Thank you for joining me here today. My name is Kevin Kumar. I am a fourth-year neurosurgery resident at Stanford. Today, I'll be presenting our work entitled Repopulation of Microglia Using Bone Marrow Transplantation as a Novel Therapeutic Platform for Neurological Disorders. Just an outline of what we'll be discussing. First, some background on microglia, followed by the experimental goals of the project and our results, including development of microglia replacement using bone marrow transplantation, enhancement of transplantation efficiency, applying hematopoietic lineages responsible for microglia replacement, application of microglia replacement to mouse models of aging, and the use of MR-guided focus ultrasound to permeabilize the barbane barrier to allow microglia replacement. Then go through some conclusions and future directions of the work. Microglia are tissue-resident macrophages of the central nervous system. They're important for normal CNS development and function, particularly with synaptic pruning and regulating neurogenesis. There are distinct subset of microglia associated with neurodegenerative disorders and aging brains termed disease-associated microglia or DAMS. Microglia dynamically survey the brain parenchyma, with each individual cell assigned a specific brain volume with an evenly-tiled distribution throughout the CNS. Of note, microglia originate from yolk sac chromatopoiesis and populate the brain during early development. However, in the adult brain, microglia are long-lived and are not exchanged from circulating blood cells under normal physiologic conditions. However, incorporation of circulation-derived myeloid cells or CDMCs into the CNS can be achieved using bone marrow transplantation. However, this is low efficiency and variable. So the goals of our project were to first characterize the engraftment of CDMCs into the brain, improve the efficiency of microglia replacement in a non-genetic mouse model, and apply this technology for a potential therapeutic use. We found that microglia replacement after bone marrow transplantation is a slow, inefficient, and variable process. In the protocol, mice are treated with busulfan, which is a myeloablative chemotherapeutic agent, followed by whole bone marrow transplantation with GFP-positive cells. The cells were then analyzed after stacking the mice at various time points, looking at both the peripheral blood and the brain. And although there was a high degree of chimerism, close to 100%, in the peripheral blood, brain chimerism peaked at 20-40% of Ibo1 GFP-positive cells, as you can see in the bottom right. We hypothesized that a difficulty of the transplantation protocol is that the microglial niches are occupied by the endogenous microglia. So we thought to treat with PLX5622, which is a CSF1 receptor inhibitor, which would cause depletion of the endogenous niches, allowing vacant niches for the CDMCs to occupy. And it turned out to be the case, achieving a high degree of brain chimerism, greater than 90%. And if you look in this bottom right panel, you can see a high degree of GFP-positivity throughout the entire mouse brain parenchyma. We're then curious which component of the whole bone marrow transplantation had the highest capacity for generating CDMCs. So we did some direct injection experiments using particular hematopoietic precursors and found that hematopoietic stem cells, HSCs, have the highest capacity of CDMC generation, which is particularly attractive since HSCs can be cultured in vitro and engineered for a particular therapeutic application. We then sought to apply this technology to the age brain, where microglia are known to become activated and exhausted with time. So we transplanted from young mice into aged mice and vice versa. And although we were able to achieve a high degree of chimerism, we found that the young CDMCs within the age brain acquired both a expression profile and morphology of the age microglia in those particular mice, suggesting that the microenvironment of the age brain had a tremendous influence on the newly transplanted cells. One aspect limiting therapeutic application to more chronic or slowly progressive neurologic disorders is the use of busulfan, which I had mentioned is a chemotherapeutic agent. So one thought was whether we could harness the potential of focus ultrasound for permeabilization of the blood-brain barrier. This would, in theory, allow CDMCs to cross the blood-brain barrier without the use of a chemotherapeutic. We found that MR-guided focus ultrasound actually allowed the CDMCs to enter the brain. And when we delivered a eccentric focus ultrasound treatment on the right side greater than the left side, we found that the level of IBO1, GFP, CD68 positivity correlated with the delivery of the focus ultrasound, suggesting that it was able to successfully permeabilize the blood-brain barrier in that region. So in conclusion, we developed a high-efficiency bone marrow transplant protocol to repopulate microglia in the brain. This is a novel cellular delivery platform to the brain. MR-guided focus ultrasound has the ability to eliminate toxicity of busulfan, increasing translational potential of this technique. Transplantated CDMCs from young mice into age mice adopted an age brain signature. And our current studies seek to apply this technology to a mouse model of Cauchy's disease. I'd just like to acknowledge all of my colleagues in the Wernick Laboratory as well as in the Department of Neurosurgery at Stanford, as well as our funding from the NINDS. Thank you for your time.

bone marrow transplantation

neurological disorders

microglia

blood-brain barrier

MR-guided focus ultrasound