There is an error in the second sentence of the fourth paragraph in the “Design of a robust survival assay for purified human motor neurons” section of the Results. The correct sentence is: Doses of GDNF, BDNF, CNTF and IGF-1 ranging from 2 pg/mL to 10 ng/mL were first tested alone for their effects on survival at day 31+3+7 (Figure 5C to 5F).
The incorrect scale bar was used in the figure legends for Figs. 1, 2, 3, 4 and 6. The scale bar should be μm instead of μM. The authors have provided corrected versions of Figs. 1, 2, 3, 4, and 6 here.
(A) Live fluorescent human motor neurons derived from the Hb9::GFP reporter line at day 31+13 after growth with a cocktail of neurotrophic factors (NTFs). (B) Automated quantification of fluorescent cells with significant neurite outgrowth (SNO) using the Neurite Outgrowth module of MetaMorph software; cells counted are identified with a red overlay. Motor neurons were considered to have significant neurite outgrowth when their overall neurite length exceeded 75 μm (scale bar). (C) Representative image of immunostained Hb9::GFP hESC-motor neuron cultures at day 31+13 after growth with a cocktail of neurotrophic factors (NTFs). Scale bar = 50 μm. (D) Number of cells with significant neurite outgrowth (SNO) when grown with (red bars) or without (blue bars) neurotrophic factors, expressed as a percentage of numbers at day 31+1. The increase in motor neuron numbers after day 31+7 in NTF-supplemented cultures suggests ongoing neurogenesis. Surviving fluorescent GFP-positive motor neurons with SNO shown as mean ± s.e.m., n>5 (t-test, ***p<0.001, *p<0.05). (E) BrdU-positive Hb9::GFP-positive motor neurons (arrows) at day 31+15 confirming the presence of newborn human motor neurons in culture. Scale bar = 50 μm. (F) The percentage of Hb9::GFP-positive motor neurons that were BrdU-positive at day 31+15 is not changed by NTFs but (G) total numbers of BrdU-positive motor neurons are increased with NTFs. Bars indicate mean ± s.e.m., n = 3 (t-test, *p<0.05; n.s. = not significant).
(A) Screening of 160 compounds for their potential to increase the number of human motor neurons in hESC cultures at day 31+13. Compounds were tested in quadruplicate at a single concentration (10 μM). Values are plotted as mean fold difference in motor neuron numbers relative to the negative control condition (No NTFs). The Rho-kinase (ROCK) inhibitor Y-27632 was the compound showing the highest capacity to increase the number of human motor neurons. (B) Y-27632 increases the number of fluorescent hESC-motor neurons in mixed cultures in a dose-dependent manner. Cells were cultured in the absence of neurotrophic factors and in the presence of increasing concentrations of Y-27632. Values shown as mean ± s.e.m., n = 4. (C) Representative images of hESC-motor neuron cultures at day 31+13 grown under neurotrophic factor deprivation (No NTFs), neurotrophic factor supplementation (NTFs + F + I) and Y-27632 (10 μM). Scale bar = 25 μm. (D) Time-dependent increase in the number of motor neurons in the presence (green) but not absence (blue) of Y-27632 (10 μM), with a peak effect at day 31+9. Values shown as mean ± s.e.m., n>5 (t-test, *p<0.05; **p<0.01). (E) Y-27632 also increases the total number of cells in culture. Mean ± s.e.m., n = 3. (F) Hb9::GFP-positive neurons continue to express motor neuron markers HB9 and ISL1 after treatment with Y-27632 for 9 days. Scale bar = 50 μm. (G) Supplementation of cultures with Y-27632 (red line) leads to increased numbers of human motor neurons expressing endogenous ISL1 at day 31+9. Mean ± s.e.m., n = 3 (**p<0.01).
(A) Y-27632-supplemented cultures contain increased numbers of OLIG2-positive cells at day 31+9. Scale bar = 50 μm. (B) Time-dependent increase in numbers of OLIG2-expressing progenitors in the presence of Y-27632. Data normalized to control at day 31+1; mean ± s.e.m., n>5 (t-test, **p<0.01). (C) OLIG2 progenitors at day 31+9 stained for BrdU. Scale bar = 25 μm. (D) Percent of OLIG2 precursors that are BrdU-positive at day 31+9 (mean ± s.e.m., n = 4). (E) Hb9::GFP-expressing motor neurons at day 31+9 stained for BrdU. Scale bar = 25 μm. (F) Percent motor neurons that are BrdU-positive at day 31+9 (mean ± s.e.m., n = 4). (G) The total number of cells in culture is increased at day 31+9 following Y-27632 treatment of hESC RUES1 and hiPSC18c. Values are mean ± s.e.m., n≥3 (t-test, *p<0.05). (H) Numbers of OLIG2 precursors increase significantly at day 31+9 following Y-27632 treatment of hiPSC 18c. Values are mean ± s.e.m., n≥3 (t-test, *p<0.05). (I) Numbers of motor neurons identified by staining for endogenous HB9 increase significantly at day 31+9 following Y-27632 treatment of hESC RUES1 and hiPSC 18c. Values are mean ± s.e.m., n≥3 (t-test, *p<0.05). (J) Cultures from healthy control hESCs (RUES1) or hiPSCs (18c) immunostained for the motor neuron marker HB9 and the pan-neuronal marker β-III tubulin. Y-27632 increases the number of motor neurons in each case. Scale bar = 25 μm.
(A) Y-27632 supplementation for 3 days leads to a 1.8-fold increase in motor neuron yield judged by FACS analysis. Data normalized to controls without Y-27632. Values are mean ± s.e.m., n>5 (t-test, **p<0.01). (B) Nine-day treatment with Y-27632 gives a ~5-fold increase in motor neuron yield as compared to controls without Y-27632, as quantified by flow cytometry. Values are mean ± s.e.m., n>5 (t-test, **p<0.01). (C) FACS purification of Hb9::GFP motor neurons expanded with Y-27632 for 3 days. Representative FACS gating used to retrieve an almost pure (>95%) population of human motor neurons. (D) FACS-purified motor neurons at day 31+3+1 stained for GFP (green), and a combination of HB9 and ISL1 (“pan-MN”; white nuclei).>95% of the FACS-purified cells in culture are Hb9::GFP positive. Scale bar = 25 μm. (E) Even following FACS sorting, some contaminant cells were able to proliferate and form colonies that interfered with survival assays (left panel). Uridine/Fluorodeoxyuridine (U/FdU) (each at 1 μM) successfully prevented the proliferation (right panel).
(A) The plating efficiency of FACS-purified human motor neurons after 24 hours is not increased in the presence of Y-27632. (B) Y-27632 enhances the survival of FACS-purified human motor neurons in a 7-day survival assay. Scale bar = 200 μm. (C) Dose-dependent effects of Y-27632 on human motor neuron survival, expressed relative to the basal condition (0 μM). Values shown as mean ± s.e.m., n≥5 (t-test, *p<0.05; **p<0.01).
Citation: The PLOS ONE Staff (2015) Correction: Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures. PLoS ONE 10(3): e0119195. https://doi.org/10.1371/journal.pone.0119195
Published: March 18, 2015
Copyright: © 2015 The PLOS ONE Staff. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited