In vitro And In vivo Evidences for the long-term multilineage (Myeloid, b, nk & t) Reconstitution capacity of Ex vivo Expanded human CD34+ cord blood cells

We have identified clinically relevant culture conditions that support the expansion of HSC. After 14 days of stroma-free, serum-free cultures of CB CD34+ cells stimulated with SCF, FLT3-1, MGDF and G-CSF, we report expansion up to 1600-fold for total cells, 300-fold for CFC, 100-fold for CD34+ cells, 45-fold for LTC-IC and 21-fold for E-LTC-IC, with the presence of lymphoid B, NK and T progenitors as determined by cultures over MS5 cell line or embryonic thymic lobes from NOD/SCID mice (FTOC). The telomere length in CD34+ expanded populations was maintained, with a 30-fold increase of the telomerase activity compared to the initial CD34+ cells. We report that expanded cells are able to regenerate myeloid and lymphoid (B, NK and T) progeny five months after injection in sublethally irradiated NOD/SCID mice. We showed the myeloid engraftment in the marrow of the NOD/SCID mice by the presence of precursor cells, the capacity of CD45+ cells to give rise to CFC and the capacity of CD34+/CD38− cells to generate LTC-ICs, with conservation of their clonogeneic capacity. The capacity of sorted CD34+/CD19− engrafted cells to secondary give rise to CD19+ and CD56+/CD3− cells demonstrates the presence of primitive B and NK progenitor cells after long-term engraftment. We also report the capacity of T-cell differentiation from human engrafted CD34+/CD19− using FTOC. In conclusion, these experiments bring evidence that NOD/SCID mice were engrafted with an immature population able to maintain complete and sustained engraftment.