Isolation, Characterization, and Culture of Human Spermatogonia1

Abstract

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…Isolation of SSCs involves a multi-step enzymatic digestion of the testicular tissues by combinations of two or more enzymes like, hyaluronidase, collagenase, trypsin and DNase I [9,12]. The SSCs have been isolated using two enzymes in human [12,13], three enzymes in buffalo [14] and four enzymes in cattle [11] as well as goat [10].…”

Downregulation of Col1a1 induces differentiation in mouse spermatogonia

“…Pcna is expressed in proliferating spermatogonia and spermatocytes, but not in non-proliferating spermatogonia or preleptotene spermatocyte. 24 Stra8 is a vertebrate-specific gene whose expression is crucial for initiation of meiosis in both male and female germ cells 25 and its overexpression is known to promote the in vitro differentiation of mouse embryonic stem cells into spermatozoa. 21,26 Our observations suggest that in response to Col1a1 suppression, the spermatogonial proliferation appeared to be suspended, while their differentiation was trigged.…”

Spermatogonial Stem Cells: An Alternate Source of Pluripotent Stem Cells for Regenerative Medicine

“…Thus, their small numbers and the lack of specific markers are the main hurdles to their characterization. Nonetheless, significant progress has recently been made in the isolation and propagation of cells with SSCs properties from rodent Guan et al 2009;Kanatsu-Shinohara, Takehashi, and Shinohara 2008;Kubota, Avarbock, and Brinster 2004;Brinster 2006, 2008;Ogawa et al 2003;Hofmann et al 2005;Kanatsu-Shinohara et al 2010) and human testes (Conrad et al 2008;Glaser et al 2008;Golestaneh et al 2009;Kossack et al 2009;Zovoilis et al 2008;Izadyar et al 2011;He et al 2009). Human testicular tissue is currently obtained from testicular biopsies (Izadyar et al 2011;Kossack et al 2009), orchiectomies (Izadyar et al 2011) and organ donors ).…”

“…Testicular biopsies of approximately one gram can yield sufficient number of human SSCs for most clinical applications. Although our knowledge of mouse and human SSCs phenotype is still limited, studies suggest that human SSCs express proteins such as cluster of differentiation antigens 49f, 90 and 133 (CD49f, CD90, and CD133, respectively), glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), G protein-coupled receptor 125 (GPR125), melanoma antigen family A 4 (MAGE4), promyelocytic leukemia zinc finger (PLZF) and stage-specific embryonic antigen-4 (SSEA-4; Costoya et al 2004;Conrad et al 2008;He et al 2009;Izadyar et al 2011). Using these markers in magnetic-or fluorescent-activated cell sorting, SSCs can be isolated with a high degree of purity (Gassei et al 2009;Izadyar et al 2011;.…”