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Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim...

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Autores principales: Stanco, Deborah, Caprara, Christian, Ciardelli, Gianluca, Mariotta, Luca, Gola, Mauro, Minonzio, Greta, Soldati, Gianni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372228/
https://www.ncbi.nlm.nih.gov/pubmed/30753235
http://dx.doi.org/10.1371/journal.pone.0212192
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author Stanco, Deborah
Caprara, Christian
Ciardelli, Gianluca
Mariotta, Luca
Gola, Mauro
Minonzio, Greta
Soldati, Gianni
author_facet Stanco, Deborah
Caprara, Christian
Ciardelli, Gianluca
Mariotta, Luca
Gola, Mauro
Minonzio, Greta
Soldati, Gianni
author_sort Stanco, Deborah
collection PubMed
description Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.
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spelling pubmed-63722282019-03-01 Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs Stanco, Deborah Caprara, Christian Ciardelli, Gianluca Mariotta, Luca Gola, Mauro Minonzio, Greta Soldati, Gianni PLoS One Research Article Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells. Public Library of Science 2019-02-12 /pmc/articles/PMC6372228/ /pubmed/30753235 http://dx.doi.org/10.1371/journal.pone.0212192 Text en © 2019 Stanco et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Stanco, Deborah
Caprara, Christian
Ciardelli, Gianluca
Mariotta, Luca
Gola, Mauro
Minonzio, Greta
Soldati, Gianni
Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title_full Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title_fullStr Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title_full_unstemmed Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title_short Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs
title_sort tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ascs
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372228/
https://www.ncbi.nlm.nih.gov/pubmed/30753235
http://dx.doi.org/10.1371/journal.pone.0212192
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