Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Abstract

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Keywords: high-throughput screening; induced pluripotent stem cells (iPSCs); neural progenitor cells; neural stem cells; neuronal differentiation.

Figure 1.

Schematic flow diagram to depict the stages of differentiation into neurons from induced pluripotent stem cells (iPSCs). NSCs: neural stem cells; NPCs: neural progenitor cells; NLSs: neurosphere-like structures.

Figure 2.

Expression of neural stem cell markers in cellular aggregates developing into neural rosettes observed in differentiating iPSCs cultured in NP expansion medium. Forming neuronal rosettes are more clearly distinguishable in Hoechst staining where nuclei of each neural rosette are arranged radially around a central cavity. Scale bar is 50 μm.

Figure 3.

Generation of NLSs and neural rosettes. (a) NLSs generated from cellular aggregates developing into neural rosettes. (b) NLSs generated from single cell suspension of cellular aggregates in Aggrewell plates. (c) Microphotograph of neural rosettes observed 12 hours after plating purified NLSs. Scale bar is 50 μm.

Figure 4.

Characterization of iPSC-derived NSCs/eNPCs. (a) Left: Immunoblots showing the expression of markers specific for NSCs (MUSASHI, PAX6, NESTIN and SOX1), late NPCs (NEUROD1) and neurons (CALRETININ, TUJ1, and MAP2) in iPSC-derived NSCs/eNPCs. Right: Normalized expression of NSC markers. The data represent an average of 3 independent experiments. (b) Immunocytochemical analysis of iPSC-derived NSCs/eNPCs. Scale bar is 50 μm. (c) Quantification of PAX6 expression using High Content Analysis. RFU: relative fluorescence unit. A01-A03 denote biological replicates. The distribution of PAX6 labeling among NSCs/eNPCs is shown in the histograms. The histograms indicate RFU intensity in the vertical axis and number of cells at that intensity in the horizontal axis. The points outside the histograms indicate outliers. The tables below the histograms indicate the number of cells measured in the populations (Count), the average (Avg) and standard deviation (StdDev) of the overall PAX6 labeling in the population. Greater than 99% of the cells have PAX6 FITC levels above background. The average background level in the well is <400 gray levels. The minimum level in the cell is around 1,000 to 2,000. More than 99% of the cells show levels greater than 500.

Figure 5.

Immunostaining of iPSC-derived neurons with TUJ1 (a), MAP2 (b), VGLUT1 (c), and NR1 subunit of the NMDA receptor (d). The percentage of TUJ1 positive neurons was analyzed by flow cytometry; cells were permeabilized with Cytofix/Cytoperm (Becton Dickinson) and dead cells were excluded from the analysis. Filled histogram shows the fraction of TUJ1 stained cells compared to secondary antibody staining (e). Scale bar is 50 μm in a-c, and 75 μm in d.

Figure 6.

Immunostaining of iPSC-derived neurons with CALBINDIN (a), CART (b), FEZ2 (c), and HS3ST2 (d). Scale bars are 75 μm.

Figure 7.

Electrophysiological recordings of iPSC-derived neurons. Example traces of whole-cell voltage-clamp recordings from HFF1-S cells in response to application of the indicated agonists. Currents were recorded in a Mg⁺-free extracellular solution at a holding potential of −85 mV (a–c) or 15 mV (d) during approximately 5 s applications (indicated by horizontal bar above each trace) of 100 μM glutamate + 100 μM glycine (a, peak current −494.3 pA), 100 μM AMPA (b, peak current = −261.8 pA), 100 μM NMDA + 100 μM glycine (c, peak current = −324.4 pA), or 1 mM GABA (d, peak current = 851.8 pA).

Figure 8.

Targeted proteomic comparison of iPSC-derived neurons and human cortex gray matter. Expression of representative presynaptic (A) and post-synaptic (B) proteins in iPSC-derived neurons (NSCs/eNPCs cultured in neurobasal medium for 6 weeks) and human cortex gray matter. (C). Distribution of the averaged difference between the 2 groups expressed as a ratio of iPSC-derived neurons: Cortex gray matter.