Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells
Por:
Cortes, Daniel, Robledo-Arratia, Yolanda, Hernandez-Martinez, Ricardo, Escobedo-Avila, Itzel, Bargas, Jose, Velasco, Ivan
Publicada:
12 sep 2016
Categoría:
Cellular and Molecular Neuroscience
Resumen:
Embryonic stem cells (ESC) are pluripotent and thus can differentiate
into every cell type present in the body. Directed differentiation into
motor neurons (MNs) 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 (mESC) that constitutively produce glial cell line-derived
neurotrophic factor (GDNF) and also contain a GFP reporter, driven by
HB9, which is expressed only by postmitotic MNs. After lentiviral
transduction, ESC lines integrated and expressed the human GDNF (hGDNF)
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 MN
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 MNs, 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 MNs in GDNF
cultures, with lower proportions in control cells. To test if the
effects of GDNF were present at early differentiation stages, exogenous
recombinant hGDNF was added to control ESC, also resulting in enhanced
MN 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,
MNs were selected for electrophysiological recordings. MNs
differentiated from GDNF-ESC, compared to control MNs, showed greater
electrophysiological maturation, characterized by increased numbers of
evoked action potentials (APs), as well as by the appearance of rebound
APs, sag inward rectification, spike frequency adaptation and
spontaneous synaptic potentials. Upon challenge with kainate,
GDNF-overexpressing cells are more resistant to excitotoxicity than
control MNs. Together these data indicate that GDNF promotes
proliferation of MN-committed precursors, promotes neuronal
differentiation, enhances maturation, and confers neuroprotection.
GDNF-expressing ESC can be useful in studies of development and disease.
Filiaciones:
Cortes, Daniel:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Lab Reprogramac Celular, Inst Fisiol Celular, Inst Nacl Neurol & Neurol Manuel Velasco Suarez, Mexico City, DF, Mexico
Robledo-Arratia, Yolanda:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Hernandez-Martinez, Ricardo:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Escobedo-Avila, Itzel:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Bargas, Jose:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Velasco, Ivan:
Univ Nacl Autonoma Mexico, Inst Fisiol Celular Neurociencias, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Lab Reprogramac Celular, Inst Fisiol Celular, Inst Nacl Neurol & Neurol Manuel Velasco Suarez, Mexico City, DF, Mexico
|