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Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

Hewitt KJ, Shamis Y, Hayman RB, Margvelashvili M, Dong S, Carlson MW, Garlick JA - PLoS ONE (2011)

Bottom Line: However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear.We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts.Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Human induced pluripotent stem (hiPS) cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM)-production (COL1A1) by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ), revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

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Changes in DNA methylation of gene promoter regions following differentiation shows large changes in methylation status in EDK and iPDK compared to hES and iPS.Heatmap showing 55 CpG sites which were differentially methylated by more than 70% in both EDK and iPDK when compared to their hES cell counterparts (A). In addition, the pluripotency-related genes OCT4 and REX1 showed increased CpG methylation in EDK and iPDK cells compared to hES and iPS cells (B). Analysis of Collagen promoter methylation showed that the COL1A1, COL3A1 and COL4A2 promoters were demethylated upon differentiation towards fibroblasts while collagen genes expressed in epithelial cell types, such as COL17A1, remained methylated in differentiated cell types (C). Fibroblast-related gene PDGFR-β was demethylated in differentiated cell types (D). Differentially-methylated Hox genes, grouped by cell type, showed a distinct methylation of Hox genes related to mesenchymal lineage commitment (E).
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pone-0017128-g003: Changes in DNA methylation of gene promoter regions following differentiation shows large changes in methylation status in EDK and iPDK compared to hES and iPS.Heatmap showing 55 CpG sites which were differentially methylated by more than 70% in both EDK and iPDK when compared to their hES cell counterparts (A). In addition, the pluripotency-related genes OCT4 and REX1 showed increased CpG methylation in EDK and iPDK cells compared to hES and iPS cells (B). Analysis of Collagen promoter methylation showed that the COL1A1, COL3A1 and COL4A2 promoters were demethylated upon differentiation towards fibroblasts while collagen genes expressed in epithelial cell types, such as COL17A1, remained methylated in differentiated cell types (C). Fibroblast-related gene PDGFR-β was demethylated in differentiated cell types (D). Differentially-methylated Hox genes, grouped by cell type, showed a distinct methylation of Hox genes related to mesenchymal lineage commitment (E).

Mentions: We next examined the genes that showed the greatest difference in methylation status between undifferentiated pluripotent and differentiated fibroblast cell types, and generated a heat map to compare the average methylation status of EDK (n = 3), iPDK (n = 3), ES (n = 1), iPS (n = 3) HFF (n = 1) and NHK n = 1). We identified a total of 55 gene-specific methylation sites that showed greater than a 70% difference in methylation between ES and EDK, and a high degree of similarity to iPDK and HFF (Figure 3A). Only 51 gene promoter regions are represented in this dataset, as MSX1 (3 times) and CDKN2B (2 times) appeared on multiple occasions in the array. Methylation of specific promoter regions of genes associated with pluripotency were also assessed using this screen. The OCT4 and REX1 promoter CpG sites showed consistently elevated levels of methylation upon differentiation towards fibroblasts, indicating epigenetic silencing of pluripotency genes as cells underwent commitment towards stable, differentiated cell types (Figure 3B). We then analyzed the methylation status of a group of promoter regions associated with the expression of collagen genes, which are linked to the phenotypic properties of extracellular matrix (ECM)-producing fibroblasts. Several of the collagen promoter regions were demethylated in EDK and iPDK cells when compared to hES and hiPS (COL1A1, 3A1, 4A2) (Figure 3C). Epithelial-related collagens, such as COL17A1, remained unchanged in methylation status in EDK and iPDK cells, and were only found to be demethylated in mature keratinocytes. Significantly, we found that platelet-derived growth factor-β (PDGFRβ) was consistently hypomethylated in all cells with properties of stromal fibroblasts, EDK and iPDK cells, indicating a commitment to a fibroblast lineage fate (Figure 3D). Several homeobox genes, CDX1, MSX1, and ALX homeobox 4 (ALX4), known to regulate various stages of mesenchymal differentiation, all showed increased promoter methylation in EDK and iPDK cells when compared to hES cells, and were methylated at a comparable level to that seen in mature fibroblasts (Figure 3E). CDX1 promoter has been previously identified as a hypermethylated region in fibroblasts derived from hES [27], while MSX1 is involved in craniofacial development and methylation at this site appears to be highly correlated to mesenchymal cell types [29]. The occurrence of DNA methylation in EDK and iPDK at multiple sites within the MSX1 promoter may indicate an extended region of hypermethylation.


Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

Hewitt KJ, Shamis Y, Hayman RB, Margvelashvili M, Dong S, Carlson MW, Garlick JA - PLoS ONE (2011)

Changes in DNA methylation of gene promoter regions following differentiation shows large changes in methylation status in EDK and iPDK compared to hES and iPS.Heatmap showing 55 CpG sites which were differentially methylated by more than 70% in both EDK and iPDK when compared to their hES cell counterparts (A). In addition, the pluripotency-related genes OCT4 and REX1 showed increased CpG methylation in EDK and iPDK cells compared to hES and iPS cells (B). Analysis of Collagen promoter methylation showed that the COL1A1, COL3A1 and COL4A2 promoters were demethylated upon differentiation towards fibroblasts while collagen genes expressed in epithelial cell types, such as COL17A1, remained methylated in differentiated cell types (C). Fibroblast-related gene PDGFR-β was demethylated in differentiated cell types (D). Differentially-methylated Hox genes, grouped by cell type, showed a distinct methylation of Hox genes related to mesenchymal lineage commitment (E).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046119&req=5

pone-0017128-g003: Changes in DNA methylation of gene promoter regions following differentiation shows large changes in methylation status in EDK and iPDK compared to hES and iPS.Heatmap showing 55 CpG sites which were differentially methylated by more than 70% in both EDK and iPDK when compared to their hES cell counterparts (A). In addition, the pluripotency-related genes OCT4 and REX1 showed increased CpG methylation in EDK and iPDK cells compared to hES and iPS cells (B). Analysis of Collagen promoter methylation showed that the COL1A1, COL3A1 and COL4A2 promoters were demethylated upon differentiation towards fibroblasts while collagen genes expressed in epithelial cell types, such as COL17A1, remained methylated in differentiated cell types (C). Fibroblast-related gene PDGFR-β was demethylated in differentiated cell types (D). Differentially-methylated Hox genes, grouped by cell type, showed a distinct methylation of Hox genes related to mesenchymal lineage commitment (E).
Mentions: We next examined the genes that showed the greatest difference in methylation status between undifferentiated pluripotent and differentiated fibroblast cell types, and generated a heat map to compare the average methylation status of EDK (n = 3), iPDK (n = 3), ES (n = 1), iPS (n = 3) HFF (n = 1) and NHK n = 1). We identified a total of 55 gene-specific methylation sites that showed greater than a 70% difference in methylation between ES and EDK, and a high degree of similarity to iPDK and HFF (Figure 3A). Only 51 gene promoter regions are represented in this dataset, as MSX1 (3 times) and CDKN2B (2 times) appeared on multiple occasions in the array. Methylation of specific promoter regions of genes associated with pluripotency were also assessed using this screen. The OCT4 and REX1 promoter CpG sites showed consistently elevated levels of methylation upon differentiation towards fibroblasts, indicating epigenetic silencing of pluripotency genes as cells underwent commitment towards stable, differentiated cell types (Figure 3B). We then analyzed the methylation status of a group of promoter regions associated with the expression of collagen genes, which are linked to the phenotypic properties of extracellular matrix (ECM)-producing fibroblasts. Several of the collagen promoter regions were demethylated in EDK and iPDK cells when compared to hES and hiPS (COL1A1, 3A1, 4A2) (Figure 3C). Epithelial-related collagens, such as COL17A1, remained unchanged in methylation status in EDK and iPDK cells, and were only found to be demethylated in mature keratinocytes. Significantly, we found that platelet-derived growth factor-β (PDGFRβ) was consistently hypomethylated in all cells with properties of stromal fibroblasts, EDK and iPDK cells, indicating a commitment to a fibroblast lineage fate (Figure 3D). Several homeobox genes, CDX1, MSX1, and ALX homeobox 4 (ALX4), known to regulate various stages of mesenchymal differentiation, all showed increased promoter methylation in EDK and iPDK cells when compared to hES cells, and were methylated at a comparable level to that seen in mature fibroblasts (Figure 3E). CDX1 promoter has been previously identified as a hypermethylated region in fibroblasts derived from hES [27], while MSX1 is involved in craniofacial development and methylation at this site appears to be highly correlated to mesenchymal cell types [29]. The occurrence of DNA methylation in EDK and iPDK at multiple sites within the MSX1 promoter may indicate an extended region of hypermethylation.

Bottom Line: However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear.We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts.Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell, Molecular and Developmental Biology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Human induced pluripotent stem (hiPS) cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM)-production (COL1A1) by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ), revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

Show MeSH
Related in: MedlinePlus