| Summary: | <p>As greater therapeutic potential is realized with human Mesenchymal stromal cells (hMSCs), the demand for hMSCs continues to grow. In order to improve the in vitro production rate and consistency, the effect of two key culture parameters, oxygen and culture surface, needs to be better understood. This study utilized biological experiments, mathematical modelling and RNA-sequencing to advance the knowledge on how in vitro hypoxia, and surface topography and hydrophobicity affect hMSCs growth rate.</p> <p>Simulation and experimental results showed that severe hypoxia reduced, while mild hypoxia enhanced hMSCs growth rate. A novel linkage on how different oxygen levels regulate the cell cycle mechanism is proposed - through the rivalry effect of HIF1α and HIF2α. The highest growth rate was experimentally observed under 1% oxygen in two sources (one immortalized and one primary) of bone marrow-derived hMSCs.</p> <p>Under normoxia, a shift in metabolism (represented by lactate production) was observed between slow-growing and fast-growing hMSCs. A flux-balance, genome-scale mathematical model was utilized with the incorporation of the proteomic resource allocation theory to capture the observed shift. Simulation results suggested the phenomenon was caused by the tricarboxylic acid cycle becoming interrupted at higher growth rates. Under hypoxia (1%), this phenomenon was not observed. Lactate production was found to positively correlate with growth rate with the interrupted Tricarboxylic acid cycle under all simulated growth rates.</p> <p>Surfaces with different topographies and chemistry treatments were synthesized to culture hMSCs and were processed through RNA sequencing. hMSCs cultured on fibrous surfaces showed significantly lower growth rates and differentially regulated transcriptomes. The effect of different chemistry treatments was small, but its synergistic effect with topographical changes could be observed. Among the biomarkers commonly used for classifying hMSCs, many were significantly down-regulated on fibrous surfaces. Network and cluster analysis yielded a list of biomarkers that are not sensitive to changes in surface conditions. In addition, surface topography was found to significantly affect the expression of HIF2A and many HIF2A-correlated downstream targets. This finding suggests the possibility of designing future biomaterials to selectively activate or suppress certain hypoxic responses.</p> <p>The findings from this work advance the understanding of how hypoxia and culture surfaces regulate the growth rate of hMSCs and encourages future studies to engineer an optimized set of culture conditions for hMSCs expansion in vitro.</p>
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