Stage-Specific Embryonic Antigen 4 Is A Membrane Marker For Enrichment Of Porcine Spermatogonial Stem Cells

Background Spermatogonial stem cells (SSCs), as tissue-specific stem cells, are capable of both self-renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub-fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. Objectives The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. Methods To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co-immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c-KIT, and SOX9) in the 7-, 90-, and 150-day-old porcine testicular tissues. SSEA4-positive cells were isolated from the 90-day-old porcine testes by flow cytometry. Immunofluorescent, RNA-sequencing, and transplantation analysis were used to reveal that SSEA4-positive fraction holds the stem cell capacity. Results We found that SSEA4 was expressed in a rare sub-fraction of porcine undifferentiated spermatogonia, and RNA-sequencing analysis revealed that the genes for stem cell maintenance and SSC-specific markers (ID4 and PAX7) were up-regulated in the SSEA4-sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4-positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti-SSEA4 antibody resulted in a 2.5-fold enrichment of SSCs compared with the germ cell gate group, and 21-fold enrichment of SSCs compared with the SSEA4-negative spermatogonia group. Conclusions Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.