Differential Gene Expression Profiling of Enriched Human Spermatogonia after Short- and Long-Term Culture

Abstract

This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+ matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.

De novo sequencing and comparative analysis of testicular transcriptome from different reproductive phases in freshwater spotted snakehead Channa punctatus

“…punctatus during spawning. Among other upregulated genes of spawning phase, rgs4 , alcam and cxcl2 have been associated with gonadal stem cells [66–68] while slc6a1 and hipk1 have been reported in spermatids and spermatozoa [69,70]. Nonetheless, it is difficult to decipher the precise role of these genes in regulation of testicular events attributed to spawning phase in C .…”

<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.…”

Characteristic Features of Male Germline Development in Primates

“…In the primate, it is still a matter of debate whether long-term culture and the expansion of spermatogonial stem cells, i.e., the culture of functional stem cells, is possible. While some studies could detect human [75,76] and marmoset monkey [77,78] spermatogonia only for very limited periods in culture, other reports described the long-term culture of human spermatogonia [79][80][81][82] and even their differentiation into haploid germ cells [83] . However, since some markers used in human long-term spermatogonial culture studies are not unequivocal germ cell markers, it remains a matter of conten-tion at present whether the cultured cells are really functional germ cells.…”