Three-Dimensional Epithelial Tissues Generated from Human Embryonic Stem Cells

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 twodimensional 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 b-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.

PDGFRβ Expression and Function in Fibroblasts Derived from Pluripotent Cells is Linked to DNA Demethylation

“…The H9 line of ESCs used in this study were purchased from the WiCell Institute (Madison, WI). Differentiation of cells was performed as described previously (Hewitt et al, 2009). Briefly, iPSCs (iPS-1, passage 12, iPS-2, passage 30) or ESCs were plated as aggregates onto fixed MEFs, and cultured in medium consisting of 3:1 DMEM:F12 (Invitrogen, Carlsbad, CA), 5% FCII (Hyclone, Logan, UT), 0.18 mM adenine, 0.5 mg/ml hydrocortisone, 10 210 M cholera toxin, 10 ng/ml EGF, 5 mg/ml insulin for 7 days and supplemented on days 4-7 with 0.5 nM BMP-4.…”

“…We have done this by performing detailed characterization of the methylation status of the PDGFRB promoter before and after differentiation from both ESCs and iPSCs, and by assaying PDGFRb-mediated functions in the fibroblasts derived from them. We have previously shown that ESC-and iPSC-derived fibroblasts exhibit the function of stromal fibroblasts as demonstrated by their capacity to support the development of 3D skin equivalent tissues in vitro (Hewitt et al, 2009), and to enhance the repair of these tissues through their production of paracrine factors . We now report that PDGFRb expression is increased in ESC-and iPSCderived fibroblasts, and knockdown of PDGFRb impaired the ability of ESC-and iPSC-derived fibroblasts to assemble 3D stroma-like ECM, and limited their cellular migration in response to PDGF stimulation, which are both essential functions for tissue regeneration and maintenance.…”

Strategies for Oral Mucosal Repair by Engineering 3D Tissues with Pluripotent Stem Cells

“…41 Both iPSCand hESC-derived cells displaying characteristics of stromal fibroblasts have also been shown to support complex, epithelial tissue development and to enhance re-epithelialization of wounds in skin equivalent tissues. 3,6 The ability of iPSCderived fibroblasts to direct the development of engineered skin in an in vitro tissue model that mimics many of the in vivo features of the tissue supports the feasibility of constructing complex, stratified epithelial tissues with iPSC-derived cells for future oral regenerative therapies. 22 Generation of iPSC-derived fibroblasts and keratinocytes may each provide functional features that can be well-suited for specific clinical indications and wound healing functions.…”

“…Tissues containing iPSCderived fibroblasts have been shown to support the development of a well-differentiated, stratified squamous epithelium 6 and stimulate reepithelialization after wounding. 3 Constructing in vivo-like tissues using iPSC-derived fibroblasts will provide a platform that can facilitate the clinical testing and optimization of repaircompetent cells that can accelerate oral wound healing through regenerative therapy approaches.…”

“…Specifically, these differentiation approaches can now generate cells with properties of repair-competent fibroblasts using well-defined growth conditions. 3,6,22 The stromal fibroblast would be a valuable cell type to derive from iPSC for regenerative strategies targeted to the oral mucosa. Fibroblasts represent a diverse population of mesenchymal stromal cells that play a central role in regulating Figure 1.…”