Genome-Wide Epigenetic Analysis of Cancer Stem Cells (CSCs) In Acute Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3640-3640 ◽  
Author(s):  
Jumpei Yamazaki ◽  
Marcos R Estecio ◽  
Jaroslav Jelinek ◽  
David Graber ◽  
Yue Lu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Population ◽  
Progenitor Cell ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Significant Difference

Abstract Abstract 3640 Background & Aims: The hypothesis that cancer is driven by Cancer Stem Cells (CSCs or Cancer-Initiating Cell) has recently attracted a great deal of attention. Epigenetic mechanism such as DNA methylation and histone modification play an important role in cancer cells and also in normal stem cells. However, their role remains unclear in CSCs. We sought to determine if CSCs have distinct epigenetic patterns in acute myeloid leukemia (AML). Methods: Peripheral blood samples in AML patients were separated to obtain stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) by magnetic cell sorting (MACS®, Myltenyi biotec). To study DNA methylation in CSCs in AML, we performed genome wide screening using methylated CpG island microarray (MCAM), which detects 7202 promoter CpG islands, 1348 non-promoter CpG islands, and 632 non-CpG island promoter methylation. MCAM was performed on 4 AML patient samples Next, we evaluated the methylation status of 7 genes which showed apparent higher DNA methylation in stem cells or progenitor cells in MCAM analysis, using a quantitative bisulfite-pyrosequencing for each population of stem cell, progenitor cell, and mature cells (CD34-) from peripheral blood samples in 6 AML patients. For histone modification analysis, we used Chromation immuprecipitation followed by massively parallel sequencing (ChIP-Seq) for stem cell and progenitor cell populations for H3K4me3 which is known to be a marker for activated genes. Results: By MCAM, we found minimal differences between stem cells and progenitor cells present in 2 out of 4 AML patients. Those few genes (<1%) which were shown to have higher DNA methylation in stem or progenitor cells by MCAM analysis were likely false positives, as no significant difference was found when analyzed by quantitative bisulfite-pyrosequencing. DNA methylation status for stem cell-related gene (OCT4, SOX2, MYC, HOXB4, and KLF4) also showed no significant difference. By ChIP-seq analysis, we found differences in 2362 genes between stem cells and progenitor cells. In stem cells, H3K4me3 was enriched in genes (Bmi1, Notch1, Wnt1, and etc) which are known to be important for stem cell function, but they were not enriched in the progenitor cell population. In pathway analysis of the H3K4me3 data, Hypoxia-Inducible Factor signaling, NFkB signaling, and p53 signaling are found to be enriched specifically in the stem cell population whereas Cellular Growth and Cell Cycle, and DNA Damage Response signaling are found in the progenitor cell population. Conclusions: There is no significant difference in DNA methylation between stem cell, progenitor cell or mature cell populations in AML. DNA methylation of promoter CpG islands is unlikely to explain tumor hierarchy in AML. Rather, histone modifications seem to have a greater significance in this regard. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Contribution of Resident Stem Cells to Liver and Biliary Tree Regeneration in Human Diseases

2018 ◽  
Vol 19 (10) ◽  
pp. 2917 ◽  
Author(s):  
Diletta Overi ◽  
Guido Carpino ◽  
Vincenzo Cardinale ◽  
Antonio Franchitto ◽  
Samira Safarikia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Human Liver ◽  
Progenitor Cell ◽  
Biliary Tree ◽  
Tree Regeneration ◽  
Multipotent Stem Cells ◽  
Resident Stem Cells ◽  
Stem Cell Niches

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

scholarly journals DNA Methylation of Endoglin Pathway Genes in Pregnant Women With and Without Preeclampsia

2020 ◽  
Vol 13 ◽  
pp. 251686572095968
Author(s):  
Allison H Rietze ◽  
Yvette P Conley ◽  
Dianxu Ren ◽  
Cindy M Anderson ◽  
James M Roberts ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Control Participant ◽  
Sample Collection ◽  
Promoter Regions ◽  
Participant Group

Objective: We compared blood-based DNA methylation levels of endoglin ( ENG) and transforming growth factor beta receptor 2 ( TGFβR2) gene promoter regions between women with clinically-overt preeclampsia and women with uncomplicated, normotensive pregnancies. Methods: We used EpiTect Methyl II PCR Assays to evaluate DNA methylation of CpG islands located in promoter regions of ENG (CpG Island 114642) and TGFβR2 (CpG Island 110111). Preeclampsia was diagnosed based on blood pressure, protein, and uric acid criteria. N = 21 nulliparous preeclampsia case participants were 1:1 frequency matched to N = 21 nulliparous normotensive control participants on gestational age at sample collection (±2 weeks), smoking status, and labor status at sample collection. Methylation values were compared between case and control participant groups [( ENG subset: n = 20 (9 cases, 11 controls); TGFβR2 subset: n = 28 (15 cases, 13 controls)]. Results: The majority of the preeclampsia cases delivered at ⩾34 weeks’ gestation (83%). Average methylation levels for ENG ([M ± (SD)]; Case Participant Group = 6.54% ± 4.57 versus Control Participant group = 4.81% ± 5.08; P = .102) and TGFβR2 (Case Participant Group = 1.50% ± 1.37 vs Control Participant Group = 1.70% ± 1.40; P = .695) promoter CpG islands did not differ significantly between the participant groups. Removal of 2 extreme outliers in the ENG analytic subset revealed a trend between levels of ENG methylation and pregnancy outcome (Case Participant Group = 5.17% ± 2.16 vs Control Participant Group = 3.36% ± 1.73; P = .062). Conclusion: Additional epigenetic studies that include larger sample sizes, investigate preeclampsia subtypes, and capture methylation status of CpG island shores and shelves are needed to further inform us of the potential role that ENG and TGFβR2 DNA methylation plays in preeclampsia pathophysiology.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2569-2577 ◽  
Author(s):  
Huei-Mei Huang ◽  
Jian-Chiuan Li ◽  
Yueh-Chun Hsieh ◽  
Hsin-Fang Yang-Yen ◽  
Jeffrey Jong-Young Yen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Stem Cell Differentiation ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Progenitor Cell Line

Abstract In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5R is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2569-2577 ◽  
Author(s):  
Huei-Mei Huang ◽  
Jian-Chiuan Li ◽  
Yueh-Chun Hsieh ◽  
Hsin-Fang Yang-Yen ◽  
Jeffrey Jong-Young Yen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Stem Cell Differentiation ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Progenitor Cell Line

In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5R is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

scholarly journals p38β - MAPK11 and its role in female cancers

2020 ◽  
Author(s):  
Periklis Katopodis ◽  
Rachel Kerslake ◽  
Athanasios Zikopoulos ◽  
Nefeli Eirini Beri ◽  
Vladimir Anikin
Keyword(s):  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Her2 Status ◽  
Tissue Specific ◽  
Cancer Data ◽  
Signalling Molecules ◽  
Significant Difference

Abstract Background The p38MAPK family of Mitogen Activated Protein Kinases are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38β, remains fairly elusive. Recent studies suggest a possible role of p38β in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38β (MAPK11) in female cancers using an in-silico approach. Methods A detailed gene expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed.Results Data using cBioportal and CanSAR suggest that expression of p38β is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the genes’ two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. Conclusion This data provides an overview of the expression of p38β in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38β MAPK expression and offering insight for future in-vitro investigation and therapeutic application.

scholarly journals Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1960-1967 ◽  
Author(s):  
S Neben ◽  
K Marcus ◽  
P Mauch
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cell Content ◽  
Blood Stem Cells ◽  
Stimulating Factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony- stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.

Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity

2006 ◽  
Vol 290 (2) ◽  
pp. G189-G193 ◽  
Author(s):  
Neil D. Theise
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Stem Cell Niche ◽  
Progenitor Cell ◽  
Stem Cell Biology ◽  
Cell Plasticity ◽  
Self Renewal ◽  
Cell Niche

This essay will address areas of liver stem/progenitor cell studies in which consensus has emerged and in which controversy still prevails over consensus, but it will also highlight important themes that inevitably should be a focus of liver stem/progenitor cell investigations in coming years. Thus concepts regarding cell plasticity, the existence of a physiological/anatomic stem cell niche, and whether intrahepatic liver stem/progenitor cells comprise true stem cells or progenitor cells (or both) will be approached in some detail.

Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 492-492
Author(s):  
Wei-Gang Tong ◽  
William G. Wierda ◽  
Neby Bekele ◽  
Shao-Qing Kuang ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cpg Islands ◽  
Human Leukemia ◽  
Dna Hypomethylation ◽  
Line P ◽  
Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

scholarly journals Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1366-1373 ◽  
Author(s):  
Heike Kroeger ◽  
Jaroslav Jelinek ◽  
Marcos R. H. Estécio ◽  
Rong He ◽  
Kimie Kondo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Leukemia Cell ◽  
Transcription Start Sites ◽  
Remission Maintenance ◽  
Acute Myeloid

AbstractDNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

Abstract 1492: Epigenetic Control of the eNOS Promoter by DNA Methylation in Vasculogenic Progenitor Cell Populations

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Georgios J Vlachojannis ◽  
Carmen Urbich ◽  
Andreas M Zeiher ◽  
Stefanie Dimmeler
Keyword(s):  
Dna Methylation ◽  
Stem Cell ◽  
Endothelial Cell ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Promoter Region ◽  
Progenitor Cell ◽  
Cell Types ◽  
Proximal Promoter ◽  
Enos Gene

DNA methylation has been shown to play an essential role in both the transcriptional regulation and endothelial cell-specific expression of the human endothelial nitric oxide synthase (eNOS) gene. Further, recent data emphasizes an important role of eNOS in stem cell biology in particular with regard to progenitor cell mobilization and vasculoprotective properties. We assessed the hypothesis that stem and vasculogenic progenitor cells will exhibit different DNA methylation patterns of the eNOS promoter region dependent on their vascular fate. Endothelial progenitor cells (EPCs), mesangioblasts, CD34+, HUVECs and microvascular endothelial cells (MVECs) were cultivated and genomic DNA was subjected to sodium bisulfite treatment. The final PCR products were subcloned and sequenced (5–10 clones). Whereas the eNOS proximal promoter was either devoid or very lightly methylated in the human endothelial cell types including HUVECs and MVECs, the promoter was heavily methylated in the examined progenitor and stem cell types namely CD34+ and mesangioblasts. Surprisingly, EPCs also exhibit a profound methylation of the eNOS promoter (see Figure ). In conclusion, we have demonstrated that progenitor and stem cells including EPCs, CD34+ and mesangioblasts - although committed to a vascular fate - are in contrast to endothelial cell types heavily methylated in the promoter region of the eNOS gene suggesting epigenetic silencing at this level of maturation. The functional importance of this finding in particular regarding vasculoprotective potency and the modulation of methylation during progenitor cell maturation is subject of future studies. Methylation pattern of the eNOS promoter

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