Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures

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 (EC50 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.

Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules

“…PKC inhibition improves naive human pluripotent stem cells and induces neuritogenesis of Neuro-2a cells (Gafni et al, 2013;Miñ ana et al, 1990). Y-27632 assists in the maintenance of pluripotent stem cells and neuron survival (Lamas et al, 2014;Xi et al, 2013). Therefore, the chemical cocktail VCRFSGY erases fibroblast-specific gene expression of the initial cells, specifically upregulating neuronal gene expression and facilitating the neuronal conversion of HAFs ( Figure S4).…”

Fat mass and obesity associated (FTO) gene influences skeletal muscle phenotypes in non-resistance trained males and elite rugby playing position

“…As such, for predeterminant neuronal cells to differentiate into a progenitor motor neuron domain and subsequently motor neurons, it appears IRX3 must be repressed by the microRNA mir-17-3p in order for OLIG2 to regulate the development of ventral spinal motor neurons [45]. Thus, as the expression of OLIG2 increases, the yield of motor neurons increases in tandem [46]. Considering FTO T allele carriers have a lower embryonic expression of IRX3, T allele carriers may have a predisposition for greater LM through enhanced life-long motor neuron availability via OLIG2 expression and therefore, may be at an advantage for certain forms of athletic ability and associated performance phenotypes (Tables 2, 3 and 4; Fig.…”

Modeling ALS with motor neurons derived from human induced pluripotent stem cells

“…Further refinement could be achieved by the use of tamoxifen-regulated Cre fused to a modified fragment of the estrogen receptor (Cre-ERT2) instead of basic Cre. While the efficiency of recombination and MN labeling might be decreased, the ability to “pulse-label” MNs will provide a much-needed tool to study long-term MN survival by eliminating the confounding effects of ongoing MN genesis in human cultures 75 . In addition to these efforts to improve the available repertoire of florescent MN reporters, the identification or engineering of new MN-specific cell surface antigens for magnetic-assisted cell sorting paradigms will facilitate the purification of MNs from mixed cultures with less mechanical stress than traditional flow-cytometry approaches.…”