The piwi family genes are highly conserved during evolution and play a crucial role in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to humans. Piwil2, also known as Hili, is one of the four human homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell (SSCs) selfrenewal and formation. Therefore, as a bid for infertility treatment, we examined the piwil2 potential to trans-differentiate human fibroblasts, as terminally differentiated cells, to germ cell lineage.
Materials and methods
Primary Human Neonatal Foreskin Fibroblasts (Nff) were transfected by plasmid overexpressing hpiwil2 under the control of CMV promoter. After establishing stable cell line, semi-quantitative RT-PCR analysis was then applied to assess the expression level of our target genes including those related to pre-meiotic and late meiotic germ cell lineage.
Our semi-quantitative gene expression analysis on the human fibroblasts ectopically expressing hpiwil2 demonstrated an increase in the expression level of many premeiotic germ cell markers including HSP90, CD49f, C-kit, Stella, Fragilis, Spoc1, RBM. However, the expression of late meiotic markers such as Scp3, Pgk2 or Prm1 were not detectable under the standard cell growth condition which were applied in our study. notably, in line with the pluripotent characteristics of premeiotic spermatogonial stemm cells (SSCs), the hpiwil2 expressing human fibroblasts demonstrated an elevated level of pluripotency markers including Oct4, C-myc, Klf4 and Nanog as well.
Our resalts clearly indicated the capability of hpiwil2 gene in reprogramming of human fibroblasts to premeiotic germ cell lineage like SSCs, providing novel approach for treatment of male infertility