Limits...
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.

Show MeSH

Related in: MedlinePlus

Morphology of ES and iPS cells during sequential stages of differentiation.hES and hiPS cells were induced to differentiate in parallel, using identical differentiation procedures, and monitored for cell morphology at various stages. Representative images of hES and 2 iPS lines during differentiation showed similar morphology at days 1, 10, and 28 of differentiation (A). iPS differentiation resulted in fewer surviving cells at each stage, yet occurred on a similar time course to hES differentiation. Stable cell lines also had similar morphology at passage 8 of differentiation (B). All images taken at 10X magnification. Immunohistochemistry during the early stages of differentiation showed the progressive loss of pluripotency-related protein Oct-4, and Oct-4 was undetectable in differentiated cell types (C). Images taken at 20X magnification.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046119&req=5

pone-0017128-g001: Morphology of ES and iPS cells during sequential stages of differentiation.hES and hiPS cells were induced to differentiate in parallel, using identical differentiation procedures, and monitored for cell morphology at various stages. Representative images of hES and 2 iPS lines during differentiation showed similar morphology at days 1, 10, and 28 of differentiation (A). iPS differentiation resulted in fewer surviving cells at each stage, yet occurred on a similar time course to hES differentiation. Stable cell lines also had similar morphology at passage 8 of differentiation (B). All images taken at 10X magnification. Immunohistochemistry during the early stages of differentiation showed the progressive loss of pluripotency-related protein Oct-4, and Oct-4 was undetectable in differentiated cell types (C). Images taken at 20X magnification.

Mentions: hiPS cells were initially generated and characterized by the Hochedlinger lab using retroviral transduction of BJ fibroblasts (ATCC, Inc., Manassas, VA) with five retroviral reprogramming factors (BJ hiPS#1) [28], and additional cells were also used that were reprogrammed using four retroviral reprogramming factors (BJ iPS #2, LD iPS). We derived cells from iPS (iPDK2 derived from BJ hiPS #1 and iPDK3 and iPDK4 from BJ iPS #2) using a differentiation protocol that was previously used to generate cells with properties of human fibroblasts from hES cells (H9 hES cell line) [9]. In parallel, hES cells were used to derive new fibroblast cell lines (EDK6 and EDK7) using the same differentiation conditions. Cells derived from both hES and iPS demonstrated a similar appearance during the sequential stages of differentiation as seen 10 and 28 days after initiation of differentiation (Figure 1A) and 4, 7, 14, and 21 days following differentiation (Figure S1). At 28 days after differentiation, iPDK and EDK cell lines showed similar fibroblast morphology, characterized by cells with elongated and stellate shapes that maintained this morphology over prolonged passage (Figure 1B). During differentiation, we observed the progressive loss of pluripotency marker OCT4 in both hES and iPS cells, and this marker was absent in fully-differentiated cells (Figure 1C). Additionally, cell surface markers of hES and iPS cells, TRA-1-60 and SSEA4, were lost upon induction of differentiation, and were undetectable in stable EDK and iPDK cell types (Figure S1B). All four cell populations (EDK6, EDK7, iPDK2, iPDK4) showed similar expression of CD markers, CD10, CD13, CD44, CD73, CD90, and CD166, characteristic of MSCs and fibroblasts (Table 1). These surface markers were found in greater than 90% of EDK6, EDK7, iPDK2, iPDK4 cells and were comparable to the percentage of cells expressing these markers in mature BJ fibroblasts, suggesting a biological potential for hES and iPS-derived cells that is similar to stromal fibroblasts. The growth of these cells was stable over long-term culture as seen by the maintenance of cell numbers upon serial passage and was similar to growth seen in normal fibroblasts (Figure S2). Taken together, this data indicates that hiPS cells can readily and reproducibly differentiate to cells with a gene expression profile of fibroblasts in a similar manner to that identified for hES cells.


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)

Morphology of ES and iPS cells during sequential stages of differentiation.hES and hiPS cells were induced to differentiate in parallel, using identical differentiation procedures, and monitored for cell morphology at various stages. Representative images of hES and 2 iPS lines during differentiation showed similar morphology at days 1, 10, and 28 of differentiation (A). iPS differentiation resulted in fewer surviving cells at each stage, yet occurred on a similar time course to hES differentiation. Stable cell lines also had similar morphology at passage 8 of differentiation (B). All images taken at 10X magnification. Immunohistochemistry during the early stages of differentiation showed the progressive loss of pluripotency-related protein Oct-4, and Oct-4 was undetectable in differentiated cell types (C). Images taken at 20X magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046119&req=5

pone-0017128-g001: Morphology of ES and iPS cells during sequential stages of differentiation.hES and hiPS cells were induced to differentiate in parallel, using identical differentiation procedures, and monitored for cell morphology at various stages. Representative images of hES and 2 iPS lines during differentiation showed similar morphology at days 1, 10, and 28 of differentiation (A). iPS differentiation resulted in fewer surviving cells at each stage, yet occurred on a similar time course to hES differentiation. Stable cell lines also had similar morphology at passage 8 of differentiation (B). All images taken at 10X magnification. Immunohistochemistry during the early stages of differentiation showed the progressive loss of pluripotency-related protein Oct-4, and Oct-4 was undetectable in differentiated cell types (C). Images taken at 20X magnification.
Mentions: hiPS cells were initially generated and characterized by the Hochedlinger lab using retroviral transduction of BJ fibroblasts (ATCC, Inc., Manassas, VA) with five retroviral reprogramming factors (BJ hiPS#1) [28], and additional cells were also used that were reprogrammed using four retroviral reprogramming factors (BJ iPS #2, LD iPS). We derived cells from iPS (iPDK2 derived from BJ hiPS #1 and iPDK3 and iPDK4 from BJ iPS #2) using a differentiation protocol that was previously used to generate cells with properties of human fibroblasts from hES cells (H9 hES cell line) [9]. In parallel, hES cells were used to derive new fibroblast cell lines (EDK6 and EDK7) using the same differentiation conditions. Cells derived from both hES and iPS demonstrated a similar appearance during the sequential stages of differentiation as seen 10 and 28 days after initiation of differentiation (Figure 1A) and 4, 7, 14, and 21 days following differentiation (Figure S1). At 28 days after differentiation, iPDK and EDK cell lines showed similar fibroblast morphology, characterized by cells with elongated and stellate shapes that maintained this morphology over prolonged passage (Figure 1B). During differentiation, we observed the progressive loss of pluripotency marker OCT4 in both hES and iPS cells, and this marker was absent in fully-differentiated cells (Figure 1C). Additionally, cell surface markers of hES and iPS cells, TRA-1-60 and SSEA4, were lost upon induction of differentiation, and were undetectable in stable EDK and iPDK cell types (Figure S1B). All four cell populations (EDK6, EDK7, iPDK2, iPDK4) showed similar expression of CD markers, CD10, CD13, CD44, CD73, CD90, and CD166, characteristic of MSCs and fibroblasts (Table 1). These surface markers were found in greater than 90% of EDK6, EDK7, iPDK2, iPDK4 cells and were comparable to the percentage of cells expressing these markers in mature BJ fibroblasts, suggesting a biological potential for hES and iPS-derived cells that is similar to stromal fibroblasts. The growth of these cells was stable over long-term culture as seen by the maintenance of cell numbers upon serial passage and was similar to growth seen in normal fibroblasts (Figure S2). Taken together, this data indicates that hiPS cells can readily and reproducibly differentiate to cells with a gene expression profile of fibroblasts in a similar manner to that identified for hES cells.

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