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Hematopoietic differentiation of human iPSC is hardly affected by knockouts in DNMT3A exons despite loss of de novo DNA methylation [RNA-seq]

ABSTRACT: DNA methyltransferase 3A (DNMT3A) is the most frequently mutated gene in clonal hematopoiesis, indicating that it may be essential for hematopoietic differentiation. We therefore addressed the functional relevance of DNMT3A for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) by knocking out either exon 2, 19, or 23. Directed differentiation towards mesenchymal stromal cells or hematopoietic progenitor cells (iHPCs) was only slightly reduced in exon 19-/- lines. Notably, exon 19-/- and exon 23-/- lines revealed absence of almost the entire de novo DNA methylation during differentiation. Despite the drastic effects on DNA methylation, there was no significant impact on gene expression of iHPCs. Notably, DNA methylation differences in acute myeloid leukemia with/without DNMT3A are related to iHPCs with/without DNMT3A knockout. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A, while it has little impact on early cell-fate decisions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180403 | GEO | 2022/05/31

REPOSITORIES: GEO

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Hematopoietic differentiation of human iPSC is hardly affected by knockouts in DNMT3A exons despite loss of de novo DNA methylation [DNA methylation]

Project description:DNA methyltransferase 3A (DNMT3A) is the most frequently mutated gene in clonal hematopoiesis, indicating that it may be essential for hematopoietic differentiation. We therefore addressed the functional relevance of DNMT3A for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) by knocking out either exon 2, 19, or 23. Directed differentiation towards mesenchymal stromal cells or hematopoietic progenitor cells (iHPCs) was only slightly reduced in exon 19-/- lines. Notably, exon 19-/- and exon 23-/- lines revealed absence of almost the entire de novo DNA methylation during differentiation. Despite the drastic effects on DNA methylation, there was no significant impact on gene expression of iHPCs. Notably, DNA methylation differences in acute myeloid leukemia with/without DNMT3A are related to iHPCs with/without DNMT3A knockout. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A, while it has little impact on early cell-fate decisions.

2022-05-31 | GSE180402 | GEO

Tracking of epigenetic changes during hematopoietic differentiation of induced pluripotent stem cells

Project description:Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modelling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive. In this study, we compared genome wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. Furthermore, we analyzed if additional co-culture for two weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward hematopoietic lineage. After 20 days of in vitro differentiation cells revealed typical hematopoietic morphology, CD45 expression and colony forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall very distinct from normal hematopoiesis. Co-culture with MSCs or iMSCs enhanced proliferation of iHPCs and maintenance of CFU potential. However, morphology, immunophenotype, and DNAm profiles did not indicate that additional culture expansion with stromal support increases hematopoietic differentiation. In conclusion, differentiation of iPSCs towards hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.

2019-01-24 | GSE119079 | GEO

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [Undifferentiated human induced pluripotent stem cells]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Undifferentiated human induced pluripotent stem cells (N = 23)

2016-06-29 | E-GEOD-60809 | biostudies-arrayexpress

de novo DNA Methylation Balances Hematopoietic Stem Cell Self-Renewal and Differentiation

Project description:Cytosine methylation is an epigenetic mark usually associated with gene repression. Despite a requirement for de novo DNA methylation for differentiation of embryonic stem cells, its role in somatic stem cells is unknown. Using conditional ablation, we show that loss of either, or both, Dnmt3a or Dnmt3b, progressively impedes hematopoietic stem cell (HSC) differentiation during serial in vivo passage. Concomitantly, HSC self-renewal is immensely augmented in absence of either Dnmt3, particularly Dnmt3a. Dnmt3-KO HSCs show upregulation of HSC multipotency genes and downregulation of early differentiation factors, and the differentiated progeny of Dnmt3-KO HSCs exhibit hypomethylation and incomplete repression of HSC-specific genes. HSCs lacking Dnmt3a manifest hyper-methylation of CpG islands and hypo-methylation of genes which are highly correlated with human hematologic malignancies. These data establish that aberrant DNA methylation has direct pathologic consequences for somatic stem cell development, leading to inefficient differentiation and maintenance of a self-renewal program. Reduced representation bisulfite sequencing (MspI,~40-220bp size fraction) of secondarily-transplanted wild-type and Dnmt3a conditional knockout hematopoietic stem cells. We used microarrays to detail the global expression of genes in secondarily-transplanted control-HSCs and Dnmt3a-KO-HSCs.

2011-09-30 | E-GEOD-27322 | biostudies-arrayexpress

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [iPSC_ESC_lines]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Human iPSCs after hematopoietic differentiation (n = 2), human iPSCs after neural differentiation (n = 1), human iPSCs with different culture conditions (n = 8), and human iPSC line forced to express IGF2 gene (n = 1), and its control (n = 1).

2016-06-29 | E-GEOD-75096 | biostudies-arrayexpress

De novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells to a pluripotent state

Project description:Induced pluripotent stem cells (iPSCs) are generated from somatic cells by the transduction of defined transcription factors and involves dynamic changes in DNA methylation. While the reprogramming of somatic cells is accompanied by de-methylation of pluripotency genes, the functional importance of de novo DNA methylation has not been clarified. Here, using loss-of-function studies, we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b. These iPSCs reactivated pluripotency genes, underwent self-renewal and showed restricted developmental potential which was rescued upon re-introduction of Dnmt3a and Dnmt3b. We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells. RNA levels of Dnmt3ab deficient iPSC cell lines were compared to control iPSC cell lines

2011-05-16 | E-GEOD-28629 | biostudies-arrayexpress

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [parental_lines_expression]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Expression analysis of parental somatic tissues of human iPSCs used for the present study.

2016-06-29 | E-GEOD-73629 | biostudies-arrayexpress

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [Human pluripotent stem cells-derived hematopoietic precursor cells]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. human pluripotent stem cell-derived hematopoietic precursor cells (n = 33)

2016-06-29 | E-GEOD-60810 | biostudies-arrayexpress

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [PSCderived_HPCs_methylation]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Bisulfite converted genomic DNA lysates from human pluripotent stem cell-derived hematopoietic precursor cells (CD34+CD38-CD43+ lineage marker-) were hybridized to Illumina HumanMethylation450 BeadChip.

2016-06-29 | E-GEOD-60811 | biostudies-arrayexpress

Reprogramming-associated aberrant DNA methylation determines hematopoietic differentiation capacity of human induced pluripotent stem cells [PSCusedfor_iPSDMRs_methylation]

Project description:The variation among induced pluripotent stem cells (iPSCs) in their differentiation capacity to specific lineages is frequently attributed to somatic memory. In this study, we compared hematopoietic differentiation capacity of 35 human iPSC lines derived from four different tissues and four embryonic stem cell lines. The analysis revealed that hematopoietic commitment capacity (PSCs to hematopoietic precursors) is correlated with the expression level of the IGF2 gene independent of the iPSC origins. In contrast, maturation capacity (hematopoietic precursors to mature blood) is affected by iPSC origin; blood-derived iPSCs showed the highest capacity. However, some fibroblast-derived iPSCs showed higher capacity than blood-derived clones. Tracking of DNA methylation changes during reprogramming reveals that maturation capacity is highly associated with aberrant DNA methylation acquired during reprogramming, rather than the types of iPSC origins. These data demonstrated that variations in the hematopoietic differentiation capacity of iPSCs are not attributable to somatic memories of their origins. Bisulfite converted genomic DNA lysates from human pluripotent stem cell-derived hematopoietic precursor cells (CD34+CD38-CD43+ lineage marker-) were hybridized to Illumina HumanMethylation450 BeadChip.

2016-06-29 | E-GEOD-60816 | biostudies-arrayexpress

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