TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS.
Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive....
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-09-01
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| Series: | Frontiers in Cellular Neuroscience |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fncel.2016.00217/full |
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| author | Daniel Édgar Cortés Daniel Édgar Cortés Yolanda Robledo-Arratia Ricardo Hernández-Martínez Itzel Escobedo-Avila Jose Bargas Ivan Velasco Ivan Velasco |
| author_facet | Daniel Édgar Cortés Daniel Édgar Cortés Yolanda Robledo-Arratia Ricardo Hernández-Martínez Itzel Escobedo-Avila Jose Bargas Ivan Velasco Ivan Velasco |
| author_sort | Daniel Édgar Cortés |
| collection | DOAJ |
| description | Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC that constitutively produce Glial cell-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic motor neurons. After lentiviral transduction, ESC lines integrated and expressed the human GDNF gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study motor neuron induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal motor neurons, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of motor neurons in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant human GDNF was added to control ESC, also resulting in enhanced motor neuron differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, motor neurons were selected for electrophysiological recordings. Motor neurons differentiated from GDNF-ESC, compared to control motor neurons, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials, as well as by the appearance of rebound action potentials, sag inward rectification, spike frequency adaptation and spontaneous synaptic potentials. Upon challenge with kainate, GDNF-overexpressing cells are more resistant to excitotoxicity than control motor neurons. Together these data indicate that GDNF promotes proliferation of motor neuron-committed precursors, promotes neuronal differentiation, enhances maturation, and confers neuroprotection. GDNF-expressing ESC can be useful in studies of development and disease. |
| first_indexed | 2024-12-13T09:49:16Z |
| format | Article |
| id | doaj.art-092dc544498445ccac11fb4fddd299ea |
| institution | Directory Open Access Journal |
| issn | 1662-5102 |
| language | English |
| last_indexed | 2024-12-13T09:49:16Z |
| publishDate | 2016-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular Neuroscience |
| spelling | doaj.art-092dc544498445ccac11fb4fddd299ea2022-12-21T23:51:57ZengFrontiers Media S.A.Frontiers in Cellular Neuroscience1662-51022016-09-011010.3389/fncel.2016.00217209636TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS.Daniel Édgar Cortés0Daniel Édgar Cortés1Yolanda Robledo-Arratia2Ricardo Hernández-Martínez3Itzel Escobedo-Avila4Jose Bargas5Ivan Velasco6Ivan Velasco7Instituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoLaboratorio de Reprogramación Celular del IFC-UNAM en el Instituto Nacional de Neurología y Neurología “Manuel Velasco Suárez”.Instituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoInstituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoInstituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoInstituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoInstituto de Fisiología Celular-Neurociencias, Universidad Nacional Autónoma de MéxicoLaboratorio de Reprogramación Celular del IFC-UNAM en el Instituto Nacional de Neurología y Neurología “Manuel Velasco Suárez”.Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC that constitutively produce Glial cell-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic motor neurons. After lentiviral transduction, ESC lines integrated and expressed the human GDNF gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study motor neuron induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal motor neurons, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of motor neurons in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant human GDNF was added to control ESC, also resulting in enhanced motor neuron differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, motor neurons were selected for electrophysiological recordings. Motor neurons differentiated from GDNF-ESC, compared to control motor neurons, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials, as well as by the appearance of rebound action potentials, sag inward rectification, spike frequency adaptation and spontaneous synaptic potentials. Upon challenge with kainate, GDNF-overexpressing cells are more resistant to excitotoxicity than control motor neurons. Together these data indicate that GDNF promotes proliferation of motor neuron-committed precursors, promotes neuronal differentiation, enhances maturation, and confers neuroprotection. GDNF-expressing ESC can be useful in studies of development and disease.http://journal.frontiersin.org/Journal/10.3389/fncel.2016.00217/fullNeuroprotectionexcitotoxicityneural precursorsspinal motor neurongdnfElectrophysiological maturation |
| spellingShingle | Daniel Édgar Cortés Daniel Édgar Cortés Yolanda Robledo-Arratia Ricardo Hernández-Martínez Itzel Escobedo-Avila Jose Bargas Ivan Velasco Ivan Velasco TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. Frontiers in Cellular Neuroscience Neuroprotection excitotoxicity neural precursors spinal motor neuron gdnf Electrophysiological maturation |
| title | TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. |
| title_full | TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. |
| title_fullStr | TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. |
| title_full_unstemmed | TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. |
| title_short | TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS. |
| title_sort | transgenic gdnf positively influences proliferation differentiation maturation and survival of motor neurons produced from mouse embryonic stem cells |
| topic | Neuroprotection excitotoxicity neural precursors spinal motor neuron gdnf Electrophysiological maturation |
| url | http://journal.frontiersin.org/Journal/10.3389/fncel.2016.00217/full |
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