Epithelial basement membrane of human decellularized cornea as a suitable substrate for differentiation of embryonic stem cells into corneal epithelial-like cells

Thaís Maria da Mata Martins; Pricila da Silva Cunha; Michele Angela Rodrigues; Juliana Lott de Carvalho; Joyce Esposito de Souza; Junnia Alvarenga de Carvalho Oliveira; Dawidson Assis Gomes; Alfredo Miranda de Goes

doi:10.1016/j.msec.2020.111215

Epithelial basement membrane of human decellularized cornea as a suitable substrate for differentiation of embryonic stem cells into corneal epithelial-like cells

Mater Sci Eng C Mater Biol Appl. 2020 Nov:116:111215. doi: 10.1016/j.msec.2020.111215. Epub 2020 Jun 20.

Authors

Thaís Maria da Mata Martins¹, Pricila da Silva Cunha², Michele Angela Rodrigues³, Juliana Lott de Carvalho⁴, Joyce Esposito de Souza², Junnia Alvarenga de Carvalho Oliveira⁵, Dawidson Assis Gomes², Alfredo Miranda de Goes³

Affiliations

¹ Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte 31270-901, Minas Gerais, Brazil. Electronic address: thaismmmartins@gmail.com.
² Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte 31270-901, Minas Gerais, Brazil.
³ Department of Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte 31270-901, Minas Gerais, Brazil.
⁴ Department of Genomic Sciences and Biotechnology, Catholic University of Brasilia, QS 07 - Lote 01, EPCT - Taguatinga, Brasília, Distrito Federal 71966-700, Brazil; Faculty of Medicine, University of Brasilia, Campus Universitário Darcy Ribeiro, Brasília, Distrito Federal 70910-900, Brazil.
⁵ Department of Microbiology, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte 31270-901, Minas Gerais, Brazil.

PMID: 32806330
DOI: 10.1016/j.msec.2020.111215

Abstract

The ability to decellularize and recellularize the corneas deemed unsuitable for transplantation may increase the number of available grafts. Decellularized corneas (DCs) may provide a natural microenvironment for cell adhesion and differentiation. Despite this, no study to date has evaluated their efficacy as a substrate for the induction of stem cell differentiation into corneal cells. The present study aimed to compare the efficiency of NaCl and NaCl plus nucleases methods to decellularize whole human corneas, and to investigate the effect of epithelial basement membrane (EBM) of whole DCs on the ability of human embryonic stem cells (hESCs) to differentiate into corneal epithelial-like cells when cultured in animal serum-free differentiation medium. As laminin is the major component of EBM, we also investigated its effect on hESCs differentiation. The decellularization efficiency and integrity of the extracellular matrix (ECM) obtained were investigated by histology, electron microscopy, DNA quantification, immunofluorescence, and nuclear staining. The ability of hESCs to differentiate into corneal epithelial-like cells when seeded on the EBM of DCs or laminin-coated wells was evaluated by immunofluorescence and RT-qPCR analyses. NaCl treatment alone, without nucleases, was insufficient to remove cellular components, while NaCl plus nucleases treatment resulted in efficient decellularization and preservation of the ECM. Unlike cells induced to differentiate on laminin, hESCs differentiated on DCs expressed high levels of corneal epithelial-specific markers, keratin 3 and keratin 12. It was demonstrated for the first time that the decellularized matrices had a positive effect on the differentiation of hESCs towards corneal epithelial-like cells. Such a strategy supports the potential applications of human DCs and hESCs in corneal epithelium tissue engineering.

Keywords: Cornea; Corneal epithelial cells; Decellularization; Differentiation; Human embryonic stem cell.

MeSH terms

Animals
Basement Membrane
Cell Differentiation
Cornea
Embryonic Stem Cells*
Epithelial Cells
Epithelium, Corneal*
Humans