Hans Keirstead - US grants

Demyelination represents a unique injury to the adult CNS in that it is followed in many instances by repair. Regions of demyelination that are capable of remyelinating are not characterized by an astroglial scar. We have demonstrated that regions of immunological demyelination, produced by the intraspinal injection of anti-galactocerebroside antibodies plus complement proteins, are capable of remyelination by endogenous oligodendrocytes, and that astroglial hypertrophy is absent within and around regions of immunological demyelination. Furthermore, our preliminary studies indicate that astrocytes within regions of immunological demyelination do not become hypertrophic following axonal injury. Thus, immunological demyelination provides us with a means to manipulate the response of reactive cells to axonal injury and investigate the ways in which that response differs from that following the majority of CNS injuries, which result in scarring. We will use integrated optical density measurements to determine whether the astrocyte and/or microglial/macrophage response to injury is eliminated, suppressed or delayed by immunological demyelination. These studies, which constitute the last two AIMS of this proposal, may lead to novel means of manipulating astrocytes such that astroglial activation or scarring is prevented. Likely associated with the absence of astroglial hypertrophy, regions of immunological demyelination promote long distance homogeneous redistribution of transplanted cells throughout the region of immunological demyelination. This provides a unique opportunity to create a PNS-like terrain within the CNS that is not bordered by an astroglial scar. This experimental paradigm allows us to study the ability to injured axons to regenerate from such a PNS-like terrain into the CNS environment without confronting a sclerotic border zone. Our regenerative analysis will be conducted using anterograde tract tracing. These studies constitute the first AIM of this proposal. There are three specific aims; 1) test the hypothesis that regenerating dorsal column axons will extend from a PNS-like terrain into a purely CNS terrain in the absence of scarring at the interface, 2) test the hypothesis that immunological demyelination suppresses astroglial hypertrophy following axonal injury, and 3) test the hypothesis that immunological demyelination alters the magnitude and duration of microglial/macrophage activation following axonal injury.

Title: 3D retina-RPE constructs for vision restoration in new rat retinal degeneration model In patients with advanced degenerative diseases, both retinal pigment epithelium (RPE) and photoreceptors are lost. If new cells can restore the function of the lost cells, a degenerating retina might be repaired and eyesight restored. Our team targets vision restoration by transplanting sheets of retinal progenitor and RPE cells derived from human embryonic stem cells (hESCs) to recipients that have lost photoreceptors and retinal pigment epithelium (RPE) cells. Transplants of freshly harvested sheets of retinal progenitor cells (delivered by a unique procedure and instrument) have shown to develop photoreceptors with normal morphology, synaptic connections with the host and to restore lost visual responses in several retinal degeneration models and in human patients. This cannot be achieved by injecting dissociated cells into the retina. Although other approaches exist, the majority are restricted to rescue of endogenous retinal cells of the recipient by a 'nursing' role of the implanted cells, an approach which does not restore lost function. The supply of fresh fetal-derived neuroblastic tissue is limited. Differentiation of hESCs into sheets of photoreceptor progenitors and RPE cells would create an unlimited tissue supply for clinical and research use. Several laboratories have recently developed procedures to differentiate pluripotent stem cells into optic vesicles and optic cup-like structures that develop retinal cell types with some degree of lamination. We want to use this approach for producing laminated retinal progenitor sheets with RPE for transplantation. Our team has developed 1) a protocol to derive early retinal progenitor tissue from hESC and 2) a new immunodeficient rodent model of retinal degeneration. Additional pre-clinical animal research is required to develop a therapeutically viable and sustainable supply of stem cell derived material. A three-year project is proposed to test the hypothesis that (1) hESC-derived photoreceptor progenitors, transplanted together with a hESC-derived RPE sheet to a new immunodeficient rat model of RD, will develop mature photoreceptor markers and integrate with the degenerating host retina; (2) hESC-derived retinal progenitors can restore visual responses in this rodent model to the same extent as transplants of fetal retina with RPE, as shown by optokinetic testing and electrophysiology. Transplantation of intact ESC-derived retinal progenitor layers with RPE in rats with retinal degeneration provides an excellent model to answer an important question for developing retinal therapies. Proof of concept will provide a new tissue source to restore and improve vision in patients with retinal diseases.