Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System - PubMed (original) (raw)

Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System

Laura I Hudish et al. Stem Cell Reports. 2020.

Erratum in

Abstract

Human motor neuron (MN) diseases encompass a spectrum of disorders. A critical barrier to dissecting disease mechanisms is the lack of appropriate human MN models. Here, we describe a scalable, suspension-based differentiation system to generate functional human MN diseases in 3 weeks. Using this model, we translated recent findings that mRNA mis-localization plays a role in disease development to the human context by establishing a membrane-based system that allows efficient fractionation of MN cell soma and neurites. In response to hypoxia, used to mimic diabetic neuropathies, MNs upregulated mitochondrial transcripts in neurites; however, mitochondria were decreased. These data suggest that hypoxia may disrupt translation of mitochondrial mRNA, potentially leading to neurite damage and development of neuropathies. We report the development of a novel human MN model system to investigate mechanisms of disease affecting soma and/or neurites that facilitates the rapid generation and testing of patient-specific MN diseases.

Keywords: cell compartments; diabetic neuropathies; fractionation; hypoxia; neurites; soma; stem cell-derived human motor neurons.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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Graphical abstract

Figure 1

Figure 1

Suspension Culture-Based Direct Differentiation Approach of Pluripotent Stem Cells into Human Motor Neurons (A) Schematic outlining the direct differentiation approach. (B) Representative images taken at the indicated magnification at key stages of the differentiation protocol. Scale bar, 200 μm. (C and D) Immunofluorescence analysis for neuronal markers ISL (C), SMI-32 (C'), merge (C”) as well as HB9 (D), βIII-TUBULIN (D') and merge (D”) of d20 MNs. Scale bar, 20 μm. (E) Quantification of HB9 and ISL1 positive cells over DAPI at d20, n = 3–4 independent experiments.

Figure 2

Figure 2

Direct Differentiation Results in Functional, Spatially Defined Human Motor Neurons (A) Global gene expression analysis of differentiated MNs in comparison with previously published datasets of hESC-derived motor neurons (Ziller et al., 2018). Gene expression values from each sample were correlated. Values represent Spearman rho correlation coefficients. (B) Quantitative PCR analysis of pluripotency markers NANOG and OCT4 normalized to TBP at subsequent stages of differentiation. n = 3–4 independent experiments; data are presented as standard error of the mean (SEM). (C) Quantitative PCR reveals expression of mature MN markers OLIG2 and MNX1 at d14, which is maintained at d23, at which point ISL1 expression also becomes highly enriched. n = 3–4 independent experiments; data are presented as SEM. (D–D″) Representative images of calcium imaging before, during, and after depolarization. Scale bar, 50 μm. (E) Whole-cell patch clamp recordings of cultured MNs. An example of evoked action potentials with a 50 pA step. Four sweeps overlaid. Y axis, millivolts; X axis, milliseconds. (E′ and E″) Additional example of evoked single or multiple action potentials from MNs. Action potentials were evoked by 25–50 pA steps. Y axis, millivolts; X axis, milliseconds. (F) Quantitative PCR analysis for HOX gene expression of mature MNs reveals a strong enrichment of HOXC8. Schematic showing the developmental distribution of HOX genes in humans. n = 4–8 independent experiments; data are presented as SEM.

Figure 3

Figure 3

Global Analysis of Motor Neuron Soma and Neurite-Enriched RNAs (A) Fractionation schematic. Neurons are plated on porous membranes that allow neurite growth through the membrane but restrict soma to the top of the membrane. Cells are then mechanically fractionated, and RNA and protein from each fraction are isolated and analyzed. (B) Dot blot of soma and neurite protein fractions from three independent fractionation experiments. Beta-actin is present in both fractions while histone H3 is restricted to the soma fraction. (C and D) (C) Hierarchical clustering of gene expression values from motor neuron RNA fractionations from ESC-derived MNs and (D) control iPSCs and T1D iPSC MNs. Values represent Spearman rho correlation coefficients. (E) Comparison of gene expression values in soma and neurite samples for ESCs, iPSC controls (E′), and T1D iPSCs (E″). Genes significantly enriched (p < 0.01, log2(fold change) >1.5) in either fraction are represented in purple. Three known neurite-enriched genes (NGRN, ACTB, and RANBP1) are highlighted and are neurite enriched in the data. (F) LR values for genes from two gene ontology categories: electron transport chain and structural constituent of ribosome in all three cell types (F, F″,F″). These ontology categories have been previously observed to contain many neurite-enriched genes. (G) Comparison of LR values from motor neuron data with previously published LR values from E15.5 mouse cortical neurons. Only one-to-one human-to-mouse orthologs are considered.

Figure 4

Figure 4

Hypoxia Induces Significant Changes in Gene Expression and RNA Localization in Motor Neurons (A) Principal component analysis of gene expression values from soma and neurite samples of normoxia and hypoxia-treated ESC MNs, (A′) iPSC controls, and (A″) iPSC T1D MNs. (B) LR values from normoxia and hypoxia-treated MNs. Genes with significant (p < 0.05) changes in LR between conditions are colored in purple. (C–E) Nuclear-encoded genes related to mitochondrial function, including those involved in the electron transport chain and the mitochondrial translation machinery are more localized to neurites upon hypoxia treatment, while those involved in cytosolic translation show no change in localization. In ESC-derived MNs (C), mitochondrially encoded genes are significantly less localized to neurites but show no significant changes in control iPSCs (D) and T1D iPSC MNs (E). p values are Wilcoxon rank sum values. (F and G) (F) Representative images (scale bar, 20 μm; inset, 50 μm) and (G) quantification of mitochondrial staining in normoxic and hypoxic conditions show a significant reduction of mitochondria stain intensity in the neurites cultured under hypoxic conditions n = 2 independent experiments, n = 8 technical replicates.

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