In this work, we developed a microcarrier (MC)-based culture system that allows for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells (hNPCs) under defined conditions. In turn, this MC approach was implemented in a rotating wall vessel (RWV) bioreactor for the large-scale expansion and neuronal differentiation of hNPCs. This work is of significance as it overcomes current limitations of conventional two dimensional (2-D) culture systems to enable the generation of hNPCs and their neuronal derivatives in quantities required for downstream applications in disease modeling, drug screening, and regenerative medicine.
Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cells (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular ‘raw material’ needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.
Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery.
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