Age-Related Transcription Levels of KU70 and BIK in CD34+ Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4205-4205
Author(s):  
Wolf C. Prall ◽  
Akos Czibere ◽  
Franck Grall ◽  
Luiz F. Zerbini ◽  
Markus Jaeger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Expression Patterns ◽  
Cell Aging ◽  
Elderly Persons ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Age Related ◽  
Dna Repair Gene ◽  
Stem And Progenitor Cells

Abstract There is a higher incidence of hematological clonal stem cell disorders in elderly persons. Age-related alterations of hematopoietic stem and progenitor cells (HSC) may represent one factor underlying this observation. However, the molecular changes related to stem cell aging are largely unknown. Therefore, we scrutinized gene expression patterns of HSC from umbilical cord blood (CB) as well as bone marrow from young (BM-Y, mean age 32,8, SD 12,4) and old (BM-O, mean age 88,8, SD 4,4) healthy donors. CD34+ HSC were isolated via immuno-magnetic separation and CD34+ purity was evaluated by FACS analysis. Thereafter we performed cDNA array analyses on a first set of samples (n=18). We found that the BCL2-interacting killer gene (BIK) and the gene encoding KU Antigen 70kD (KU70) show age-related mRNA expression levels. BIK is a proapoptotic gene and its expression was positively correlated with donor’s age, i.e. lowest in CB, 1.8-fold higher in BM-Y and 4.2-fold higher in BM-O. KU70 is a DNA repair gene and part of the DNA dependent protein kinase (DNA-PK). Its expression was negatively correlated with donor’s age showing highest expression levels in CB, 2.2-fold lower levels in BM-Y and 5.3-fold lower levels in BM-O. These findings were confirmed with a second set of samples (n=16) by means of quantitative RT-PCR. Elucidation of age-dependent molecular alteration in healthy HSC might facilitate a better understanding of hematological malignancies in elderly persons.

scholarly journals Age-Related Changes in Methylome and Transcriptome of Hematopotietic Stem Cells Underlie Natural Genetic Variations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2660-2660
Author(s):  
Ying Liang
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Aging Process ◽  
Expression Patterns ◽  
Genetic Variations ◽  
Cell Aging ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Age Related Changes

The aging of hematopoietic stem cells (HSCs) contributes to the aging of blood system and perhaps the whole organism. The aging process is coordinately determined by both genetic and epigenetic factors, and demonstrates inter-individual variations. We used high-throughput sequencing methods to study the age-dependent changes of genome-wide DNA methylation and gene expression patterns in HSCs of C57BL/6 (B6) and DBA/2 mouse strains, which have shown natural variations in HSC aging process. We observed global age-associated decrease of DNA methylation in both strains, but D2 HSCs have a stronger loss of epigenetic control than B6 stem cells during aging. Majority age-related changes of DNA methylation occur from young to mid-aged stages. We identified stable strain-specific differentially methylated regions (DMRs) that overlap with cis-eQTLs. Moreover, transcription factor binding site motifs are more likely to be disrupted in the DMRs, suggesting the potential impact of genetic variations on epigenetic regulation of HSC aging. We further demonstrated that strain-specific DMRs have more profound effects on the aging of B6 HSCs than D2 stem cells. Transposons are differentially regulated by the DMRs in the two strains, in which D2 HSCs are prone to transposon insertion. This study comprehensively investigated the effects of natural genetic and epigenetic variations on HSC aging. Loss of DNA methylation is an epigenetic signature of stem cell aging, and DNA methylation variations correlates with genetic variations, both contributing to inter-individual differences in stem cell and perhaps organismal aging. Disclosures No relevant conflicts of interest to declare.

Identification of a Novel Candidate Regulator of HSC Aging.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1345-1345
Author(s):  
Erin J. Oakley ◽  
Gary Van Zant
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Secretory Pathway ◽  
Cell Function ◽  
Mammalian Species ◽  
Cell Aging ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. We have previously mapped a quantitative trait locus (QTL) to murine Chr 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In C57BL/6 (B6) mice the HSC population steadily increases with age, whereas in DBA/2 mice, this population declines. A QTL regulating the natural variation in lifespan between the two strains was mapped to the same location on mouse Chr 2, thus leading to the hypothesis that stem cell function affects longevity. B6 alleles, associated with expansion of the stem cell pool, are also associated with a ~50% increase in lifespan. Using a congenic mouse model, in which D2 alleles in the QTL interval were introgressed onto a B6 background, genome wide gene expression analyses were performed using sorted lineage negative hematopoietic cells, which are enriched for primitive stem and progenitor cells. Three variables were examined using Affymetrix M430 arrays:the effect of strain--congenic versus background;the effect of age--2 months versus 22 months; andthe effects of 2 Gy of radiation because previous studies indicated that congenic animals were highly sensitive to the effects of mild radiation compared to B6 background animals. Extensive analysis of the expression arrays pointed to a single strong candidate, the gene encoding ribosome binding protein 1 (Rrbp1). Real-time PCR was used to validate the differential expression of Rrbp1 in lineage negative, Sca-1+, c-kit+ (LSK) cells, a population highly enriched for stem and progenitor cells. Further analysis revealed the presence eight non-synonymous, coding single nucleotide polymorphisms (SNPs), and at least one of them because of its location and nature may significantly alter protein structure and function. The Rrbp1 gene consists of 23 exons in mouse and is highly conserved among mammalian species including mouse, human, and canine. The Rrbp1 protein is present on the surface of the rough endoplasmic reticulum where it tethers ribosomes to the membrane, stabilizes mRNA transcripts, and mediates translocation of nascent proteins destined for the cell secretory pathway. It is well established that the interaction of HSCs with microenvironmental niches in the bone marrow is crucial for their maintenance and self-renewal, and that this interaction is mediated in part by the molecular repertoires displayed on the cell surfaces of both HSCs and niche stromal cells. Therefore, we hypothesize that age and strain specific variation in Rrbp1, through its role in the secretory pathway, affects the molecular repertoire at the cell surface of the HSC, thus altering the way stem cells interact with their niches. This altered microenvironmental interaction could have profound effects on fundamental properties relevant to stem cell aging such as pluripotency, self-renewal, and senescence.

Identification Of Leukemia Suppressive Genes By Inducible Overexpression Of The DNA Methyltransferase DNMT3B During Leukemogenesis In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2493-2493
Author(s):  
Isabell Schulze ◽  
Petra Tschanter ◽  
Christian Rohde ◽  
Annika Krause ◽  
Heinz Linhart ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Expression Patterns ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Serial Transplantation

Abstract DNA methyltransferases (DNMT) play an important role in regulation of DNA methylation and mutations of DNMT3A are frequently found in AML. In previous studies using a tetracycline-inducible DNMT3B mouse model, we could show that overexpression of DNMT3B affected leukemia initiation and maintenance upon retroviral transduction and serial transplantation of hematopoietic stem and progenitor cells with MSCV-MLL-AF9-GFP and MSCV-cmyc-bcl2-mcherry oncogenic vectors, respectively. Sublethally irradiated recipient mice of DNMT3B overexpressing MLL-AF9 and cmyc/bcl2 leukemic cells developed leukemia with a prolonged latency when compared to recipients of wildtype cells. We performed serial transplantation assays of MLL-AF9 leukemic stem cells, which were sorted for high expression of ckit. The life-prolonging effect of DNMT3B expression was stem cell-specific, as the potential to initiate leukemia was maintained upon serial retransplantation and recipients of DNMT3B overexpressing leukemic stem cells also died significantly later in secondary (p<0.001) and tertiary transplantations (p<0.001). Analysis of global DNA methylation levels in MLL-AF9 ckit+ leukemic stem cells and cmyc/bcl2 leukemic cells via Reduced Representation Bisulfite Sequencing (RRBS) revealed a strong hypermethylation in DNMT3B overexpressing cells, independent of the oncogene used for leukemia induction. Differentially methylated CpG sites were defined as CpGs with at least 20% methylation difference between wildtype and DNMT3B overexpressing samples. Hypermethylation in MLL-AF9 leukemic cells directly correlated with observed hypermethylation in cmyc/bcl2 leukemic cells and inversely correlated with hypomethylation in cmyc/bcl2 cells, indicating that in both leukemias, the same sites are prone to DNMT3B induced DNA methylation. To investigate, if these changes in DNA methylation resulted in different gene expression patterns, we performed microarray analysis of the same MLL-AF9 leukemic wildtype and DNMT3B expressing samples which were also used for DNA methylation analysis. In microarray analyses, we could identify several genes differentially expressed in DNMT3B overexpressing cells when compared to wildtype samples. Interestingly, changes in expression levels could not be attributed to differential DNA methylation in promoter regions. Instead, hypermethylation in exons and gene bodies resulted in downregulation of the respective genes, whereas genes with hypomethylated exons and gene bodies showed higher expression levels. Genes downregulated in DNMT3B overexpressing cells, were mainly cancer-associated genes, which are known to have functions in cellular growth and proliferation, as well as in the hematopoietic system development and maintenance. Gene Set Enrichment Analysis (GSEA) of wildtype cells revealed a strong enrichment of genes upregulated in different stages of hematopoietic stem and progenitor cells as well as in leukemic stem cells, whereas DNMT3B overexpressing samples were enriched in genes which have been shown to be downregulated in hematopoietic and leukemic stem cells and upregulated in mature hematopoietic cells. This strengthens our hypothesis that DNMT3B induced DNA methylation mainly influences the phenotype and function of hematopoietic stem cells and thereby, exerts its inhibitory function on leukemia initiation and maintenance. Taken together, these findings demonstrate that DNMT3B exerts its anti-leukemic effect mainly via induction of aberrant DNA methylation in hematopoietic and leukemic stem cells, thereby changing expression patterns of genes known to be important for stem cell function. The identification of differentially expressed DNMT3B target genes could help to find promising targets for new therapeutic strategies in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (4) ◽  
pp. 1479-1487 ◽  
Author(s):  
Ying Liang ◽  
Gary Van Zant ◽  
Stephen J. Szilvassy
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Negative Effects ◽  
Hematopoietic Stem ◽  
Stem Cell Homing ◽  
Age Related ◽  
Cell Clones ◽  
Stem And Progenitor Cells ◽  
Rate Of Recovery ◽  
Effects Of Aging

AbstractTo test the hypothesis that aging has negative effects on stem-cell homing and engraftment, young or old C57BL/6 bone marrow (BM) cells were injected, using a limiting-dilution, competitive transplantation method, into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age, the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly, the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However, the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage, indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes, erythrocytes, and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively, these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients. (Blood. 2005; 106:1479-1487)

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

The GEF Lsc Regulates Hematopoietic Stem Cell Motility, Mobilization and Recruitment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1346-1346
Author(s):  
Isabelle Petit ◽  
Prashant Kaul ◽  
Daniel J. Lerner ◽  
Shahin Rafii
Keyword(s):  
Stem Cell ◽  
Cell Migration ◽  
Tumor Growth ◽  
Cell Motility ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Tumor Angiogenesis ◽  
Progenitor Cell ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Lsc is a Rho GTPase guanine nucleotide exchange factor (RhoGEF) that physically and functionally links G-protein coupled receptors (GPCR) to the monomeric GTPase RhoA in mature hematopoietic and other cells. Lsc−/− (LscKO) mice have a peripheral leukocytosis, abnormal neutrophil and B cell motility, and immune response deficiencies. Although Lsc is required for neutrophil homeostasis, its role in hematopoietic stem and progenitor cells is unknown. In this study, we have used LscKO mice to determine if Lsc is required for normal stem cell motility and mobilization. Initially, we used immunofluorescence labeling to demonstrate that hematopoietic stem and progenitor cells express Lsc. This suggested that Lsc may be required for normal hematopoietic stem and progenitor cell migration. Stromal-cell derived factor-1 (SDF-1) is a potent chemokine for hematopoietic stem cells and activates the CXCR4 GPCR. It has been reported that Lsc is not required for SDF-1-stimulated migration of mature murine T and B cells. However, using a bare-filter transwell assay, we found that while LscKO Sca-1+ cells and Sca-1+Lin- cells have normal spontaneous migration, they have significantly increased SDF-1-stimulated migration compared to their wild-type (WT) counterparts, 1.4 and 2.3 fold, respectively. We then demonstrated that adhesion of LscKO Sca-1+ cells to bone marrow (BM) stromal MS-5 cells was normal, indicating that impaired adhesion was not responsible for the abnormal SDF-1-stimulated migration. Using colony assay, we demonstrated that LscKO mice have a normal number of circulating peripheral stem and progenitor cells. Strikingly, after 5 days of G-CSF administration, LscKO mice have 1.6 fold and 2.3 fold the number of peripheral mature WBC and stem and progenitor cells (colony forming units), respectively, compared to WT mice. Recruitment of BM CXCR4+ pro-angiogenic stem and progenitor cells has been linked to enhanced tumor angiogenesis. Because LscKO BM cells had abnormal SDF-1-stimulated migration and mobilization, we hypothesized that Lsc might regulate tumor angiogenesis as well. To this end, we assessed tumor growth in LscKO mice by injecting congenic Lewis lung carcinoma cells subcutaneously into LscKO mice and WT controls. Preliminary experiments revealed that tumors were 3.3 times larger in the LscKO mice as compared to WT mice. Quantification of the tumor vessels with anti-CD31 staining demonstrated that the tumors in LscKO mice were 1.4 fold more vascularized than controls. In summary, our results demonstrate that the Rho GEF Lsc is essential for normal hematopoietic stem cell migration and mobilization. In addition, we propose that absence of Lsc facilitates tumor growth by promoting BM stem and progenitor cell recruitment to the neo-angiogenic vessels, possibly augmenting tumor vascularization.

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1174-1174
Author(s):  
Taito Nishino ◽  
Atsushi Iwama
Keyword(s):  
Stem Cell ◽  
Natural Product ◽  
Progenitor Cells ◽  
Small Molecule ◽  
Inhibitory Activity ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cells Cultured

Abstract Abstract 1174 Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) have recently been explored to optimize autologous and allogeneic HSPC transplantation and shown to be effective in the field of stem cell biology. However, to our knowledge, identification of culture conditions that allow HSPCs expansion and long-term hematopoietic reconstitution have remained incomplete, and clinical methods to expand human HSPCs have yet to be realized. In this study, we assumed that some small molecule compounds may preferentially activate signals that are required for optimal HSPC expansion and facilitate self-renewal of hematopoietic stem cells (HSCs). Thus, we evaluated the effects of several biologically active compounds on the ex vivo expansion of CD34+ hematopoietic stem and progenitor cells from human cord blood (hCB) and identified Garcinol, a plant-derived natural product as a novel modulator of HSPC proliferation. We cultured hCB CD34+ cells in serum-free medium supplemented with human thrombopoietin, human stem cell factor and Garcinol for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with Garcinol was similar to those cultured without Garcinol, the cultures with Garcinol showed >2-fold increase in the number of CD34+CD38- hematopoietic stem and progenitor cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to control cultures. Correspondingly, SCID-repopulating cells (SRCs) were increased 2-fold during a 7-day culture with Garcinol compared to cultures without Garcinol. These findings suggest that Garcinol efficiently promotes the net expansion of HPSCs. To investigate the structure-activity relationship of Garcinol, we synthesized the chemical derivatives of Garcinol and evaluated the effect of Garcinol and its derivatives, Isogarcinol and O, O'-dimethylisogarcinol, on the proliferation of CD34+CD38- cells. Although Isogarcinol exhibited almost the same activity as Garcinol, O, O'-dimethyl isogarcinol was scarcely effective in the CD34+CD38- cell proliferation. Correspondingly, O, O'-dimethylisogarcinol had no effect on numbers of HPP-CFCs. These results indicate that dihydroxybenzoyl moiety is crucial for the positive effect of Gacinol on HSPCs.Garcinol has been reported to be a potent inhibitor of histone acetyltransferases (HAT). Thus, we estimated the HAT activity in cells treated with Garcinol and its derivatives. Garcinol and Isogarcinol inhibited HAT activity while O, O'-dimethylisogarcinol showed much less HAT inhibitory activity as compared to Garcinol and Isogarcinol, which suggested that HAT inhibitory activity of Garcinol is correlate with the expansion of HPSCs. We are now investigating gene expression profiling in cells cultured with Garcinol using DNA microarray analysis and Q-PCR. In conclusion, we have identified Garcinol, a plant-derived small-molecule compound, which exhibits inhibitory effect on HAT activity, as a novel stimulator of HSPC expansion. The results reported here indicate that Garcinol would be applied as a useful tool for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures: No relevant conflicts of interest to declare.

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