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Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

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Abstract

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.

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Acknowledgments

This work was supported by Cluster “Handicap Vieillissement NeuroSciences” (HVN) (Décryptage des interactions des cellules souches avec la matrice extracellulaire nécessaires à leur conversion en chondrocytes) from the Région Rhône-Alpes. The authors would like to thank Dr L J Sandell (Washington University School of Medicine, St Louis, MO, USA) for rabbit antiserum against recombinant human type IIA and Sylviane Guerret (Novotec, Lyon, France) for her expertise in histological analysis. The Analyse Génétique and Platim platforms of IFR 128 are gratefully acknowledged for the use of the iCycler iQ (BioRad) and the Nikon E600 microscope.

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Authors and Affiliations

  1. Institut de Biologie et Chimie des Protéines, Université Lyon 1, Univ Lyon, CNRS FRE 3310, IFR128 BioSciences Gerland-Lyon Sud, 7 Passage du Vercors, 69367, Lyon Cedex 7, France

    Emeline Perrier, Marie-Claire Ronzière, Frédéric Mallein-Gerin & Anne-Marie Freyria

  2. Inserm Unité 577, Université « Victor Segalen », Bordeaux 2, 33076, Bordeaux, France

    Reine Bareille

  3. Laboratoire de Physiopathologie, Pharmacologie et Ingénierie Articulaires, UMR 7561 CNRS-Nancy Université, 9, avenue de la Forêt de Haye, 54505, Vandœuvre-Lès-Nancy, France

    Astrid Pinzano

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  1. Emeline Perrier
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  2. Marie-Claire Ronzière
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  3. Reine Bareille
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  4. Astrid Pinzano
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  5. Frédéric Mallein-Gerin
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  6. Anne-Marie Freyria
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Corresponding author

Correspondence to Anne-Marie Freyria.

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Supplementary Fig. 1

Effect of FGF-2 during MSC expansion on chondrogenic gene expression during subsequent pellet culture. Effects were measured by real-time PCR on total RNA isolated on days 1, 12 and 24 and expressed in relative quantity. Collagen genes levels associated with the chondrogenic phenotype (COL1A1, COL2A1, COL6A1, COL9A1, COL10A1, COL11A1, COL11A2, COL12A1, COL27A1) were analyzed in MSCs isolated from 3 donors and amplified in the absence or presence of FGF-2 (+) during the fourth passage and further induced towards chondrogenesis in pellet culture in the absence (CTL) or presence of BMP-2 and TGF-β3 (BT). For reference, the values measured in human knee articular chondrocytes (Ach) cultured for 36 h at P0 for the different genes, are presented. Abbreviation: genes are listed in Table 1. Error bars show mean ± SE and ***= p<0.001; **=p<0.05 and *=p<0.01 (PDF 54 kb)

Supplementary Fig. 2

Effect of FGF-2 during MSC expansion on chondrogenic gene expression during subsequent pellet culture. Effects were measured by real-time PCR on total RNA isolated on days 1, 12 and 24 and expressed in relative quantity. Gene levels associated with the formation of chondrogenic and osteogenic extracellular matrix components (AGC1, SOX9 and MMP13) were analyzed in MSCs isolated from 3 donors and amplified in the absence or presence of FGF-2 (+) during the fourth passage and further induced towards chondrogenesis in pellet culture in the absence (CTL) or presence of BMP-2 and TGF-β3 (BT). For reference, the values measured in human knee articular chondrocytes (Ach) cultured for 36 h at P0 for the different genes are presented. Abbreviation: genes are listed in Table 1. Error bars show mean ± SE and ***= p<0.001; **=p<0.05 and *=p<0.01 (PDF 36 kb)

Supplementary Fig. 3

Expression of the IIA and IIB isoforms of the COL2A1 gene in pellet culture. MSCs obtained from donors 2 and 3 were treated in the same way as those from donor 1, presented in Figure 2a. Total RNA were extracted from the pellets after 12 and 24 days of culture and measured by conventional PCR using specific primers. GAPDH was used as the housekeeping gene (PDF 530 kb)

Supplementary Fig. 4

Deposition of extracellular matrix components in MSC pellet culture. Pellets were obtained after 24 days of culture, as described in Figure 4a, then fixed and immunostained for aggrecan and types I, II and VI collagens. Staining shows different amounts deposited for each ECM component, as well as areas with condensed cells or loose matrix. Results are obtained from one representative of three independent experiments. Bar = 100 μm (PDF 10503 kb)

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Perrier, E., Ronzière, MC., Bareille, R. et al. Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells. Biotechnol Lett 33, 2091–2101 (2011). https://doi.org/10.1007/s10529-011-0653-1

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