Date: November 10, 2003
Subject: Making A Difference In The Lives Of Children: Stem Cells And Repair Of The Developing Brain
Location: The Society for Neuroscience 33rd Annual Meeting, New Orleans, Louisiana
Attendees: Top neuroscience researchers , including Dr. Evan Snyder of Harvard Medical School, Dr. Nicholas Gaiano of Johns Hopkins School of Medicine, Baltimore, Dr. Martha Windrem, of Weill Cornell Medical College, and representatives of the nonprofit community such as Ms. Fia Richmond.
Recap: Pediatric Brain Foundation hosted a panel discussion on how stem cell research could offer new avenues for recovery from pediatric brain injury and disease. Speakers provided examples through specific models of disease.
The workshop was universally hailed as a success. While there was incredible optimism that scientists are on the cusp of an extraordinary breakthrough in their ability to treat childhood neurological disorders with stem cells, there was also a sense that much work must be done before victory can be declared in earnest. Participants left with the resolve to do whatever is necessary to bring these therapies to children as quickly as possible.
Highlights From the Annual Meeting of the American Society of Neuroscience; November 8-12, 2003; New Orleans, Louisiana
Conference Report – Stem Cells and Neurologic Repair
–Sara M. Mariani, MD, PhD
Stem cells, neurogenesis, and repair — this powerful combination of words announces how far neuroscience has come in the past few years in the search for new venues of treatment, [1-4] forecasting tissue repair in brains damaged by stroke or in spinal cords injured by trauma.  Replacement of selected cell populations is also being proposed and investigated for Parkinson’s disease or Alzheimer’s disease, [6-8] neurodegenerative diseases affecting mostly adults in the later decades of their lives. But what are we doing for infants and children with cerebral palsy or developmental disability, congenital defects of their central nervous system (CNS) that severely impair execution of many functions?
Notwithstanding all the efforts of their caregivers, quality of life for these young subjects can be, at times, substantially compromised; even more so when they age and develop secondary complications. Many advances have been made in their management and rehabilitation. Yet, often these children’s life expectancies are lower than those of their peers. 
Cerebral palsy and developmental disabilities are conditions that affect children from all countries and all ethnic backgrounds. It is estimated that there are at least 14 million children living with these diseases in the United States alone. Is there a way to give them a second chance?
These issues were addressed at a symposium held during the 33rd Annual Meeting of the Society for Neuroscience, in New Orleans, Louisiana, entitled: “Stem Cells and Pediatric Disorders: Forging New Paths to Progress” under the auspices of the Children’s Neurobiological Solutions foundation. Neuroscience is, nowadays, certainly one of the “hottest” research fields in biomedicine. Will the hopes raised indeed meet their promise?
Three leading researchers who are experts in cutting-edge research on brain repair using stem cells, Dr. Evan Snyder, Dr. Nick Gaiano, and Dr. Martha Windrem, gave an overview of the state of the art in this field, outlining successes and limitations of the strategies that are currently being evaluated in experimental animal systems.
Intrauterine Delivery of Vectors and Cells
First of all, one wonders: can we deliver biologic agents or even cells into the brain? And if indeed this is possible, when is it best to do so?
Viral vectors as well as cells can be delivered into the CNS via intrauterine surgery using ultrasound imaging. Dr. Nicholas Gaiano,  of Johns Hopkins School of Medicine, Baltimore, Maryland, and colleagues are optimizing a procedure to deliver recombinant vectors directly into the developing brain. 
Studies in experimental animals have shown that delivery of viral vectors under ultrasound guidance is feasible in the forebrain of mice at the embryonal stages E9.5 through E13.5. At this stage of development, animals are immunotolerant — they do not develop an immune response to antigenic determinants expressed by transduced proteins or by the vector itself. In addition, initial experiments have shown that the earlier the injection, the higher the number of cells successfully manipulated.
Of all delivery options — by intravenous, intraperitoneal, intraparenchymal, or intraventricular injections — only the latter 2 are viable options for delivery to the CNS, as it is far more difficult for cells to reach their homing site through the other routes, even provided that they can successfully cross the blood-brain barrier. On day E9.5, ultrasound guidance allows very targeted delivery to the embryos, [12,13] with injection of stem cells prior to the onset of neurogenesis, using a needle of 30 to 40 micron that is inserted directly into the ventricular system of the forebrain.
Delivery of Cells
In the past few years, Fishell and colleagues  have shown that dissociated cells can integrate into host tissues following intra-CNS delivery, and that they can differentiate in response to local cues in the striatum and the neocortex. The surrounding microenvironment, however, has to be ready to accept and “accommodate” the transplanted cells. Very similar results have been obtained with intraventricular injections.
Other studies using intraparenchymal cell transplantation under ultrasound image guidance [15,16] showed substantial expression by grafted cells of an alkaline phosphatase reporter gene. Cells were widely dispersed in the CNS (e.g., in the cortex and in the hippocampus), as they had been selected to display this intrinsic property. Cell source and final localization are critical factors in all these approaches if specific targeting is being sought.
Delivery of Vectors
Among the potential candidate vectors for intrauterine delivery are murine retroviruses, lentiviruses, adenoviruses, and adenoviral associated viruses. Proteins can be effectively expressed by transduced retroviruses, but this strategy is limited by the ability of retroviruses to infect only dividing cells. Lentiviruses, on the other hand, can infect both dividing and postmitotic cells. Adenoviruses are believed to have a lower transforming potential, but they can mediate expression of exogenous proteins only in an acute, transient fashion, as they do not integrate into the genome of the host cells.
Most studies in the CNS have been performed, so far, using retroviral vectors. Injection at day E8.5 resulted in a widespread distribution in host tissues. Inclusion of an internal promoter helped to overcome the retroviral silencing seen in the developing brain.  Expression of an alkaline reporter gene injected on day E9.5 was easily detectable on day E12.5 using an EF1a promoter in a virus containing conserved late elements. Intraembryonal injection of adenoviral vectors into progenitor cells of the forebrain yielded an even more widespread distribution of a fluorescent green protein used as a tracking molecule.
Notch in Brain Development
While the technical aspects of intrauterine delivery and vector design are being further refined, Gaiano and colleagues  are using this approach to study brain development, and in particular the role of Notch signaling. Notch is a transcription factor that activates CBF1 and other transcriptional targets in the nucleus, including Hes-5 and Hesr-1.
Notch is usually expressed in proliferating zones of the developing CNS, suggesting a critical role in this process. Consistently, studies with Notch mutants have shown that in the presence of gain-of-function mutations, activated Notch inhibited neuronal differentiation and perturbed cell-fate specification in the immature brain.
Conversely, interactions between Notch and delta in other cell types may lead to cell differentiation. In fact, in vivo activation of Notch promoted radial glial morphology at E12.5/E14.5, while constitutive activation was associated with an excess formation of oligodendrocyte progenitor cells (OPCs).  In addition, others have shown that, postnatally, Notch-expressing progenitors become dispersed astrocytes and periventricular cells. 
The technical challenges in using CNS-delivered stem cells or vectors for brain repair are still significant, as outlined by Dr. Gaiano in his conclusion, and they include:
- Achieving targeted delivery with limited tissue damage during intrauterine injection
- Controlling differentiation of transduced cells into specific cell types
- Determining and selecting source and purity of donor cell populations
- Achieving efficacy of viral gene delivery and desired length of expression (short- or long-term).
Engraftment of Oligodendrocyte Precursors
A number of neurologic diseases are associated with defects in myelination and in neuronal homeostasis and function. Among them we find leukodystrophies and leukomalacia. Leukodystrophies are degenerative processes leading to progressive demyelination, whereas leukomalacia is associated with damage of premyelinating oligodendrocytes. Of note, periventricular leukomalacia (linked to preterm brain injury) may be associated with damages of the pyramidal tract fibers and motor dysfunction in children affected by spastic cerebral palsy. 
Dr. Martha Windrem,  of Weill Cornell Medical College, New York, reviewed how progenitor cells can be selected for in vivo brain repair, and discussed the latest results obtained in this field with OPCs.
Almost 3% of white matter progenitor cells selected via CNP2 (a phosphohydrolase) can give rise to oligodendrocytes in vitro, but the transfection efficiency in these cells is very low. Dr. Windrem, on the other hand, showed how monoclonal antibody A2B5 identifies more effectively a similar population in adult human brain tissues. Immunofluorescent and magnetic cell sorting yielded a subset of cells (approximately 4% of the starting population) able to generate oligodendrocytes in vitro. Positive selection of cells from the fetal human ventricular zone (late second semester) for the A2B5 determinant, plus negative selection for PSA-NCAM, led to a 16.3% enrichment in OPCs (04-positive cells). 
To evaluate the regeneration potential associated with these cells, the researchers performed studies in Shiverer mice, which represent a very useful experimental dysmyelination model, as this defect is associated with lack of myelin basic protein (MBP) and hypomyelination.  Usually, mice carrying the shi mutation are severely ataxic by 4 months of age.
Following intraparenchymal injection, selected fetal A2B5+/PSA-NCAM- cells were found to migrate extensively, with migration and proliferation occurring predominantly in the white matter (corpus callosum). A few cells were found to migrate also into the gray matter (e.g., striatum and neocortex). The proportion of viable, myelinating cells substantially increased over time.  In quantitative terms, 50,000 OPCs were injected on each side of the brain in a very small volume of liquid, for a total of 100,000 cells per treated mouse.
Can these fetal OPCs indeed remyelinate extensive regions of the brain in Shiverer mice? By immunofluorescence microscopy, fetal OPCs appeared differentiated in myelin-producing cells in the cerebral cortex, but they remained in the precursor stage when infiltrating the striatum. 
Following differentiation, the OPCs gave rise to oligodendrocytes able to ensheath “naked” axons in the host, with production of MBP. Electron microscopy revealed that the newly formed myelin was compact in nature, with major dense lines.
Adult-derived OPCs were found to mature more rapidly than fetally derived OPCs. They also successfully engrafted in the white matter and differentiated as myelinating oligodendrocytes. However, adult-derived OPCs were less likely than fetally derived cells to differentiate into astrocytes, thus showing a more restricted potential.  Adult xenografts were less dense, but a higher proportion of cells expressed MBP, thus ensheathing more native axons per donor cell (approximately 5 times more). Even taking into account in vivo differentiation as a correction factor, each adult-derived oligodendrocyte ensheathed more native axons: approximately twice as many. 
The myelin newly produced by the grafted human cells interacted with the native axons to form Caspr+ paranodes and physiologically functional nodes of Ranvier. Of note, while untreated Shiverer mice are severely compromised at 4 months of age, mutant mice xenografted with human OPCs showed a significantly prolonged survival, comparable to that of wild-type mice, suggesting a functional and long-term correction of their myelination defect. [21,24]
As pointed out by Dr. Windrem, these results are very encouraging and suggest that this may represent, in the future, after further refinements, a feasible strategy for the correction of leukodystrophies and possibly of leukomalacia.
Neurogenesis and Brain Repair
The mechanisms by which the CNS shifts developmental patterns to achieve homeostasis are the subject of many ongoing studies. As outlined by Dr. Evan Snyder,  of Harvard Medical School, Boston, Massachusetts, investigation of the perturbations occurring in developmentally less mature brains, such as those of children, may provide a temporal/spatial window on neurogenesis.
More insights into these complex processes may also provide, in the future, ways to repair brains that have undergone significant damages in the pre- or postnatal period, or that might have incurred significant alterations of a developmentally well-regulated process. Studies done in brains from infants who have suffered from a stroke show that, fortunately, pediatric brains can compensate in ways that are precluded in adult brains.
Complex environmental interactions between components of the white and gray matter, as well as nonneuronal elements such as vessels, contribute to brain homeostasis. A few years ago, Snyder’s group  showed that stem cells recovered from the brain were able to differentiate into progenitor cells able to generate neuroblasts as well as glioblasts, using lacZ as a tracking gene. These finding raised considerable interest and paved the way for further studies on the regeneration potential of the brain, and how to harness it using transplantation and/or manipulation of stem cells.
Examples of diseases amenable to this therapeutic approach are the lysosomal storage diseases (e.g., Tay Sachs disease), in which congenital deficiency of key metabolic enzymes leads to abnormal intracellular accumulation of unprocessed substrates, and ensuing cellular toxicity and death. If affected cells can be engineered to produce the missing enzyme or the end product, this would lead to normal or reduced production of the toxic substrate.
Pilot studies in suitable animal models showed that intraventricular injection into newborn mice was associated with expression of the therapeutic enzyme all over the brain, and elimination of the storage disease. For example, expression of hexoaminidase-beta alpha chain (with green fluorescent protein tracking) in the brains of Sandhoff mice (an experimental model of Tay Sachs disease with abnormal accumulation of neutral glycosphingolipids) successfully shifted the survival curve in treated mice vs. controls, indicating a beneficial effect on overall survival. 
Neuronal Replacement in the Brain
If cells could be grafted into sick tissues to correct a genetic defect, then, researchers argued, stem cells or progenitor cells could potentially be used, after appropriate selection and in vivo differentiation, to restore myelination in brains that had an intrinsic deficit in this critical developmental process.
And, indeed, similarly to the results of the studies presented by Dr. Windrem, transplantation of progenitor cells in Shiverer mice, which suffer from a congenital defect in myelination, led to significant reduction in shivering following in vivo remyelination by grafted cells. 
In Twitcher mice, which represent an experimental model of Krabbe disease (or globoid cell leukodystrophy, that originates from an enzymatic defect),  things turned out to be far more complex than initially hoped. Following transplantation of progenitor cells, production of myelin could be detected in association with myelinated axons, but there was no improvement in symptoms and survival of treated animals, even though significant amounts of myelin had been replaced. 
Toxicity of the excess psychosine produced in this disease was, in fact, not corrected by the remyelination process induced in vivo. Thus, one more corrective step — induction of resistance to psychosine — should be envisioned. And, although somewhat counterintuitive, this is possible, in principle, because neuronal stem cells (NSCs) are already, intrinsically, more resistant to psychosine toxicity than mature cells. Of note, brain repair has been more frequently correlated with the use of immature cells, donor-derived oligodendrocytes, and other donor-derive support cells, suggesting that complex intercellular interactions are needed to repair substantial in vivo tissue damages. 
Recent results obtained in vivo, in a brain injury model, showed that cells grafted in the injured area or infarction cavity built a new network of proliferating cells with neuronal extensions and neoangiogenesis.  This process, however, appeared more as a “filling process” than a successful restoration of the damaged zone to the original parenchymal architecture. Functional data are not yet available; thus, it is not known whether this “abnormal” repair may in some way be associated with a functional recovery. One element has, however, been defined: filling of the parenchyma by the grafted cells was associated with reduction in the inflammatory infiltrates and an overall decrease in glio-scarring.
Neuronal Replacement in the Spinal Cord
Can NSCs be of help in repairing defects of the spinal cord by cell replacement? Studies in rats with extensive spinal resections have shown a certain degree of functional recovery following cell replacement in vivo.  But the surprising finding was that, apparently, the cells responsible for this improvement were derived from the host, not from the donor.
Thus, it seems that grafted cells may actually affect neurologic repair in more than one way, not only by differentiation and proliferation in vivo, but also by inducing some level of neuroprotection in vivo, possibly by mitigating the loss of connectivity associated with damage or resection, and by reducing formation of scar tissue.
A dialogue between donor and recipient cells may thus be initiated following grafting of NSCs,  in which different interactions can be envisioned: donor NSCs-NSCs; donor NSCs-host cells; and host cells-donor NSCs. Molecular studies are underway to identify which cytokines, chemokines and/or trophic factors might be involved in these complex interactions.  Transforming growth factor beta and brain-derived neurotrophic factor are among the potential candidates.
Preliminary studies are ongoing to evaluate feasibility and potential benefits deriving from intrauterine delivery of NSCs in the subventricular zone of fetal monkeys, but far more experiments and refinements are needed before this strategy may offer a reasonable therapeutic option in humans. As stated by Dr. Evans, “we can’t go beyond the biology of stem cells,” and too little is known so far to fully exploit their potential in vivo.
In short, in most cases of congenital or acquired defects involving the CNS, we still don’t know :
- Which molecular or cellular defects need to be fixed
- Which cells are in need of replacement (multiple cell types might be needed)
- How to reconstruct a complex cellular and extracellular environment
- How manipulation of one component may affect other endogenous systems or networks
- How the dynamic nature of a disease is indeed susceptible to permanent correction of a specific defect.
At the moment, these are the settings in which future applications of neurologic cell repair might find clinical applicability :
- Rescue of cells and pre-existing circuits (subacute stroke, spinal cord injury)
- Decrease in inflammatory scarring (subacute stroke, spinal cord injury)
- Remyelination in demyelinating diseases
- Correction of amyotrophic lateral sclerosis disease
- Correction of Parkinson’s disease.
In conclusion, we can quote the question posed by Ms. Fia Richmond, president of the CNS Foundation, that is still awaiting an answer: “How many neuroscientists does it take to heal a brain?”
Would it be fair to guess tens of thousands?
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