Directed differentiation of human pluripotent stem cells into epidermal keratinocyte-like cells

Summary The human pluripotent stem cell (hPSC) differentiation has allowed for the generation of in vitro models to study human diseases in a dish. This protocol describes the generation of keratinocyte-like cells from hPSCs in chemically defined media. Treating hPSCs with retinoic acid and BMP4 induced the generation of keratinocyte progenitors, which further differentiated into mature keratinocytes in the presence of calcium. The keratinocytes generated with this protocol could be used to study keratinocyte biology, drug screening, and skin-related diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).


SUMMARY
The human pluripotent stem cell (hPSC) differentiation has allowed for the generation of in vitro models to study human diseases in a dish. This protocol describes the generation of keratinocyte-like cells from hPSCs in chemically defined media. Treating hPSCs with retinoic acid and BMP4 induced the generation of keratinocyte progenitors, which further differentiated into mature keratinocytes in the presence of calcium. The keratinocytes generated with this protocol could be used to study keratinocyte biology, drug screening, and skin-related diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).

BEFORE YOU BEGIN
For the execution of this protocol, we used human pluripotent stem cells (hPSCs), including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). The cells were cultured and maintained in mTeSR1 medium in a humified 37 C incubator and 5% CO2. The cultured media, solutions, and matrix-coated plates were prepared before initiating the differentiation protocol. The growth factors and small molecules were added to the medium before use. Prior to the beginning of differentiation protocol, prepare media, solutions, and matrix-coated culture dishes. All media should be brought to 37 C prior to adding on the cells.

Geltrex coating
1. Thaw Geltrex at 4 C for 24 h. 2. Dilute Geltrex in cold knockout DMEM/F12 as per dilution factor mentioned on the datasheet (final concentration of 100 mg/mL). 3. Add the diluted Geltrex to the dish to cover the entire surface. 4. Incubate at 37 C for at least 60 min. The coated dish can be store at 4 C and is stable for two weeks.

Timing: 2 weeks
This section provides detailed description of obtaining keratinocytes from hPSCs. The first stage is the differentiation of hPSCs into keratinocyte progenitor-like cells by using retinoic acid (RA) to promote ectodermal fate (Bain et al., 1995) and bone morphogenetic protein 4 (BMP4) to block neural fate (Gambaro et al., 2006), which together can induce epithelial differentiation from hPSCs.
Note: Ensure the quality of hESCs before starting the differentiation by checking the cell morphology and expression of key pluripotency markers, such as OCT4, NANOG, and SOX2. The cells should have a confirmed normal karyotype, free of differentiated cells, and mycoplasma contamination.
1. On day 0, remove the culture medium from the cells and wash with 1 mL PBS to remove dead cells. Treat the cells with RelesR for 4-5 min at 37 C, then remove ReLeSR and detach the cells with media by gentle pipetting. Collect the cells in a 15 mL Falcon tube, centrifuge at 130 g for 5 min at 22 C and plate on Geltrex (1:100) coated plates as small clumps in mTeSR1 containing 10 mM Rock inhibitor. Split one confluent well of 6-well plate into two wells of a 6-well plate (1:2 ratio) to attain a confluency of 90-100% next day. The cells can be maintained in culture until they reach 90-100% confluency. 2. Remove the hESC medium and wash the cells with 1 mL of DMEM/F12 basal medium. Start the differentiation by adding 2.5 mL of unconditioned medium (UCM) supplemented with 1 mM RA and 20 ng/mL BMP4. 3. Continue to culture the cells for five days in UCM containing 1 mM RA and 20 ng/mL BMP4 with media change every day. 4. On day 5, treat the cells with ReLeSR for 4-5 min at 37 C, and then completely remove the dissociating reagent. Dissociate the cells with media and pipette gently to make small clumps of approximately 10-20 cells, centrifuge at 130 g for 5 min at 22 C, and plate the cells at 1:2 ratio onto Geltrex-coated plates. For RNA extraction and immunostaining, one well of 6-well is splitted into 4 wells of a 12-well plate and 3 wells of a 24-well plate. Add 10 mM Rock inhibitor during passaging the cells in addition to RA and BMP4. 5. Switch the media stepwise from UCM to N2 medium. On day 6, mix UCM and N2 medium at 1:1 ratio and add to the cells for stepwise transition. Change the media completely to N2 medium on day 7 and continue to treat the cells with RA and BMP4 until day 8 with media change every day. 6. On day 8, withdraw RA and BMP4 and add 10 ng/mL EGF to the N2 medium. 7. Continue to culture the cells until day 14 with media change every day to differentiate the cells into keratinocyte progenitors (Figure 1). 8. On day 14, check the keratinocyte progenitor cells for the expression of progenitor markers using RT-PCR and immunostaining (Figures 2A and 2B). Extract RNA from one well of a 12-well plate to check the expression of progenitor markers and use the cells grown in a 24-well plate for immunostaining.

Differentiation of keratinocyte progenitor cells into mature keratinocyte-like cells
Timing: $2 weeks 9. On day 14, wash the keratinocyte progenitor cells with 1 mL PBS and treat with TrypLE for 8-10 min at 37 C. Gently remove the TrypLE and detach the cells with N2 medium. Following centrifugation at 130 g for 5 min at 22 C, plate the cells at 1:2 ratio onto Geltrex coated plate. Add 10 mM Rock inhibitor to the fresh media during passaging the cells. 10. On day 15, withdraw the Rock inhibitor and add N2 medium containing 10 ng/mL EGF. 11. Continue to grow the cells in N2 medium containing 10 ng/mL EGF for 8-days with media change every other day. 12. On day 23, add 1.2 mM CaCl2 to the N2 medium containing 10 ng/mL EGF for one week to induce maturation of keratinocytes and change media every other day. 13. On day 30, check the expression of mature keratinocyte markers using Western blotting and immunostaining. The mature keratinocytes can be passaged and kept in culture until day 45 (Figure 1). 14. For passaging keratinocytes, treat the cells with TrypLE for 4-5 min at 37 C. Gently remove the TrypLE and detach the cells with N2 medium. Following centrifugation at 130 g for 5 min at 22 C, culture the keratinocytes onto Geltrex coated plates in N2 medium containing 10 ng/ mL EGF. Change the culture media every other day until day 45 ( Figure 5).

EXPECTED OUTCOMES
Stem cell-derived keratinocytes offer a satisfactory human skin specific in vitro model to study disease pathogenesis, host-pathogen interaction, skin function, and drug screening. This protocol describes successful generation of mature keratinocyte-like cells from hPSCs that express markers of all skin layers as shown in Figures 2, 3, and 4. The hPSCs were differentiated using RA and BMP4, which are regulators of keratinocyte differentiation and proliferation and disrupt neuronal differentiation and induce ectodermal fate (Bilousova and Roop, 2013;Metallo et al., 2008). Treatment of hPSCs with RA and BMP4 induced the expression p63 and KRT18, resembling epithelial progenitors, while the expression of pluripotency markers (OCT4 and NANOG) was down-regulated (Figures 2A and  2B). Further differentiation and maturation of progenitors was achieved with calcium, which is the major regulator of keratinocyte differentiation in vitro and in vivo. A calcium gradient within the epidermis promotes the sequential differentiation of keratinocytes as they traverse the different layers of the epidermis to form the permeability barrier of the stratum corneum (Bikle et al., 2012). The hPSC-derived mature keratinocyte-like cells express markers of stratum basale (p63 and KRT14), stratum spinosum (KRT1), stratum granulosum (FILAGGRIN), and stratum corneum (LORICRIN and INVOLUCRIN) (Eichner et al., 1986;Metallo et al., 2010;Sebastiano et al., 2014) (Figures 3 and 4).

OPEN ACCESS
LIMITATIONS This protocol has been tested for the generation of keratinocyte-like cells in 2D culture condition using H1-hESCs and iPSCs derived from healthy controls and patients with psoriasis (Ali et al., 2020). However, the reproducibility of the protocol may vary with other hPSC lines and may need initial adjustment of passaging and cell density. The keratinocyte-like cells produced using this protocol express specific markers for mature keratinocytes; however, further characterization of these cells would be necessary by comparing with primary keratinocytes. Furthermore, the use of this protocol for the preparation of 3D iPSC-derived skin organoid needs to be optimized.

Potential solution
To overcome spontaneous differentiation and cell death, hPSCs should not be allowed to overgrow and passaging frequency needs to be optimized depending on the cell type. Aliquot the media containing supplements and store at À20 C to keep the activity of the growth factors. Use fresh culture media and change the media daily.

Potential solution
We observe the progenitor cells were hard to detach during passaging. To overcome this, treat the progenitor cells with TrypLE for 8-10 min at 37 C. Remove the dissociating solution, add fresh culture media and pipette the cells several times to detach from the surface. Centrifuge the cells at 130 g for 5 min at 22 C and plate at 1:2 ratio onto Geltrex-coated plate.

Potential solution
Seed the cells at high density and include 5 mM Rock inhibitor during passaging for 24 h.

Potential solution
Optimize treatment time with cell dissociation reagent and detach keratinocytes with the medium by gentle pipetting. Seed the cells at high density and add 5 mM Rock inhibitor during passaging for 24 h to increase cell survival rate.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Essam M. Abdelalim (emohamed@hbku.edu.qa).

Materials availability
Materials associated with this protocol are available upon request from the lead contact.
Data and code availability This paper does not report datasets or original code.