Hedgehog Protocol Turns Supersonic in Faster Route to Motor Neurons

It takes sonic hedgehog, retinoic acid, and more than a month to turn stem cells into a few motor neurons for study or, eventually, transplantation. For researchers keen to understand amyotrophic lateral sclerosis and other motor neuron conditions, making just a few motor neurons over months hampers their studies. A team from Nationwide Children’s Research Institute in Columbus, Ohio, has developed a faster way. To the standard procedure, they added three transcription factors that sped up the differentiation. The group, led by senior author Brian Kaspar and first author Mark Hester, report in the July 19 Molecular Therapy online that they halved the time to produce the desired neurons, and simultaneously doubled the percentage of mature motor neurons resulting from the protocol.

The slow route to motor neurons has been a “bottleneck,” wrote Vania Broccoli of the San Raffaele Scientific Institute in Milan, Italy, in an e-mail to ARF. “This is a great advance that will likely speed up research on in-vitro modeling of motor neuron diseases.”

The classic method to turn stem cells or induced pluripotent cells into motor neurons involves a 10-day protocol to convert them to neural progenitors, then treatment with retinoic acid and the signaling protein sonic hedgehog to make a motor phenotype (see ARF related news story on Wichterle et al., 2002 and ARF related news story on Dimos et al., 2008). With human cells, this protocol takes six to eight weeks, and “you are lucky to get 30 percent motor neurons,” Kaspar lamented.

The researchers sought a shortcut, looking to transcription factors that are activated to mediate motor neuron differentiation and maturation once the cells respond to retinoic acid and sonic hedgehog. Collaborating with Samuel Pfaff of the Salk Institute in La Jolla, California, they determined that a heterotrimer of transcription factors is key to the motor neuron pathway. Neurogenin 2, islet-1, and LIM/homeobox protein 3 work together to transform neural precursors into full-fledged motor neurons. In particular, they turn on a further transcription factor, HB9, which controls much of the motor neuron transcriptome.

Normally, progenitors make the three transcription factors two or three weeks into the differentiation process. To jump-start differentiation, Hester used an adenovirus vector to deliver genes for the three at the same time as he added sonic hedgehog and retinoic acid. Within just 11 days, the cells contained HB9 as well as choline acetyltransferase (CHAT), another motor neuron marker. Another bonus was that nearly 70 percent of cells went down the motor neuron pathway. By 15 days, the scientists confirmed that the neurons could maintain action potentials, and possessed the strong sodium currents typical of motor neurons.

To further test function, the researchers cultured their homemade motor neurons with muscle cells, looking for the formation of neuromuscular junctions. They were pleased to see that motor axons mingled with the muscle, co-localizing with acetylcholine receptors and producing synaptic markers. Thus, the authors concluded that they had produced mature motor neurons inside of a month.

Using transcription factors to control fate is a good idea, said Zhiping Ping of Stanford University in Palo Alto, California, but as to “whether the cells are mature or not, I am not quite convinced,” he said. He noted that the authors did not provide evidence that the motor neurons' signal is actually received by the muscle cells. Moreover, they have not yet shown that the motor neurons can receive upstream signals, from interneurons, for example.

There are hundreds of different kinds of motor neurons in the spinal cord. Following this recipe, Hester made motor neurons that express homeobox transcription factors matching those in cervical motor neurons, which innervate upper body muscles. Motor neurons made with retinoic acid and sonic hedgehog typically connect with the axial muscles of the trunk and head, Kaspar said. However, with this “proof of concept” in hand, he suggested other transcription factors could make other kinds of neurons. For example, researchers have used the transcription factor Lmx1a to force neurons toward a dopaminergic fate. “A similar approach is conceivable for a fast-track generation of other neuronal subtypes,” Broccoli agreed. In the meantime, Hester can spend more time studying motor neurons and less time making them.—Amber Dance

Comments

  1. This paper by Kaspar and colleagues aimed to accelerate the generation of motor neurons from induced pluripotent stem (iPS) cells.
    Indeed, this is currently a bottleneck for all stem cell biologists interested in using iPS-derived motor neurons
    for in-vitro disease modeling. Currently, iPS cells need to be differentiated in vitro for at least 42 days
    in order to get functional motor neurons. This is a long time, especially if you want to use large numbers of these neurons
    for drug screenings or high-throughput procedures.

    Kaspar and colleagues conceived to accelerate cell differentiation by expressing a set of three transcription factors that are
    critical to promote this neuronal lineage during development. Interestingly, their expression is sufficient to boost
    neuronal differentiation in time and number, generating functional motor neurons in as few as 11 days.
    The authors provided results for only this single combination of master genes, leaving open the possibility
    to further increase the cell differentiating output with new combinations of related transcription factors.

    This is a great advance that will likely speed up research on in-vitro modeling of motor neuron diseases. Importantly,
    these results indicate that a similar approach is conceivable for a fast-track generation of other neuronal sub-types.
    Thus, hopefully, new studies will follow the experimental strategy designed by these authors.

    View all comments by Vania Broccoli

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References

News Citations

  1. Turning Stem Cells into Motor Neurons
  2. ALS: Predicting Prognosis, Banking on Pluripotent Stem Cells

Paper Citations

  1. Wichterle H, Lieberam I, Porter JA, Jessell TM. Directed differentiation of embryonic stem cells into motor neurons. Cell. 2002 Aug 9;110(3):385-97. PubMed.
  2. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008 Aug 29;321(5893):1218-21. PubMed.

Further Reading

Papers

  1. Wyatt TJ, Rossi SL, Siegenthaler MM, Frame J, Robles R, Nistor G, Keirstead HS. Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss. Stem Cells Int. 2011;2011:207230. PubMed.
  2. Wilmut I, Sullivan G, Chambers I. The evolving biology of cell reprogramming. Philos Trans R Soc Lond B Biol Sci. 2011 Aug 12;366(1575):2183-97. PubMed.
  3. 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.
  4. Inoue H. Neurodegenerative disease-specific induced pluripotent stem cell research. Exp Cell Res. 2010 Oct 1;316(16):2560-4. PubMed.
  5. Courtois ET, Castillo CG, Seiz EG, Ramos M, Bueno C, Liste I, Martínez-Serrano A. In vitro and in vivo enhanced generation of human A9 dopamine neurons from neural stem cells by Bcl-XL. J Biol Chem. 2010 Mar 26;285(13):9881-97. PubMed.
  6. Silani V, Calzarossa C, Cova L, Ticozzi N. Stem cells in amyotrophic lateral sclerosis: motor neuron protection or replacement?. CNS Neurol Disord Drug Targets. 2010 Jul;9(3):314-24. PubMed.

Primary Papers

  1. Hester ME, Murtha MJ, Song S, Rao M, Miranda CJ, Meyer K, Tian J, Boulting G, Schaffer DV, Zhu MX, Pfaff SL, Gage FH, Kaspar BK. Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. Mol Ther. 2011 Oct;19(10):1905-12. PubMed.

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