12 Oct 2012

As scientists celebrate the 2012 Nobel Prize in Physiology or Medicine for the development of induced pluripotent cells, researchers are looking to traditional and modern stem cell sources to treat diseases of the brain in both young and aging people. An old standby, fetal brain cells, provides a potential treatment for a missing myelin disorder, according to two papers in the October 10 Science Translational Medicine. The authors treated four children, in addition to mouse models, with neural stem cells banked by StemCells, Inc., of Newark, California. The treatment was safe, and the team saw evidence that the transplants morphed into the needed oligodendrocytes that myelinate naked axons.

Another potential source of stem cells treatments is the body’s own cells. In the September PLoS One, researchers from Seoul National University describe how they transplanted easily obtained adipose-derived stem cells into Alzheimer’s model mice, and achieved an improvement in learning and memory. Senior author Yoo-Hun Suh believes the transplanted stem cells provided cytokines and growth factors that eliminated amyloid-β and supported sickly neurons.

It’s in the Bank
StemCells, Inc., maintains a collection of neural stem cells (NSCs) expanded and frozen from a donated fetal brain (Uchida et al., 2000). Researchers are testing this cell population in a variety of conditions, such as macular degeneration, spinal cord injury and Batten disease, a rare childhood neurodegenerative disorder. The clinical trial is the first human safety study with these cells to be published, said senior author David Rowitch of the University of California, San Francisco.

Rowitch, first author Nalin Gupta, and colleagues injected the NSCs into four young boys with a rare disorder called Pelizaeous-Merzbacher disease, or PMD. Affecting one in 200,000-500,000 children, this extremely rare condition is one of many leukodystrophies. These diseases damage the heavily myelinated white matter of the brain. PMD results from a recessive mutation in the gene for proteolipid protein 1 (PLP1), a key myelin component. Children with PMD often require machines to breathe and eat, and many cannot sit up, crawl, walk or talk, Rowitch said. Those with the severest disease will die from it.

Animal studies showed that StemCells’ banked NSCs happen to be particularly good at turning into oligodendrocytes, making PMD an ideal test case. Plus, since the kids make almost no myelin, their brains would provide minimal background signal in a magnetic resonance image (MRI) for myelin. Any evidence of myelination would have to come from the transplanted cells.

Gupta injected three million NSCs into each boy, dividing the cells between four shallow sites in the frontal lobe. Over the next year, the researchers examined the children and performed MRIs. “We saw signals that are suggestive of myelin production,” Rowitch said of the images. (They did not want to subject the children to a biopsy to confirm it.)

Three of the boys improved in skills such as walking and eating on their own. However, this was probably not due to the injection of the small stem cell populations at a few sites, Rowitch said, but merely their natural development. In this Phase I safety study, all the researchers were aiming for is that the cells should do no harm, which they did not. The team will continue to follow the participants for four more years, and Rowitch is pondering the design for a controlled, Phase II trial to look for benefit from the treatment.

Gupta’s work depended on a preclinical mouse study, performed before the trial but published alongside it in Science Translational Medicine. Researchers at StemCells, Inc., led by first author Nobuko Uchida, grafted NSCs from the bank into the corpus callosum, fornix, and cerebellum of young shiverer mice. Lacking the gene for myelin basic protein, these mice and are a good phenocopy of human PMD, said senior author Stephen Back of the Oregon Health & Science University in Portland, where most of the animal analysis was performed.

The researchers implanted newborn and three-week-old mice. The latter had begun to suffer tremors because their oligodendrocytes encircled their axons with loosely wound, dysfunctional myelin. No matter the age, the NSCs found their niche. Sixty to 70 percent differentiated into oligodendrocytes, migrated past the injection site, and produced myelin sheaths with normal gaps, or nodes of Ranvier, along the axons. Electrophysiology on brain slices indicated that neural transmission sped up due to the new myelin. “They form nice oligodendrocytes,” said James Weimann of Stanford Medical School in Palo Alto, California, who was not involved with the study (see full comment below). “The interspacing of the nodes looks great, as does the conduction velocity.”

Weimann was pleased to see that five to seven percent of the transplanted NSCs expressed the cell proliferation marker Ki67. This indicated the cells were dividing and keeping up a good-sized pool of progenitor cells. But they did not proliferate so much as to form tumors, which bodes well for long-term treatment. The team could not measure transplant half life, since shiverer mice are quite ill, and they died within 10 weeks. However, in other experiments, transplanted oligodendrocyte progenitors have persisted for more than a year (Windrem et al., 2008).

The key challenge in translating these results to people, Weismann suggested, will be encouraging the new cells to migrate and populate the brain. While human transplants travel well in mouse brains, it is not known if they will do the same in human brains.

If the researchers are successful, people with PMD could be the first of many to benefit from their efforts. “This raises the possibility that stem cell therapy may be part of the tools we use to repair or slow injury to the white matter,” Back said. “The challenge is to fine-tune the therapy to the unique characteristics of [each] particular disease.” In children, cerebral palsy might be a possible target, he suggested. People with demyelinating conditions such as multiple sclerosis and vascular dementia might also benefit—but it could take decades to reach that point, Rowitch noted.

Auto-Harvest
In the PLoS One paper, the Korean team focused not on the white matter but on the grey, in the hopes of designing a treatment for Alzheimer’s. They used a different source, namely human stem cells from people undergoing liposuction. Obtaining these adipocyte-derived cells is “very simple and convenient,” said study author Suh. He envisions someday transplanting a person’s own fat-derived cells into the brain, thus eliminating the possibility of immune rejection. Plus, he noted, using one’s own cells is more ethically palatable than cells from donated fetal tissue. Scientists have tested adipose-derived stem cells in models of Huntington’s disease (Lee et al., 2009) and stroke (Bhang et al., 2009).

Joint first authors Saeromi Kim and Keun-A Chang used the stem cells to treat Tg2576 mice expressing mutated human amyloid precursor protein. They injected the stem cells intravenously in one set of mice, and intracranially in another. In a water maze test, both sets of treated animals matched wild-type mice in recalling the place where a hidden platform used to be. “Learning and memory almost recovered to normal,” Suh said.

The researchers counted fewer amyloid plaques, and measured less amyloid-β overall, in the brains of transplanted animals. Suh believes the benefits stem from the upregulation of the anti-inflammatory cytokine IL-10, and neurotrophic growth factors such as VEGF, that the researchers saw in the brains of treated animals. The team noticed no negative side effects. Suh is planning a clinical trial.

“Adipocytes are a great resource for stem cell collection,” commented Tsuneya Ikezu of Boston University, who was not involved in the study (see full comment below). At the same time, he expressed some reservations about the preclinical data. He was not convinced that the stem cells actually crossed from the circulatory system into the brain. Suh, in an email to Alzforum, responded that the stem cells and neurons must be in direct contact to promote symptom improvement he observed. In addition, Ikezu suggested that the beneficial molecules such as IL-10 could have arisen due to a peripheral immune response to the foreign human cells, not because of the cells’ protective activity. Suh believes the mice did not mount an immune response to the human cells, because the anti-inflammatory cytokine IL-1β was actually decreased in the animals.

While transplants appear to be a simple way to provide new stem cells where they are needed, another option is to keep the whole treatment process inside the body. Transdifferentiation, as it is called, shepherds cells from one differentiated fate to another, skipping the Nobel prize-winning step of induced pluripotency (reviewed in Vierbuchen and Wernig, 2011). Researchers at the Ludwig-Maximilians University in Munich, Germany, recently reported that they reprogrammed human pericytes directly into neurons. They obtained the pericytes from tissue samples donated by people who had brain surgery to remove a lesion or treat epilepsy. “Our data provide strong support for the notion that neuronal reprogramming of cells of pericytic origin within the damaged brain may become a viable approach to replace degenerated neurons,” the authors concluded (Karow et al., 2012).—Amber Dance

Comments

- James Weimann
  Stanford Medical School
- Posted: 12 Oct 2012
This is very exciting work. The paper by Uchida etal provides the animal model and proof in principle for the clinical trial. Although the “shiverer” mouse has been used extensively to study myelination with many types of transplanted cells obtained from rodents and humans, this study clearly demonstrates that fetal derived HuCNS-stem cells can integrate into existing axon fiber tracts and myelinate axons.

Several points in the Uchida paper are of interest:

1) The low proliferation rate of 5-7 percent of the human cells after transplantation suggests that these stem cells are generating more progeny and may provide more oligodendrocytes over time. The problem with the shiverer mouse model is that the mice do not live long enough to do behavioral studies after transplantation and to assess the extent of continuous production of myelinating cells. The data presented suggest that these cells are a good source for myelin producing oligodendrocytes.

One worry on everyone’s mind is always whether these cell will proliferate in an uncontrolled manner to produce tumors. It appears from these and other studies using these HuCNS cells that this is not the case, which is critical for human trials.

2) It is very interesting that the human cells appear to be responding to the same signals as the endogenous mouse cells by proliferating and making more healthy human oligodendrocytes. This suggests that the signals originating in the diseased brain to make more myelin producing cells are interpreted in a similar fashion by both human and mouse oligodendrocytes. What is really interesting is that the human stem cells seem to prefer to differentiate into predominantely myelin producing oligodendrocytes over the production of neurons and glial cells. This results in the generation of the needed cell type and minimizes the appearance of the “wrong” cell types.

The clinical study by Rowitch’s group shows that these human HuCNS-SCs can be transplanted into young human brains and engraft. There does not appear to be any overgrowth of transplanted cells, thus minimizing the concern of tumor formation. The data using magnetic resonance imaging methods suggest that these cells are contributing to the generation of new myelin in and around the transplantation sites. It does not appear that the cells migrate as extensively in the human brain as they do in the rodent brain, which is consistent with many other transplant studies that compare the migration of human cells and rodent cells. Human cells can migrate great distances in the rodent brain. Whether this is true in the human brain is less certain.

Nonetheless, this phase 1 study provides exciting new data that these HuCNS-SCs are well tolerated and point to future trials. One would expect that when larger regions of the brain are infused with healthy cells a better outcome could be achieved. Intervention earlier in the disease process may also produce a better outcome. The authors note that since this was a phase 1 trial, no subjects were transplanted as controls. Therefore the minor behavioral improvements observed, while very encouraging, are hard to interpret. This study should lead to another trial that may be performed on younger subjects.

View all comments by James Weimann

- Tsuneya Ikezu
  Mayo Clinic Florida
- Posted: 12 Oct 2012
I am cautiously optimistic about these results. We have also found that hippocampal viral gene delivery of FGF2 or IL-10 is beneficial in spatial learning and reducing amyloid load in APP+PS1 mice. However, it is surprising to see that xenogenetic immune rejection is not an issue by intravenous injection of human stem cells to immunocompetent mice for 13 times over 3 months in this study.

Figure S1 shows the migration of fluorescent particles labeled hASC into the brain, but it is unclear if they transmigrated across the blood brain barrier into brain parenchyma, or still stayed in the cerebrovasculature. It is also unclear if the same result was obtained after repetitive injection of hASC. The fluorescent signal could be directly from the particles but not from hASC. Some immunofluorescence or electron microscopic imaging would be helpful for future studies.

As for IL-10 or VEGF upregulation in brain after hASC injection, these factors could be from peripheral blood or the cerebrovascularture (especially VEGF is highly abundant in endothelium). If so these are rather indicative of peripheral inflammation due to hASC xenotransplantation. A comprehensive characterization of peripheral immune activation would be desirable. As for the in-vitro study, I am not sure what is reconstituted in vitro without detection of hASC-neuron-microglia interaction in Tg2576 mouse brain. It is likely that hASC injection may contribute to some beneficial effects to hippocampal function in a peripheral manner, not necessarily via migration in to the CNS. I am curious if mouse ASC from the original host would have the same effect in this study.

Nonetheless, stem cell approaches should be encouraged to be rigorously characterized to understand their therapeutic applications.

References:

Kiyota T, Ingraham KL, Jacobsen MT, Xiong H, Ikezu T. FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders. Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):E1339-48. PubMed.

Kiyota T, Ingraham KL, Swan RJ, Jacobsen MT, Andrews SJ, Ikezu T. AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice. Gene Ther. 2012 Jul;19(7):724-33. PubMed.

View all comments by Tsuneya Ikezu

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References

Paper Citations

Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14720-5. PubMed.
Windrem MS, Schanz SJ, Guo M, Tian GF, Washco V, Stanwood N, Rasband M, Roy NS, Nedergaard M, Havton LA, Wang S, Goldman SA. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell. 2008 Jun 5;2(6):553-65. PubMed.
Lee ST, Chu K, Jung KH, Im WS, Park JE, Lim HC, Won CH, Shin SH, Lee SK, Kim M, Roh JK. Slowed progression in models of Huntington disease by adipose stem cell transplantation. Ann Neurol. 2009 Nov;66(5):671-81. PubMed.
Bhang SH, Cho SW, Lim JM, Kang JM, Lee TJ, Yang HS, Song YS, Park MH, Kim HS, Yoo KJ, Jang Y, Langer R, Anderson DG, Kim BS. Locally delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs. Stem Cells. 2009 Aug;27(8):1976-86. PubMed.
Vierbuchen T, Wernig M. Direct lineage conversions: unnatural but useful?. Nat Biotechnol. 2011 Oct;29(10):892-907. PubMed.

Stem Cells Make New Myelin, Show Potential for AD

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