(F1228) DIRECTED DIFFERENTIATION OF MRNA-REPROGRAMMED IPSCS VIA BIOREACTORS GENERATES CLINICALLY RELEVANT YIELDS OF NEURAL STEM AND PROGENITOR CELLS THAT CAN BE ENGINEERED TO SECRETE THERAPEUTIC PROTEINS
Associate Scientist Factor Bioscience Inc. Cambridge, Massachusetts, United States
Abstract: Neural stem and progenitor cells (NSPCs) hold potential as a potent cell therapy because of their unique ability to provide trophic support, modulate neuroinflammation, and further differentiate into neurons and glia to restore structure and function to damaged areas of the CNS. Allogeneic brain-derived NSPCs engineered with viral vectors have been explored as a therapeutic modality; however, this approach faces major challenges that hinder its clinical translation, including limited source material and risk of viral integration. Here, we demonstrate a scalable process for differentiation of mRNA-reprogrammed induced pluripotent stem cells (iPSCs) to iNSPCs. Differentiation in a 0.1L bioreactor generated 1.9 x 10^6 iNSPCs with a percent yield >2-fold higher than a static differentiation performed in parallel. Bioreactor-derived iNSPCs were passaged in static and expanded approximately 17-fold over nine days, implying a theoretical yield of 3.2 x 10^7 cells from a 0.1L bioreactor (or 3.2 x 10^8 cells per liter), exceeding the number of cells administered in recent or ongoing clinical trials for ALS and retinitis pigmentosa, which ranges from 0.3 – 10.5 x 10^6 NSPCs per patient. Key iNSPC markers SOX1, nestin, and PAX6 were upregulated in both bioreactor- and static-derived iNSPCs; however, bioreactor-derived iNSPCs displayed approximately 4.6-fold higher expression of PAX6 and 2-fold lower expression of SOX10 (a negative iNSPC marker) than static-derived iNSPCs. After one additional passage and cryopreservation, bioreactor-derived iNSPCs were positive for SOX1 (90%) and nestin (95%), and negative for pluripotency markers TRA-1-60 and TRA-1-81 ( < 1%). Additionally, we demonstrated that iNSPCs could be non-virally engineered to overexpress neurotrophic factors. Electroporation with mRNA encoding glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) resulted in >1ng/mL of secreted protein, compared to undetectable GDNF levels and >25-fold lower BDNF levels in untransfected iNSPCs. Our results suggest that mRNA-reprogrammed iPSCs can be effectively differentiated using a scalable process to generate engineered iNSPCs, which could accelerate the clinical translation of cell therapies for neurodegenerative and neuroinflammatory diseases.
