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TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertr...

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Autores principales: Pfeifer, Christian G., Karl, Alexandra, Kerschbaum, Maximilian, Berner, Arne, Lang, Siegmund, Schupfner, Rupert, Koch, Matthias, Angele, Peter, Nerlich, Michael, Mueller, Michael B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Korean Society for Stem Cell Research 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457698/
https://www.ncbi.nlm.nih.gov/pubmed/30836731
http://dx.doi.org/10.15283/ijsc18088
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author Pfeifer, Christian G.
Karl, Alexandra
Kerschbaum, Maximilian
Berner, Arne
Lang, Siegmund
Schupfner, Rupert
Koch, Matthias
Angele, Peter
Nerlich, Michael
Mueller, Michael B.
author_facet Pfeifer, Christian G.
Karl, Alexandra
Kerschbaum, Maximilian
Berner, Arne
Lang, Siegmund
Schupfner, Rupert
Koch, Matthias
Angele, Peter
Nerlich, Michael
Mueller, Michael B.
author_sort Pfeifer, Christian G.
collection PubMed
description BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions. METHODS AND RESULTS: Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4. CONCLUSIONS: Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling.
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spelling pubmed-64576982019-04-19 TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation Pfeifer, Christian G. Karl, Alexandra Kerschbaum, Maximilian Berner, Arne Lang, Siegmund Schupfner, Rupert Koch, Matthias Angele, Peter Nerlich, Michael Mueller, Michael B. Int J Stem Cells Original Article BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions. METHODS AND RESULTS: Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4. CONCLUSIONS: Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling. Korean Society for Stem Cell Research 2019-02-28 /pmc/articles/PMC6457698/ /pubmed/30836731 http://dx.doi.org/10.15283/ijsc18088 Text en Copyright © 2019 by the Korean Society for Stem Cell Research This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Pfeifer, Christian G.
Karl, Alexandra
Kerschbaum, Maximilian
Berner, Arne
Lang, Siegmund
Schupfner, Rupert
Koch, Matthias
Angele, Peter
Nerlich, Michael
Mueller, Michael B.
TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title_full TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title_fullStr TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title_full_unstemmed TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title_short TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation
title_sort tgf-β signalling is suppressed under pro-hypertrophic conditions in msc chondrogenesis due to tgf-β receptor downregulation
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457698/
https://www.ncbi.nlm.nih.gov/pubmed/30836731
http://dx.doi.org/10.15283/ijsc18088
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