Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Abstract

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 [1][2][3][4] . However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol 5 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 6 . FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity [7][8][9] . 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 1 . Using this robust protocol, we achieved 51±0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test) 5 . 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. Video LinkThe video component of this article can be found at

Differentiation of Wharton’s Jelly-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells on Three-Dimensional Collagen-Grafted Nanofibers

“…In order to differentiate Wharton's jelly stem cell into motor neuron-like cells in this study, RA was utilized along with Shh similar to those carried out by Liqing et al [44]. The combination of RA and Shh are commonly used for inducing the expression of transcription factors which play important roles in motor neuronal differentiation [45,46]. Differentiated neurons in different groups were then characterized morphologically and for the expression of motor neuron markers including Nestin, PAX6, NF-H, Islet-1, HB9, and Chat in mRNA levels and protein by quantitative reverse transcription PCR and immunocytochemistry, respectively.…”

Stepwise acquirement of hallmark neuropathology in FUS-ALS iPSC models depends on mutation type and neuronal aging

“…heat stress) (Dormann and Haass, 2013;Dormann et al, 2012;Dormann et al, 2010). Of note, all these data were derived from non-neuronal models and/or by heterologous expression of the mutant FUS variants (Wu et al, 2012).…”

Directed Migration of Embryonic Stem Cell-derived Neural Cells In An Applied Electric Field

“…Wu et al reported a two-step differentiation protocol that improved the motor neuron differentiation efficiency to 51 %. In that study, the first step was “neutralization” by adding Noggin and fibroblast growth factors (FGFs) to the cell culture medium [3]. The second step was to induce motor neuron specification.…”