Investigating Combinatorial Extrinsic Regulation of Pluripotent and Neural Stem Cells for Applications in Regenerative Medicine

The potential for a biological stem cell to replenish itself as well as create diverse functional cell types leads to many applications in the field of regenerative medicine, such as disease modeling or cell replacement therapies. However, many of these applications rely on clear understanding and efficient control of regulatory processes that govern expansion and differentiation of stem cells. In its natural environment, a stem cell resides in a complex niche that presents multiple forms of instructive or selective signals simultaneously that act as “inputs” for cellular biochemical processes. Interestingly, combinatorial presentation of multiple signals have reported synergistic effects that cannot be predicted from linear addition of the signals in isolation. Here, we apply a novel in vitro culture system to systematically and effectively dissect combinatorial signaling environments for adult hippocampal neural stem cells and pluripotent stem cells to advance our fundamental understanding of these cellular systems as well as derive insights to aid in the clinic translation of potential cell replacement therapies.Numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between FGF-2, TGF-beta, and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-beta and BMP-4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell based therapies for neurodegenerative diseases. Additionally, the promising outlook for hPSC-derived cell therapies leads many to consider the development of manufacturing processes to meet the patient demand for such therapeutics. Toward this aim, 3D culture systems for hPSC differentiation are emerging because of their potential for higher expansion and yield of target cell types compared to 2D culture systems. Therefore, the ability to screen through a multifactorial parameter space of exogenous chemical cues for 3D hPSC cultures would greatly accelerate the pace of discovery and development of efficient in vitro differentiation protocols for target cell types of interest. Here, we demonstrate the advanced capabilities of a 3D micro-culture platform that we employ to screen through more than 1000 unique combinations of 12 independent 3D culture parameters to derive Olig2+Nkx2.2+ oligodendrocyte progenitor cells (OPCs) from hPSCs with 0.2% of the reagent volumes used in 96-well plates. We leverage novel fluorescent hPSC reporter cell lines to live-monitor proliferation and differentiation for over 80 days in the 3D micro-culture system. The robust data set enabled statistical modeling of the OPC differentiation process to uncover interactions and differential sensitivities to culture parameters such as hPSC seeding density, Retinoic Acid dose, Wnt pathway agonist CHIR dose and duration, SHH pathway agonist SAG dynamics, and combinations thereof. To show the generalizability of the platform, we then applied it to simultaneously assay 90 unique differentiation protocols to derive TH+ midbrain dopaminergic neurons from hPSCs. Overall, we demonstrate a strong methodology for upstream microscale screening/optimization to inform downstream scale-up processes to improve 3D production strategies of hPSC-derived CRTs.Finally, as stem cell therapeutics continue to emerge in the clinic, upscaling processes must be developed to bridge the gap between microscale screening and patient demand. Toward this aim, we describe a pilot study to assess a stirred bioreactor configuration for the upscaling of 3D thermoreversible gel-encapsulated OPC production from hPSCs. Initially, we use computational modeling to predict and quantify the effect of stirred agitation on glucose concentration and shear stress profiles at a cross-section of the bioreactor vessel. Then, we use a pilot bioreactor system to measure hPSC viability, proliferation, and OPC differentiation with and without stirred agitation. Coupled together, screening and upscaling processes serve to accelerate the pace of discovery and development of much needed CRTs