Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons

Abstract

Controlled differentiation of embryonic stem (ES) cells into clinically relevant cell types is a fundamental goal of stem cell research. This unit describes one of the most efficient protocols for conversion of mouse ES cells into a defined type of nerve cells, the spinal motor neurons. ES cells are separated from feeder mouse embryonic fibroblasts and aggregated to form embryoid bodies (EBs). Two days after the withdrawal of growth factors, EBs reach a stage at which they are responsive to patterning signals and can be effectively induced with retinoic acid (RA) to differentiate into spinal nerve cells. Nascent neural cells become responsive to the ventralizing signal sonic hedgehog (Hh) that controls expression of ventral spinal progenitor markers and initiates the genetic program of motor neuron differentiation. Curr. Protoc. Stem Cell Biol. 5:1H.1.1‐1H.1.9. © 2008 by John Wiley & Sons, Inc.

Cytochrome P450 Cyp26a1 Alters Spinal Motor Neuron Subtype Identity in Differentiating Embryonic Stem Cells

“…1 ϫ 10 3 units/ml leukemia inhibitory factor (LIF2010, Millipore-Chemicon (Temecula, CA)) was added prior to use. ESCs were cultured in ADFNK medium to form embryoid bodies (EBs) and subsequently HB9 ϩ motor neurons as described (15). ADFNK medium is made as follows: 250 ml of Advanced DMEM/F-12 (catalog no.…”

“…All-trans-retinoic acid (Sigma) was dissolved in 100% ethanol and diluted in the appropriate cell medium to obtain final concentrations. The sonic hedgehog agonist (Hh-Ag1.3 (SHH), Curis, Cambridge, MA) was dissolved in DMSO and diluted in ADFNK medium (15) to obtain the final concentration. Doxycycline was from Sigma-Aldrich.…”

“…Fixation, Cryosectioning, and Immunostaining-EBs were fixed, sectioned, and processed for immunocytochemistry as described (15). For immunostaining, slides were thawed at room temperature for ϳ10 min.…”

“…Factor Profile-To examine the role of RA metabolism in spinal motor neuron differentiation and subsequent motor column specification, we utilized murine wild type (WT) and Cyp26a1 Ϫ/Ϫ ESCs (14) in a previously established differentiation protocol (15) (Fig. 1A).…”

“…To test the function of the cytochrome p450 enzyme Cyp26a1 (Gene ID: 13082) in specifying MNs, we subjected Cyp26a1 Ϫ/Ϫ ESCs (14) to an MN differentiation protocol (15) and assessed the molecular phenotype of the resulting MNs. Our results show a significant role for Cyp26a1 and RA in the specification of MN subtype identities and also provide insight into how Cyp26a1 inhibition could be used for cell differentiation therapies to generate a reliable source of specified LMC MNs.…”