In humans, injury or diseases resulting in impairment of oligodendrocyte (OL) function leads to myelin loss or defects manifesting as developmental and/or adult disabilities and mental retardation. At the present time remyelination of the human CNS is not yet possible. Oligodendrocytes synthesize and maintain myelin, the sheath that insulates axons for a fast nervous impulse transmission in the central nervous system (CNS). Myelin deficiency whether inherited or acquired, like Pelizaeus Merzbacher’s disease and Multiple Sclerosis in humans, results from OL dysfunction. These are progressive degenerative disorders, resulting in deterioration of sensory and motor functions. Neural stem/progenitor cells are present in the embryonic, postnatal and adult brain and they represent a potential source of cells for therapy. Yet, the CNS appears not to have all the elements to successfully sustain autonomous remyelination. Human embryonic stem cells offer a great potential to identify appropriate neurotrophic factors or molecules necessary for these cells to acquire neural characteristics as a source of specific cell types such as Neural Stem Cell. Human embryonic stem cells provide novel prospects for cellular replacement strategies because of their ability to provide seemingly unlimited stem cell numbers in vitro, their flexibility to genetic engineering, and their broad developmental capacity. hES-derived oligodendrocytes could potentially address the needs to benefit the outcome of demyelinating diseases by helping to promote survival, growth of host tissue, and/or replacement of cells lost. The work we propose here is to study the conditions to obtain homogeneous OL populations (in the absence of other cell types) in high numbers that will allow us to understand the needs of these cells as they develop from a progenitor (young) cell into a mature and functional cell able to myelinate naked axons. The data derived from the studies we are proposing, will have direct relevance on the long term development of standardized methodology that would be made available for future clinical research using cell replacement therapies in cases of myelin deficient CNS of both inherited and acquired disorders, e.g. Pelizaeus Merzbacher’s disease, spinal cord injury, traumatic brain injury, AIDS dementia, periventricular leucomalacia, cerebral palsy, autism, and multiple sclerosis.
Statement of Benefit to California:
Myelin is a sheet of fat (80%) and proteins (20%) that wraps around axons, speeding messages through the brain by insulating these neural connections. Although myelinogenesis takes place in perinatal stages, in humans, myelination continues through childhood and well into adulthood ( Bartozkis, Neurobiology of Aging, January 2004). New evidence points to disruption of myelination as a key neurobiological component behind childhood developmental disorders. Many factors can disrupt myelination and contribute to or worsen disorders such as autism, attention deficit/hyperactivity disorder (ADHD) and schizophrenia. (Bartzokis, G., 2005). Other disorders like Multiple Sclerosis and trauma result in myelin loss with a concomitant reduction or even impairment of the central nervous system (CNS) function. Approximately 1 in 700 or 0.14% or 388,571 people in the United States are afflicted with Multiple Sclerosis (source NIAID). Therefore, besides autism, ADHD, and schizophrenia, environmental toxins and drugs of addiction may contribute to demyelination. Moreover, in Periventricular Leucomalacia (PVL), the white matter can be prevented from being formed in the fetus if infection, inflammation or other alterations of the mother’s metabolism occur during gestation, preventing the progression of oligodendrocyte progenitors (OLPs) into mature oligodendrocytes (OL) and therefore resulting in myelin deficit. Although babies with PVL generally have no outward signs or symptoms of the disorder, they are at risk for motor disorders, delayed mental development, coordination problems, and vision and hearing impairments. PVL may be accompanied by a hemorrhage or bleeding in the periventricular-intraventricular area (the area around and inside the ventricles), and can lead to cerebral palsy. It is estimated that some 500,000 children and adults in the United States manifest one or more of the symptoms of cerebral palsy. Currently, about 8,000 babies and infants are diagnosed with the condition each year. To date there is no cure for myelin diseases in the sense that no treatment is aimed at myelination/remyelination but rather treatments are directed to alleviate the symptoms. The work proposed here will allow us to acquire the knowledge necessary to obtain a homogeneous source of OL derived from human embryonic stem cells (ES) in adequate numbers by identifying the specific factors and conditions that are needed to obtain ES-derived OLPs in a reproducible and cost efficient manner. The implementation of a successful propagation and cryopreservation method for OL committed cells in defined conditions will allow us to obtain a dependable source of OL to be used for potential cell therapy. With these progresses and other pertinent issues overcome, considerable enthusiasm continues to be generated about treating disorders of white matter degeneration; diseases once thought to be a totally incurable condition.
SYNOPSIS: The major aim is to derive large numbers of oligodendrocytes from mouse and human ES cells for myelin repair purposes, in defined, serum-free media. Mouse ES cells will be purchased from ACT and human cells (hESC’s) from D. Melton, Harvard. The applicant will use defined media devised by them which is serum-free to produce a more homogenous population of oligodendrocytes and their progenitors in rodents, and devise similar approaches to produce pure populations of these cells from human ESC’s. They will use an extensive battery of antibodies to characterize and define the cells throughout their differentiation. To test the myelinating potential of these cells they will use three approaches; 1) A co-culture system in which ESC’s are added to rat dorsal root ganglia. 2) Transplant the cells into the early, developing corpus callosum prior to host myelination. 3) Transplant the cells into demyelinated lesions created by focal injection of lysolecthicin. SIGNIFICANCE AND INNOVATION: Devising a technique to generate large numbers of human oligodendrocytes in defined serum-free media is a worth-while goal, and if successful, could be useful in supplying cells for the treatment of a variety of human myelin disorders. However, the proposal lacks great innovation in regard to the methods to be used. The idea of generating OLPs from both mouse and hES cells, using serum-free methods and developing better cryopreservation methods is important. The PI has experience with working in demyelinating-remyelinating animal models, and will attempt to further refine culture protocols for establishing OLP lines for future applications in human white matter diseases. This is of course significant toward using myelin-producing precursor cells to restore neurological function in these debilitating diseases, and the field does have interest in the use of precursor cells for replacement therapies. STRENGTHS: Extensive experience of the PI in the field of the in vitro culturing of oligodendrocytes and their progenitors, and in the use of neural precursor cells to potentially replace at-risk or lost OLPs and oligodendrocytes in demylenating disease models. This is a rather straightforward proposal to study mouse and human embryonic stem cells in a culture system that he has developed. Both in vitro and in vivo studies are proposed to test the abilities of both the mouse and hES-generated OLPs for their ability to survive and integrate in a demyelinated CNS. It is not unreasonable to propose the generation and comparison of both mouse (J1 line) and hES-derived oligodendrocyte precursors since the PI has worked with the mouse and is now re-gearing up to work with the human cells; mouse cells do offer a nice model system for guidance and comparison when working with the hES cells. The PI has laid out a series of immunophenotyping experiments to establish controlled in vitro development of the precursor cells. WEAKNESSES: • Lack of truly innovative approaches • Appearance of a “fishing-trip” approach in the elucidation of oligodendrocyte development from mouse ES cells, or the lack of justification as to why all these markers will be used • Specific Aim 3 is hard to follow the way it is presented. They have a lack of experience here as evidenced by their choice of multiple time points at which they will analyze animals. It would be better to choose to do these experiments in the spinal cord • The proposal is poorly prepared and written, with confusing sentences, grammatical errors and numerous spelling mistakes. The proposal should be much more carefully prepared and proof-read. • There is little or no documented data from the ongoing mouse ES and ES-derived OLP studies • There is no reason to believe that, due to truly inherent differences in mouse ES and hES cells, the defined serum free STM and OSM media that is touted to work on the mouse cells will work at all with the hES cells • The applicant should consider working only on human ES cells. The mouse work may not greatly help the applicant and the ultimate goal is to produce human cells. DISCUSSION: There was no further discussion following the reviewers' comments.