Chromatin-modifying Agents Research Articles

Expansion of hematopoietic stem/progenitor cells

Hematopoietic stem/progenitor cells (HSPCs) transplantation is hampered by the low number of stem cells per sample. To tackle this obstacle, several protocols for expansion of HSPCs in vitro are currently in development, such as the use of cytokine cocktails, coculture with mesenchymal stem cells as feeder cells, and cell culture in bioreactors. With the progress in the understanding of the molecular and cellular mechanisms regulating HSPCs maintenance and expansion, more recent approaches have involved transcription regulation, cell cycle regulation, telomerase regulation, and chromatin-modifying agents. The potential clinical application and safety issues relevant to the expanded HSPCs are also discussed in this review.

Pluripotency Associated Genes Are Reactivated by Chromatin-Modifying Agents in Neurosphere Cells

Chromatin architecture in stem cells determines the pattern of gene expression and thereby cell identity and fate. The chromatin-modifying agents trichostatin A (TSA) and 5-Aza-2'-deoxycytidine (AzaC) affect histone acetylation and DNA methylation, respectively, and thereby influence chromatin structure and gene expression. In our previous work, we demonstrated that TSA/AzaC treatment of neurosphere cells induces hematopoietic activity in vivo that is long-term, multilineage, and transplantable. Here, we have analyzed the TSA/AzaC-induced changes in gene expression by global gene expression profiling. TSA/AzaC caused both up- and downregulation of genes, without increasing the total number of expressed genes. Chromosome analysis showed no hot spot of TSA/AzaC impact on a particular chromosome or chromosomal region. Hierarchical cluster analysis revealed common gene expression patterns among neurosphere cells treated with TSA/AzaC, embryonic stem (ES) cells, and hematopoietic stem cells. Furthermore, our analysis identified several stem cell genes and pluripotency-associated genes that are induced by TSA/AzaC in neurosphere cells, including Cd34, Cd133, Oct4, Nanog, Klf4, Bex1, and the Dppa family members Dppa2, 3, 4, and 5. Sox2 and c-Myc are constitutively expressed in neurosphere cells. We propose a model in which TSA/AzaC, by removal of epigenetic inhibition, induces the reactivation of several stem cell and pluripotency-associated genes, and their coordinate expression enlarges the differentiation potential of somatic precursor cells.

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

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.

Effects of Chromatin-Modifying Agents on CD34+ Cells from Patients with Idiopathic Myelofibrosis

Idiopathic myelofibrosis (IM) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes that silence critical genes that control cell proliferation, differentiation, and apoptosis. We have explored the effects of the sequential treatment with the DNA methyltransferase inhibitor, decitabine [5-aza-2'-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), on the behavior of IM CD34(+) cells. Unlike normal CD34(+) cells where 5azaD/TSA treatment leads to the expansion of CD34(+) cells and marrow-repopulating cells, treatment of IM CD34(+) cells results in a reduction of the number of total cells, CD34(+) cells, and assayable hematopoietic progenitor cells (HPC). In IM, HPCs are either heterozygous or homozygous for the JAK2V617F mutation or possess wild-type JAK2 in varying proportions. Exposure of IM CD34(+) cells to 5azaD/TSA resulted in a reduction of the proportion of JAK2V617F-positive HPCs in 83% of the patients studied and the reduction in the proportion of homozygous HPCs in 50% of the patients. 5azaD/TSA treatment led to a dramatic reduction in the number of HPCs that contained chromosomal abnormalities in two JAK2V617F-negative IM patients. IM is characterized by constitutive mobilization of HPCs, which has been partly attributed to decreased expression of the chemokine receptor CXCR4. Treatment of IM CD34(+) cells with 5azaD/TSA resulted in the up-regulation of CXCR4 expression by CD34(+) cells and restoration of their migration in response to SDF-1. These data provide a rationale for sequential therapy with chromatin-modifying agents for patients with IM.

The therapeutic uses of chromatin-modifying agents

In contrast to genetic aberrations, epigenetic aberrations can be reversed by the use of histone acetyltransferase (HAT), histone deacetylase (HDAC), SIRT, or histone methyltransferase (HMT) inhibitors. A well-known HDACi, suberoylanilide hydroxamic acid, has been recently approved for the treatment of cutaneous T cell lymphoma, and a number of HDACi are in clinical trials as anticancer drugs. In addition, HDACi could be useful in antimalarial and antifungal therapies and can reactivate the HIV-1 expression in latent cellular reservoirs, thus suggesting the use in a combination therapy with highly active antiretroviral therapy. HDACi have also been reported to have anti-inflammatory effects through inhibition of cytokines and key transcription factors, and to ameliorate the phenotypes in animal models of neurological disorders. HDACi can also reactivate the γ-globin gene for the treatment of β-thalassaemia, and recently were shown to relieve morphological and functional effects of muscular dystrophia. Dysfunction of HAT enzymes is also often associated with several diseases, including cancer; thus, the HATi can represent new chemical entities for the development of new drugs. Only a few HMTi have been described to date, but these small molecules could be a useful scaffold to discovering new highly active and enzyme-selective compounds to develop as therapeutics.

Chromatin-modifying agents permit human hematopoietic stem cells to undergo multiple cell divisions while retaining their repopulating potential

AbstractHuman hematopoietic stem cells (HSCs) exposed to cytokines in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program that determines the properties of an HSC. We have attempted to reverse the silencing of this HSC genetic program by the sequential treatment of human cord blood CD34+ cells with the chromatin-modifying agents, 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA). We determined 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 only cytokines. When CD34+CD90+ cells that have undergone extensive number of cell divisions (5-10) in the presence of cytokines alone were transplanted into immunodeficient mice, donor cell chimerism was not detectable. By contrast, 5azaD/TSA-treated cells that have undergone similar numbers of cell divisions retained their marrow repopulating potential. The expression of several genes and their products previously implicated in HSC self-renewal were up-regulated in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. These data indicate that HSC treated with chromatin-modifying agents are capable of undergoing repeated cell divisions in vitro while retaining their marrow-repopulating potential.

Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents

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/TSA-treated CD34(+)CD90(+) cells, but not CD34(+)CD90(-) cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice. 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.

The vast majority of bone-marrow-derived cells integrated into mdx muscle fibers are silent despite long-term engraftment

Bone-marrow-derived cells can contribute nuclei to skeletal muscle fibers. However, serial sectioning of muscle in mdx mice implanted with GFP-labeled bone marrow reveals that only 20% of the donor nuclei chronically incorporated in muscle fibers show dystrophin (or GFP) expression, which is still higher than the expected frequency of "revertant" fibers, but there is no overall increase above controls over time. Obviously, the vast majority of incorporated nuclei either never or only temporarily turn on myogenic genes; also, incorporated nuclei eventually loose the activation of the beta-actin::GFP transgene. Consequently, we attempted to enhance the expression of dystrophin. In vivo application of the chromatin-modifying agents 5-azadeoxycytidine and phenylbutyrate as well as local damage by cardiotoxin injections caused a small increase in dystrophin-positive fibers without abolishing the appearance of "silent" nuclei. The results thus confirm that endogenous repair processes and epigenetic modifications on a small-scale lead to dystrophin expression from donor nuclei. Both effects, however, remain below functionally significant levels.

In vivo haematopoietic activity is induced in neurosphere cells by chromatin-modifying agents

Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (neurospheres) can be modified by destabilizing their epigenotype, neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2'-deoxycytidine (AzaC) (TSA/AzaC neurosphere cells) yielded long-term, multilineage and transplantable neurosphere-derived haematopoietic repopulation. Untreated neurosphere cells exhibited no haematopoietic repopulation activity. The neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of neurosphere cells followed by transplantation enables the generation of neurosphere-derived haematopoietic cells.