A proteomic analysis of chondrogenic, osteogenic and tenogenic constructs from ageing mesenchymal stem cells

Abstract

Background: Mesenchymal stem cells (MSCs) have prospective applications in regenerative medicine and tissue engineering but to what extent phenotype and differentiation capacity alter with ageing is uncertain. Consequently, any loss in functionality with age would have profound consequences for the maintenance of tissue viability and the quality of tissues. Proteomics enables the set of proteins responsible for a particular cell phenotype to be identified, as well as enabling insights into mechanisms responsible for age-related alterations in musculoskeletal tissues. Few proteomic studies have been undertaken regarding age-related effects on tissue engineered into cartilage and bone, and none for tendon. This study provides a proteome inventory for chondrogenic, osteogenic and tenogenic constructs synthesised from human MSCs, and elucidates proteomic alterations as a consequence of donor age.

Methods: Human bone-marrow derived MSCs from young (n = 4, 21.8 years ± 2.4SD) and old (n = 4, 65.5 years ± 8.3SD) donors were used to make chondrogenic, osteogenic and tenogenic tissue-engineered constructs. We utilised an analytical method relying on extracted peptide intensities as a label-free approach for peptide quantitation by liquid chromatography-mass spectrometry. Results were validated using western blotting.

Results: We identified proteins that were differentially expressed with ageing; 128 proteins in chondrogenic constructs, 207 in tenogenic constructs and four in osteogenic constructs. Differentially regulated proteins were subjected to bioinformatic analysis to ascertain their molecular functions and the signalling pathways. For all construct types, age-affected proteins were involved in altered cell survival and death, and antioxidant and cytoskeletal changes. Energy and protein metabolism were the principle pathways affected in tenogenic constructs, whereas lipid metabolism was strongly affected in chondrogenic constructs and mitochondrial dysfunction in osteogenic constructs.

Conclusions: Our results imply that further work on MSC-based therapeutics for the older population needs to focus on oxidative stress protection. The differentially regulated proteome characterised by this study can potentially guide translational research specifically aimed at effective clinical interventions.

Keywords: Ageing; Chondrogenesis; Label-free quantification; Mesenchymal stem cells; Osteogenesis; Oxidative stress; Tenogenesis.

Figures

Fig. 1

Histochemical and gene expression analysis of chondrogenic, osteogenic and tenogenic lineage differentiation for MSCs. Images are representative of all experiments. a MSC pellets cultured in control or chondrogenic media were fixed and stained with Alcian Blue (scale bar = 100 μm). Gene expression of b aggrecan, cCOL2A1 and dSOX9 following chondrogenic differentiation. Statistical evaluation was undertaken using Mann–Whitney U test (n = 6). e Osteogenic differentiation from MSCs was confirmed with Alizarin Red S staining at day 21 to visualise mineralised bone matrix following extraction of the calcified mineral from the stained monolayer at low pH. f Box and whisker plot showing quantitative results of Alizarin red staining. Statistical significance, Mann–Whitney U test p < 0.001 (n = 12). g Gene expression of RUNX2 following osteogenesis. h Histology images of a tendon construct made from MSCs stained with Masson’s Trichrome to identify collagenous matrix. Image was captured at ×4 magnification and ×10 magnification (inset, upper image) (scale bar = 100 μm). Example of more organised areas of collagen is marked on the inset image (red). (Lower image) Ultrastructural analysis using scanning TEM. Presence of aligned extracellular collagen fibrils (A) and less organised collagen (B) are inset (red) (scale bar = 1 μm). Tenogenic differentiation was also evaluated using gene expression of iCOL1A1, jSERPINF1 and kTHBS4. For gene expression, data are represented as 2–ΔCT compared with GAPDH. Statistical evaluation was undertaken using Mann–Whitney U test (n = 8) with data represented as 2–ΔCT compared with GAPDH. MSC mesenchymal stem cell

Fig. 2

Coomassie-stained one-dimensional SDS-PAGE of the guanidine-soluble protein extracts of chrondrogenic and osteogenic constructs and Rapigest™ extracts of tenogenic constructs compared with Rapigest™ extract of MSCs. Images are representative of all experiments. Equal protein loading by weight (30 μg per well) allowed a qualitative and grossly quantitative comparison of soluble protein extracts. Vertical black line indicates independent gels. Lines indicate digital splicing. MSC mesenchymal stem cell

Fig. 3

Pie charts depicting protein classification of DE proteins in ageing constructs using PANTHER. Proteins were demonstrated as DE when quantified with ProgenesisQI™ with at least two unique peptides, a 2-fold change in expression and q < 0.05. First row, chondrogenic constructs; second row, tenogenic constructs. a Biological processes, b molecular functions and c cellular components. Because osteogenic constructs had only four DE proteins with the filters of a 2-fold change in expression. p < 0.05 and q < 0.05, PANTHER analysis was undertaken on DE proteins quantified with ProgenesisQI™ with at least two unique peptides, a 2-fold change in expression and p < 0.05 (third row)

Fig. 4

IPA generated networks derived from the proteins with different abundance in the chondrogenic, tenogenic and osteogenic constructs derived from young and old MSCs. IPA identified that lipid metabolism signalling pathways were predominant in chondrogenic constructs (a). In tenogenic constructs, signalling pathways were enriched for glucose metabolic processes (b). In osteogenic constructs, the principle signalling pathway was mitochondrial dysfunction (c). One of the principle functions associated with the DE proteins in tenogenic constructs was also protein metabolism (d). Green nodes, greater protein abundance in young; red nodes, greater protein abundance in old; white nodes, proteins not differentially abundant between young and old. Intensity of colour is related to higher fold-change. Key to the main features in the networks is shown

Fig. 5

Western blotting validations of mass spectrometry results. COMP, biglycan and SOD1 abundance were confirmed by western blotting. Representative western blots for tenogenic constructs (a COMP and c biglycan) and chondrogenic constructs (e SOD1). Abundance of each protein is expressed semi-quantitatively (b, d, f) relative to total protein content. Statistical differences were assessed with age in the respective construct and antibody analysis using Mann–Whitney tests. Significant differences represented with p ≤ 0.05