Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture
Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this...
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| Veröffentlicht in: | PloS one 2023-02, Vol.18 (2), p.e0281404-e0281404 |
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| creator | Daniele, Elena Bosio, Lorenzo Hussain, Noor Ahmed Ferrari, Barbara Ferrari, Stefano Barbaro, Vanessa McArdle, Brian Rassu, Nicolò Mura, Marco Parmeggiani, Francesco Ponzin, Diego |
| description | Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy. |
| doi_str_mv | 10.1371/journal.pone.0281404 |
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Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0281404</identifier><identifier>PMID: 36745611</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Atomic force microscopy ; Biology and Life Sciences ; Care and treatment ; Cell adhesion ; Cell culture ; Cell Culture Techniques ; Cell Differentiation - genetics ; Cell Line ; Cell morphology ; Cell proliferation ; Cell survival ; Cell therapy ; Cellular therapy ; Clinical trials ; Collagen ; Cornea ; Cytology ; Denudation ; Denudation (Geology) ; Descemet Membrane ; Diagnosis ; Electron microscopy ; Endothelium ; Enzymes ; Epithelial Cells - metabolism ; Epithelium ; Gene expression ; Growth factors ; Histology ; Human Embryonic Stem Cells ; Humans ; Inserts ; Integrity ; Localization ; Macular degeneration ; Maturation ; Medicine and Health Sciences ; Membranes ; Methods ; Microscopy ; Monolayers ; Patient outcomes ; Photoreceptors ; Physical characteristics ; Pluripotency ; Polyethylene terephthalate ; Prevention ; Protein seeding ; Research and Analysis Methods ; Retina ; Retinal cells ; Retinal degeneration ; Retinal Diseases - metabolism ; Retinal pigment epithelium ; Retinal Pigment Epithelium - metabolism ; Risk factors ; Scaffolding ; Scanning electron microscopy ; Secretion ; Stem cells ; Substrates ; Thermolysin ; Thermolysin - metabolism ; Tight junctions ; Tissue culture ; Topology ; Transplantation ; Transplants & implants ; Vascular endothelial growth factor ; Vision disorders</subject><ispartof>PloS one, 2023-02, Vol.18 (2), p.e0281404-e0281404</ispartof><rights>Copyright: © 2023 Daniele et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2023 Public Library of Science</rights><rights>2023 Daniele et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2023 Daniele et al 2023 Daniele et al</rights><rights>2023 Daniele et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-bb7849fbd6c799f579e09526fada3908cacb7042dc891ebaa1da33a01dab45e63</citedby><cites>FETCH-LOGICAL-c692t-bb7849fbd6c799f579e09526fada3908cacb7042dc891ebaa1da33a01dab45e63</cites><orcidid>0000-0001-5283-5366 ; 0000-0001-9900-2767</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9901769/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9901769/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36745611$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Daniele, Elena</creatorcontrib><creatorcontrib>Bosio, Lorenzo</creatorcontrib><creatorcontrib>Hussain, Noor Ahmed</creatorcontrib><creatorcontrib>Ferrari, Barbara</creatorcontrib><creatorcontrib>Ferrari, Stefano</creatorcontrib><creatorcontrib>Barbaro, Vanessa</creatorcontrib><creatorcontrib>McArdle, Brian</creatorcontrib><creatorcontrib>Rassu, Nicolò</creatorcontrib><creatorcontrib>Mura, Marco</creatorcontrib><creatorcontrib>Parmeggiani, Francesco</creatorcontrib><creatorcontrib>Ponzin, Diego</creatorcontrib><title>Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.</description><subject>Analysis</subject><subject>Atomic force microscopy</subject><subject>Biology and Life Sciences</subject><subject>Care and treatment</subject><subject>Cell adhesion</subject><subject>Cell culture</subject><subject>Cell Culture Techniques</subject><subject>Cell Differentiation - genetics</subject><subject>Cell Line</subject><subject>Cell morphology</subject><subject>Cell proliferation</subject><subject>Cell survival</subject><subject>Cell therapy</subject><subject>Cellular therapy</subject><subject>Clinical trials</subject><subject>Collagen</subject><subject>Cornea</subject><subject>Cytology</subject><subject>Denudation</subject><subject>Denudation (Geology)</subject><subject>Descemet Membrane</subject><subject>Diagnosis</subject><subject>Electron microscopy</subject><subject>Endothelium</subject><subject>Enzymes</subject><subject>Epithelial Cells - metabolism</subject><subject>Epithelium</subject><subject>Gene expression</subject><subject>Growth factors</subject><subject>Histology</subject><subject>Human Embryonic Stem Cells</subject><subject>Humans</subject><subject>Inserts</subject><subject>Integrity</subject><subject>Localization</subject><subject>Macular degeneration</subject><subject>Maturation</subject><subject>Medicine and Health Sciences</subject><subject>Membranes</subject><subject>Methods</subject><subject>Microscopy</subject><subject>Monolayers</subject><subject>Patient outcomes</subject><subject>Photoreceptors</subject><subject>Physical characteristics</subject><subject>Pluripotency</subject><subject>Polyethylene terephthalate</subject><subject>Prevention</subject><subject>Protein seeding</subject><subject>Research and Analysis Methods</subject><subject>Retina</subject><subject>Retinal cells</subject><subject>Retinal degeneration</subject><subject>Retinal Diseases - metabolism</subject><subject>Retinal pigment epithelium</subject><subject>Retinal Pigment Epithelium - 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Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Daniele, Elena</au><au>Bosio, Lorenzo</au><au>Hussain, Noor Ahmed</au><au>Ferrari, Barbara</au><au>Ferrari, Stefano</au><au>Barbaro, Vanessa</au><au>McArdle, Brian</au><au>Rassu, Nicolò</au><au>Mura, Marco</au><au>Parmeggiani, Francesco</au><au>Ponzin, Diego</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2023-02-06</date><risdate>2023</risdate><volume>18</volume><issue>2</issue><spage>e0281404</spage><epage>e0281404</epage><pages>e0281404-e0281404</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>36745611</pmid><doi>10.1371/journal.pone.0281404</doi><tpages>e0281404</tpages><orcidid>https://orcid.org/0000-0001-5283-5366</orcidid><orcidid>https://orcid.org/0000-0001-9900-2767</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2023-02, Vol.18 (2), p.e0281404-e0281404 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2773650418 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Analysis Atomic force microscopy Biology and Life Sciences Care and treatment Cell adhesion Cell culture Cell Culture Techniques Cell Differentiation - genetics Cell Line Cell morphology Cell proliferation Cell survival Cell therapy Cellular therapy Clinical trials Collagen Cornea Cytology Denudation Denudation (Geology) Descemet Membrane Diagnosis Electron microscopy Endothelium Enzymes Epithelial Cells - metabolism Epithelium Gene expression Growth factors Histology Human Embryonic Stem Cells Humans Inserts Integrity Localization Macular degeneration Maturation Medicine and Health Sciences Membranes Methods Microscopy Monolayers Patient outcomes Photoreceptors Physical characteristics Pluripotency Polyethylene terephthalate Prevention Protein seeding Research and Analysis Methods Retina Retinal cells Retinal degeneration Retinal Diseases - metabolism Retinal pigment epithelium Retinal Pigment Epithelium - metabolism Risk factors Scaffolding Scanning electron microscopy Secretion Stem cells Substrates Thermolysin Thermolysin - metabolism Tight junctions Tissue culture Topology Transplantation Transplants & implants Vascular endothelial growth factor Vision disorders |
| title | Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture |
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