Pluripotent Stem Cell Differentiation Toward Functional Basal Stratified Epithelial Cells
In this chapter, an efficient feeder-free protocol of differentiating human pluripotent stem cells (hPSCs) toward the epidermal lineage to generate induced epidermal keratinocytes (iKCs) is described. The iKCs are able to terminally differentiate supra-ba
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Pluripotent Stem Cell Differentiation Toward Functional Basal Stratified Epithelial Cells Eduardo Soares and Huiqing Zhou Abstract In this chapter, an efficient feeder-free protocol of differentiating human pluripotent stem cells (hPSCs) toward the epidermal lineage to generate induced epidermal keratinocytes (iKCs) is described. The iKCs are able to terminally differentiate supra-basally. This hPSC-to-iKC differentiation can serve as a useful model to study epidermal development and disease as well as for therapeutic applications. Key words hPSCs, Keratinocytes, BMP-4, Retinoic acid, Single cell passaging, Differentiation
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Introduction Pluripotent stem cells (PSCs) have been extensively used to model development and disease [1]. Given that PSCs can be frozen, expanded and genetically modified, they offer great potential to generate transplantable tissues such as the epidermis. In addition, the dissection of molecular mechanisms underlying abnormal epidermal commitment and differentiation can serve as a reference to the design of therapeutic options for patients with epidermal abnormalities [2]. So far, the use of hPSCs as a research tool to understand epidermal diseases has made a tremendous impact on the dermatologic field, paving the way for alternative disease treatments. For examples, the use of the small molecule APR-246 and mesodermal inhibitors (suramin, valproic acid, and heparin) can enhance epithelial differentiation of hPSCs carrying p63 mutations [2–4]; and the use of gene editing, via TALEN or CRISPR/Cas9, has also been explored to correct COL7A1 mutations in hPSCs and corresponding iKCs derived from patients with epidermolysis bullosa (EB) [5, 6]. To this end, we and others have used mouse or human PSCs to establish and study epidermal commitment (3–12). In the early attempts, feeder layer of mouse embryonic fibroblasts (MEFs) have commonly been applied in the differentiation procedures [7– 13]. Recent protocols have used more chemically controlled
Eduardo Soares and Huiqing Zhou
a
Pluripotent
Start induction
b
Simple epithelium
Basal epithelium
Passaging
Stratified epithelium
Optional passaging
iKCs
Timeline E8(Flex)
Day -2
KSFM + 1μM RA + 10ng/ml BMP-4
Day 0
Day 7
KSFM
Day 15
Day 30
Coating: Geltrex
Fig. 1 (a) The in vitro commitment of PSCs toward the stratified epithelia fate involves initial induction toward simple epithelium, subsequent basal epithelium maturation and formation of a stratified epithelium. (b) Schematic view of the in vitro commitment of PSCs toward the stratified epithelia fate, small molecules are indicated in the figure
Fig. 2 (a) Bright-field pictures of normal PSC colonies, (b) induced keratinocytes (iKCs), (c) primary keratinocytes (pKCs) (Scale bars bright-field ¼ 100 μm), (d) immunofluorescence staining of the basal epithelial marker KRT14 (green) in iKCs, (e) and pKCs. Cell nuclei are stained with DAPI (blue) (Scale bars immunofluorescence ¼ 100 μm)
conditions [14–16], facilitating epigenomic analyses and increasing reproducibility and the tran
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