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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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Purified human motor neurons show a potent response to known neurotrophic factors.(A) Whole-well imaging of live motor neurons labeled with calcein-AM captured using the Plate Runner (left two panels). Surviving human motor neurons were counted in whole culture wells in an automated manner using MetaMorph (red tracing, right two panels). Scale bar = 200 µm. (B) Y-27632-expanded motor neurons show enhanced survival in the presence of a cocktail of neurotrophic factors. Values shown as mean ± s.e.m., n>5 (t-test, ***p<0.001). (C) GDNF, (D) BDNF and (E) CNTF alone (blue lines) enhance the survival of expanded FACS-purified human motor neurons. The addition of F+I significantly potentiates the survival-inducing activity of GDNF at high concentrations. Values shown as mean ± s.e.m., n>4 (t-test, *p<0.05; **p<0.01; ***p<0.001). Asterisks on individual points represent significance of difference with No-NTF control (white rectangle in the curve); asterisks on bars represent significant differences between a given concentration of NTF and the corresponding value for NTF + F+ I. (F) The cocktail of neurotrophic factors (NTFs) enhances the survival of expanded FACS-purified human motor neurons in a dose-dependent manner in the presence of 10 µM forskolin plus 100 µM IBMX. Values shown as mean ± s.e.m., n≥5 (t-test, **p<0.01; ***p<0.001).
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pone-0110324-g005: Purified human motor neurons show a potent response to known neurotrophic factors.(A) Whole-well imaging of live motor neurons labeled with calcein-AM captured using the Plate Runner (left two panels). Surviving human motor neurons were counted in whole culture wells in an automated manner using MetaMorph (red tracing, right two panels). Scale bar = 200 µm. (B) Y-27632-expanded motor neurons show enhanced survival in the presence of a cocktail of neurotrophic factors. Values shown as mean ± s.e.m., n>5 (t-test, ***p<0.001). (C) GDNF, (D) BDNF and (E) CNTF alone (blue lines) enhance the survival of expanded FACS-purified human motor neurons. The addition of F+I significantly potentiates the survival-inducing activity of GDNF at high concentrations. Values shown as mean ± s.e.m., n>4 (t-test, *p<0.05; **p<0.01; ***p<0.001). Asterisks on individual points represent significance of difference with No-NTF control (white rectangle in the curve); asterisks on bars represent significant differences between a given concentration of NTF and the corresponding value for NTF + F+ I. (F) The cocktail of neurotrophic factors (NTFs) enhances the survival of expanded FACS-purified human motor neurons in a dose-dependent manner in the presence of 10 µM forskolin plus 100 µM IBMX. Values shown as mean ± s.e.m., n≥5 (t-test, **p<0.01; ***p<0.001).

Mentions: To develop a survival assay based on neurotrophic factor deprivation [31], [38], [39], FACS-sorted motor neurons were seeded in 96-well plates and stained using the vital dye calcein-AM. This had the advantage that it stained cell bodies and neurites more intensely than live imaging of GFP, which was no longer required to identify motor neurons. Numbers of surviving hESC-MNs were counted in whole culture wells in an automated manner using MetaMorph (Figure 5A). We first asked whether the survival of purified motor neurons was dependent on trophic support in these conditions. At day 31+3+7, motor neuron survival was enhanced ∼2.5-fold by a cocktail of NTFs (BDNF, CNTF, GDNF, IGF-1, each at 10 ng/ml) with F (10 µM) plus IBMX (100 µM) (Figure 5B), similar to published results using cultures of primary rodent motor neurons [39]–[41]. We tested forskolin and IBMX alone and found that they showed only slight innate neurotrophic activity (not shown).


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)

Purified human motor neurons show a potent response to known neurotrophic factors.(A) Whole-well imaging of live motor neurons labeled with calcein-AM captured using the Plate Runner (left two panels). Surviving human motor neurons were counted in whole culture wells in an automated manner using MetaMorph (red tracing, right two panels). Scale bar = 200 µm. (B) Y-27632-expanded motor neurons show enhanced survival in the presence of a cocktail of neurotrophic factors. Values shown as mean ± s.e.m., n>5 (t-test, ***p<0.001). (C) GDNF, (D) BDNF and (E) CNTF alone (blue lines) enhance the survival of expanded FACS-purified human motor neurons. The addition of F+I significantly potentiates the survival-inducing activity of GDNF at high concentrations. Values shown as mean ± s.e.m., n>4 (t-test, *p<0.05; **p<0.01; ***p<0.001). Asterisks on individual points represent significance of difference with No-NTF control (white rectangle in the curve); asterisks on bars represent significant differences between a given concentration of NTF and the corresponding value for NTF + F+ I. (F) The cocktail of neurotrophic factors (NTFs) enhances the survival of expanded FACS-purified human motor neurons in a dose-dependent manner in the presence of 10 µM forskolin plus 100 µM IBMX. Values shown as mean ± s.e.m., n≥5 (t-test, **p<0.01; ***p<0.001).
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Related In: Results  -  Collection

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

pone-0110324-g005: Purified human motor neurons show a potent response to known neurotrophic factors.(A) Whole-well imaging of live motor neurons labeled with calcein-AM captured using the Plate Runner (left two panels). Surviving human motor neurons were counted in whole culture wells in an automated manner using MetaMorph (red tracing, right two panels). Scale bar = 200 µm. (B) Y-27632-expanded motor neurons show enhanced survival in the presence of a cocktail of neurotrophic factors. Values shown as mean ± s.e.m., n>5 (t-test, ***p<0.001). (C) GDNF, (D) BDNF and (E) CNTF alone (blue lines) enhance the survival of expanded FACS-purified human motor neurons. The addition of F+I significantly potentiates the survival-inducing activity of GDNF at high concentrations. Values shown as mean ± s.e.m., n>4 (t-test, *p<0.05; **p<0.01; ***p<0.001). Asterisks on individual points represent significance of difference with No-NTF control (white rectangle in the curve); asterisks on bars represent significant differences between a given concentration of NTF and the corresponding value for NTF + F+ I. (F) The cocktail of neurotrophic factors (NTFs) enhances the survival of expanded FACS-purified human motor neurons in a dose-dependent manner in the presence of 10 µM forskolin plus 100 µM IBMX. Values shown as mean ± s.e.m., n≥5 (t-test, **p<0.01; ***p<0.001).
Mentions: To develop a survival assay based on neurotrophic factor deprivation [31], [38], [39], FACS-sorted motor neurons were seeded in 96-well plates and stained using the vital dye calcein-AM. This had the advantage that it stained cell bodies and neurites more intensely than live imaging of GFP, which was no longer required to identify motor neurons. Numbers of surviving hESC-MNs were counted in whole culture wells in an automated manner using MetaMorph (Figure 5A). We first asked whether the survival of purified motor neurons was dependent on trophic support in these conditions. At day 31+3+7, motor neuron survival was enhanced ∼2.5-fold by a cocktail of NTFs (BDNF, CNTF, GDNF, IGF-1, each at 10 ng/ml) with F (10 µM) plus IBMX (100 µM) (Figure 5B), similar to published results using cultures of primary rodent motor neurons [39]–[41]. We tested forskolin and IBMX alone and found that they showed only slight innate neurotrophic activity (not shown).

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