Long-term expansion of directly reprogrammed keratinocyte-like cells and in vitro reconstitution of human skin

Jie Zheng,Junghyun Park,Gwonhwa Song,Seungkwon You,Phil Jun Kang,Wonjin Yun,In Yong Kim,Gilju Lee

doi:10.1186/s12929-020-00642-1

Jie Zheng, Junghyun Park + Show 6 more

Open Access

https://doi.org/10.1186/s12929-020-00642-1

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Abstract

BackgroundHuman keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting.MethodsInduced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition.ResultsBN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro.ConclusionsThis study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.

Highlights

Human keratinocytes and derived products are crucial for skin repair and regeneration
Based on a previous report of NP63α and KLF4 (NK)-driven conversion of human neonatal foreskin fibroblasts into induced keratinocyte like cell (iKC) [12], we first infected human urine cells, with retroviruses encoding NK and cultured them in 2% FKGM with 3 T3-J2 feeder cells (Fig. 1a, S2A)
Considering that KLF4 is highly expressed during induction into terminal differentiated keratinocytes [31, 32] and △NP63α-triggered epithelial-mesenchymal transition of normal primary human epidermal keratinocytes [33], we hypothesized that BMI1, rather than KLF4, would improve reprogramming of urine cells into iKCs and acquisition of epidermal stemness

Summary

Introduction

Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. Major skin injuries and diseases caused by thermal burns, surgical incisions, infection, trauma, or chronic ulcers require medical intervention to heal properly and can be life-threatening in severe cases. Zheng et al Journal of Biomedical Science (2020) 27:56 accessibility of material for wound closure. Their clinical use is limited by the recipient’s immune response, which leads to cellular destruction, and these grafts are only used as temporary biologic dressings [4]. Much attention has been paid to generating an unlimited and immunocompatible graft material that provides therapeutic benefit as a skin replacement. Proof-of-principle studies demonstrated that these cells induce rapid re-epithelialization; the functional and aesthetic outcomes, clinical feasibility, and cost-benefit relationship of remote culture facilities remain unclear [7, 8]

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