Efficient differentiation of mouse embryonic stem cells into motor neurons.
Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.
Nemours Biomedical Research, Alfred I. duPont Hospital for Children, USA. wu@nemoursresearch.org
Chia-Yen
CY
Wu
Dosh
D
Whye
Robert W
RW
Mason
Wenlan
W
Wang
D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002795:Choline O-Acetyltransferase / Q000096:biosynthesis; D003584:Cytological Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051524:Fibroblast Growth Factor 8 / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000032:analysis; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors; D060850:LIM-Homeodomain Proteins / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists; D014157:Transcription Factors / Q000096:biosynthesis; D014212:Tretinoin / Q000494:pharmacology; D000099176:Noggin Protein
D002352:Carrier Proteins; D002795:Choline O-Acetyltransferase; D016222:Fibroblast Growth Factor 2; D051524:Fibroblast Growth Factor 8; D049452:Green Fluorescent Proteins; D053823:Hedgehog Proteins; D060850:LIM-Homeodomain Proteins; D043562:Receptors, G-Protein-Coupled; D014157:Transcription Factors; D014212:Tretinoin; D000099176:Noggin Protein; C063843:insulin gene enhancer binding protein Isl-1