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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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FACS-sorting of amplified cultures yields a pure preparation of viable human motor neurons.(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).
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pone-0110324-g004: FACS-sorting of amplified cultures yields a pure preparation of viable human motor neurons.(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).

Mentions: Our overall goal was to study the trophic requirements of human motor neurons. Bulk day 31 cultures were therefore dissociated and grown in the presence of Y-27632 for 3 days or 9 days before FACS analysis, leading to a ∼2-fold increase in the total yield of motor neurons after 3 days (Figure 4A; p<0.01) and a nearly 4-fold increase after 9 days (Figure 4B; p<0.01). For all subsequent experiments, expanded human motor neurons from the day 31+3 time point were used.


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)

FACS-sorting of amplified cultures yields a pure preparation of viable human motor neurons.(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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110324-g004: FACS-sorting of amplified cultures yields a pure preparation of viable human motor neurons.(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).
Mentions: Our overall goal was to study the trophic requirements of human motor neurons. Bulk day 31 cultures were therefore dissociated and grown in the presence of Y-27632 for 3 days or 9 days before FACS analysis, leading to a ∼2-fold increase in the total yield of motor neurons after 3 days (Figure 4A; p<0.01) and a nearly 4-fold increase after 9 days (Figure 4B; p<0.01). For all subsequent experiments, expanded human motor neurons from the day 31+3 time point were used.

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