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Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

Lamas NJ, Johnson-Kerner B, Roybon L, Kim YA, Garcia-Diaz A, Wichterle H, Henderson CE - PLoS ONE (2014)

Bottom Line: First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures.GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM).Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

View Article: PubMed Central - PubMed

Affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America; Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Minho, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Minho, Portugal.

ABSTRACT
Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

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Related in: MedlinePlus

Y-27632 enhances proliferation of motor neuron progenitors in hESC- and hiPSC-derived motor neuron cultures.(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.
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pone-0110324-g003: Y-27632 enhances proliferation of motor neuron progenitors in hESC- and hiPSC-derived motor neuron cultures.(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.

Mentions: To better understand the level at which Y-27632 exerts its effect, we next examined the expansion of motor neuron progenitors (pMNs), using OLIG2 as a marker [26]. Treatment with Y-27632 increased the number of OLIG2-positive cells ∼3.6-fold compared to controls by day 31+9 (Figures 3A and 3B, p<0.05) comparable to the ∼3.3-fold increase in DAPI-stained cells over controls over the same period [Figure 2E, p>0.05; Ratio DAPI/OLIG2 = 6.8∶1 (CONTROL) vs. 6.2∶1 (Y-27632)]. Application of BrdU from day 31 to day 31+9 led to nuclear labeling of 86% of OLIG2-positive cells, indicating that they are actively proliferating progenitors (Figures 3C and 3D). Accordingly, 74% of GFP-positive motor neurons on day 31+9 were BrdU-positive, indicating that they were born during the period of Y-27632 treatment (Figures 3E and 3F). Similar percentages were observed using fresh, unfrozen motor neuron preparations (not shown). Therefore Y-27632 non-selectively enhances cell proliferation in hESC-derived cultures, resulting in a ∼3.5-fold increase in the number of motor neuron progenitors that is likely to contribute significantly to the observed increase in postmitotic hESC-MNs.


Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

Lamas NJ, Johnson-Kerner B, Roybon L, Kim YA, Garcia-Diaz A, Wichterle H, Henderson CE - PLoS ONE (2014)

Y-27632 enhances proliferation of motor neuron progenitors in hESC- and hiPSC-derived motor neuron cultures.(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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206291&req=5

pone-0110324-g003: Y-27632 enhances proliferation of motor neuron progenitors in hESC- and hiPSC-derived motor neuron cultures.(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.
Mentions: To better understand the level at which Y-27632 exerts its effect, we next examined the expansion of motor neuron progenitors (pMNs), using OLIG2 as a marker [26]. Treatment with Y-27632 increased the number of OLIG2-positive cells ∼3.6-fold compared to controls by day 31+9 (Figures 3A and 3B, p<0.05) comparable to the ∼3.3-fold increase in DAPI-stained cells over controls over the same period [Figure 2E, p>0.05; Ratio DAPI/OLIG2 = 6.8∶1 (CONTROL) vs. 6.2∶1 (Y-27632)]. Application of BrdU from day 31 to day 31+9 led to nuclear labeling of 86% of OLIG2-positive cells, indicating that they are actively proliferating progenitors (Figures 3C and 3D). Accordingly, 74% of GFP-positive motor neurons on day 31+9 were BrdU-positive, indicating that they were born during the period of Y-27632 treatment (Figures 3E and 3F). Similar percentages were observed using fresh, unfrozen motor neuron preparations (not shown). Therefore Y-27632 non-selectively enhances cell proliferation in hESC-derived cultures, resulting in a ∼3.5-fold increase in the number of motor neuron progenitors that is likely to contribute significantly to the observed increase in postmitotic hESC-MNs.

Bottom Line: First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures.GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM).Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

View Article: PubMed Central - PubMed

Affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America; Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Minho, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Minho, Portugal.

ABSTRACT
Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

Show MeSH
Related in: MedlinePlus