Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jun 27;6(3):103919.
doi: 10.1016/j.xpro.2025.103919. Online ahead of print.

Protocol for differentiation of human embryonic stem cells into surface epithelium and functional keratinocytes

Jiafeng Liu  1 , Huaxing Huang  2 , Hong Ouyang  3
Affiliations

Protocol for differentiation of human embryonic stem cells into surface epithelium and functional keratinocytes

Jiafeng Liu et al. STAR Protoc. .

Abstract

The normal development and function of the epidermis and other ectodermal appendages depend on the proper formation of the surface epithelium (SE). Here, we present a protocol to differentiate human embryonic stem cells (hESCs) into SE and subsequently into epidermal keratinocyte progenitors. We then detail procedures for the passage and expansion of keratinocyte progenitors, followed by 2D validation of terminal differentiation and 3D assessment of stratification potential. This protocol represents a promising advancement for stem cell-based regenerative medicine. For complete details on the use and execution of this protocol, please refer to Liu et al.1 and Huang et al.2.

Keywords: Cell Biology; Cell culture; Stem Cells.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Directed differentiation of hESCs into SE (A) Phase contrast images showing the differentiating hESCs during 7 days of RA induction. Scale bar: 100 μm. (B) Immunostaining for KRT18 and TP63 in the differentiated cells on D7. Scale bar: 100 μm. (C) qRT-PCR analysis of representative genes in hESCs and cells after seven days of differentiation. qRT-PCR values were normalized to the values in hESC group. Values are presented as means ± SD (n=3 biological replicates; ∗∗P<0.01; ∗∗∗P<0.001, t test).
Figure 2
Figure 2
Induction of SE cells into mature keratinocyte progenitors (A) Left panel: Phase contrast images of the induced keratinocyte progenitors after 45 days of differentiation and 4 days after passaging. Scale bar: 100 μm. Right panel: Immunostaining for KRT14 and TP63 in the induced keratinocyte progenitors. Scale bar: 100 μm. (B) qRT-PCR analysis of representative genes in SE and induced keratinocyte progenitors. qRT-PCR values were normalized to the SE group. Values are presented as means ± SD (n=3 biological replicates; ∗∗P<0.01; ∗∗∗P<0.001, t test).
Figure 3
Figure 3
Terminal differentiation of keratinocyte progenitors (A) Immunostaining for KRT1 and KRT10 in the keratinocytes after 7 days of CaCl2 treatment. Scale bar: 100 μm. (B) qRT-PCR analysis of representative genes in keratinocyte progenitors and keratinocytes after 7 days of CaCl2 treatment. qRT-PCR values were normalized to the keratinocyte progenitor group. Values are presented as means ± SD (n=3 biological replicates; ∗P<0.05; ∗∗P<0.01, t test).
Figure 4
Figure 4
Air-lifting assay of keratinocyte progenitors (A) Schematic diagram illustrating the procedure of the air-lifting culture assay. (B) Immunostaining for KRT1, KRT10 and TP63 in the stratified keratinocytes after 10 days of air-lifting induction. Scale bar: 100 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Liu J., Huang H., An F., Wu S., Guo H., Wang B., Han Z., Tan J., Lin Z., Fang Y., et al. FOXO4-SP6 axis controls surface epithelium commitment by mediating epigenomic remodeling. Stem Cell Rep. 2025;20 doi: 10.1016/j.stemcr.2025.102445. - DOI - PMC - PubMed
    1. Huang H., Liu J., An F., Wu S., Guo H., Wang B., Mo K., Huang Y., Tan J., Zhu J., et al. Retinoic acid drives surface epithelium fate determination through the TCF7-MSX2 axis. Cell. Mol. Life Sci. 2024;82:16. doi: 10.1007/s00018-024-05525-4. - DOI - PMC - PubMed
    1. Biggs L.C., Mikkola M.L. Early inductive events in ectodermal appendage morphogenesis. Semin. Cell Dev. Biol. 2014;25–26:11–21. doi: 10.1016/j.semcdb.2014.01.007. - DOI - PubMed
    1. Huang H., Liu J., Li M., Guo H., Zhu J., Zhu L., Wu S., Mo K., Huang Y., Tan J., et al. Cis-regulatory chromatin loops analysis identifies GRHL3 as a master regulator of surface epithelium commitment. Sci. Adv. 2022;8 doi: 10.1126/sciadv.abo5668. - DOI - PMC - PubMed
    1. Li L., Wang Y., Torkelson J.L., Shankar G., Pattison J.M., Zhen H.H., Fang F., Duren Z., Xin J., Gaddam S., et al. TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment. Cell Stem Cell. 2019;24:271–284.e8. doi: 10.1016/j.stem.2018.12.012. - DOI - PMC - PubMed

LinkOut - more resources