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Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas

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Abstract

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

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Acknowledgments

The authors thank MG Transplantes Eye Tissue Bank/FHEMIG and Sociedade Oftamológica-MG. The microscopic data was obtained using the microscopes of “Centro de Aquisição e Processamento de Imagens” (CAPI-ICB/UFMG) and Center of Microscopy/UFMG (http://www.microscopia.ufmg.br).

Funding

This work was supported by CAPES, CNPq (167447/2017–3/404326/2012–9/471732/2012–5, 304188/2019–0), FAPEMIG (RED-00570-16, APQ-03132-18), INCT-Regenera (Brazil).

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Author notes

  1. Dawidson Assis Gomes and Alfredo Miranda de Goes contributed equally to this work.

Authors and Affiliations

  1. Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil

    Thaís Maria da Mata Martins

  2. Department of Genomic Sciences and Biotechnology, Catholic University of Brasilia, QS 07 – Lote 01, EPCT - Taguatinga, Brasília, Distrito Federal, 71966-700, Brazil

    Juliana Lott de Carvalho

  3. Faculty of Medicine, University of Brasilia, Campus Universitário Darcy Ribeiro, Brasília, Distrito Federal, 70910-900, Brazil

    Juliana Lott de Carvalho

  4. Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil

    Pricila da Silva Cunha & Dawidson Assis Gomes

  5. Department of Biology, Minas Gerais State University, Avenida Olegário Maciel, 1427, Ubá, Minas Gerais, 36502-002, Brazil

    Pricila da Silva Cunha

  6. Department of Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-901, Brazil

    Alfredo Miranda de Goes

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  1. Thaís Maria da Mata Martins
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  2. Juliana Lott de Carvalho
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  3. Pricila da Silva Cunha
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  4. Dawidson Assis Gomes
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Contributions

Thaís Martins: Conceptualization, Methodology, Formal analysis, Investigation, Writing-original Draft. Juliana Carvalho: Conceptualization, Resources, Writing-review & editing. Pricila Cunha: Investigation, Formal analysis. Dawidson Gomes: Resources, Writing – review & editing. Alfredo Goes: Supervision, Conceptualization, Project administration, Resources. All authors approved the final version of the manuscript.

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Correspondence to Thaís Maria da Mata Martins.

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Ethics Approval

The Research Ethics Committee of the Universidade Federal de Minas Gerais approved the study (ETIC-UFMG n° 49967715.0.0000.5149), and it was conducted in accordance with the Declaration of Helsinki.

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Informed consent was obtained from the donors’ relatives to use corneal tissue for research purposes. The orbital fat tissues and blood were collected after obtaining informed consent from the donors.

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Not applicable.

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The authors indicate no potential conflicts of interest.

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da Mata Martins, T.M., de Carvalho, J.L., da Silva Cunha, P. et al. Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas. Stem Cell Rev and Rep 18, 2522–2534 (2022). https://doi.org/10.1007/s12015-022-10356-6

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  • DOI: https://doi.org/10.1007/s12015-022-10356-6

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