[Show abstract][Hide abstract] ABSTRACT: Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.
PLoS ONE 01/2013; 8(12):e83755. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The controlled differentiation of induced pluripotent stem cells (iPSC) towards clinically-relevant cell types has benefitted from epigenetic profiling of lineage-specific markers to confirm the phenotype of iPSC-derived cells. Mapping epigenetic marks throughout the genome has identified unique changes which occur in the DNA methylation profile of cells as they differentiate to specific cell types. Beyond characterizing the development of cells derived from pluripotent stem cells, the process of reprogramming cells to iPSC resets lineage-specific DNA methylation marks established during differentiation to specific somatic cell types. This property of reprogramming has potential utility in reverting aberrant epigenetic alterations in nuclear organization that are linked to disease progression. Since DNA methylation marks are reset following reprogramming, and contribute to restarting developmental programs, it is possible that DNA methylation marks associated with the disease state may also be erased in these cells. The subsequent differentiation of such cells could result in cell progeny that will function effectively as therapeutically-competent cell types for use in regenerative medicine. This suggests that through reprogramming it may be possible to directly modify the epigenetic memory of diseased cells and help to normalize their cellular phenotype, while also broadening our understanding of disease pathogenesis.
Molecular Aspects of Medicine 09/2012; · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet-derived growth factor receptor-beta (PDGFRβ) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRβ in fibroblasts derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and showed that this expression is important for cellular functions such as migration, extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRβ following differentiation from ESCs and iPSCs, we analyzed the DNA methylation status of a region of the PDGFRB promoter that contains multiple CpG sites, before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally regulated, differentially methylated region linked to PDGFRβ expression. Understanding the epigenetic regulation of genes such as PDGFRB, and identifying sites of active DNA demethylation, is essential for future applications of iPSC-derived fibroblasts for regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of L: -ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.
[Show abstract][Hide abstract] ABSTRACT: Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation.
In this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs.
While both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF.
Our findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation.
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2011; 6(2):e17128. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.
Tissue Engineering Part A 05/2009; 15(11):3417-26. · 4.64 Impact Factor