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Expansion and differentiation of human pluripotent stem cells to neural progenitors: a simplified bioreactor process replacing Noggin with 2 small molecules

      Scaling up the production of human pluripotent stem cells (hPSCs) holds the key to the future realization of cell therapy. Conventionally, the cultivation and expansion of hPSCs is still based on the static tissue culture plate with limited surface area and requires repetitive passaging for expansion. We have developed a microcarrier based process for the expansion and differentiation of hPSCs to neural lineage. Using this platform, hPSCs in serum free medium can be expanded to 3×106 cells/ml with 15 fold expansion in a 100ml spinner flask. These expanded hPSCs were differentiated to neural progenitors (NPCs) using two small molecules (Dorsomorphin and SB 431542). The process was simple as medium exchange without manual manipulation required for embryoid body formation. By replacing recombinant protein Noggin with these two small molecules, the neural differentiation was shortened by 4 days to generate above 90% PSA-NCAM+ NPCs, achieving a higher cell concentration of 15×106 NPCs/ml (398 NPCs/hPSCs seeded) as compared to 6.1×106 NPCs/ml (163 NPCs/hPSCs seeded) achieved using Noggin based protocol. These NPCs can be further differentiated to dopaminergic neurons with positive expression of tyrosine hydroxylase. In conclusion, the microcarrier platform is robust and scalable. hPSCs and NPCs generated are significant higher than the conventional 2D platform. It is our long term goal to further develop this microcarrier based hPSC expansion and differentiation to specific cell types for cell therapy, disease studies, drug screening, and tissue engineering.
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