(193o) Metabolomics As a Quality Control Tool for Chondrogenic Differentiation in Spheroids: From Microaggregates to Microtissues

INTRODUCTION: Tissue Engineering aims to replace lost or regenerate malfunctioning tissues by man-made biological substitutes¹, holding great promise to provide effective therapies for diseases that were untreatable until now. The use of cartilage intermediate templates that will ossify upon implantation following endochondral ossification is becoming a promising bone tissue engineering strategy for the healing of large defects^2,3. Recently, the use of 3D cellular microaggregates has been introduced, as this format mimics the in vivo environment more closely allowing cell-cell and cell-extracellular matrix interactions⁴. The use of microaggregates for differentiation protocols allows a more controlled differentiation cascade to take place, due to their small size (exhibiting diameters <200µm). However, this promising process is not fully characterized as single parameter measurements such as evaluation of a few genes cannot robustly predict the final product quality while no mechanistic insight is gained. Considering the role of metabolism as a key regulator of stem cell fate^5-9 and the inherent capability of metabolomics to provide an extensive, high resolution and quantitative view of metabolic networks, the proposed approach in this study can highlight novel critical biochemical pathways and sensitive biomarkers indicative of functional bone tissue engineered constructs. In particular, we focus on the use of metabolomics for the comprehensive characterization of chondrogenic differentiation using microaggregates, improving the current conventional set of markers for monitoring cell and tissue quality.

METHODS: In this study, we present the development of a robust and optimized workflow for the exometabolome profiling of microaggregate culture and chondrogenic differentiation of hPDCs (human periosteum derived stem cells). HPDCs are mesenchymal stem cells that can be isolated from the periosteum. Subcultured cells were seeded in a 24 well plate containing microwell agarose insertsto create aggregates composed of 250 cells according to a previously established protocol¹⁰ that allowed high throughput production of microaggregates with controlled cell density per aggregate. To induce chondrogenic differentiation , the microwells were filled with 2 ml of an in-house developed chemically defined chondrogenic differentiation medium¹¹. Twice a week 50% of the medium was refreshed. Samples of medium supernatant were collected at Day 0 (5h), Day 7 , Day 14 and Day 21 as these time points capture the transition of (hPDC derived) chondrocyte phenotypes from proliferating to prehypetrophic and finally hypertrophic (based on gene expression of specific markers). Microaggregates were harvested at each of the aforementioned time points and total DNA content was quantified to determine the cell number (normalization strategy). Three biological replicates were carried out per time point. In this study we used LC-MS (liquid chromatography-mass spectrometry) metabolic profiling, enabling an accurate screening of metabolites in the extracellular environment, including amino acids, glucose, lactate among other substrates and byproducts.

RESULTS & DISCUSSION: Our results showed that hPDC microaggregates after the 1st week of culture presented a highly glycolytic metabolism, with high glucose consumption and lactate production rates. The profiles observed for glucose consumption and lactate synthesis suggest a complete conversion of pyruvate, generated as final product of glycolysis, to lactate. In the 1^st week we observed a significant increase (p<0.001) in the concentration of glutamine and glutamate in the spent media, while in the 2^nd week and towards the 3^rd week we observed a significant decrease (p<0.01). Additionally, α-ketoglutarate showed an increase (p<0.01) during the 1^st week, while we observed a significant decrease (p<0.01) towards the 3^rd week. Proline and hydroxyproline showed significant increase (p<0.01) during the process. As reported in other studies^12,13 glutamine can be converted to glutamate and then to α-ketoglutarate that catalyzes the formation of hydroxyproline, which is crucial for the formation of the collagen triple helix. A-ketoglutarate contributes to facilitate collagen synthesis by increasing the pool of proline residues¹². Through this improved proline and hydroxyproline formation α-ketoglutarate is believed to enhance bone tissue formation. We indeed noticed significant secretion of GAF rich extracellular matrix (ECM) after the 1^st week and these observations might be linked with the transition of hPDCs in ECM-synthesizing phenotypic and metabolic states. Serine had the most significant decrease (p<0.001) during the first 1 week where we observed cell growth (DNA content, EdU staining, ki67 gene expression). This consumption of serine could explain the significant increase of glycine (p<0.001) during the same culture time as these amino acids are biosynthetically linked and together are essential precursors for the synthesis of proteins, nucleic acids and lipids, crucial for cell growth¹⁴. Future experiments will include the use of ¹³C labeled nutrients, such as glutamine, for gaining insights into the mechanisms of chondrogenic differentiation in a 3D microaggregate setting.

CONCLUSION: The approach applied in this study enables an accurate metabolic profiling of chondrogenic differentiation of hPDCs cultured as microaggregates. To the best of our knowledge, this is the first secretome analysis of hPDCs cultured as microaggregates. We envisage that the application of the aforementioned metabolomics strategy will provide thorough understanding of the endochondral ossification process in this 3D culture set up, leading to a shift from empirical to rational approaches for the production of chondrogenic microtissues.

 

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