Genetic and Epigenetic Evaluation of Human Spermatogonial Stem Cells Isolated by MACS in Different Two and Three-Dimensional Culture Systems.

Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this
study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in
propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS).
Materials and Methods: In this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse.
Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm.
Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of
spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.