Histone Deacetylase Inhibitor And Methylase Inhibitor Repress Synergistically Differentiation Of HSC

Epigenetic events play important roles in many biologic functions,including transcriptional regulation .This critical biologic process has beenlinked to events including aging, neoplasia, and a growing number ofdevelopmental disorders in man. Histone acetylation has also been shown tohave a profound effect on the normal transition from a fetal to an adulthematopoietic cellular differentiation program during ontogeny. There isevidence that early HSCs express a promiscuous set of transcription factorsand an open chromatin structure required to maintain theirmultipotentiality,which is progressively quenched as these cells progressdown a particular pathway of differentiation.We hypothesized that theseprocesses associated with differentiation may be mediated by silencing ofgenome which sustain HSC characters and silencing of genome associatedwith DNA methylation and histone deacetylation.To date, attempts to create an in vitro environment that favors HSCself-replication rather than commitment and differentiation has resulted inlimited success. All of the hematopoietic growth factors promote HSCself-replication as well as commitment and differentiation .So,scientistsattempt to expand largely the amount of HSC ,at the least maintain theirmultipotentiality during in vitro long term of culture. DNA methylaseinhibitors and histone deacetylase inhibitors in combination can reverseepigenetic event and be capable of synergistically reactivatingdevelopmentally silenced genes.We have developed the investigation to assessthe role of 5aza -2-deoxycytidine (5azaD)-a DNA methylase inhibitorsand trichostatinA(TSA)-a histone deacetylase inhibitors in inhibit HSC/HPCdifferentiation.Fresh mononuclear cells, isolated from bone marrow aspirated fromhealthy human volunteers after informed consent. Next, we investigated thephenotype of marrow CD34+ cells prior to and after treatment with cytokinesin vitro for a period of 9 days. A 2-step culture system was set up. The cellswere initially cultured in cytokines that promote HSC division to ensure theircycling and thus allow theoretically for the incorporation of 5azaD which wasadded after 16 hours of this culture. At 48 hours,TSA was added and thecytokines were changed to a combination which promotes terminaldifferentiation. This was done in order to detect a change in phenotype andfunction in the presence of an extreme external milieu favoring differentiationrather than HSC proliferation and self-renewal. During culture in cytokinesalone, a progressive decline in the percentage and absolute number ofCD34+cells was observed as well as the percentage of CD34+ cells expressinga primitive HSC phenotype. In particular there was a significant decrease inthe absolute number of CD34+CD90+ cells in these cultures. By contrast, theCD34+ cells exposed to the same cytokines and sequential 5azaD and TSAretained the primitive HSC phenotype resulting in an expansion ofCD34+CD90+cell numbers. Cells exposed to cytokines and to either 5azaD orTSA alone had a equal CD34+CD90+ cell numbers to primitive CD34+ cell.To show that this effect could not be attributed merely to cytotoxicity, 5-FUwas added to cultures instead of the 5azaD and TSA. 5-FU treatment haspreviously been utilized to select for primitive human progenitor cells invitro.30 The CD34+CD90+ cell number in the 5-FU–treated culturesdecreased significantly after 9 days so that by day 9 no CD34+CD90+cellswere observed. Marrow CD34+cells following 9 days of culture exposed to5azaD and TSA resulted in a -fold expansion of the CD34+CD90+ cellpopulation. To ensure that a discordance between phenotype and function of thedrug-treated progenitor cells did not exist, we evaluated the functionalproperties of these cells using CFC assays, which are used to quantitate thenumber of differentiated and primitive HPCs, respectively. I CD34+ cellstreated with cytokines alone for 9 days contained dramatically reducednumbers of assayable progenitors in comparison to primary cells. By contrast,cells from cultures treated with cytokines and sequential 5azaD and TSA haddramatically greater numbers of HPCs than cultures receiving cytokines alone.Significantly, the drug-treated cultures contained greater numbers ofmultilineage progenitor cells than even theprimary cells. By contrast, cellsassayed from the cultures treated with cytokines and 5-FU contained noassayable HPCs. The plating efficiency of CD34_ cells assayed immediatelyafter selection (primary CD34+ cells) was %, whereas that of theunfractionated cells, derived from the primary CD34+ cells,after 9 days ofculture in cytokines alone was %. The plating efficiency of the cultured cells,derived from the primary CD34+ cells, exposed to cytokines in combinationwith 5azaD and TSA was %, which was comparable to that of the primaryCD34+ –selected cell population. Our studies show that sequential addition of 5azaD and TSA in thepresence of cytokines, which favor differentiation, results rather in theretention of stem cell phenotype, number, and function.With regard tohematopoietic stem cell transplantation which use expanded hematopoietic...