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The Journal of Neuroscience, March 22, 2006, 26(12):3256-3268; doi:10.1523/JNEUROSCI.5537-05.2006

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Development/Plasticity/Repair
Motoneurons Derived from Embryonic Stem Cells Express Transcription Factors and Develop Phenotypes Characteristic of Medial Motor Column Neurons

Prabakaran Soundararajan,¹ Gareth B. Miles,¹ Lee L. Rubin,³ Robert M. Brownstone,^1,2 and Victor F. Rafuse¹

¹Departments of Anatomy and Neurobiology and ²Surgery, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 1X5, and ³Curis, Inc., Cambridge, Massachusetts 02138

Correspondence should be addressed to either Dr. Victor F. Rafuse or Dr. Robert M. Brownstone, Department of Anatomy and Neurobiology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada, B3H 1X5. Email: vrafuse{at}dal.ca or rob.brownstone{at}dal.ca

Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 ± 0.7% of the differentiated eGFP⁺ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMC_m), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP⁺ motoneurons migrated to the MMC_m, expressed Lhx3, projected axons to the appropriate target for MMC_m motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3⁺ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMC_m motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties.

Key words: development; sonic hedgehog; chemotropic; Lhx3; axon guidance; transcription factor; spinal cord; stem cells; regeneration; neuromuscular; motoneuron (motor neuron)

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Received Aug. 3, 2005; revised Feb. 3, 2006; accepted Feb. 3, 2006.

Correspondence should be addressed to either Dr. Victor F. Rafuse or Dr. Robert M. Brownstone, Department of Anatomy and Neurobiology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada, B3H 1X5. Email: vrafuse{at}dal.ca or rob.brownstone{at}dal.ca

This article has been cited by other articles:

				D. C. Yohn, G. B. Miles, V. F. Rafuse, and R. M. Brownstone Transplanted Mouse Embryonic Stem-Cell-Derived Motoneurons Form Functional Motor Units and Reduce Muscle Atrophy J. Neurosci., November 19, 2008; 28(47): 12409 - 12418. [Abstract] [Full Text] [PDF]

				C. K. Franz, U. Rutishauser, and V. F. Rafuse Intrinsic neuronal properties control selective targeting of regenerating motoneurons Brain, June 1, 2008; 131(6): 1492 - 1505. [Abstract] [Full Text] [PDF]

				G. W. Yeo, N. Coufal, S. Aigner, B. Winner, J. A. Scolnick, M. C.N. Marchetto, A. R. Muotri, C. Carson, and F. H. Gage Multiple layers of molecular controls modulate self-renewal and neuronal lineage specification of embryonic stem cells Hum. Mol. Genet., April 15, 2008; 17(R1): R67 - R75. [Abstract] [Full Text] [PDF]

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