Neurodifferentiation/Stem Cell Unit

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Summary: Our unit is committed to new approach methodologies (NAMs) and poised to serve NIH with these NAMs including patient specific iPSC derived brain organoids, MPS (micro physiological system, i.e. brain-on-a-chip). We aimed to improve the scalability and reproducibility using automatic culture assistance and state of art technologies together with our Translational Neuroscience Center collogues. Specific aim 1: To develop in vitro 2D neuronal and 3D brain organoid models derived from human adult peripheral CD34+ cells for studying neural development and degeneration and infectious diseases involving human brain. We have published our protocol on microglia incorporated 3D brain organoid and produced a video protocol on J Vis Exp which has drawn a lot of interests of collaboration from the neuroscience research field. We have facilitated the collogues to learn these techniques by either studying onsite or through online consultation. We expect more collogues to use our technologies in the near future to study the immune and inflammation associated neurological disorders and eventually benefit the patients. One of the major technical hurdles in using 3D brain organoids to model the human brain is the lack of blood vessels in the 3D organoids. We have developed a protocol to generate endothelial cell derived-blood vessel like structures in the brain organoids and helped our collogues in NIH intramural to use this model to study several neural logical disorders which asked for complicated human brain models. We have also uploaded the protocol online as a bioRxiv preprint so the broader field can assess it. Besides that, we further researched alternative ways to generate blood vessels in culture, and the preliminary results showed that the endothelial cell marker CD31+ capillary could be derived from human iPSC colonies cultured in cell culture plates. We are working on refining the procedures and incorporating the capillary into brain organoids to make assembloids. We have also started incorporating liquid/oil barriers technology into our cell culture system aiming to make neuronal-muscular junction microfluidic cultures. The technology compared to existing microfluidic culture vessels is easier for re-modification during cell culture and will provide a versatile tool for us to study motor neuron damage. Specific aim 2: To study the roles of HERV-K on brain development. Using our iPSC models, we have collaborated with Dr. Steiner from TNC and Dr. Jack Mashall from SINS lab/NINDS to study and screen compounds for HERV-K LTR activation. We have found two compounds dramatically activate HERV-K LTR. This finding provides us a very critical tool/marker to study the role of HERV-K activation during human brain development. We will study the effect of the compounds in our 3D brain organoids to study its effect on brain development. Using the data, TNC has submitted a DOD grant to also use it for drug screening for HERV-K associated ALS study. We also studied cGAS/Sting gene expressions during pluripotent stem cells through motor neuron differentiation and their relations to HERV-K Env activation. The preliminary result showed a significant correlation between cGAS and HERV-K Env expression levels during the neuronal differentiation, indicating a possible association of the critical immune critical pathway and neuronal differentiation. Specific aim 3: To study the association of HERV-K and motor neuron degeneration like ALS. We have previously confirmed C9orf72 iPSC derived motor neurons produced phosphorylated-TDP43, a protein observed in degenerated motor neurons and antisense Oligonucleotides (ASOs) targeting HERV-K Env or C9orf72 repeats treatments decreased the phos-TDP43 products significantly. We further used this model to show that both DNA repairing mechanisms and defective DNA sensing mechanisms are involved in the HERV-K activation and TDP-43 pathogenesis. In addition to delineating the pathways that C9orf72 gene mutation leading to HERV-K activation and ALS pathology, these results also indicates that our C9orf72 derived motor neurons could be used as a valuable tool to study ALS and potentially for therapeutic development. We presented the result in the 2025 ISSCR annual meeting in Hong Kong. Specific aim 4: To facilitate research and therapeutic developments for neurological disorders using our models and methods. We have trained more colleagues from NIH intramural groups as well as extramural labs. In collaboration with Dr. Farinaz Safavi, we generated 3D brain organoids from BTK gene mutated iPSCs to study its effect on human brain development. We also provided motor neuron samples and help Dr. Marta Garcia Montojo from Twilight Bioscience to develop the C9orf72 repeat expansion PolyGA detection assay. We continued the collaboration with Dr. Pankaj Seth from India National Brain Research Center on studying ASOs treatment in Dengue virus infection in human brain. In addition, we started the collaboration with Dr. Suresh Kuchipudi from University of Pittsburgh School of Public Health to study the potential infection of bird flu virus H5N1 on human brain cells using the 3D brain organoids. In collaboration with Dr. In Hing Yang, we studied the magnetic/electronic stimulations on brain organoid maturation and found certain stimulations may help oligodendroglia maturation. This research may provide potential insights into the relations between earth physics and human brain.