Small Molecule Based Ex Vivo Expansion of CD34⁺CD90⁺CD49f⁺ Hematopoietic Stem & Progenitor Cells from Non-Enriched Umbilical Cord Blood Mononucleated Cells

Umbilical cord blood (UCB) transplantation in adults have slow hematopoietic recovery compared to bone marrow (BM) or peripheral blood grafts mainly due to low number of total nucleated cells (TNC) and hematopoietic stem & progenitor cells (HSPC). Current investigational clinical strategies focus on increasing HSPC dosage by expanding CD34 enriched grafts that have resulted in early neutrophil recovery followed by long term hematopoietic reconstitution.

In an effort to expand HSPC, specifically those expressing primitive phenotype (CD34⁺CD90⁺CD49f⁺) from non-enriched UCB, we developed a proprietary library of 50 small molecules using structure-activity-relationship studies. Freshly-thawed UCB-mononucleated cells (MNC), were cultured in serum or animal component free expansion medium supplemented with optimal concentration of respective compound. The effects of the expansion protocol were measured based on phenotypic and functional assays. Cell cultures with basal cytokines served as control.

Screening of the small molecule library showed negligible acute adverse effects on CD45⁺ leukocyte population and its viability within 72 hours compared to cytokine control. In long term expansion cultures lasting up to 11 days, one specific structural analog, C7, resulted in 1195.8±71.7-folds increase of absolute CD45⁺CD34⁺CD38^-CD45RA^- progenitors which was at least 9.2-folds higher than control cultures (P<0.01; n=4). Colony forming unit assay showed significant increase of granulocyte-macrophage colonies from C7 treated cells compared to cytokine control (P<0.01; n=6) although TNC expansion was comparable between the culture conditions. It was necessary for the cytokine cocktail to comprise of at least stem cell factor, thrombopoietin and Fms-related tyrosine kinase 3 ligand for mediating HSPC expansion in presence of C7, although further addition of insulin like growth factor binding protein 2 marginally boosted expansion (P<0.001; n=3). Majority of HSPC expansion occurred between the seventh and tenth day of the culture period. In MNC initiated cultures, addition of C7 boosted primitive HSPC (CD45⁺CD34⁺CD38^-CD45RA^-CD90⁺CD49f⁺) by 633.3±8.5-folds over 10 days which was at least 7.4-folds higher than control cultures (P<0.001; n=3). In cultures initiated with purified CD34⁺CD38^- cells, there was at least 15.9-folds higher expansion of HSPC defined by CD45⁺CD34⁺CD38^-CD45RA^-CD90⁺ in presence of C7 compared to cytokine cultures (P<0.05; n=3). Expansion of HSPC by C7 was at least 2-folds higher in comparison to mesenchymal stromal co-culture system which is the only known clinical protocol that allows for UCB expansion without prior CD34/CD133 selection (P<0.001; n=6). Transplantation of C7 expanded UCB grafts (n=11) at equivalent dosage of 2.5x10⁷ cells/kg to sub-lethally irradiated NOD SCID Gamma (NSG) mice resulted in 3.21- and 2.09-folds higher engraftment of human CD45⁺ cells in the peripheral blood by day 21 compared to non-expanded (P=0.0030; n=6) and cytokine expanded grafts (P=0.0005; n=12) respectively. The frequency of SCID repopulating cells contributing to early peripheral blood engraftment was 2.48-folds higher in C7 expanded graft compared to unmanipulated graft. C7 expanded graft sustained human cell engraftment over 19 weeks which were primarily myeloid cells (CD33⁺/CD15⁺) as opposed to non-expanded graft which consisted of CD3⁺ T cells. Analysis of NSG BM at week 19 post-transplantation, showed significantly better (P<0.0001) chimerism of human CD45 cells in female (n=15) recipient compared to male (n=14) irrespective of graft type (transplantation dosage: 2.5x10⁷ cells/kg). C7 expanded graft gave comparable level of human CD45 and CD34 progenitor cell engraftment as that of the non-expanded grafts. Multi-lineage reconstitution of NSG BM comprising of both myeloid and lymphoid human cells could be achieved with the C7 expanded graft. At higher transplantation dosage of 5.0x10⁷ cells/kg, the expanded grafts had a higher survival rate of 75% compared to 50% for non-expanded graft mainly due to lower incidence of graft-versus-host-disease.

In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment or stromal cell co-culture. The expanded UCB consists of phenotypically defined primitive HSPC that maintains in vitro and in vivo functionality.