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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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The ROCK inhibitor Y-27632 increases human motor neuron numbers in hESC-derived motor neuron cultures.(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).
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pone-0110324-g002: The ROCK inhibitor Y-27632 increases human motor neuron numbers in hESC-derived motor neuron cultures.(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).

Mentions: Compounds (10 µM in quadruplicate wells) were added on the day of seeding and motor neurons were counted at day 31+13, the time point at which the greatest differences in human motor neuron numbers between control and NTF-supplemented cultures were observed (Figure 1C, p<0.001). Most compounds showed no effect, and a significant number resulted in lower motor neuron numbers than the negative control condition (Figure 2A). In contrast, two compounds increased motor neuron numbers by>1.4 fold compared to basal conditions (Figure 2A). The most significant increase (1.9-fold) was induced by the Rho kinase (ROCK) inhibitor Y-27632 (Figure 2A, Y-27632 vs. No NTFs, p<0.05).


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)

The ROCK inhibitor Y-27632 increases human motor neuron numbers in hESC-derived motor neuron cultures.(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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206291&req=5

pone-0110324-g002: The ROCK inhibitor Y-27632 increases human motor neuron numbers in hESC-derived motor neuron cultures.(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).
Mentions: Compounds (10 µM in quadruplicate wells) were added on the day of seeding and motor neurons were counted at day 31+13, the time point at which the greatest differences in human motor neuron numbers between control and NTF-supplemented cultures were observed (Figure 1C, p<0.001). Most compounds showed no effect, and a significant number resulted in lower motor neuron numbers than the negative control condition (Figure 2A). In contrast, two compounds increased motor neuron numbers by>1.4 fold compared to basal conditions (Figure 2A). The most significant increase (1.9-fold) was induced by the Rho kinase (ROCK) inhibitor Y-27632 (Figure 2A, Y-27632 vs. No NTFs, p<0.05).

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