In-Vitro Innervation: Stem Cell-Derived Motor Neurons Meet Muscles

Scientists have made strides turning stem cells into motor neurons, but a motor neuron by itself is not much use—it’s only when it innervates a muscle that it can get things moving. Now, for the first time, researchers claim that stem cell-derived motor neurons make electrically active connections with co-cultured muscle cells. Researchers who reported the new model system in the May 4 PLoS ONE are interested in using it to study spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), both of which involve impairment at the neuromuscular junction, said senior author Bennett Novitch, University of California, Los Angeles.

With the advent of stem cell and induced pluripotent stem cell (iPSC) technology, scientists are studying many neurological diseases in culture (see ARF related news story). However, researchers have mainly examined neurons in isolation or in co-culture with glia, Novitch said. While stem cell-derived motor neurons can innervate the muscles of mice and chick embryos (Yohn et al., 2008; Peljto et al., 2010), it has been challenging to evaluate the electrical function of these new, stem cell-based junctions. The tightly packed tissue makes it hard to isolate a single neuron-muscle pair to analyze. Novitch teamed with faculty colleague and first author Joy Umbach to create neuromuscular junctions in vitro so they could measure junction function.

The researchers tested many different parameters to find those that best nurtured cell survival and synapse formation. For Umbach and Novitch’s goal of studying the electrophysiology of the synapse, the most crucial part of the protocol was to plate the neurons and muscle cells sparsely. Sparse turns out to be approximately 24,000 cells per 35-millimeter dish. Higher densities made it difficult to patch-clamp an isolated pair of cells and obtain clear recordings, Novitch said.

Umbach and colleagues found that where the motor neurons and muscles met, they made synapses that included many of the proteins found in natural neuromuscular junctions, such as acetylcholine receptors and synaptic vesicle proteins. Stimulating the neurons created an action potential and, a few milliseconds later, an excitatory current in the associated muscle cells. Examining the kinetics, “they seem to be remarkably similar to what one gets with [natural] tissues,” Novitch said, such as isolated nerve-muscle preps (Fatt and Katz, 1951).

The researchers next plan to test some diseased cell types in their system, such as iPS-based neurons from people with ALS or SMA. Novitch hopes the culture could be used to screen for medicines that would block denervation, the process that robs people with ALS of their ability to control their skeletal muscle. The neuromuscular junction has not been closely examined in studies of motor neuron disease, and may turn out to be an important site of action, commented Arnold Kriegstein of the University of California, San Francisco, who was not involved with the study.

However, Kriegstein suggested that the current in-vitro model is not very robust, because the junctions only lasted for a week. This is the most important limitation of the artificial system, Novitch agreed; natural synapses, of course, last much longer. Novitch suspects that adding more elements of the neural circuit—such as Schwann cells, glia, and interneurons—will help stabilize the connections. The team has also found, in not-yet-published experiments, that neuromuscular junctions derived from human embryonic stem or iPS cells last for several weeks, suggesting the mouse synapses, in particular, are unstable.—Amber Dance

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References

News Citations

  1. Where in the World Are the iPS Cells?

Paper Citations

  1. Yohn DC, Miles GB, Rafuse VF, Brownstone RM. Transplanted mouse embryonic stem-cell-derived motoneurons form functional motor units and reduce muscle atrophy. J Neurosci. 2008 Nov 19;28(47):12409-18. PubMed.
  2. Peljto M, Dasen JS, Mazzoni EO, Jessell TM, Wichterle H. Functional diversity of ESC-derived motor neuron subtypes revealed through intraspinal transplantation. Cell Stem Cell. 2010 Sep 3;7(3):355-66. PubMed.
  3. FATT P, Katz B. An analysis of the end-plate potential recorded with an intracellular electrode. J Physiol. 1951 Nov 28;115(3):320-70. PubMed.

Further Reading

Papers

  1. Park HW, Cho JS, Park CK, Jung SJ, Park CH, Lee SJ, Oh SB, Park YS, Chang MS. Directed induction of functional motor neuron-like cells from genetically engineered human mesenchymal stem cells. PLoS One. 2012;7(4):e35244. PubMed.
  2. Lunn JS, Sakowski SA, Federici T, Glass JD, Boulis NM, Feldman EL. Stem cell technology for the study and treatment of motor neuron diseases. Regen Med. 2011 Mar;6(2):201-13. PubMed.
  3. Soundararajan P, Miles GB, Rubin LL, Brownstone RM, Rafuse VF. Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons. J Neurosci. 2006 Mar 22;26(12):3256-68. PubMed.
  4. Wichterle H, Przedborski S. What can pluripotent stem cells teach us about neurodegenerative diseases?. Nat Neurosci. 2010 Jul;13(7):800-4. PubMed.
  5. Karumbayaram S, Kelly TK, Paucar AA, Roe AJ, Umbach JA, Charles A, Goldman SA, Kornblum HI, Wiedau-Pazos M. Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS. Dis Model Mech. 2009 Mar-Apr;2(3-4):189-95. PubMed.
  6. Glass JD, Boulis NM, Johe K, Rutkove SB, Federici T, Polak M, Kelly C, Feldman EL. Lumbar intraspinal injection of neural stem cells in patients with amyotrophic lateral sclerosis: results of a phase I trial in 12 patients. Stem Cells. 2012 Jun;30(6):1144-51. PubMed.

Primary Papers

  1. Umbach JA, Adams KL, Gundersen CB, Novitch BG. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons. PLoS One. 2012;7(5):e36049. PubMed.

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