8: Glycogen Synthase Kinase-3β Regulates Ex-vivo Expansion and Engraftment of Umbilical Cord Blood Hematopoietic Stem Cells through Activation of WNT Signalling

Abstract

We investigated whether the addition of two chromatin-modifying agents, 5-aza-2’-deoxycytidine (5azaD) and trichostatin A (TSA), to cord blood (CB) CD34( ) cells in culture results in expansion of the numbers of severe combined immunodeficient (SCID) repopulating cells (SRC). Human CB CD34( ) cells were cultured with cytokines in the presence or absence of 5azaD/TSA. After 9 days of culture, the fold expansion of CD34( ) and CD34( )CD90( ) cell numbers, colony-forming unit (CFU)-mix, cobblestone area-forming cell (CAFC), and SRC numbers were determined. A 12.5-fold expansion of CD34( )CD90( ) cells was observed in the 5azaD/TSA-treated cultures in comparison to the input cell numbers. Expansion of CD34( )CD90( ) cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold increase in CAFC. 5azaD/TSA treatment of the CB CD34( ) cells resulted in a 9.6-fold expansion of the absolute number of SRC following 9 days of culture as determined by limiting dilution analysis. Expansion of cells maintaining CD34( )CD90( ) phenotype was not due to the retention of a quiescent population of cells because all of the CD34( )CD90( ) cells in the culture had undergone cellular division. 5azaD/TSAtreated CD34( )CD90( ) cells, but not CD34( )CD90( ) cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice. CONCLUSION: Ex vivo expansion strategy using chromatin-modifying agents provides a potential avenue by which to expand the number of hematopoietic stem cells (HSC) with a single CB unit for use as an alternative source of HSC grafts for adult recipients.