An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).