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.

Keywords: epidermal biology; iPSC; iPSC-derived keratinocytes; induced pluripotent stem cell; keratinocytes; skin diseases.

Figure 1

Timeline for iPSC‐K generation. (A) Annotated timeline for the generation of iPSC‐K. Numbers on the timeline reflect days. (B) Morphology as well as numbers of cells and plates generated using this protocol.

Figure 2

Phase‐contrast images showing morphology of human iPSC colonies. (A) Density at which iPSC colonies are passaged (approximately 60% colony density). (B) Higher magnification of a normal iPSC colony. Note the smooth edge of the colony and the high nuclear‐to‐cytoplasmic ratio. Both characteristics are signs of an undifferentiated healthy iPSC colony. (C,D) Normal iPSC colonies surrounded by scattered spontaneously differentiating cells (circles).

Figure 3

Phase‐contrast images showing early stages of iPSC‐K differentiation (days 5‐10). (A) Healthy colonies at day 5 after start of iPSC‐K differentiation (arrowheads). (B) Mix of healthy and unhealthy colonies at day 5 of differentiation. Dashed arrows point to colonies that did not properly attach to the plate. Note the darker (brown) cell clumps. These colonies will die. Solid arrows point to colonies that do not look ideal, but that may differentiate into keratinocytes. Arrowheads point to colonies with the expected epithelial morphology. (C) Expanding early‐stage differentiating colony at day 7. (D) Higher magnification of a day 10 colony. Note the emerging cobblestone appearance, a typical feature of epithelial cells. Arrow points to cells that have acquired the expected cobblestone morphology.

Figure 4

Phase‐contrast images showing colony morphologies observed during the selection phase. (A,B) Day 20 colonies. Arrow in (A) points to a colony with abnormal morphology. (B) Day 20 colony with the expected cobblestone cell morphology (C,D) Day 26 cells with emerging keratinocyte morphology. Note the shiny edge in the colony shown in (C), a sign that the cells are ready to be passaged.

Figure 5

Fully differentiated iPSC‐K. (A) Phase‐contrast image of day 45 iPSC‐K. The cells show typical cobblestone keratinocyte morphology. A few cells appear to differ in morphology. These cells are mostly likely migratory leading to the elongated appearance. (B) Example of a day 43 culture labelled with TP63 and KRT14 antibodies.

Figure 6

Expression of keratinocyte markers in iPSC‐K. iPSC‐K and primary human epidermal keratinocytes were exposed to 1.3 mM Ca²⁺ for 48 hr. (A‐F) Immunofluorescence staining and (G) Western blot analysis demonstrates normal expression and localization of desmosomal proteins (DSC3, DSG3) and an adherens junction protein (β‐catenin). Antibodies used for immunofluorescence staining are: DSG3 (clone 5H10; courtesy of Dr. James K Wahl III, PhD, University of Nebraska Medical Center, Lincoln, NE), DSC3 (Progen cat. no. 61093), and β‐catenin (Santa Cruz cat. no. sc‐7963). Antibodies used forlotting are: DSG3 (Invitrogen cat. no. MA5‐16025), DSC3 (Progen cat. no. 61093), and β‐ catenin (Santa Cruz cat. no. sc‐7963).