Newly published recipes direct neural stem cell research
By Cynthia Fox
How do you make a neuron? Nowadays, that depends on how you like your neurons. Perhaps you're having problems expanding your neural stem cells (NSCs) to large numbers because after repeated passaging, they lose the phenotype or go into crisis. Read the June Nature Biotechnology, where National Institute of Neurological Disorders and Stroke neurobiologist Ron McKay offers a recipe for making dopaminergic and GABAnergic neurons from rat embryonic stem (ES) cells.1 Because they often proliferate more rapidly than NSCs, ES cells do much of your expansion work for you.
Out of ES cells? Make your own. This month in Current Biology, Stem Cell Sciences offers a recipe for making rejection-free, designer mouse ES cells out of somatic cells, via nuclear transfer.2 Or check out recipes in two journals last month for turning adult bone marrow stromal cell (BMSC) precursors into neurons,3 or any number of recent papers on committed progenitors, which do much of your differentiation work for you.
Or just sit back and watch. For the Food and Drug Administration recently approved one of the country's first NSC clinical trials. "There's going to be an explosion in stem cell therapy," neurosurgeon Michel Levesque of Cedars-Sinai Medical Center in Los Angeles has predicted.
Neuroscientists have been making neurons from neural precursors for years. Petri dishes worldwide have teemed with the sprawling, spidery things. "It's not crazy anymore to think we'll understand the molecular controls of neurogenesis," says Harvard University neurologist Jeffrey Macklis.
Lately, optimism has been fostered in part by Macklis' group, which showed that the neurons that researchers are creating may be making or facilitating functional connections. Many papers offer powerful preliminary evidence that NSC transplants are functional. For example, adult parkinsonian rats can regain function after receiving dopaminergic neurons created in vitro out of fetal rat neural precursors.4 Newborn mice whose neurons lack myelin sheaths stop shivering after transplanted immortalized newborn mouse NSCs form new oligodendrocytes.5
Lab coups occur daily. But it's been difficult to demonstrate that these are due to electrophysiological connections, not trophic factors. Adds University of California neurobiologist Hans Keirstead, "The greatest stem cell transplantation successes have been transplants into experimental models of demyelination. Such clean 'lesions' with intact axons and cells direct differentiation to glial cells, which repair the lesion. However, such an environment never exists in human disease."
In February, however, Macklis and Rockefeller University neuroscientists Constance Scharff and Fernando Nottebohm went a long way toward demonstrating that the brain is capable of being coaxed into functional regeneration. When they lesioned neurons that naturally turn over at very low levels in adult songbirds, the birds permanently stopped singing. But when they induced a slow, selective apoptosis similar to Alzheimer's degeneration, the birds eventually sang again. Labeling revealed that they had recruited a dramatic increase in neuronal replacement.6
In June, Macklis' group showed that neurons can even regenerate in the cortex, if endogenous precursors are exposed to the correct molecular signals. When the team initiated a slow apoptosis to specific projection neurons deep in cortical layer 6 of adult mice near endogenous stem cells, markers revealed the growth of new cortical neurons, some with axons reaching to the appropriate targets in the thalamus, suggesting a hook-up with damaged circuitry.7
The Big Question
Still, says Macklis, "There are many molecular steps involved, and we're applying these all at once, not under individual control. We're not doing it perfectly. It will take a while to more fully understand the critical signals involved." But while fetal cells presently seem more flexible, the fact that adult and fetal NSCs can respond to similar cues has "massive implications. We don't have to start over in the adult brain," says McKay. So the big question is often asked: How well to understand NSCs before using them?
Certainly, no one knows if they have the NSC yet. Many cells seem to meet basic requirements: the ability to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. But the cortex alone contains hundreds of neuron types. Which cell makes all? Also, the cells are often cocultured, so it's hard to gauge which ones self-renew. And no unique markers are known. A National Institutes of Health panel is devising a group of markers--and lack thereof--that may suffice. Not until all markers are in can researchers decide, for example, if the BMSCs in recent experiments are true BMSCs, or rare wandering ES-like cells. If the cells are BMSCs and form true neurons, their accessibility makes them a jackpot for neurologists.
Furthermore, while neurons overrun petri dishes, the brain can have other ideas. "We run into a major problem when we transplant stem cells in vivo," says Keirstead. "The CNS [central nervous system] dictates the fate of transplanted stem cell populations. The researcher does not. The Parkinson's and Alzheimer's fields will tell you that whatever stem cell population you put into a CNS lesion, it always generates glial cells." Others note that no one has published the perfect recipe for dopaminergic neurons--a Holy Grail for some--from adult NSCs, key if a new funding ban on embryo work is levied.
But many are getting greater neuron yields in vivo by tirelessly altering growth-factor doses, cell-to-cell contact, and cell sources. Others pursue ES work like McKay's, which Keirstead calls "a terrific example of modifying tissue culture conditions in subtle and sequential steps to increase amplification and direct differentiation." Still others turn to committed progenitors, the NSCs' more differentiated daughters. "We can get around our ignorance by transplanting predifferentiated cell populations," says Keirstead. "That field will have a very bright future." As things stand, such cells in vivo may retain the phenotype better and may expand and migrate more easily. "The implications are huge," says University of Utah neurobiologist Mahendra Rao.
How to Avoid Mutation Accumulation?
A nagging concern is that many cancers spring from stem or progenitor cells whose DNA has mutated.8 Because these cells make up the bulk of the body's dividing cells, they are likely to fall prey to DNA replication errors, which cause mutation. Two to six mutations may turn a cell malignant.
Furthermore, though progenitors may divide more transiently than stem cells, if an error or a carcinogenic hit occurs in a growth-driving gene, progenitors can keep dividing, increasing the odds of mutation accumulation. NSCs are rarer and may replicate more slowly in vivo, but they may do so indefinitely. So NSCs may pump out altered daughter cells endlessly.
Indeed, stem cells are leading oncologists to new conclusions. Oligodendroma, once assumed to come from a single mature brain cell, is now believed to be made of both oligodendrocytes and astrocytes, an indication that it arises from a bipotent progenitor.9 Medulloblastomas are now thought to arise in committed progenitors; many neuroblastomas are believed to come from the blocked differentiation of neural crest stem cells. Growth factors used to differentiate stem cells are being tried as cancer therapy.
The cell's repair mechanisms, however, correct the vast majority of DNA errors. NSCs are more active in the adult CNS than once thought--yet only 2 percent of cancers occur there. And NSC researchers are aware. Occasionally they see tumors, especially when cells are overpassaged. "Because we're trying to remove some of the cells' in vivo proliferative restrictions," says Macklis, "we're potentially setting up the possibility for uncontrolled .proliferation."
So many who expand NSCs epigenetically, changing the environment by adding substances such as growth factors but not altering the cells via gene transfer, are in hot pursuit of just-so cells--not too committed, not too flexible, and in many cases, not too different from cells in target areas. Says Albert Einstein University neurologist Mark Mehler, "We have significant experience with transplanted NSCs that exhibit unrestricted growth and [both] variable and lineage-selective neural differentiation in vivo. We are exploring the specific environmental conditions and intrinsic signals that differentially mediate these cellular events." In July, University of Cambridge neuroscientist Clive Svendsen offered the first report that fetal human neuroblasts, mixes of NSCs and progenitors, contain telomerase levels 50 times lower than a lung cancer line in the first 20 doublings, none later.10 Many cells, he says, may be best transplanted shortly after 20 doublings. He is also studying the cells' telomeres, to gauge the length offering optimal safety and efficacy. "Concerns are valid but need to be tested ..., which is what we are in the process of doing."
An alternative offered by Harvard neurobiologist Evan Snyder, among others, is NSCs that are immortalized--in Snyder's case, via the retroviral transfer of the Vmyc gene, which is expressed in some cancers. Snyder's mouse cells form whatever cells are needed in a huge variety of impaired mice--and he's seen no tumors in hundreds. More homogenous than primary cell controls, because they divide five to 10 times faster and need less time in culture, similar fetal human cells stop dividing after implant or growth factor removal, says Autonomous University of Madrid molecular biologist Alberto Martinez-Serrano.11 But such cells worry many. Two exciting papers showed this year that immortalized precursor cells--engineered to produce therapeutic agents--can track and, in one paper, shrink tumors.12 Yet Nature Medicine asked that the feat be repeated with epigenetically expanded cells, says one author. (It was.) Caution is justified, says Martinez-Serrano, who finds cells can still express Vmyc post-implant. He says suicide genes may be crucial for immortalized NSCs until scientists understand them better.
Benefits Outweigh Risks?
The FDA, however, apparently believes researchers know enough about Cedars Sinai's cells for benefits to outweigh risks, so a trial is brewing. In 1998, 10 to 15 NSCs were taken from the frontal lobe of a Parkinson's patient during an electrode implant at Cedars-Sinai. Due partly to fibroblast growth factor and the cells' telomerase, Levesque has said, they proliferated merrily. Six million were made dopaminergic (with factors he can't name for patent reasons). In March 1999, a mix of NSCs and neurons was placed in the patient's left putamen.
Pretransplant, the patient was "quite impaired" by rigidity, akinesia, dyskinesia, and depression, according to Levesque. But within weeks, "he improved to the point that we halved his medication. On a UPDRS [Unified Parkinson Disease Rating Scale] motor scale he improved 40-50 percent in certain tasks. His dopamine uptake increased 62 percent where we transplanted the cells. The rigidity, akinesia, and dyskinesia were gone." Also gone was the patient. He took off for Indonesia (scuba diving), Africa, and Egypt. "This guy doesn't stop." The hospital will release details of the planned trial after institutional review board review.
There have been few related trials. Although 300 patients have had fetal tissue transplants (which prompted 120 improvements and at least one death from accessory cell overgrowth), the grafts aren't comparable--they are mixes of unknown cells. And from 1998 to 1999, 12 stroke patients received Layton Bioscience (LBS) neurons made from stem cells. But unlike NSCs, the LBS cells were derived from embryonic tumor (teratocarcinoma) cells and had chromosome abnormalities. LBS treated them with differentiation agents and metabolic inhibitors (retinoic acid, cytosine arabinoside, fluorodeoxyuridine, and uridine). But there are factors in cells that can reverse or block the effects of some of the agents. No assay detects all cancerous cells. Though no tumors were spied in the one stroke-induced rat study published pretrial, rats live 24 months. Cancer can take years to develop. So while LBS has good news--half the patients saw improved PET scans--its trial isn't relevant, many say.
Meanwhile, cornea precursor trials are doing well.13 And this month, Harvard assistant scientist Michael Young reports that NSC transplant patients may not need cyclosporine. When he placed neurons from one rat into the kidney capsules of another, they were rejected. But NSCs weren't, and neither were the neurons they formed. Analysis revealed that NSCs may express few "self" antigens.14 How they may "train" hosts to accept mature cells may give immunologists clues to the blood/brain barrier. In many fields, says McKay, "The cellular revolution has begun."
Cynthia Fox is a freelance science writer in New York.
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