A Small Molecule Inhibitor of Src Family Kinases Promotes Simple Epithelial Differentiation of Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA) – induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4− simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications.

Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells

“…hPSCs were maintained in E8 medium on Synthemax according to previously published methods (Chen et al, 2011; Lian et al, 2012, 2013a, 2013b). …”

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

“…Here, this first step of simple epithelial cell differentiation is initiated using a Src family kinase inhibitor, SU6656, which modulates β-catenin localization [9]. The second step in this process involves the maturation of simple epithelial cells to epidermal keratinocyte progenitors, which can then form terminally-differentiated epidermal keratinocytes, marked by the expression of several proteins, including cytokeratin 10 (K10).…”

“…To generate populations of somatic cells to be used for such applications, it is imperative to design differentiation systems that are robust and produce high purity populations of cells. While there are different strategies to obtain epithelial populations from hPSCs [4-8], a recent study demonstrated how epithelial differentiation can be modulated by β-catenin localization, providing insight as to what mechanisms are involved in governing the epithelial differentiation process [9]. Here, we describe a method to produce simple epithelial cells and, subsequently, epidermal keratinocyte progenitor populations by exploiting this mechanism using a Src family kinase inhibitor.…”

Hair Follicle Reconstruction and Stem Cells

“…For example, the derivation of functional DP-like cells from human embryonic stem cells was recently reported [102]. Derivation of multipotent progenitors of the epithelial lineage [103,104] gives an opportunity to combine trichogenic cells at their early stages of commitment with hopefully beter results.…”