Abstract
Human pluripotent stem cells (hPSCs) have potential to differentiate to unlimited number of neural cells, which provide powerful tools for neural regeneration. To date, most reported protocols were established with an animal feeder system. However, cells derived on this system are inappropriate for the translation to clinical applications because of the introduction of xenogenetic factors. In this study, we provided an optimized paradigm to generate region-specific forebrain neurons from hPSCs under a defined system. We assessed five conditions and found that a vitronectin-coated substrate was the most efficient method to differentiate hPSCs to neurons and astrocytes. More importantly, by applying different doses of purmorphamine, a small-molecule agonist of sonic hedgehog signaling, hPSCs were differentiated to different region-specific forebrain neuron subtypes, including glutamatergic neurons, striatal medium spiny neurons, and GABA interneurons. Our study offers a highly defined system without exogenetic factors to produce human neurons and astrocytes for translational medical studies, including cell therapy and stem cell-based drug discovery.
Highlights
Dissociated with dispase was the most efficient condition to differentiate to neurons
The Human pluripotent stem cells (hPSCs)-derived neuroepithelial cells were patterned to nearly pure dorsal forebrain (PAX6 + ), lateral ganglionic eminence (MEIS2 + ), or medial ganglionic eminence (NKX2.1 + ) progenitors under xeno-free condition, which further matured to cerebral cortex glutamatergic neurons, medium spiny neurons (MSNs), or GABA interneurons, respectively
Vitronectin improves the efficiency of neural differentiation from hPSC under defined conditions
Summary
Dissociated with dispase was the most efficient condition to differentiate to neurons. By using the optimized conditions and embryoid body (EB) protocol, hPSCs were robustly differentiated to forebrain neurons (FOXG1 + ) with high efficiency (> 90%). We specified hPSCs to region-specific forebrain neurons by applying different doses of purmorphamine (Pur), a substitute for SHH. The hPSC-derived neuroepithelial cells were patterned to nearly pure dorsal forebrain (PAX6 + ), lateral ganglionic eminence (MEIS2 + ), or medial ganglionic eminence (NKX2.1 + ) progenitors under xeno-free condition, which further matured to cerebral cortex glutamatergic neurons, MSNs, or GABA interneurons, respectively. Our current research provides an opportunity that effectively generates region-specific neurons under xeno-free condition,moving forward hPSC-derived neurons for translational applications concerning the treatment of forebrain neuron-associated diseases
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