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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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Gene expression of EDK and iPDK are similar to normal fibroblasts and distinct from pluripotent cells.hES and hiPS cells as well as EDK, iPDK, HFF and BJ were analyzed by real-time RT-PCR (n = 3) and values were normalized to expression in hES cells. The pluripotency gene OCT4, SOX2 and NANOG were expressed in ES and iPS cells, and were absent in all ES and iPS-derived cells, and EDK and iPDK cells did not express neural stem cell marker Nestin (A). In contrast, genes characteristic of fibroblasts, including vimentin (VIM) Type I collagen (COLIA1) and platelet-derived growth factor-beta (PDGFRB) were all upregulated in EDK and iPDK cell lines compared to pluripotent cells from which they were derived (B).
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pone-0017128-g004: Gene expression of EDK and iPDK are similar to normal fibroblasts and distinct from pluripotent cells.hES and hiPS cells as well as EDK, iPDK, HFF and BJ were analyzed by real-time RT-PCR (n = 3) and values were normalized to expression in hES cells. The pluripotency gene OCT4, SOX2 and NANOG were expressed in ES and iPS cells, and were absent in all ES and iPS-derived cells, and EDK and iPDK cells did not express neural stem cell marker Nestin (A). In contrast, genes characteristic of fibroblasts, including vimentin (VIM) Type I collagen (COLIA1) and platelet-derived growth factor-beta (PDGFRB) were all upregulated in EDK and iPDK cell lines compared to pluripotent cells from which they were derived (B).

Mentions: To further study the properties of iPDK and EDK cells during their differentiation towards a fibroblast lineage fate, we analyzed patterns of gene expression in EDK6, EDK7, iPDK2, iPDK4 after eight passages on Type I Collagen-coated plates and compared them to undifferentiated hiPS and hES cells from which they were derived and to foreskin-derived fibroblasts (BJ and HFF). Expression of OCT4, SOX2 and NANOG were significantly down-regulated during differentiation in all EDK and iPDK cell lines when compared to hES and iPS cells, indicating loss of pluripotency upon differentiation (Figure 4A). Additionally, expression of the neural precursor gene nestin was significantly down-regulated in all EDK and iPDK cells upon their differentiation, suggesting they did not undergo commitment to a neural fate (Figure 4A). In contrast, the expression of genes commonly associated with mesenchymal cells and fibroblasts, including vimentin (VIM) and platelet-derived growth factor receptor-β (PDGFRβ), were consistently upregulated in EDK and iPDK cells following differentiation (Figure 4B). This data correlates with the results from the methylation screen that showed a decrease in methylation of the PDGFRβ promoter when both iPS and hES are differentiated to iPDK and EDK cell lines (Figure 3C). Type I collagen (COLIA, COLIA2) was also significantly upregulated in iPS- and hES-derived cell lines following their differentiation (Figure 4B), consistent with a decrease in the promoter methylation of COL1A1 seen in differentiated cell types (Figure 3D). Expression levels of VIM and COL1A1/2 were also comparable to those seen in mature fibroblasts (Figure 4B), while the expression levels of PDGFRβ was lower in EDK and iPDK when compared to fibroblasts. Another marker of fibroblasts, Thy-1, showed higher expression in the hES-derived cells compared to iPS-derived lines, but we also observed significant variability in expression in different lines of mature fibroblasts that were tested.


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)

Gene expression of EDK and iPDK are similar to normal fibroblasts and distinct from pluripotent cells.hES and hiPS cells as well as EDK, iPDK, HFF and BJ were analyzed by real-time RT-PCR (n = 3) and values were normalized to expression in hES cells. The pluripotency gene OCT4, SOX2 and NANOG were expressed in ES and iPS cells, and were absent in all ES and iPS-derived cells, and EDK and iPDK cells did not express neural stem cell marker Nestin (A). In contrast, genes characteristic of fibroblasts, including vimentin (VIM) Type I collagen (COLIA1) and platelet-derived growth factor-beta (PDGFRB) were all upregulated in EDK and iPDK cell lines compared to pluripotent cells from which they were derived (B).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046119&req=5

pone-0017128-g004: Gene expression of EDK and iPDK are similar to normal fibroblasts and distinct from pluripotent cells.hES and hiPS cells as well as EDK, iPDK, HFF and BJ were analyzed by real-time RT-PCR (n = 3) and values were normalized to expression in hES cells. The pluripotency gene OCT4, SOX2 and NANOG were expressed in ES and iPS cells, and were absent in all ES and iPS-derived cells, and EDK and iPDK cells did not express neural stem cell marker Nestin (A). In contrast, genes characteristic of fibroblasts, including vimentin (VIM) Type I collagen (COLIA1) and platelet-derived growth factor-beta (PDGFRB) were all upregulated in EDK and iPDK cell lines compared to pluripotent cells from which they were derived (B).
Mentions: To further study the properties of iPDK and EDK cells during their differentiation towards a fibroblast lineage fate, we analyzed patterns of gene expression in EDK6, EDK7, iPDK2, iPDK4 after eight passages on Type I Collagen-coated plates and compared them to undifferentiated hiPS and hES cells from which they were derived and to foreskin-derived fibroblasts (BJ and HFF). Expression of OCT4, SOX2 and NANOG were significantly down-regulated during differentiation in all EDK and iPDK cell lines when compared to hES and iPS cells, indicating loss of pluripotency upon differentiation (Figure 4A). Additionally, expression of the neural precursor gene nestin was significantly down-regulated in all EDK and iPDK cells upon their differentiation, suggesting they did not undergo commitment to a neural fate (Figure 4A). In contrast, the expression of genes commonly associated with mesenchymal cells and fibroblasts, including vimentin (VIM) and platelet-derived growth factor receptor-β (PDGFRβ), were consistently upregulated in EDK and iPDK cells following differentiation (Figure 4B). This data correlates with the results from the methylation screen that showed a decrease in methylation of the PDGFRβ promoter when both iPS and hES are differentiated to iPDK and EDK cell lines (Figure 3C). Type I collagen (COLIA, COLIA2) was also significantly upregulated in iPS- and hES-derived cell lines following their differentiation (Figure 4B), consistent with a decrease in the promoter methylation of COL1A1 seen in differentiated cell types (Figure 3D). Expression levels of VIM and COL1A1/2 were also comparable to those seen in mature fibroblasts (Figure 4B), while the expression levels of PDGFRβ was lower in EDK and iPDK when compared to fibroblasts. Another marker of fibroblasts, Thy-1, showed higher expression in the hES-derived cells compared to iPS-derived lines, but we also observed significant variability in expression in different lines of mature fibroblasts that were tested.

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