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

## TL;DR

This paper describes a protocol to turn human embryonic stem cells into surface epithelium and functional keratinocytes, which could help regenerative medicine.

## Contribution

A new protocol for differentiating human embryonic stem cells into surface epithelium and keratinocyte progenitors is introduced.

## Key findings

- A protocol successfully differentiates hESCs into surface epithelium and keratinocyte progenitors.
- 2D and 3D methods validate terminal differentiation and stratification potential of keratinocytes.
- The protocol could advance stem cell-based regenerative medicine.

## 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

•Protocol for differentiating surface epithelium from human embryonic stem cells•Differentiation steps from surface epithelium to keratinocyte progenitors•Calcium-mediated terminal differentiation in SE-derived keratinocytes•Air-liquid interface culture system recapitulates keratinocyte stratification

Protocol for differentiating surface epithelium from human embryonic stem cells

Differentiation steps from surface epithelium to keratinocyte progenitors

Calcium-mediated terminal differentiation in SE-derived keratinocytes

Air-liquid interface culture system recapitulates keratinocyte stratification

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.