Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes

Abstract

Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create “designer” iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC‐derived keratinocytes (iPSC‐K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™400 6‐well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells

Dynamic 3D Combinatorial Generation of hPSC‐Derived Neuromesodermal Organoids With Diverse Regional and Cellular Identities

“…However, due to the asynchronous nature of cell division, the resulting embryoid bodies are heterogenous in size and shape. To overcome this limitation, methods exist to separately aggregate pluripotent stem cells as individual spheroids in static multi-well plates as described in previously published Current Protocols articles (Koch et al, 2022;Lewis-Israeli et al, 2022). Still, this approach can pose technical challenges for downstream handling and manipulation of these aggregates once formed.…”

“…Several methods for the culture of hPSCs have been established that utilize either undefined animal‐origin products or defined xeno‐free products. The utility of these products can be found in previously published Current Protocols articles (Gogolou, Frith, & Tsakiridis, 2021; Koch, Webb, Gugger, Salois, & Koster, 2022; Lewis‐Israeli, Abdelhamid, Olomu, & Aguirre, 2022). The differentiation protocol presented here is compatible with many different hPSC culture systems; however, we acknowledge that greater reproducibility can be achieved when using reagents that are free from the influence of animal derivatives.…”

Rare diseases of ectoderm: Translating discovery to therapy

“…Further, genetic variants can be corrected in iPSC, allowing for the generation of pairs of cells that share the same genetic background, and differ only by the presence or absence of the variant. Using specialized protocols, iPSC can be differentiated into relevant somatic cell types, for example into cells of the skin, eye, or lacrimal gland (Kasal et al, 2022; Koch et al, 2022; Sareen et al, 2014). Patient‐derived iPSC‐based models have been used extensively in studies aimed at dissecting the cellular and molecular basis of skin defects in patients affected by AEC and EEC, as well as different forms of EB, and have led to important insights into the respective disease mechanisms (Coutier et al, 2022; Dinella et al, 2018; Jackow et al, 2019; Shalom‐Feuerstein et al, 2013).…”

Effects of TP63 mutations on keratinocyte adhesion and migration

“…6,15 We used a well-established protocol that relies on BMP4 and retinoic acid to differentiate iPSC into iPSC-derived keratinocytes (iPSC-K). 6,16 We previously demonstrated that these iPSC-K express marker proteins of normal human keratinocytes. 6,16 Further, using these AEC and GC iPSC-K, we identified major defects in the expression and subcellular distribution of desmosomal proteins.…”

“…6,16 We previously demonstrated that these iPSC-K express marker proteins of normal human keratinocytes. 6,16 Further, using these AEC and GC iPSC-K, we identified major defects in the expression and subcellular distribution of desmosomal proteins. 6 These defects were also observed in AEC patient skin, further validating AEC iPSC-K as a model for AEC skin.…”