Abstract
Limited access to human neurons, especially motor neurons (MNs), was a major challenge for studying neurobiology and neurological diseases. Human pluripotent stem cells (hPSCs) could be induced as neural progenitor cells (NPCs) and further multiple neural subtypes, which provide excellent cellular sources for studying neural development, cell therapy, disease modeling and drug screening. It is thus important to establish robust and highly efficient methods of neural differentiation. Enormous efforts have been dedicated to dissecting key signalings during neural commitment and accordingly establishing reliable differentiation protocols. In this study, we refined a step-by-step strategy for rapid differentiation of hPSCs towards NPCs within merely 18 days, combining the adherent and neurosphere-floating methods, as well as highly efficient generation (~90%) of MNs from NPCs by introducing refined sets of transcription factors for around 21 days. This strategy made use of, and compared, retinoic acid (RA) induction and dual-SMAD pathway inhibition, respectively, for neural induction. Both methods could give rise to highly efficient and complete generation of preservable NPCs, but with different regional identities. Given that the generated NPCs can be differentiated into the majority of excitatory and inhibitory neurons, but hardly MNs, we thus further differentiate NPCs towards MNs by overexpressing refined sets of transcription factors, especially by adding human SOX11, whilst improving a series of differentiation conditions to yield mature MNs for good modeling of motor neuron diseases. We thus refined a detailed step-by-step strategy for inducing hPSCs towards long-term preservable NPCs, and further specified MNs based on the NPC platform.
Highlights
The emerging technology of human-induced pluripotent stem cells reprogrammed directly from adult somatic cells such as primary fibroblasts opened a new era of stem cell biology, which has been rapidly applied in both basic science and translational medicine with the concerted efforts of biologists, clinic doctors and entrepreneurs
The differentiated neural progenitors and neurons are versatile for studying the molecular biology of neural induction [2,3,4] and transplantationbased cell therapy [5,6], as well as modeling the pathogenesis of diseases [7,8,9,10] and drug screening [11,12]. Those endeavors are especially useful in studying neurodegenerative diseases since murine models might not always truly recapitulate the human pathogenesis, due to species differences, and previous efforts have been hindered by limited access to patient neurons
MGCD0103 can greatly promote the generation of neural progenitor cells (NPCs) by 15-fold, as we have previously shown that HDACs, especially HDAC3, are critical regulators in neural differentiation [2]
Summary
Generation of Neural Progenitors and Motor Neurons from Human Pluripotent Stem Cells. Jie Ren 1,2,3 , Chaoyi Li 1,2,3 , Mengfei Zhang 1,2,3 , Huakun Wang 1,2,3 , Yali Xie 3,4 and Yu Tang 1,2,3,4,5, *. Aging Research Center, Xiangya Hospital, Central South University, Changsha 410008, China. The Biobank of Xiangya Hospital, Central South University, Changsha 410008, China
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