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Principal Investigator: David Wang
Organization: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
Fiscal Year: 2023
Award: $895,783
Funding agency: National Institute of Neurological Disorders and Stroke
Specific aim 1:
To develop in vitro 2D neuronal and 3D brain organoid models derived from human adult peripheral CD34+ cells to study neural development and degeneration and infectious diseases involving human brain. We set up a new brain-on-the-chip microfluidic culture equipment into our unit, we now can reconstitute 3D brain cultures in a precise way by combining four types of essential cells in the brain: neurons, microglia, astroglia and endothelial cells. This approach, in additional to our existing 3D brain organoid models, is very useful to delineate the role of specific cell types in the neural development or neurodegeneration. In the preliminary study, we have used iPSC-derived neurons and commercially obtained primary astroglia, endothelial cells and a microglial cell line to make the mixed brain-on-the-chip successfully. We also optimized protocols to successfully generate microglia, astrocyte and endothelial cells from iPSCs and will use these methods to generate disease-specific brain-on-the-chip in the future study. In addition, we used the home developed endothelial incorporated 3D brain model to test if SARS-CoV2 can directly infect it. Using a pseudo-virus with SARS-CoV2 spike protein, we found that the SARS-CoV2 pseudo-virus can hardly infect the brain organoids, with only scarce weak positive cells along endothelial linings, not in the inner neuronal mass. This result is agreed to the clinical observation that SARS-CoV2 is seldom detected from neurons in patient brains. Although this is different with other reports which used traditional brain organoids to show high levels of SARS-CoV2 infections. These conflicts may be due to the existence of endothelial cells and blood-brain-barrier. We have been invited to present the model and findings in the upcoming 11th IBRO World Congress of Neuroscience, Granada, Spain.
Specific aim 2: To study the roles of HERV-K on brain development and tumorigenicity. We continued the collaboration with Dr. Ashish Shah on studying the mechanism of HERV-K on the pathogenesis of glioblastoma. We used the human CD34-derived neural stem cells to generate a 3D model for astroglial growth. In transfected astroglia with plasmid containing HERV-K we detected higher level of gene transcription responsible for stemness and tumorigenesis and the organoids grow faster than controls with an irregular shape, indicating a potential for easier metastasis. These results contributed to a new publication.
Specific aim 3: To study the association of HERV-K and motor neuron degeneration like ALS. We continued our study of retro virus activation and motor neuron degeneration. For functional study, we collaborated with Dr. InHong Yang from UNC to use mitotrackers to stain the mitochondria of motor neurons. Use this method, we counted mitochondrial density from both ALS neurons and controls. While our preliminary result indicated a difference of mitochondrial staining between the two groups, more studied using rigor differentiation conditions are needed to confirm the result. We also used specifically designed ASO targeting HERV-K Env to study the effect of 3D brain development and motor neuron degeneration, we observed that ASO resulted in slower growth of 3D organoids as expected. We also used an in vitro neural culture derived from rhesus skin fibroblasts to study its susceptibility of HERV-K infection, in collaboration with Dr. Dr. Catherine Demarino from SINS, NINDS. We found that NSC derived from Rhesus skin fibroblasts express HERV-K Env binding target and high titer of HERV-K virions infects the NSC, indicating Rhesus monkey could be used as the animal model for studying HERV-K infection and neurodegeneration.
Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We Generated a new iPSC line from fibroblast of a patient with a specific FRMD4 gene variation, in collaborated with Dr. Maria Isis Atallah Gonzalez, CHUV. We trained Dr. Sanjay Yadav, a collaborator from AIIMS Raebareli, India on generating dopaminergic neurons from patient PBMCs and since generated 4 lines of iPSCs from PD patients. We continued our collaborate with Dr. Youssef Kousa by providing material support and technique advice of the iNSC/iPSC generation and 3D modeling that he has adopted in his newly established lab in National Childrens Hospital.
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