Efficient Generation of Functional Epithelial and Epidermal Cells from Human Pluripotent Stem Cells Under Defined Conditions

Abstract

Human pluripotent stem cells (hPSCs) have an unparalleled potential to generate limitless quantities of any somatic cell type. However, current methods for producing populations of various somatic cell types from hPSCs are generally not standardized and typically incorporate undefined cell culture components often resulting in variable differentiation efficiencies and poor reproducibility. To address this, we have developed a defined approach for generating epithelial progenitor and epidermal cells from hPSCs. In doing so, we have identified an optimal starting cell density to maximize yield and maintain high purity of K18+/p63+ simple epithelial progenitors. In addition, we have shown that the use of synthetic, defined substrates in lieu of Matrigel and gelatin can successfully facilitate efficient epithelial differentiation, maintaining a high (>75%) purity of K14+/p63+ keratinocyte progenitor cells and at a two to threefold higher yield than a previously reported undefined differentiation method. These K14+/p63+ cells also exhibited a higher expansion potential compared to cells generated using an undefined differentiation protocol and were able to terminally differentiate and recapitulate an epidermal tissue architecture in vitro. In summary, we have demonstrated the production of populations of functional epithelial and epidermal cells from multiple hPSC lines using a new, completely defined differentiation strategy.

Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells

“…The potential of both human ESCs and iPSCs to generate cell lineages from various tissues has fuelled extensive research in the development of methods to differentiate these cells into the specific cell types of interests. To date, differentiation of human ESCs or iPSCs into cardiac 3 , neuronal 4,5 , retinal 6 , hepatic 7 , intestinal 8 , pancreatic 9 and skin 10 have been established with varying efficiencies in the absence of selection or fractionation. With recent advances in differentiation protocols, mature cell phenotypes capable of exhibiting some functional characteristics of adult cells have been established in liver and endothelial and neuronal models 7,[11][12][13] .…”

Generation of Epithelial Cell Populations from Human Pluripotent Stem Cells Using a Small-Molecule Inhibitor of Src Family Kinases

“…These K14+ cells can be terminally-differentiated into epidermal keratinocytes using one of several previously-reported protocols or assays [6,5]. …”

Differential Effects of the Extracellular Microenvironment on Human Embryonic Stem Cell Differentiation into Keratinocytes and Their Subsequent Replicative Life Span

“…Consequently, various strategies have been reported utilizing defined culture environments to direct the differentiation of hESCs to epithelial lineage [6,8,12]. Epithelial commitment in feeder-free systems with defined culture milieu is the most promising approach to exclude or minimize the xenogeneic components.…”

“…They concluded that the cell-ECM interactions are responsible for this discrepancy in proliferation [12]. These differences in growth ability suggest the significant influence of culture microenvironment during differentiation on the subsequent replicative capacity of the keratinocytes.…”

“…These differences in growth ability suggest the significant influence of culture microenvironment during differentiation on the subsequent replicative capacity of the keratinocytes. The limited proliferative capacity of hESC-Kert derived in different culture systems [6][7][8]12] is likely to pose a major obstacle for future clinical applications of these cells. Elucidating the proliferative capacity of hESC-Kert under different culture microenvironments and the underlying signaling mechanisms leading to cellular senescence would essentially pave the development of these cells for wider applications including engineering of skin tissue grafts.…”