A Novel Population of Oct-4⁺ SSEA-1⁺ CXCR4⁺ CD34⁺ CD133⁺ Lin⁻ CD45⁻ Very Small Embryonic-Like (VSEL) Stem Cells Identified in Human Cord Blood.

Mononuclear cells (MNC) isolated from bone marrow (BM) or cord blood (CB) contributes to organ/tissue regeneration, however, the identity of the specific cell type(s) involved remains unknown. Recently we identified in murine BM a homogenous population of rare (~0.01% of BM MNC) Sca-1⁺ lin⁻ CD45⁻ cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1, highly express Rif-1 telomerase protein and display several features typical for primary embryonic stem cells such as

• a small size (~2–4 um in diameter),

• a large nuclei surrounded by a narrow rim of cytoplasm, and

• open-type chromatin (euchromatin) that is typical for embryonic stem cells (

  Leukemia 2006;20,857–869
  ).

These cells were named very small embryonic-like (VSELs) stem cells. We will present a new two step isolation procedure to purify a similar population of cells from human CB, which is based on isolation of CB mononuclear cells (CB MNC) by hypotonic lysis and multiparameter FACS sorting. Accordingly, we perform

• hypotonic lysis of CB to remove erythrocytes and to enrich for CB MNC combined with

• multiparameter sorting for CXCR4⁺AC133⁺CD34⁺lin⁻CD45⁻ CB MNC.

CB-derived VSELs (CB-VSELs) isolated this way similarly as those isolated from adult murine BM are very small (3–5 um), possess large nuclei containing unorganized euchromatin, express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. In vitro cultures CB-VSELs are able to grow neurospheres that gave rise to neuronal lineages (beta-III tubulin⁺, nestin⁺, O4⁺, MBP⁺, GFAP⁺) and cardiomyocytes (beta-myosin heavy chain⁺, alpha-sarcomeric actin. Based on this we conclude that CB contains VSELs and that the majority of these CB VSELs are lost during routine procedures employed currently for banking of CB MNC. Thus based on our observations, new more efficient methods of CB banking are needed that will enrich/preserve these cells in CB units during preparation before storage. Furthermore, we conclude that CB tissue/organ regenerating potential may be much higher than initially postulated if the proper fraction of CB MNC is employed and we are currently testing this hypothesis in animal models.