Neural Stem Cells and ALS: Delivering the Gift of Growth Factors

The motor neurons and surrounding cells damaged in amyotrophic lateral sclerosis cry out for assistance, and scientists are developing ways to home in on that need with growth factors that slow the degeneration process. In a Gene Therapy paper published online July 23, researchers from the University of British Columbia in Vancouver; Ajou University in Suwon, Korea; and Chungang University in Seoul, Korea, describe how they commandeered neural stem cells to deliver vascular endothelial growth factor (VEGF) to the spinal cord of mouse models for ALS. The researchers transplanted VEGF-overexpressing stem cells intrathecally, and at least some of those cells then migrated to the spinal cord grey matter—a key advancement over many techniques that only provide growth factors to the site of injection, said senior author Seung Kim of the University of British Columbia. The treatment delayed symptom onset by a week and prolonged survival by 12 days compared to control mice treated with phosphate-buffered saline alone.

Researchers have long wanted to provide growth factors to ALS-damaged tissue in the spinal cord, and the road to a viable ALS therapy is crowded with a variety of delivery methods. Transgene-toting viral vectors, perhaps sliding up axons via retrograde transport, are a popular choice (see ARF related news story and Azzouz et al., 2004; ARF related news story and Kaspar et al., 2003). But those vectors may only target a small area, and could induce an immune response, Kim suggested. Other approaches, both in clinical trials, include injecting the VEGF gene or the growth factor itself via a pump (Nagano et al., 2005). These therapeutics, too, may not travel far and would either degrade quickly or have to rely on the pump for a steady supply. Kim’s technique is different, he said, because the cells are attracted to damaged areas by chemokines and growth factors: “These cells will find the place of the pathology.”

In addition to VEGF, both insulin-like growth factor 1 (IGF-1) and glial cell line-derived growth factor (GDNF) show promise as ALS therapeutics, said Brian Kaspar of Nationwide Children’s Hospital in Columbus, Ohio, who was not involved in the current study. Although they cannot cure disease, the growth factors could slow degeneration by promoting cell health and survival. But in clinical trials, IGF-1 has disappointed researchers (see ARF related news story and Sorenson et al., 2008). Those failures emphasize to Kaspar the importance, regardless of delivery method, of targeting the growth factor to the right place. “This study highlights one new approach,” he said.

Kim, along with joint senior author I.S. Joo and first author D.H. Hwang of Ajou University, engineered human neural stem cells, isolated from fetal tissue and immortalized by myc expression (Kim, 2004), to express VEGF at a level nearly four times that of normal (Lee et al., 2007). They transplanted the cells into a common ALS model, mice overexpressing the G93A mutant of human superoxide dismutase 1 (SOD1), which is one cause of inherited ALS. As controls, they used stem cells without the added VEGF production, and cell-free buffer. By injecting the cells intrathecally, they were able to position them near their target but avoid direct damage to the spinal cord. When the researchers sacrificed the animals four weeks after transplantation, they found human cells along the spinal cord, with the majority (64 percent) along the meninges and some (12 percent) in the parenchyma as well.

The mice received the therapy before symptoms normally appear. In the animals treated with VEGF-boosted cells, poor performance on the standard rotarod test was delayed, first appearing at 122 days of age instead of 115 in both sets of control animals. Paw grip endurance and extension reflexes also remained normal longer in the VEGF treatment group. The treated mice lived for 145 days, versus 141 in the control cell population and 133 in the buffer population. The differences between the VEGF treatment group and the buffer group were statistically significant.

The positive effects could be a result of the added VEGF protecting cells, or the transplanted cells might actually replace damaged motor neurons. The researchers found evidence for both. Some grafted cells expressed the neural marker MAP2+, indicating they might have differentiated into motor neurons. However, there is not yet evidence that the new cells joined up with the old in functioning neural networks. In addition, the scientists looked at apoptosis markers in the spinal cords of transplanted mice. The pro-apoptotic proteins caspase 3 and Bax were reduced, and the anti-apoptotic markers Bcl-2 and Bcl-XL increased, in the treated mice compared to those injected with buffer alone.

Any cell-based therapy will only work as long as the cells survive. VEGF clearly was of benefit in this respect: VEGF-producing cells survived longer than control grafted cells, with 64 percent of VEGF cells still present at four weeks versus 38 percent of the parent cell line. The VEGF cells may have enhanced their own survival via the excess growth factor, but they still died over time. Part of the problem, Kim suggested, might be that the human cells were a xenograft; human-to-human transplants might last longer. The cell line used in this study would be inappropriate for human use, Kim said, because it expresses the myc oncogene. He suggested that cell lines based on patients’ stem cells or induced pluripotent cells might be a better approach in the clinic.

The current study is hardly the first to use stem cells to treat ALS. Some previous approaches have stalled disease (Corti et al., 2007); others have not (Park et al., 2009). “This approach of using neuronal stem cells to carry the genes is, for now, the best way to go,” Kim said. But other techniques certainly have merit, Kaspar said, and there is more than one contender in the race to the clinic. Those approaches that do succeed, stem cell-based or otherwise, seem to slow the disease by similar amounts, Kaspar said, suggesting the growth factor works in the same way when delivered appropriately. As for those techniques that fail, he speculated the growth factor might need to achieve a certain threshold level before assisting struggling neurons.

In the case of stem cells, questions remain about how many cells migrate to the right site and how long they will last there. There is also the risk that the grafted cells will proliferate and cause cancer. Kim suggested as a failsafe, the cells could be engineered to carry an inducible “suicide” gene that doctors could activate with, say, tetracycline in the case of overproliferation.

Neural stem cells are an ideal delivery vehicle, Kim suggested, not only for ALS but for Parkinson disease, Huntington disease, spinal cord injury, and brain cancer (reviewed in Kim and de Vellis, 2009). The cells can carry genes tailored to each condition: enzymes that make L-dopa for Parkinson’s, for example, or an inducible killer for tumors. “This is universally applicable,” he said.—Amber Dance

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References

News Citations

  1. Viral VEGF Treats Mouse ALS
  2. Repairing Damaged Tissues—Viruses Get into the Akt
  3. IGF-1 Disappoints in Trials for AD, ALS

Paper Citations

  1. Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous KA, Kingsman SM, Carmeliet P, Mazarakis ND. VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature. 2004 May 27;429(6990):413-7. PubMed.
  2. Kaspar BK, Lladó J, Sherkat N, Rothstein JD, Gage FH. Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science. 2003 Aug 8;301(5634):839-42. PubMed.
  3. Nagano I, Shiote M, Murakami T, Kamada H, Hamakawa Y, Matsubara E, Yokoyama M, Moritaz K, Shoji M, Abe K. Beneficial effects of intrathecal IGF-1 administration in patients with amyotrophic lateral sclerosis. Neurol Res. 2005 Oct;27(7):768-72. PubMed.
  4. Sorenson EJ, Windbank AJ, Mandrekar JN, Bamlet WR, Appel SH, Armon C, Barkhaus PE, Bosch P, Boylan K, David WS, Feldman E, Glass J, Gutmann L, Katz J, King W, Luciano CA, McCluskey LF, Nash S, Newman DS, Pascuzzi RM, Pioro E, Sams LJ, Scelsa S, Simpson EP, Subramony SH, Tiryaki E, Thornton CA. Subcutaneous IGF-1 is not beneficial in 2-year ALS trial. Neurology. 2008 Nov 25;71(22):1770-5. PubMed.
  5. Kim SU. Human neural stem cells genetically modified for brain repair in neurological disorders. Neuropathology. 2004 Sep;24(3):159-71. PubMed.
  6. Lee HJ, Kim KS, Park IH, Kim SU. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS One. 2007;2(1):e156. PubMed.
  7. Corti S, Locatelli F, Papadimitriou D, Del Bo R, Nizzardo M, Nardini M, Donadoni C, Salani S, Fortunato F, Strazzer S, Bresolin N, Comi GP. Neural stem cells LewisX+ CXCR4+ modify disease progression in an amyotrophic lateral sclerosis model. Brain. 2007 May;130(Pt 5):1289-305. PubMed.

Further Reading

Papers

  1. Thonhoff JR, Ojeda L, Wu P. Stem cell-derived motor neurons: applications and challenges in amyotrophic lateral sclerosis. Curr Stem Cell Res Ther. 2009 Sep;4(3):178-99. PubMed.
  2. Appel SH, Engelhardt JI, Henkel JS, Siklos L, Beers DR, Yen AA, Simpson EP, Luo Y, Carrum G, Heslop HE, Brenner MK, Popat U. Hematopoietic stem cell transplantation in patients with sporadic amyotrophic lateral sclerosis. Neurology. 2008 Oct 21;71(17):1326-34. PubMed.
  3. Badayan I, Cudkowicz ME. Is it too soon for mesenchymal stem cell trials in people with ALS?. Amyotroph Lateral Scler. 2008 Dec;9(6):321-2. PubMed.
  4. Morita E, Watanabe Y, Ishimoto M, Nakano T, Kitayama M, Yasui K, Fukada Y, Doi K, Karunaratne A, Murrell WG, Sutharsan R, Mackay-Sim A, Hata Y, Nakashima K. A novel cell transplantation protocol and its application to an ALS mouse model. Exp Neurol. 2008 Oct;213(2):431-8. PubMed.
  5. Mazzini L, Mareschi K, Ferrero I, Vassallo E, Oliveri G, Nasuelli N, Oggioni GD, Testa L, Fagioli F. Stem cell treatment in Amyotrophic Lateral Sclerosis. J Neurol Sci. 2008 Feb 15;265(1-2):78-83. PubMed.
  6. Lee H, Shamy GA, Elkabetz Y, Schofield CM, Harrsion NL, Panagiotakos G, Socci ND, Tabar V, Studer L. Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells. 2007 Aug;25(8):1931-9. PubMed.
  7. Kim SU. Genetically engineered human neural stem cells for brain repair in neurological diseases. Brain Dev. 2007 May;29(4):193-201. PubMed.

Primary Papers

  1. Hwang DH, Lee HJ, Park IH, Seok JI, Kim BG, Joo IS, Kim SU. Intrathecal transplantation of human neural stem cells overexpressing VEGF provide behavioral improvement, disease onset delay and survival extension in transgenic ALS mice. Gene Ther. 2009 Oct;16(10):1234-44. PubMed.

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