The transcription factor octamer-binding transforming factor 4 (Oct-4) is central to the gene regulatory network responsible for self-renewal, pluripotency, and lineage commitment in embryonic stem (ES) cells and induced pluripotent stem cells (PSCs). This study was undertaken to evaluate differential localization and expression of two major transcripts of Oct-4, viz. Oct-4A and Oct-4B, in adult human testis. A novel population of 5- to 10-μm PSCs with nuclear Oct-4A was identified by ISH and immunolocalization studies. Besides Oct-4, other pluripotent markers like Nanog and TERT were also detected by RT-PCR. A(dark) spermatogonial stem cells (SSCs) were visualized in pairs and chains undergoing clonal expansion and stained positive for cytoplasmic Oct-4B. Quantitative PCR and Western blotting revealed both the transcripts, with higher expression of Oct-4B. It is proposed that PSCs undergo asymmetric cell division and give rise to A(dark) SSCs, which proliferate and initiate lineage-specific differentiation. The darkly stained nuclei in A(dark) SSCs may represent extensive nuclear reprogramming by epigenetic changes when a PSC becomes committed. Oct-4B eventually disappeared in mature germ cells, viz. spermatocytes, spermatids, and sperm. Besides maintaining normal testicular homeostasis, PSCs may also be implicated in germ cell tumors and ES-like colonies that have recently been derived from adult human testicular tissue.

Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.

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