Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs)

Abstract

Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for the generation of neural stem/progenitor cells (NSCs/NPCs) from iPSCs within 7 days and of functional mature neurons. Our method yields a PAX6-positive homogeneous cell population, a cortical NSCs/NPCs, and the resultant NSCs/NPCs can be cryopreserved, expanded, and differentiated into functional mature neurons. Moreover, our protocol will be less expensive than other methods since the protocol requires fewer neural supplements during neural induction. This article also presents the FM1-43 imaging assay, which is useful for the presynaptic assessment of the iPSCs-derived human neurons. This protocol provides a quick and simplified way to generate NSCs/NPCs and neurons, enabling researchers to establish in vitro cellular models to study brain disease pathology.

Keywords: FM1-43; Human Induced Pluripotent Stem Cell; Neural Induction; Neural Progenitor Cell; Neural Stem Cell; Neuron.

Figure 1.. Schematic diagrams of neural induction (for NSCs/NPCs and neurons) from human iPSCs.

Time points of differentiation, corresponding steps, and culture conditions are shown. A. NSCs/NPCs induction from iPSCs (days -1-7). B. Neuronal differentiation from NSCs/NPCs (neuronal day 0: N0-N56).

Figure 2.. Representative phase-contrast images of cells during neural differentiation from iPSCs into NSCs/NPCs.

A. Undifferentiated iPSC colonies kept on MEF feeder cells before starting neural induction. B. iPSC colonies on a Geltrex-coated well on the day of starting the induction. C. Day 7 of successful neural induction, P0 NSCs/NPCs displaying uniform and compact morphology are ready to be harvested and expanded. D. Day 7 of failed neural induction. The cell population consists of flat non-neural cells with a heterogeneous morphology (indicated by red arrows) and compact NSCs/NPCs (indicated by blue arrows). To purify, it is required to pick up the compact NSCs/NPCs. E. P1 NSCs/NPCs at 24 h after the harvest and a passage of P0 NSCs/NPCs. F. P2 NSCs/NPCs show uniform morphology and grow in a honeycomb-like structure. Scale bars, 500 μm.

Figure 3.. Immunofluorescence staining of NSCs/NPCs with antibodies for neural stem cell markers.

iPSC-derived NSCs/NPCs (P1) are immunostained with anti-SOX1 (green), anti-NESTIN (red) (A), and anti-PSA-NCAM (green), anti-PAX6 (red) antibodies (B). Cell nuclear was identified by Hoechst staining (blue). Scale bars, 200 μm.

Figure 4.. Immunofluorescence staining of neurons differentiated from NSCs/NPCs.

iPSC-derived neurons are immunostained with anti-Tuj1 (red) (A), and anti-MAP2 (green) antibodies (B). Cell nuclear was also stained with Hoechst (blue). Scale bars, 200 μm.

Video 1.. Immunofluorescence Tutorial.

In this tutorial, we cover the basics of immunostaining cells with primary and fluorescence-labeled secondary antibodies.

Figure 5.. Functional analysis of human neurons with FM1-43 fluorescent dye.

Mature neurons were used for functional analysis with FM1-43 imaging, a powerful tool to determine exocytotic activity. A. Bright and fluorescent images of neurons loaded with FM1-43 dye before the stimulation to induce cell depolarization. B. 60 s after the stimulation. Scale bars, 25 μm. C. The time-course of the intensity of FM1-43 fluorescence before and after the stimulation. The reduction of FM1-43 intensity can be recognized as an exocytosis. A high KCl solution (final 50 mM) was added to evoke the cell depolarization.