually may allow successful differentiation of transplanted cells.

Morphological and ultrastructural studies of human spermatogonial stem cells from patients with maturation arrest

“…Disruption of the Dmc1 gene leads to fail in homologous chromosome synapsis formation and causes meiotic cell death. It was speculated that the absence of this meiotic gene may be an indication for differentiation arrest of spermatogonia cells at the premeiotic stages in the culture system in this study (Mirzapour, Movahedin, Koruji, & Nowroozi, ). Therefore, in our study, spermatocytes and spermatids were not observed at the light and electron microscopic level.…”

“…In this situation where there is an accumulation of spermatogonia, probably apoptosis of damaged spermatogonia does eventually occur because of failure of the density regulatory system. The expression of Bax gene was observed approximately 32–35 days after culture (the results were not shown; Mirzapour et al., ). Therefore, ever time, some changes take place, which could cause a delay in cell growth and proliferation of SSC colonies.…”

<p>Role of stem cells in fertility preservation: current insights</p>

“…Subsequently, several teams attempted to find a culture system of dissociated testicular cell suspensions (TCSs) able to propagate human SSCs in vitro (Table 1). [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] Sadri-Ardekani et al adapted the protocol developed by Kanatsu-Shinohara et al for human testicular cells (TCs). Briefly, this culture system relies on the capacity of somatic cells to adhere to the plate while the germ-cell fraction stays in suspension, allowing enrichment of SSCs after differential plating.…”

“…32,33 This technique led to an 18,450-fold enrichment of adult SSCs after 64 days and to a 9.6-fold enrichment of prepubertal SSCs after 11 days of culture using xenotransplantation as the gold standard to identify SSCs able to migrate along the basement membrane of the STs, colonize their niches, and generate germ-cell colonies. Among researchers who have xenotransplanted long-term cultured human SSCs, 32,33,39,40,42,43,51 only Sadri-Ardekani et al and Nickkholgh et al quantified SSCs in STs after transplantation and demonstrated SSC enrichment. 32,33,43 However, several other teams using the same protocol could not reproduce such results due to the complexity and skills needed to distinguish between SSCs and human embryonic stem cell-like (hESC-like) cells, 34 because of low germ-cell survival and overgrowth of remaining somatic cells.…”

Grand Challenge Animal Reproduction-Theriogenology: From the Bench to Application to Animal Production and Reproductive Medicine

“…Traditionally, this has been approached through experimental designs involving live animals. The development of techniques such as specific molecular probes and xenografting of testicular tissues opened a new era of study on factors affecting spermatogenesis (8–11). …”