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Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential
BACKGROUND: The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population ide...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683518/ https://www.ncbi.nlm.nih.gov/pubmed/31383008 http://dx.doi.org/10.1186/s13287-019-1354-2 |
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author | Vattulainen, Meri Ilmarinen, Tanja Koivusalo, Laura Viiri, Keijo Hongisto, Heidi Skottman, Heli |
author_facet | Vattulainen, Meri Ilmarinen, Tanja Koivusalo, Laura Viiri, Keijo Hongisto, Heidi Skottman, Heli |
author_sort | Vattulainen, Meri |
collection | PubMed |
description | BACKGROUND: The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population identity and the regulation of LSC homeostasis is hindering the full therapeutic potential of hPSC-derived LSCs as well as primary LSCs. METHODS: The differentiation trajectory of two distinct hPSC lines towards LSCs was characterized extensively using immunofluorescence labeling against pluripotency, putative LSC, and mature corneal epithelium markers. Cell counting, flow cytometry, and qRT-PCR were used to quantify the differences between distinct populations observed at day 11 and day 24 time points. Initial differentiation conditions were thereafter modified to support the maintenance and expansion of the earlier population expressing ABCG2. Immunofluorescence, qRT-PCR, population doubling analyses, and transplantation into an ex vivo porcine cornea model were used to analyze the phenotype and functionality of the cell populations cultured in different conditions. RESULTS: The detailed characterization of the hPSC differentiation towards LSCs revealed only transient expression of a cell population marked by the universal stemness marker and proposed LSC marker ABCG2. Within the ABCG2-positive population, we further identified two distinct subpopulations of quiescent ∆Np63α-negative and proliferative ∆Np63α-positive cells, the latter of which also expressed the acknowledged intestinal stem cell marker and suggested LSC marker LGR5. These populations that appeared early during the differentiation process had stem cell phenotypes distinct from the later arising ABCG2-negative, ∆Np63α-positive third cell population. Importantly, novel culture conditions modulating the Wnt and BMP signaling pathways allowed efficient maintenance and expansion of the ABCG2-positive populations. In comparison to ∆Np63α-positive hPSC-LSCs cultured in the initial culture conditions, ABCG2-positive hPSC-LSCs in the novel maintenance condition contained quiescent stem cells marked by p27, demonstrated notably higher population doubling capabilities and clonal growth in an in vitro colony-forming assay, and increased regenerative potential in the ex vivo transplantation model. CONCLUSIONS: The distinct cell populations identified during the hPSC-LSC differentiation and ABCG2-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1354-2) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6683518 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-66835182019-08-09 Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential Vattulainen, Meri Ilmarinen, Tanja Koivusalo, Laura Viiri, Keijo Hongisto, Heidi Skottman, Heli Stem Cell Res Ther Research BACKGROUND: The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population identity and the regulation of LSC homeostasis is hindering the full therapeutic potential of hPSC-derived LSCs as well as primary LSCs. METHODS: The differentiation trajectory of two distinct hPSC lines towards LSCs was characterized extensively using immunofluorescence labeling against pluripotency, putative LSC, and mature corneal epithelium markers. Cell counting, flow cytometry, and qRT-PCR were used to quantify the differences between distinct populations observed at day 11 and day 24 time points. Initial differentiation conditions were thereafter modified to support the maintenance and expansion of the earlier population expressing ABCG2. Immunofluorescence, qRT-PCR, population doubling analyses, and transplantation into an ex vivo porcine cornea model were used to analyze the phenotype and functionality of the cell populations cultured in different conditions. RESULTS: The detailed characterization of the hPSC differentiation towards LSCs revealed only transient expression of a cell population marked by the universal stemness marker and proposed LSC marker ABCG2. Within the ABCG2-positive population, we further identified two distinct subpopulations of quiescent ∆Np63α-negative and proliferative ∆Np63α-positive cells, the latter of which also expressed the acknowledged intestinal stem cell marker and suggested LSC marker LGR5. These populations that appeared early during the differentiation process had stem cell phenotypes distinct from the later arising ABCG2-negative, ∆Np63α-positive third cell population. Importantly, novel culture conditions modulating the Wnt and BMP signaling pathways allowed efficient maintenance and expansion of the ABCG2-positive populations. In comparison to ∆Np63α-positive hPSC-LSCs cultured in the initial culture conditions, ABCG2-positive hPSC-LSCs in the novel maintenance condition contained quiescent stem cells marked by p27, demonstrated notably higher population doubling capabilities and clonal growth in an in vitro colony-forming assay, and increased regenerative potential in the ex vivo transplantation model. CONCLUSIONS: The distinct cell populations identified during the hPSC-LSC differentiation and ABCG2-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13287-019-1354-2) contains supplementary material, which is available to authorized users. BioMed Central 2019-08-05 /pmc/articles/PMC6683518/ /pubmed/31383008 http://dx.doi.org/10.1186/s13287-019-1354-2 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Vattulainen, Meri Ilmarinen, Tanja Koivusalo, Laura Viiri, Keijo Hongisto, Heidi Skottman, Heli Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title | Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title_full | Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title_fullStr | Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title_full_unstemmed | Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title_short | Modulation of Wnt/BMP pathways during corneal differentiation of hPSC maintains ABCG2-positive LSC population that demonstrates increased regenerative potential |
title_sort | modulation of wnt/bmp pathways during corneal differentiation of hpsc maintains abcg2-positive lsc population that demonstrates increased regenerative potential |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683518/ https://www.ncbi.nlm.nih.gov/pubmed/31383008 http://dx.doi.org/10.1186/s13287-019-1354-2 |
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