Specific Human Astrocytes Promote SCI Repair

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Stephen Davies, PhD, Associate Professor, Department of Neurosurgery, University of Colorado School of Medicine

Jeannette Davies, PhD, Assistant Professor, Department of Neurosurgery, University of Colorado School of Medicine

Our latest SCI study published in PLoS ONE and conducted in collaboration with Drs. Chris Proschel, Margot Mayer-Proschel and Mark Noble at University of Rochester NY, shows that transplantation of a specific sub-type of human astrocyte (a major cell type of the central nervous system) into spinal cord injured rats can promote SCI repair. Before injection into spinal cord injuries, the human astrocytes were first made in tissue culture from a type of human nervous system stem cell called glial precursor cells. To our knowledge, no previous study of transplanted human astrocytes derived from glial precursor cells has demonstrated functional recovery of the traumatically injured spinal cord. Astrocytes: Why they should be a major focus of SCI repair strategies Glia meaning "glue" in Greek is the general name given to cells other than neurons in the central nervous system. Two of the major types of glial cells are astrocytes - named for the star-like shape they commonly have in gray matter - and oligodendrocytes, the cells that form the myelin sheaths around nerve fibers. As the Greek meaning of their name suggests it was long thought that glial cells in general, and astrocytes in particular, merely provided structural support for neurons within tissues of the brain and spinal cord. However it is now recognized that these relatively large cells, that greatly outnumber neurons in the human central nervous system, are vitally important for conduction of signals within neural circuits of the brain and spinal cord. Modern studies have shown that besides providing metabolic and structural support to neurons, astrocytes can promote the growth of axons (nerve fibers) as well as regulate the formation and activity of connections (synapses) between neurons in the brain and spinal cord. Many people have heard of neurotransmitters, molecules that are released by neurons to transmit signals to other neurons within a neural circuit. Recently however scientists have discovered that astrocytes release their own "gliotransmitters" that can either promote or suppress the transmission of signals between neurons. It is estimated that just one astrocyte in the cerebral cortex of the brain can regulate the activity of up to 1 million synapses between surrounding neurons. Astrocytes are also thought to have their own signaling networks that interact with neuronal circuits. Two lay style articles published in the popular science magazines Discover and Scientific American talk about astrocytes and how they have been overlooked in terms of their importance in the normal function of the brain and spinal cord. Another article featuring astrocytes published online by NPR describes how studies of Einstein's brainrevealed that he had many more astrocytes than the average person in areas of the brain involved in complex thinking. The many newly discovered functions of astrocytes and the growing recognition that they are essential components of neural networks make astrocytes an attractive cell type for repairing the injured or diseased brain and spinal cord. However, compared to neurons, relatively little is known about the functions of individual sub-types of astrocytes within the normal nervous system, let alone the functions of different types of astrocytes that can be made from human glial precursor cells or their ability to promote spinal cord repair

The two different types of human astrocytes in tissue culture. The left image shows the beneficial hGDAsBMP, the right image shows hGDAsCNTF. (Tissue culture images by Dr. Proschel.) Images adapted from Davies et al., 2011 PLoS ONE.

Making the right astrocytes for SCI repair The discovery of glial precursor cells that could make both astrocytes and oligodendrocytes in the developing nervous system was first made by Drs. Mark Noble, Robert Miller and Martin Raff in 1983. It is now known that glial precursor cells are also found in the more mature central nervous system and are thought to be involved in replacing worn out astrocytes and oligodendrocytes throughout adult life. Scientists are also now realizing that there are different types of glial precursor cells that have different "restrictions" as to which specific types of glial cells they can make. The type of glial precursor cell used to make the different types of astrocytes tested in our studies of SCI repair was first discovered by Drs. Margot Mayer-Proschel and Mahendra Rao in 1997 and named glial restricted precursor cells (GRPs). Glial restricted precursors are a type of multi-potent neural stem cell and as their name indicates, they are restricted to making glial cells and do not make neurons. Human GRP cells were first described in a study published in 2002 led by Dr. Mayer-Proschel. The discovery of glial precursor cells that could make different types of astrocytes was therefore a vitally important step towards the development of future astrocyte transplantation based SCI therapies. Previous studies from other research groups had shown that the signaling molecules bone morphogenetic protein-4 (BMP) and cilliary neurotrophic factor (CNTF) are important for generating astrocytes during development of the central nervous system. These molecules were therefore used to control which type of astrocytes the rat or human glial precursors turned into in tissue culture. The different types of astrocytes made in this way have been named Glial precursor derived Astrocytes BMP or GDAsBMP, and in the case of the second type of astrocyte that proved not to be beneficial - GDAsCNTF. The discoveries that a specific sub-type of astrocyte -GDAsBMP - can promote robust axon growth across injury sites, protection of brain neurons and functional recovery in spinal cord injured rats - and that transplanted GDAsCNTF fail to provide these benefits and even promote pain syndromes, were first made by the Davies research team in 2006 and 2008 in collaboration with the Rochester team. Specific human astrocytes for SCI repair The 2006 and 2008 studies were conducted with rat GDAs transplanted in adult rat spinal cord injuries. Our new paper published in PLoS One shows that different sub-types of human astrocytes made by the same nervous system multi-potent stem cell (the human glial precursor cell: hGPC) can also have remarkably different effectson axon growth, protection of injured spinal cord neurons and recovery of locomotor function when transplanted into the injured adult spinal cord. For this SCI study, human glial precursor cells (hGPCs) were purified from human fetal spinal cord tissues by Dr. Chris Proschel in Rochester. The human GPCs were then sent to the Davies research team in Denver where Dr. Jeannette Davies turned them into the two different types of human astrocytes in tissue culture and with the help of other staff in the Davies lab then investigated the ability of hGDAsBMP, hGDAsCNTF and "naive" hGPCs to promote recovery in spinal cord injured rats.

Laser scanning microscope images of transplanted human GDAsBMP (left) and GDAsCNTF (right) within the center of spinal cord injuries. Far greater numbers of nerve fibers (green and viewed end on) have grown into the center of a spinal cord injury site filled with hGDAsBMP (red) than into an injury site filled with hGDAsCNTF (also red). Images adapted from Davies et al., 2011 PLoS ONE. The results of our experiments comparing the effects of specific sub-types of human astrocytes and glial precursor cells on spinal cord repair have important implications for the future development of human astrocyte and stem cell transplantation based therapies: (1) Our studies show for the first time that different sub-types of human astrocytes that can be made by the same type of human glial precursor cell can have widely different effects on growth of axons (nerve fibers), protection of injured neurons and most importantly recovery of locomotor function when transplanted into the injured spinal cord. Treatment of acute cervical spinal cord injured rats with human GDAsBMP promoted a robust recovery of targeted paw placement in a stringent test of brain control of limb movement. Spinal cord injured rats that received transplants of human GDAsCNTF however completely failed to show this locomotor recovery, similar to rats that had received no treatment at all. (2) Our paper is the first to show that transplantation of pure cell suspensions of a specific type of human astrocytes into the traumatically injured adult spinal cord can promote robust protection of spinal cord neurons. The beneficial human GDAsBMP cells promoted a remarkable ~ 70% increase in protection of motor neurons (the spinal neurons that control muscle movement) as well as robust protection of several types of spinal neurons in tissues adjacent to injury sites. This result also has important implications for the use of these cells in treating neuro-degenerative disorders such as ALS. (3) That "naive" human glial precursor cells (that were not first instructed in tissue culture as to which glial cells to turn into) failed to promote functional recovery or similarly robust protection of multiple types of spinal cord neurons when transplanted into identical spinal cord injuries. (4) Our collaboration with the Rochester team has led to the discovery of a specific type of human astrocyte - the hGDAsBMP - that is remarkably effective at promoting SCI repair. As our research continues with the University of Rochester team, we are realizing that not all types of human astrocytes that can be made from human stem cells have the same capacity to promote SCI repair - and - not all types of stem cells / glial precursor cells are necessarily competent to make specific types of beneficial astrocytes. Graph shows the numbers of mistakes made by different groups of rats with cervical spinal cord injuries that have received hGDAsBMP (black dot), hGDAsCNTF (white dot) or no treatment (black triangle). The Grid Walk / Horizontal Ladder test is a stringent test of brain controlled targeting of accurate paw placement in rats with cervical spinal cord injuries. Before injury all rats are making ~ 2 mistakes on average. At 3 days after injury all groups of rats are making around 8 mistakes. However by 28 days after injury / treatment, the scores of all SCI rats that received hGDAsBMP have recovered to near pre-injury scores. The rats treated with the other type of human astrocyte (hGDAsCNTF) had scores that were not better than untreated SCI rats at all time points. This kind of SCI experiment clearly identifies which cells are best suited for SCI repair (hGDAsBMP) and which cells are not (hGDAsCNTF). Images adapted from Davies et al., 2011 PLoS ONE. Moving to treatment of human SCI Our latest SCI studies clearly show that human GDAsBMP represent a highly promising human cell type for treating human spinal cord injuries. The challenge now is to accelerate the process of moving these cells from the lab to the clinic. Sources of human stem cells for making hGDAsBMP At present there are a variety of different sources of stem cells that could potentially be used to make the specific hGDAsBMP used in our experiments. However, although we are working with Dr. Proschel to make this type of human astrocyte from embryonic and adult (iPS) stem cells, our latest paper describes a clinically relevant means by which large numbers of beneficial hGDAsBMP can be rapidly made from fetal human GPCs that have been stimulated to undergo cell division in tissue culture. Theoretically enough human GPCs can be harvested from just one fetal spinal cord to generate enough human GDAsBMP cells to treat many people with spinal cord injuries. Deriving hGDAsBMP from fetal tissue therefore presents one approach by which this type of astrocyte can be translated from the lab to human use in the near future. Developing the best ways of using hGDAsBMP to promote SCI repair Making hGDAsBMP suitable for human clinical trials however is only half the challenge. Developing a better understanding of the likely multiple different mechanisms by which hGDAsBMP promote functional recovery is vitally important if clinicians are to use these cells in the most effective manner for treating acute and chronic SCI in humans. How best to use these cells to treat different types (transection "cuts" and contusions) and severities of SCI at different levels of the spinal cord must also be investigated in animal SCI models. These kinds of experiments are ongoing as are investigations of the ability of GDAsBMP to promote recovery in the chronically injured spinal cord. Learning how best to combine hGDAsBMP with other promising cell, drug or rehab based SCI treatments being developed by research groups around the world is also important for optimizing SCI repair. In a separate line of research, our research team in Denver is also developing the use of a molecule called Decorin for treatment of both acute and chronic spinal cord injuries. Our latest research indicates that treatment of spinal cord injuries with Decorin alone holds equal promise as an SCI therapy. Our ultimate goal however is to combine the use of hGDAsBMP and Decorin in treating acute and chronic SCI in humans.

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