Expression profile of genes identified in human spermatogonial stem cell‐like cells using suppression subtractive hybridization

Abstract

Spermatogenesis is the process by which testicular spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm in the testis. Maintaining healthy spermatogenesis requires proper proliferation of SSCs. In this study, we sought to identify factors that regulate the proliferation of SSCs. Human SSC (hSSC)-like cells were isolated from azoospermic patients by a modified culture method and propagated in vitro. After four to five passages, the SSC-like cells spontaneously ceased proliferating in vitro, so we collected proliferating (P)-hSSC-like cells at passage two and senescent (S)-hSSC-like cells at passage five. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between the P-hSSC-like and S-hSSC-like cells. We selected positive clones up-regulated in P-hSSC-like cells using SSH and functionally characterized them by reference to public databases using NCBI BLAST tools. Expression levels of genes corresponding to subtracted clones were analyzed using RT-PCR. Finally, we confirmed the differential expression of 128 genes in positive clones of P-hSSC-like cells compared with S-hSSC-like cells and selected 23 known and 39 unknown clones for further study. Known genes were associated with diverse functions; 22% were related to metabolism. Fifteen of the known genes and two of the unknown genes were down-regulated after senescence of hSSC-like cells. A comparison with previous reports further suggests that known genes selected, SPP1, may be related to germ cell biogenesis and cellular proliferation. Our findings identify several potential novel candidate biomarkers of proliferating- and senescencet-hSSCs, and they provide potentially important insights into the function and characteristics of human SSCs.

Propagation of Adult SSCs: From Mouse to Human

“…Remarkably, the spermatogonia were obtained from deceased organ donors, a reasonable source from which to obtain human SSCs from healthy donors. A recent report using testicular tissue from patients with azoospermia identified genes differentially expressed between proliferating putative human SSCs versus the senescent human SSCs that resulted from human SSC-like cells after five passages in culture [81]. This yielded a list of potential genes related to proliferation of human germ cells.…”

Expression Profiles of Subtracted Genes During Neural Differentiation from Human Embryonic Stem Cells using Suppression Subtractive Hybridization

“…Bene t of SSH is that it can nd novel genes and has a low false-positive rate (Yoo et al, 2010). In our previous study, we reported that the expression of TPX2 gradually decrease during neurogenesis (Noh et al, 2007).…”

“…On the other hand, adult stem cells can be induced from adult cells, including bone marrow, cord blood, brain, and testis. Adult stem cells also show self-renewal and multidifferentiation but are more limited in both of these functions compared with ES cells (Ramalho-Santos et al, 2002; Yoo et al, 2010). Patient-speci c pluripotent stem cells are produced by modifying gene regulation via pluripotency-related genes such as Oct4, Sox2, Klf4 and c-Myc (Takahashi et al, 2007).…”

From stem cells to male germ cells: Experimental approaches for the in vitro generation of mouse and human spermatogonial stem cells

“…The functionality of these in vitro cultured putative hSSCs could be proven by xenotransplantation to mice. This progress shows that long-term culture and in vitro propagation of hSSCs is achievable, nevertheless it also illustrates the importance of the less popular basic research to identify factors, which might determine the status of proliferating and senescent hSSCs, and helps to figure out culture and growth requirements for the long-term culture of hSSCs (Yoo et al, 2010).…”