Capture of Neuroepithelial-Like Stem Cells from Pluripotent Stem Cells Provides a Versatile System for In Vitro Production of Human Neurons

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds

All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS

Summary Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, “Optopatch,” pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation

All-optical electrophysiology for high-throughput functional characterization of a human iPSC-Derived motor neuron model of ALS

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, 'Optopatch,' pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation

Neuronal differentiation of lt-NES cells.

<p>(<b>A–B</b>) Four weeks after growth factor removal, lt-NES cells differentiated predominantly into neurons expressing beta III-tubulin (90%), MAP2ab and the neurotransmitter GABA, plus a minor fraction of GFAP-positive astrocytes (10%). Error bars represent STD. (<b>C</b>) Following growth factor withdrawal the clonal line PKa-2 gave rise to glia and neurons of preferentially GABAergic phenotype, comparable to the parental line PKa. (<b>D</b>) Specific neuronal subtypes expressing TH, 5HT and HB9 could be observed after 4 weeks of differentiation; O4-positive oligodendrocytes were detected after 10 weeks of differentiation. (<b>E</b>) AF22 lt-NES cells stained for Nestin and beta III-tubulin at day 0, 5, 8, 11, 18, 22 of differentiation. (<b>F</b>) Proliferating AF22 cells expressing GFP under the control of the Nestin enhancer (Nestin-GFP) show double labeling with an antibody to the Nestin protein. (<b>G</b>) Nestin-GFP expression in AF22 cells at day 0, 5, 7, 9, 11, 13, 15 of differentiation under control conditions or after exposure to DAPT (2 µM). Scale bars: 100 µm.</p

Long-term self-renewing neuroepithelial stem cells (lt-NES cells) derived from pluripotent stem cells of different origins.

<p>(<b>A</b>) Representative pictures of the lt-NES cell lines PKa and AF22, derived from iPSCs generated from reprogrammed adult dermal fibroblasts. The iPSCs were induced to form neural rosettes, which were isolated and expanded into lt-NES cell lines. Lt-NES cells exhibit a rosette-like growth pattern, stain positive for Sox2, Dach1, Nestin and PLZF and show apical expression of the tight junction protein ZO-1. (<b>B</b>) Endpoint RT-PCR analysis (30 cycles) reveals expression of the neural progenitor markers SOX1, PAX6, HES5 and the neural rosette markers PLZF, DACH1, MMNR1 and PLAGL1. Lt-NES cells also express telomerase and high levels of the hindbrain marker GBX2. Fetal human cortex (FC) was used as control. (<b>C</b>) Lt-NES cells exhibit a stable karyotype over extensive passaging as assessed by G-banding (line AF22 at passage 30). (<b>D</b>) Growth kinetics of the three lt-NES cell lines AF22 (dark blue), AF23 (green), and AF24 (light blue) as measured by change in % confluence over time. (<b>E</b>) The clonal lt-NES line AF22:3, derived by single cell deposition into 96-well plates, displays a morphology indistinguishable from its parental line AF22 and stains positive for Sox2 and Nestin. (<b>F</b>) The cell surface expression of CD133/PROMININ by AF23 and AF22 lt-NES cells was analyzed using flow cytometry. Debris was excluded by use of TO-PRO-3, and both non-stained cells and cells stained with only the secondary antibody were used to set up the gate (negative control). (<b>G</b>) Early passage lt-NES cells (PKb, PKc) still express forebrain markers such as FOXG1 and OTX2. However, expression is lost at higher passages. P, passage number; white scale bars: 100 µm; red scale bars: 10 µm.</p

Gene expression analysis of lt-NES cells and fetal NS cells.

<p>(<b>A</b>) A total of six cell lines representing two cell types (lt-NES and fetal NS) of different origins were profiled using TLDAs. Hierarchical cluster visualization (Pearson) shows separation into two major clusters representing the two cell types analyzed. (<b>B</b>) Correlation plots based on CT gene expression values of the individual cell lines. The lt-NES cell lines AF24 and AF22 show a higher degree of correlation than the lt-NES cell line AF24 and the NS line CB660, although the latter carry the same genome (R = 0.95 vs. R = 0.76). Red dots represent the median aggregated CT expression value of the 18S endogenous control; blue dots indicate the median aggregated CT expression values of all other genes on the TLDA. (<b>C</b>) Relative levels of differently expressed genes (corrected p-value threshold 0.05) in 9 lt-NES cell lines and 3 fetal NS cell lines. (<b>D–H</b>) Panels depicting the expression of markers representing the categories neural stem cell (NSC) markers, ventricular zone (VZ) expressed transcription factors (TF), regional position, rosette genes, and cell cycle. Data in C–H are depicted at a Log10 scale with the mean expression in fetal NS cells set to 1. No significant differences in expression levels were detected in ESC- vs. iPSC-derived lt-NES cells.</p

Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1+/+ stem cell line do not display the hyperexcitability phenotype. SOD1A4V/+ ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates

List of lt-NES cell lines, their origins, the reprogramming method used and the capacity of the pluripotent cells lines to form neural rosettes.

<p>(KSR) knock out serum replacement, (zfFGF2) zebrafish fibroblast growth factor 2, (BMP4) bone morphogenic protein 4, (LIF) leukemia inhibitory factor, (ADF) adult dermal fibroblasts, (hESC) human embryonic stem cells, (NSC) neural stem cells, (VSV-G) vesicular stomatitis virus G protein.</p

Induction of midbrain dopaminergic neurons using extrinsic factors.

<p>IPS cell derived lt-NES cells cultured for 7 days in the presence of sonic hedgehog (Shh) and FGF8 showed nuclear expression of Lmx1a and FoxA2 (<b>A</b>). (<b>B</b>) With prolonged (14 days) exposure to Shh and FGF8, the number of cells expressing FoxA2 gradually increased. Shh/FGF8-treated cells were also positive for the midbrain marker En1, whereas neither FoxA2 nor Lmx1a were expressed in control populations cultured in the absence of Shh/FGF8. (<b>C–D</b>) Growth factor removal from lt-NES cell lines grown in Shh/FGF8 for 14 days induced differentiation and appearance of cell clusters positive for FoxA2 and tyrosine hydroxylase (TH), with beta III-tubulin-positive neurons co-expressing Nurr1. In contrast, control cultures propagated in FGF2/EGF remained TH-negative. Abbreviations in upper right corners of the immunofluorescence photomicrographs denote lt-NES cell lines used. Scale bars: 50 µm.</p