Ex-Vivo Expansion of Marrow Repopulating Cells by Chromatin Modifying Agents Is Associated with Modulation of Expression of Genes Implicated in HSC Self-Renewal.

Human hematopoietic stem cells (HSCs) exposed to cytokine combinations in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program which determines the properties of HSC. In order to expand the number of HSC present in a single unit of cord blood (CB) ex vivo, self-renewal type of HSC division must occur. We hypothesize that in vitro culture conditions result in the silencing of genes crucial for HSC maintenance and that silencing of these genes can be circumvented by addition of chromatin modifying agents. We have attempted to reverse the silencing of the genes crucial for HSC self-renewal which apparently occurs during the ex vivo culture by treatment of CD34+ cells with the chromatin modifying agents, 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA). In our current studies, we have investigated the mechanism of expansion of SRC following treatment with chromatin modifying agents in the culture. We demonstrate that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to cytokines alone. CD34+CD90+ cells exposed to the chromatin modifying agents are capable of producing greater numbers of primitive multipotential progenitors and also form cobblestone areas. When CD34+CD90+ cells that had undergone extensive number of cell divisions (5–10) in vitro in the presence of cytokines alone were re-isolated by FACS and transplanted into immunodeficient mice, donor cell chimerism was not detectable (0 of 5 mice). By contrast, 5azaD/TSA treated cells that had undergone similar numbers of cell divisions retain their marrow repopulating potential (3 of 6 mice). To test whether chromatin modifying agents treated cells following culture possess long-term in vivo repopulation potential, we have performed secondary NOD/SCID assay. Five of six secondary NOD/SCID mice receiving bone marrow from primary mice engrafted with cells treated with 5azaD/TSA resulted in human cell engraftment, indicating that these cells are capable of secondary reconstitution. To understand the molecular mechanism responsible for the expansion of HSC observed following 5azaD/TSA treatment, we examined transcription levels of several genes and their products (i.e., HOXB4, Bmi-1 and P21) implicated in self-renewal of HSC using real-time quantitative PCR and Western blot. The expression of these genes and their products were up-regulated in CB cells treated with 5azaD/TSA. We have also compared the efficacy of an additional HDAC inhibitor valproic acid (VPA) in order to determine its ability to expand HSC ex vivo. VPA was capable of dramatic expansion of CD34+CD90+ cells as well as progenitor cells but was unable to expand SRC. However, unlike the culture exposed to cytokines alone VPA treatment resulted in maintenance of SRC numbers. Currently, we are investigating key candidate genes accountable for the expansion of SRC using a global microarray approach analyzing cells exposed to various chromatin modifying agents in conjunction with their in vivo functional potential. In summary, our data suggest that the loss of SRC can be circumvented by the use of chromatin modifying agents in the culture which results in a slower rate of cell division and is associated with higher expression of a group of HSC regulatory genes.