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Neural Stem Cells: Promoting Repair and Plasticity of the Nervous System
July 20-21, 1999
Co-chaired by Drs. Fred Gage of the Salk Institute and Story Landis of NINDS, this workshop was sponsored by NINDS to assess the state-of-the-science in neural stem cell research, identify the limitations in our current knowledge base in this fast-moving field, and recommend future directions in applying neural stem cells most effectively to repair the dysfunctional nervous system. These three topics were considered in the context of two treatment approaches: (I) tapping the potential of endogenous neurogenesis within the mature CNS, and (II) transplantation of stem and progenitor cells to repair and replace lost populations in the brain. I. Endogenous Neurogenesis in the mature CNS
Current state of knowledge
Neurogenesis occurs in select regions of the mature central nervous system
In recent years it has become clear that substantial neurogenesis occurs during the postnatal period in several brain areas including the dentate gyrus, olfactory bulb and cerebellum. This production of neurons continues well into adulthood in the dentate gyrus and olfactory bulb in a variety of mammalian species, including humans. However, the number of new neurons produced in the aged brain is significantly diminished.
Neurogenesis can be altered by external parameters such as experience
The production of new neurons can be altered either by affecting the proliferation of precursor cells or by altering the survival of newly generated neurons. Animal studies demonstrate that the numbers of new neurons added to the mature central nervous system can be modified by:
- Seizures or the induction of seizures
- An enriched environment
- Stress and circulating levels of glucocorticoids
- Genetic differences
Invasive parameters also modify neurogenesis in the mature brain, including:
- Injury, cell death or degeneration
- Infusion of growth and neurotrophic factors
Mechanisms of neurogenesis
- Is neurogenesis accomplished primarily through promoting proliferation or enhancing survival?
- What are the molecules and genes involved in neurogenesis?
- How many types of neural stem cells are there, how are they related, and where are they found in vivo?
- How are characteristics of neural stem cells, including proliferation, survival, lineage decisions, migration, activity, longevity and integration into existing networks regulated by environmental cues?
- Why does neurogenesis take place predominantly in a few, select brain areas? What is promoting this activity at these sites, or conversely, what inhibits neurogenesis in other parts of the adult CNS?
- Are there quiescent resident stem cells that could be activated in parts of the CNS that normally do not exhibit neurogenesis?
Migration, Differentiation and Function
- What is the function of neurogenesis in adulthood?
- How is the translocation of these new neurons from the site of proliferation to their final destinations achieved?
- How do these cells get incorporated into existing circuits?
- How does gene expression change as cells move through their lineage decisions?
- Do cells incorporated early behave differently in the same circuit than cells incorporated later?
- How do new cells get integrated into the existing networks and circuits, and once established, what are their effects?
Applications for repair of the nervous system
- Is having more neurons always beneficial? What is the potential for harm?
- Should neurogenesis be stimulated globally, or only in selective regions? How can the latter be achieved?
- How can the normal fates of cells produced by endogenous progenitors be changed? How can these cells be regulated in order to have them perform very specific functions in the brain?
- Can some of these questions be answered by many of the available transgenic animals?
Research Needs and Opportunities
- Markers with which to identify, unequivocally, stem and progenitor cells. The terms "stem" cell and "progenitor or precursor" cell are operational definitions. These cell types are only identified by their behavior or potential under defined culture conditions. No one can, as yet, identify these cells in vivo or in situ.
- Defined in vitro systems to study the conditions that regulate the behavior and potential of each variety of stem cell.
- Define the in vivo environment of the normal brain to identify the endogenous cue(s) involved with proliferation, survival and migration of resident systems.
- Review and use knock-outs, knock-ins and a variety of transgenic animal models that may already bear clues to some of the answers we seek.
- Screen genetic models of disease to see what happens to the endogenous populations of precursor cells (i.e. any aberrant behavior or ectopic movement of cells?)
- Infrastructure to harness protocols that guide utilization and standardize procedures.
Current state of knowledge
- Sources of neural stem cells
A variety of pluripotent and multipotent "stem" cells have been harvested from fetal/embryonic and adult tissue and shown to produce neuronal and glial phenotypes in culture. These include :
- Embryonic Stem (ES) cells
- Primary Cells, acutely isolated from proliferative zones in the developing and mature CNS. Primary cells can be manipulated "genetically" or "epigenetically" (by externally supplied agents to expand or instruct the cells).
- Engineered cells and cell lines (with purposefully introduced genes).
- In vivo potential of neural stem cells
Introduced into embryos, some of these multipotent cells integrate seamlessly into the developing host nervous system, producing neurons and glia in vivo. Transplanted into more mature hosts, these cells respond to as yet unknown endogenous factors expressed during phases of active neurodegeneration in the CNS by migrating and differentiating specifically to repopulate damaged regions. This has been observed in a number of settings, including hypoxic-ischemic brain injury, spinal motor neuron degeneration, neuronal apoptosis in the neocortex, & cerebellar degeneration.
- Multipotent stem cells can differentiate into site-appropriate phenotypes in neonatal animals, such as pyramidal neurons in the neocortex and granule neurons in the cerebellum, as well as into oligodendrocytes that remyelinate in mouse models of the leukodystrophies or other disorders of demyelination and dysmyelination.
- Genetically-engineered neural stem cells may serve as vehicles for gene therapy , and appear capable of treating enzyme deficiency diseases of the nervous system.
- How should cells, cell lines and procedures be standardized to assure their utility both in the research community and for future clinical applications? Standardization is a greater challenge with the use of primary cells due to problems such as variability in cell numbers, cell quality and dissection, and tissues that may arrive in many states or conditions. Engineered cells on the other hand, should be standardized for the science as well as for safety and efficacy. A better understanding of conditions such as passage number, and how the properties of the cells change at different passages is also needed. In clinical trials, great attention is paid to quality control where proteins and other biomolecules are used. Similar attention should be paid to the standardization of cell lines if they are to be used in transplants, drug discovery or any such endeavor.
- Safety is a largely unknown factor. Because stem cells are known for their plasticity and self-renewal capacity, it is crucially important to understand and control the possibility that they may become generate tumors.
- How should the question of efficacy of cell transplantation be addressed? Do these cells "actually work" to produce functional recovery?
- How can the production of desired neurons and glia be scaled up to produce large numbers of cells suitable for transplantation and other applications?
- What are the advantages and disadvantages of each of the cell types and sources for transplantation, drug efficacy and toxicity testing, and studies of neural development?
Research Needs and Opportunities
One of the advantages in using stem and progenitor cells over fetal tissue lies in the fact that they are highly reproducible and behave in a predictable fashion. The expectation is that there exist set and optimal conditions for expanding and differentiating these cells that should produce the same or a reproducible outcome in all laboratories.
- There is consensus that there is a strong need for research on adult, fetal, and embryonic human cells in this field. We are not at the stage of understanding adult stem cells to be able to eliminate embryonic stem cells as potential therapies. It is not unclear that an adult cell will have the same potential as stem cells taken from younger stages. Other important issues include the numbers and viability of cells, and the ability to obtain sufficient numbers of them. It is very unclear if studies conducted on rodent stem cells are representative of human cells. In summary, there is clear consensus that to solve the questions under discussion, access to all these cell types and sources are needed. It is premature to eliminate any particular cell type.
- Access to suitable human primary cells is a significant limiting factor.
- Standardization of cells is fundamental because the issue of reproducibility is crucial to progress in the field. Some potentially useful cells (such as neurospheres) have not been subjected to the same kind of standardization that others (such as ES cells) have. These needs included standardization of propagation and conditions that drive cells down one lineage path versus another.
- New tools, reagents and methodology needed to bridge research done in vitro and in vivo:
- Better tools to identify cells at different stages of maturation, to mark and follow cells after transplantation, and to follow transitions in cells.
- Reliable modes of cell delivery for both systemic and spacially selective delivery, and a better understanding of how different modes of delivery help achieve the desired outcome.
- There is a great need to define powerful predictive models of disease, particularly for those diseases that appear to be most promising for translational testing, including myelin diseases, Parkinson's Disease, and enzymatic deficiencies.
- The potential interactions between cell and gene therapies are important considerations that need to be addressed.
- As we enter a time when novel approaches will be attempted, it becomes even more important to build channels of communications between the scientific research enterprise and the regulatory enterprise in the federal government. If there are a variety of uses for stem cells, it will be important to have seminars, symposia and other collaborative interactions between the FDA and NINDS in order to bring in a broad base of active clinicians and scientists to air significant concerns before conducting clinical trials.
Arturo Alvarez-Buylla, Rockefeller University
Mary Ellen Cheung, NINDS
Arlene Y. Chiu, NINDS
Ian Duncan, University of Wisconsin School of Veterinarian Medicine
Fred H. Gage, The Salk Institute of Biological Studies
Steven Goldman, Cornell University Medical College
David I. Gottlieb, Washington University School of Medicine
Elizabeth Gould, Princeton University
John Kessler, AECOM
Jeffery D Kocsis, VA Medical Center
Story Landis, NINDS
Gabrielle G. LeBlanc, NINDS
Marla Luskin, Emory University School of Medicine
Daniel R. Marshak, Osiris Therapeutics Inc.,
Ronald McKay, NINDS
Robert H. Miller, Case Western Reserve University School of Medicine
Jack Parent, University of California
Mahendra Rao, University of Utah Medical School
Allan Tobin, University of California Los Angeles
Derek van der Kooy, University of Toronto
Nancy S Wexler, College of Physicians & Surgeons, Columbia University