(190av) Using Metabolomics As a High-Sensitivity Quality Control Tool for the Characterization of Chondrogenic Microtissues

The use of cartilage intermediates is a promising skeletal tissue engineering (TE) strategy for the healing of large defects through endochondral ossification. In addition, the use of 3D microtissues is becoming a standard for such approaches, as this format allows cell-cell and cell-extracellular matrix interactions. Considering the role of metabolism as a key regulator of stem cell fate and the high sensitivity of metabolomics, which is able to provide a high resolution and quantitative view of metabolic networks, this study aims to identify metabolic quality attributes indicative of a functional cartilage intermediate TE construct.

We conducted LC-MS (liquid chromatography-mass spectrometry) tracer analysis experiments to gain mechanistic insight during chondrogenic differentiation of spheroids of hPDCs (human periosteum derived stem cells). ¹³C labeled glucose, glutamine but also serine and aspartate have been used, as these metabolites showed significant differences between the time points of interest in a prior exometabolomics study. Cell suspensions were drop seeded in 24 wells containing molded microwells at the bottom, resulting in the formation of spheroids containing 250 cells each. Twice a week 50% of the medium was refreshed. Samples were analyzed at day 0, day 14 and day 21. These time points capture the transition of hPDCs-derived chondrocyte phenotypes from a proliferating to a prehypertrophic and finally a hypertrophic state, based on gene expression of specific markers. Total DNA content was used to normalize the data.

Our tracer analysis results showed progressive ¹³C glucose enrichment in palmitate from 0% at day 0 to 8% at day 14 and 22% at day 21, suggesting activation of fatty acid synthesis. Furthermore, we observed ¹³C glutamine enrichment in proline from 0 % at day 0 to 20 % at day 14 and 42,5 % at day 21 and a similar trend of ¹³C glutamine contribution to hydroxyproline (from 0 % at day 0 to 35 % at day 14 and 38 % at day 21). These data indicate that glutamine contributes progressively to proline biosynthesis, an important amino acid for collagen production. In addition, we observed significant production of GAG (glucosaminoglycans) rich extracellular matrix (ECM) after Day 7. These observations suggest that the consecutive stages of chondrogenic differentiation of hPDCs are characterized by specific metabolic adaptations. To the best of our knowledge, this is the first metabolomics analysis of chondrogenic differentiation of an adult stem cell population cultured as spheroids. We envisage that metabolomics strategies for quality control will provide thorough understanding of hPDC metabolic shifts during chondrogenic differentiation in 3D culture leading eventually to the identification of measurable potency biomarkers.