How to Make a Neuron: New Opportunities for ALS Stem Cell Therapy

CHRISTINE ZHANG

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Figure 1 shows the pathways between fibroblast to neuron conversions. The red arrow represents the conversion first to the embryonic state followed by differentiation to neurons. The blue arrow represents direct conversion to neurons. [3]

The golden age of stem cells is dawning. These cells possess the ability to solve a number of the greatest problems in medicine. Amyotrophic lateral sclerosis (ALS) is one. ALS, commonly known as Lou Gehrig’s disease, causes paralysis as motor neurons die and the brain loses control of muscle movement. More than 30,000 Americans suffer from ALS at any time and the disease has a low survival rate, with less than 50% of patients living two years or more post-diagnosis. There is no present cure and the only treatment option is Riluzole, a drug that can prolong one’s lifespan by a few months but provides limited relief. We are hoping that stem cells will be able to replace the neurons lost to the disease.

Stem cells have the remarkable ability to transform into many different cell types. Endogenous stem cells, taken from an individual’s own body, hold much promise as a self-repair solution compatible with one’s immune system. The 2012 Nobel Prize in Physiology or Medicine was awarded jointly to Dr. Gurdon and Dr. Yamanaka for their breakthrough on converting adult cells–cells that have developed into specific cell types–back into the embryonic, pre-differentiated state. Embryonic cells are pluripotent, able to differentiate into any human body cell type. In contrast, adult stem cells are multipotent; their differentiation is limited to a narrow range of cells. Thus, inducing the embryonic state confers a great advantage.

Dr. Yamanaka discovered that through gene regulation, he could cause adult cells to revert to their embryonic state.1 However, his experimental design involved viruses, a method that would raise health concerns including the destabilization of DNA/RNA and increased likelihood of tumors. In response, chemical procedures have been created to replace the need for viruses. From converting adult cells to the embryonic state to differentiating the induced embryonic cells into neurons, the full procedure can span over a month (red arrow in diagram).2 Directly converting adult cells to neurons, bypassing the embryonic state, would provide increased efficiency (blue arrow). However, direct conversion through chemicals has not yet been achieved.

Dr. Kevin Eggan is co-head of the Stem Cell and Regenerative Biology department and his lab specializes in using stem cells to treat neurodegenerative diseases, particularly ALS. I have been fortunate to work with one of his graduate students, Feng Tian, on novel treatment options. Our experiment consists of using chemicals to discover a pathway of direct conversion to create neurons from other somatic cell types, a technique known as trans-differentiation (blue arrow).

The first stage of the experiment consisted of screening for chemicals that would best stimulate specialization into neurons. A combination of chemicals controlling DNA methylation and histone de-acetylation proved to be the most successful. This was followed by the second stage of conversion, which promoted neuron maturation and stabilization. Afterwards, we established the similarities between the chemically induced neurons and standard neurons by comparing DNA base pairs, electrical activities, and expression of neuronal markers.

Our experiment consists of using chemicals to discover a pathway of direct conversion to create neurons from other somatic cell types, a technique known as trans-differentiation.”

Using chemicals in place of viruses not only removes the ethical and health concerns that viruses pose, it also presents its own benefits. The chemical procedure is shorter, more cost-effective, and more adaptable for large-scale processing. Moreover, a tremendous procedural advantage is that directly converted neurons (blue arrow) possess fewer artifacts than neurons produced from induced embryonic cells (red arrow). Each time an adult cell transforms identity, it carries over remnants from its past. In this case, neurons could still retain behavioral patterns of their previous role, which could interfere with their functionality as neurons. With fewer such artifacts, direct conversion produces neurons that are more stable and viable for long-term use. 4

The greatest implication of this discovery is that it provides a powerful tool to combat diseases including ALS. A cell sample from a patient with ALS would display a mix of healthy and diseased cells. In our experiments, we were able to cause healthy cells to proliferate and convert into neurons, while diseased cells do not. We compared chemically induced neuron growth (blue arrow) in the fibroblasts (cells that are transformed into neurons) derived from mice who had the ALS-related mutation against chemically induced neuron growth in fibroblasts derived from their healthy littermates.5 Consistently, cell lines from mice with the ALS-related mutation expressed significantly lower neuron yields. Furthermore, neurons displayed poor survival rates when cultured on ALS-related mutated glia, cells that support neurons. So when we chemically induce  neurons, diseased neurons can be distinguished from healthy neurons and only the healthy ones will multiply.

After we collect the patient’s cells and chemically transform these into neurons, this batch of neurons accumulates and, over time, becomes more robust. These neurons then display greater potential for in vivo medicinal applications. For ALS patients who have lost motor neurons, our goal is to have our healthy neurons replace the damaged ones and restore motor movement. We hope this could become a treatment that improves patients’ quality of life and ultimately increases their survival rate.

Works Cited:

  1. Yamanaka, S., and Takahashi, K. (2006). Induction of pluripotent stem cells from mouse fibroblast cultures. Tanpakushitsu kakusan koso Protein, nucleic acid, enzyme 51, 2346-2351.
  2. Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J., Li, H., Zhao, T., Ye, J., Yang, W., Liu, K., et al. (2013). Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. New York, NY 341, 651-654.
  3. Davis-Dusenbery BN, Williams LA, Klim JR, Eggan K. How to make spinal motor neurons. Development. 2014 Feb; 141(3):491-501.
  4. Dimos, J.T., Rodolfa, K.T., Niakan, K.K., Weisenthal, L.M., Mitsumoto, H., Chung, W., Croft, G.F., Saphier, G., Leibel, R., Goland, R., et al. (2008). Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. New York, NY 321, 1218-1221.
  5. Di Giorgio, F.P., Carrasco, M.A., Siao, M.C., Maniatis, T., and Eggan, K. (2007). Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nature neuroscience 10, 608-614.

Special acknowledgements to Dr. Kevin Eggan for providing me the opportunity to work in his lab and to my mentor, Feng Tian, Dr. Eggan’s graduate student, for his support and guidance.

Christine Zhang, College ’18, is currently conducting research in the Eggan Laboratory of SCRB. She can be reached at christinezhang@college.harvard.edu