In vitro differentiation of human male germ cells from spermatogonial stem cells derived from umbilical cord blood

Abstract

Rationale: Spermatogenesis is a unique, complex and orchestrated process that requires a period of amplification of spermatogonial stem cells (SSCs proliferation), the reduction, division (meiosis), and the morphological transformation of round spermatids into spermatozoa (spermiogenesis). Objective: Our objective was to recapitulate this process in vitro using umbilical cord blood derived mesenchymal stem cells (UCB-MSCs). Methodology: Blood was collected from the veins of umbilical cords and, mono nuclear cells (MNCs) were separated using the density gradient centrifugation over Ficoll-Hypaque. The separated cells were propagated in monolayer culture system. Cells were characterized using MSCs specific markers, and differentiation potential in to adipogenic and osteogenic lineages. At the second passage cells were induced with 10 flM all trans retinoic acid (ATRA) for two weeks. Stage specific genes expressed or suppressed at pre-meiotic, meiotic and post-meiotic stages were detected using RT-PCR technique. Possibility of arising primitive stages of SSCs were determined by alkaline phosphatase activity (ALP) and GPR ] 25 gene expression. Putative germ cells were cultured with rat Sertoli cells for further four weeks. Chromosome spreading was used to observe meiotic reduction. Development of advanced stages of germ cells were detected using periodic acid Schiff stain and aniline blue stain. Results: The number offibroblastoid colony forming units (CFU-f) was very low in UCB samRles. However, the rate of proliferation was high, and reached 70% confluence within six weeks. Cells were positive for MSCs and pluripotent makers; CD44, CD 73, CD 90, CD 105 and OCT4. OCT4 a stem cell marker, and PLZF an early SSCs marker, were suppressed during the induction period. Expression of other germ cell markers; pre-meiotic (STRAS), meiotic (SCP3) and post-meiotic (ACR, PRMl) were increased. Mild ALP activity and lack ofGPR ]25 expression confirmed the absence of PGCs and early stages of SSCs. Around ] 5% transformed in to haploid cells after induction. Different stages of acrosome formation and replacement of histone from protamine were observed in post meiotic cells. Conclusion: Human cord blood derived MSCs can be differentiated in to male germ cells up to round spermatids stage without genetic manipulations. Further studies are needed to improve the culture techniques to transform spermatids into mature and functional sperm.