Monya Baker
Electron microscopy reveals functioning synapses
[Editor's note: It's not easy to assess a study's ramifications from press releases, three of which caught my eye this month. I asked Phil Schwartz, a neural stem cell expert at Children's Hospital of Orange County, California, to help me understand the gap between study and therapy. I also asked authors from each paper to respond. This article is one of three resulting from this process.]
Research summary by Nature Reports Stem Cells: For many types of cell therapy, getting transplanted cells to integrate into a patient's tissue will be harder than making the cells. Researchers led by Leyan Xu of Johns Hopkins Medical Institutions, in Baltimore, Maryland had previously shown that grafts of human neural stem cells (NSCs) could relieve symptoms in a rat model of amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease).
To try to understand what was happening, the researchers grafted human neural stem cells into the spinal cords of 18 rats with and without superoxide dismutase 1 (SOD1) mutations, a mutation which causes the rats to display many symptoms of the disease. Forty days later, they carefully labelled the human cells and examined the tissue. The fetally derived neural stem cells had developed into into interneurons within the spinal cord and were forming structurally mature synapses with the rat motor neurons in both the normal and the SOD1-mutated rats1.
News coverage: A press release describing the article was sent out to a public relations newswire by Neuralstem Inc., located in Rockville, Maryland
It's worth noting that Neuralstem plans to use the cells in a human clinical trial for ALS that is under review at the US Food and Drug Administration (FDA) in Silver Spring, Maryland. Karl Johe, the company's chief scientific officer and an author on the paper, says that Neuralstem has addressed all of the FDA's concerns, and that, optimistically, the company could receive permission to go ahead with the trial in less than a month.
Schwartz's take: It's a very interesting paper. Clearly the researchers show that these human-derived fetal neural stem cells do engraft nicely in these rats.
But these are very short-term experiments. It would be interesting to see longer-term experiments for one major reason: two recent papers in Cell Stem Cell showed some interesting findings from SOD1-mutated cell lines2, 3. What they showed was that if you isolated embryonic stem cell lines and grew them, the neurons grown by themselves survived quite nicely, but if you grew them in the presence of their compatriot glia, they didn't. Furthermore, if you took neurons from normal embryonic stem cells and grew them in the presence of those SOD1 knockout glia, those neurons died. So this suggests that the cause of neuronal death is not inherent to the neurons themselves but is [in] the environment in which the neurons find themselves — the glial environment.
This is the basic issue with all of these transplantation studies: you really have to know what your disease is and what your target should be if you're going to use neural stem cells. This is an interesting study, but it really begs the question as to whether it's the right study. Especially since it was short term, there was no way to determine whether the neurons would actually survive long term in this toxic environment.
Author reply:
Nature Reports Stem Cells (NRSC): My understanding is that recent cell-based work indicates that the cause of ALS is that the supporting glial cells poison the neurons. If so, won't the introduced neurons get poisoned too, rendering the benefits temporary at best? How can you tell?
Leyan Xu, Johns Hopkins Medical Institutions: Motor neuron degeneration is a complicated process that involves intrinsic (autonomous) as well as extrinsic (non-cell autonomous) mechanisms. Toxicity caused by glial cells may be contributory. The fact is that, in rodents with SOD1 mutations that are supposed to confer glia toxicity, neurons differentiating from our human stem cell grafts do just fine. Some of the grafted NSCs differentiate into glial cells, which may also help alleviate potential damage [caused] by mutant glial cells. Whatever the case, there is no question that motor neuron degeneration in ALS is a complex process that may require multiple therapeutic approaches and interventions.
NRSC: What is the main hurdle that this paper overcomes?
Xu: This is a triple hurdle. First, we show that new neurons differentiated from stem cell grafts can make synapses with neurons of the host nervous system. Second, we show that this can be done in the spinal cord, which normally has little, if any, potential for endogenous regeneration. Third, we show that this [synapse formation] can be done between neurons differentiating from human stem cells — that is, human neurons and neurons in rodent hosts (rodent neurons). All these are amply demonstrated in the manuscript.
NRSC: How can you know the synapses are functional after the tissue has been removed from the rats and stained?
Xu: The ideal way to test functionality is with electrophysiological methods. However, this would be extremely difficult to do in the spinal cord in vivo, i.e. firing graft-derived neurons with one electrode and recording from host neurons via a second electrode. The trans-synaptic retrograde transfer of PRV (a virus used here as tracer) and the ultrastructural maturity of these new synapses are strong indicators that the synapses are functional.
NRSC: [The paper describes the neurons as inhibitory.] Why does it matter that the neurons are inhibitory?
Xu: First, because this is the functional role of one of the most abundant types of neurons in the spinal cord — interneurons. In other words, grafted human cells seem to become interneurons or interneuron-like cells. Second, excitotoxicity (that is, excessive glutamate effects on neurons) is one of the mechanisms of cell death in motor neuron disease; thus, neurons differentiated from these grafts may have a greater therapeutic potential.
[Editor's note: Schwartz's comments are transcribed from an interview. Xu's responses reflect e-mailed replies to e-mailed questions.]
[Editor’s note: an earlier version of this article indicated that the human cells became motor neurons, the cells that communicate with muscle, rather than interneurons, the cells that communicate between neurons. That has been fixed.]