CD45⁻/ALDH^low/SSEA-4⁺/Oct-4⁺/CD133⁺/CXCR4⁺/Lin⁻ Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (

• smaller than erythrocytes;

• SSEA-4⁺/Oct-4⁺/CD133⁺/CXCR4⁺/Lin⁻/CD45⁻;

• responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin).

Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133⁺/CD45⁻/ALDH^high and CD133⁺/CD45⁻/ALDH^low sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45⁺ cells when replated into methylcellulose cultures. Furthermore, while CD133⁺/CD45⁻/ALDH^high VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133⁺/CD45⁻/ALDH^low VSELs was delayed. The data indicate that both populations of CD45⁻ cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133⁺/CD45⁻/ALDH^low cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133⁺/CD45⁻/ALDH^high VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133⁺/CD45⁻/ALDH^low exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133⁺/CD45⁻/ALDH^low are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45⁻ ALDH^low VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity.

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