Abstract
Human pluripotent stem cells (hPSCs) play important role in studying the function of human glutamatergic neurons and related disease pathogenesis. However, the current hPSC-derived cortical system produced a significant number of inhibitory GABAergic neurons that reduced the purity of excitatory neurons. In this study, we established a robust hPSC-derived cortical neurogenesis system by applying the SHH inhibitor cyclopamine. Cyclopamine specified the dorsal cortical fate in a dose-dependent manner and enhanced the generation of cortical glutamatergic neurons, expressing PAX6, TBR1, TBR2, CTIP2, SATB2, and vesicular glutamate transporters (vGLUT). In contrast, the ventral patterning was inhibited and the GABAergic neurons were significantly reduced to 12% with the treatment of cyclopamine. In addition, we applied our current method to generate trisomy 21 iPSC-derived glutamatergic neurons that showed a robust reduction of vesicular glutamate transporters in the glutamatergic neurons with trisomy 21, revealing the developmental deficits in cortical glutamatergic neurons. Our method enriched the generation of cortical glutamatergic neurons which may facilitate the study of human neurological diseases and cell therapy.
Highlights
Cortical glutamatergic neuron originates from dorsal telencephalon and comprises the major excitatory network in the central nervous system[1]
Results human embryonic stem cells (hESCs)-derived telencephalic progenitors partially differentiated into ventral neurons by default differentiation. Human pluripotent stem cells (hPSCs) differentiated into dorsal telencephalic progenitors and subsequently largely became glutamatergic neurons without additional morphogens[9, 10, 27,28,29]
We observed that dorsal PAX6+ progenitors were mixed with the lateral ganglionic eminence (LGE) progenitors that expressing MEIS2 (Supplementary Fig. S2a) or ventral cells that expressing ISLET1 (Supplementary Fig. S2c)
Summary
Cortical glutamatergic neuron originates from dorsal telencephalon and comprises the major excitatory network in the central nervous system[1]. Inhibition of ventral patterning may enrich the population of glutamatergic neurons that derived from hPSCs. Sonic hedgehog (SHH) plays a key role in regulating the development of ventral telencephalon[3, 21]. The SHH inhibitor cyclopamine was applied to efficient inhibit ventral patterning during neuronal differentiation from mouse embryonic stem cells[22, 23]. We applied the SHH inhibitor cyclopamine to promote the differentiation of glutamatergic neurons from hPSCs. We showed that cyclopamine reduced the population of ventral GABAergic neurons, while enriched the cortical glutamatergic neurons significantly. We showed that cyclopamine reduced the population of ventral GABAergic neurons, while enriched the cortical glutamatergic neurons significantly Following this method, we applied cyclopamine www.nature.com/scientificreports/. Cyclopamine treated trisomy 21-glutamatergic neurons exhibited robust reduction of vesicular glutamate transporters in comparison to the treated euploid control, which were not observed in untreated excitatory neurons
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
