Easy and Rapid Differentiation of Embryonic Stem Cells into Functional Motoneurons Using Sonic Hedgehog‐Producing Cells

Abstract

Directing embryonic stem (ES) cells to differentiate into functional motoneurons has proven to be a strong technique for studying neuronal development as well as being a potential source of tissue for cell replacement therapies involving spinal cord disorders. Unfortunately, one of the mitogenic factors (i.e., sonic hedgehog agonist) used for directed differentiation is not readily available, and thus this technique has not been widely accessible. Here, we present a novel and simple method to derive motoneurons from ES cells using readily attainable reagents. ES cells were derived from a mouse in which enhanced green fluorescent protein (eGFP) was linked to a motoneuron specific promoter. The cells were plated onto a monolayer of 293 EcR-Shh cells that carry an integrated construct for the expression of sonic hedgehog (Shh) under ecdysone-inducible control. To initiate motoneuron differentiation, 293 EcR-Shh:ES cell cocultures were treated with ponasterone A (PA) and retinoic acid for 5 days. PA induces ecdysone, and thus drives Shh expression. To assess differentiation, putative ES cellderived motoneurons were studied immunocytochemically and cultured on chick myotubes for functional analysis. We found that ES cells differentiated into eGFP ؉ cells that expressed transcription factors typical of motoneurons. Furthermore, ES cell-derived motoneurons were capable of forming functional connections with muscle fibers in vitro. Finally, when transplanted into the developing chick spinal cord, ES cell-derived motoneurons migrated to the ventral horn and projected axons to appropriate muscle targets. In summary, this simple treatment paradigm produces functional motoneurons that can be used for both developmental and preclinical studies.

Guidance of Postural Motoneurons Requires MAPK/ERK Signaling Downstream of Fibroblast Growth Factor Receptor 1

“…Interestingly, recent studies indicate that mouse ES cell-derived motoneurons represent a distinct subpopulation of motoneurons that may be used to study axon guidance. In particular, mouse HBG3 ES cells differentiate into a single population of Lhx3 ϩ motoneurons when treated in culture with RA and an Shh agonist (Wichterle et al, 2002;Soundararajan et al, 2006) or RA and Shh (Soundararajan et al, 2007). HBG3 ES cells differentiate into eGFP ϩ motoneurons because they express eGFP under control of the mouse Hb9 promotor (Wichterle et al, 2002).…”

“…ES cells were directed to differentiate into motoneurons as described previously (Soundararajan et al, 2007). Briefly, sonic hedgehog (Shh) expressing HEK293 cells (293 EcR-Shh cells; obtained from American Type Culture Collection) were attached to the external bottom surface of collagen-coated membrane inserts (3 m pore size; catalog #3494; Corning) placed in wells containing DFK10 media.…”

“…Approximately 120 eGFP ϩ ES cell-derived MMCm motoneurons were transplanted into ventral region of the neural tube. We chose this number of cells because our previous studies (Soundararajan et al, 2006(Soundararajan et al, , 2007 showed that 100% of the transplanted ES cell-derived motoneurons innervated epaxial muscles when ϳ100 motoneurons were transplanted into the neural tube at this stage in development. If Ͼ250 ES cell-derived MMCm motoneurons are transplanted in ovo, many will mistarget to the limbs (Soundararajan et al, 2006).…”

“…Although subpopulations of motoneurons cannot be easily purified from embryos, recent studies have shown that mouse embryonic stem (ES) cells can be directed to differentiate into a pure population of Lhx3 ϩ MMCm motoneurons (Wichterle et al, 2002;Soundararajan et al, 2006Soundararajan et al, , 2007. Here, we use this unique population to show that MMCm motor axon guidance in vitro is mediated through MAPK/extracellular signal-regulated kinase (ERK) signaling pathway downstream of FGF8 activation of FGF receptor 1 (FGFR1).…”

Stem Cells: Current Approach and Future Prospects in Spinal Cord Injury Repair

“…These progenitor domains are established mainly by interaction of Class I and Class II homeodomain proteins, which are inhibited or induced by the graded secreted inductive factors, such as SHH (Kuh et al, 2005). Previous studies showed that human stem cells can differentiate to spinal motor neurons in an adherent culture by applying retinoid acid (RA) and SHH with 20% efficiency (Shin et al, 2007;Soundararajan et al, 2007), similar to that from mouse ESCs. However, this efficiency is not ideal for a variety of analyses, and the identity of nearly 80% of the differentiated cells in the culture remains unknown.…”