HEMATOPOIETIC DIFFERENTIATION OF UMBILICAL CORD BLOOD-DERIVED VERY SMALL EMBRYONIC/EPIBLAST-LIKE STEM CELLS

A population of CD133(+)lin(−)CD45(−) very small embryonic-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). In order to speed up isolation of these cells, we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45(−)/GlyA(−)/CD133(+)/ALDH(high) and CD45(−)/GlyA(−)/CD133(+)/ALDH(low) cells, which are enriched for VSELs, and CD45(+)/GlyA(−)/CD133(+)/ALDH(high) and CD45(+)/GlyA(−)/CD133(+)/ALDH(low) cells, which are enriched for hematopoietic stem/progenitor cells (HSPCs). While freshly isolated CD45(−) VSELs did not grow hematopoietic colonies, the same cells, when activated/expanded over OP9 stromal support, acquired hematopoietic potential and grew colonies composed of CD45(+) hematopoietic cells in methylcellulose cultures. We also observed that CD45(−)/GlyA(−)/CD133(+)/ALDH(high) VSELs grew colonies earlier than CD45(−)/GlyA(−)/CD133(+)/ALDH(low) VSELs, which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility, real-time PCR analysis confirmed that, while freshly isolated CD45(−)/GlyA(−)/CD133(+)/ALDH(high) VSELs express more hematopoietic transcripts (e.g., c-myb), CD45(−)/GlyA(−)/CD133(+)/ALDH(low) VSELs exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4). More importantly, hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice 4–6 weeks after transplantation. Overall, our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB.