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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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Ongoing birth of motor neurons in hESC-derived cultures is stimulated by neurotrophic factors.(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).
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pone-0110324-g001: Ongoing birth of motor neurons in hESC-derived cultures is stimulated by neurotrophic factors.(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).

Mentions: To determine whether hESCs differentiated in vitro to a mixed spinal cord identity exhibit prolonged motor neurogenesis as in the fetal human spinal cord, we first examined changes in numbers of hESC-derived motor neurons (hESC-MNs) in mixed spinal cultures over a 15-day period using an hESC reporter line that expresses green fluorescent protein (GFP) under the control of the motor neuron-specific murine homeobox gene 9 (Hb9) promoter [23]. We and others previously showed using a range of other markers and functional assays that GFP-positive neurons generated from this line possess many properties of postmitotic motor neurons [6], [23], [30]. Motor neurons were differentiated from hESCs using a standard protocol involving exposure of embryoid bodies (EBs) to retinoic acid (RA) and recombinant sonic hedgehog protein (SHH) (see Methods) [4], [6]. After 31 days, EBs were dissociated and cryopreserved to allow multiple experiments to be performed on identical aliquots; however, similar data were obtained using fresh, unfrozen cells (not shown). Cell suspensions were thawed and plated in 96-well plates and automated counts of live motor neurons, defined as GFP+ neurons with significant neurite outgrowth (SNO, total neurite length >75 µm), were performed (Figures 1A and 1B) [31]–[33]. In standard culture medium without neurotrophic support motor neuron numbers decreased over the first 7 days, reaching a plateau that was maintained until day 31+13 (Figure 1C and 1D). This did not reflect a loss of reporter expression since a similar decrease was seen when motor neurons were identified by staining for endogenous HB9 (not shown). In contrast, when the medium was supplemented with four neurotrophic factors [NTFs; brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor 1 (IGF-1) at 10 ng/mL] in addition to the cAMP-elevating compounds forskolin (F; 10 µM) and isobutylmethylxanthine (I; 100 µM), after an initial decrease in motor neuron numbers by day 31+7, there was a subsequent increase in the number of hESC-MNs, which reached nearly starting levels by day 31+13 (Figure 1C and 1D).


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)

Ongoing birth of motor neurons in hESC-derived cultures is stimulated by neurotrophic factors.(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).
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Related In: Results  -  Collection

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

pone-0110324-g001: Ongoing birth of motor neurons in hESC-derived cultures is stimulated by neurotrophic factors.(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).
Mentions: To determine whether hESCs differentiated in vitro to a mixed spinal cord identity exhibit prolonged motor neurogenesis as in the fetal human spinal cord, we first examined changes in numbers of hESC-derived motor neurons (hESC-MNs) in mixed spinal cultures over a 15-day period using an hESC reporter line that expresses green fluorescent protein (GFP) under the control of the motor neuron-specific murine homeobox gene 9 (Hb9) promoter [23]. We and others previously showed using a range of other markers and functional assays that GFP-positive neurons generated from this line possess many properties of postmitotic motor neurons [6], [23], [30]. Motor neurons were differentiated from hESCs using a standard protocol involving exposure of embryoid bodies (EBs) to retinoic acid (RA) and recombinant sonic hedgehog protein (SHH) (see Methods) [4], [6]. After 31 days, EBs were dissociated and cryopreserved to allow multiple experiments to be performed on identical aliquots; however, similar data were obtained using fresh, unfrozen cells (not shown). Cell suspensions were thawed and plated in 96-well plates and automated counts of live motor neurons, defined as GFP+ neurons with significant neurite outgrowth (SNO, total neurite length >75 µm), were performed (Figures 1A and 1B) [31]–[33]. In standard culture medium without neurotrophic support motor neuron numbers decreased over the first 7 days, reaching a plateau that was maintained until day 31+13 (Figure 1C and 1D). This did not reflect a loss of reporter expression since a similar decrease was seen when motor neurons were identified by staining for endogenous HB9 (not shown). In contrast, when the medium was supplemented with four neurotrophic factors [NTFs; brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor 1 (IGF-1) at 10 ng/mL] in addition to the cAMP-elevating compounds forskolin (F; 10 µM) and isobutylmethylxanthine (I; 100 µM), after an initial decrease in motor neuron numbers by day 31+7, there was a subsequent increase in the number of hESC-MNs, which reached nearly starting levels by day 31+13 (Figure 1C and 1D).

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