Unveiling the Influence of Culture Conditions on Mesenchymal Stem Cells: A Transcriptome Sequencing Study.

Aims: To optimize the culture process of Mesenchymal Stem Cells (MSCs) and enhance their biological functions.

Background: MSCs have shown great potential in treating various diseases due to their low immunogenicity and potent paracrine effects. However, the inherent heterogeneity of MSC populations, which can vary depending on the culture conditions, may challenge large-scale clinical application.

Objective: This study investigates the inconsistency of MSCs cultured in different media, from the transcriptional level to biological functions.

Method: RNA sequencing was used to identify different expressed genes of MSCs separated and expanded in three media, which were then validated with qPCR. In vitro assays, including proliferation, tube formation, wound healing, multilineage differentiation, paracrine secretome and injured hepatocyte protection assay, were performed to verify the potential differences among three groups.

Result: MSCs cultured in platelet lysate-containing medium exhibited high expression of genes involved in extracellular matrix regulation, collagen metabolic processes, and angiogenesis, whereas those cultured in serum-free medium demonstrated high expression of genes associated with DNA replication and chromosome segregation. MSCs cultured under serum-containing medium indicated high levels of genes associated with extracellular matrix regulation, cartilage development, and chemotaxis. The results of functional comparative experiments were consistent with the differences in their gene expression patterns. Notably, MSCs cultured in the serum- containing system exhibited greater protective effect against hepatocyte activity.

Conclusion: Different culture conditions affect the biological functions of MSCs. Optimal conditions should be investigated for applications. Next, an in vivo model should be established to evaluate differences in MSC tissue repair function under various culture conditions.

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