Chemogenetic Regulation in Reprogrammed Stem Cell-derived Precursor Cells in Treating Neurodegenerative Diseases

Our research focuses on illustrating the pathogenic mechanism underlying neurodegenerative disease, while integrating advanced technologies to develop effective cell therapy techniques for clinical applications. Our recent work transplanted chemogenetically modified stem cells in Parkinson disease mouse models. We modulate neuron activities of one cell with designer drugs, so electrophysiological monitoring and behavioral evaluation. We primarily utilize CRISPR for genetic engineering, electrophysiology to monitor neural activity, and behavioral assay to evaluate function recovery in Parkinson's disease models. The efficiency of graft cells rise on proper indication, survival, and functional contribution to the host tissue in stem cell-based therapy, treating neurodegenerative disease. Uncontrolled cellular activity related to negative consequences, including tumor genesis.

Leveraging technology in human reprogrammed stem cell and driver neurons provides the means to precisely control neuronal activities via administration of the designer drug CNO.

[Instructor] To begin, gently mix 50 to 100 nanograms of the linearized vector, fragment one and fragment two, in a one to three to three molar ratio, along with 2X cloning mix. Incubate the mixture at 55 degrees Celsius for one hour to facilitate homologous recombination using Gibson assembly. Insert the guide RNA targeting the AAVS1 site into the BBS1 site of the PX458 vector. Maintain the reprogrammed stem cells in culture media, aiming for a cell density between two to 5 million cells per milliliter. Now, mix two to 5 million cells, two micrograms of each donor plasmid, and two micrograms of GRNA plasmid in 100 microliters of electroporation buffer. Transfer the cell and plasmid mixture into a cuvette to perform electroporation. Next, transfer the electroporated cells into six-well plates that have been coated with poly-D-lysine and laminin. Distribute approximately 500,000 cells per well in two milliliters of culture medium. After 72 hours of electroporation, use a flow cytometer to sort fluorescent positive cells. Configure the sorter with the FITC channel for ZsGreen detection. Include untreated induced neural stem cells as negative controls to define the baseline fluorescence. Plate single ZsGreen-positive cells into 96-well plates pre-coated with poly-D-lysine and laminin and add 200 microliters of culture medium per well. Maintain the culture for seven to 14 days. Isolate single clones exhibiting fluorescence using an inverted fluorescence microscope equipped with a green fluorescent protein filter set. After expanding the positive cells on coated 48-well plates, split them into two parts, one for continued culture, and the other for genotyping. Extract genomic DNA from the candidate clones. Prepare PCR mix using two prime repairs, one to confirm insertion of the gene of interest, and another to distinguish between homozygous and heterozygous clones. Digest the cells, wash them in PBS, and centrifuge at 250 G for three minutes. Conduct unilateral stereotaxic injections of three microliters of 6-hydroxydopamine at a concentration of five micrograms per microliter on the anesthetized animal. Target the right corpus striatum using the correct coordinates. Administer an intraperitoneal injection of apomorphine at one milligram per kilogram using a 0.5 milligram per milliliter solution dissolved in saline four weeks after the surgery to validate the lesions. Administer a stereotaxic injection of four microliters of the prepared cell suspension into the Parkinson's disease mouse model. Perform the cylinder test at multiple time points following cell transplantation. Place a glass beaker with 20 centimeter diameter and 30 centimeter height on a flat, non-reflective surface. Then, position a top view camera above the cylinder to capture the full diameter. Place each mouse in the center of the cylinder and initiate simultaneous video recording for three minutes per session. After the session, return the mouse to its home cage. Assess upper limb movement of the mice during the three minute session. Calculate the number of wall contacts made by the impaired forelimb relative to the total contacts made by both forelimbs. Administer either saline or Clozapine N-oxide at a dosage of 1.2 milligrams per kilogram via intraperitoneal injection. Conduct the cylinder test again to assess behavioral modulation in the Parkinson's disease mouse model following Clozapine N-oxide administration. After extracting the brain from the anesthetized animal, and immediately place it in ice cold sucrose-based artificial cerebrospinal fluid to preserve cellular integrity Using a vibratome, slice the brain into sections of 300 to 400 micrometers thickness. Adjust gas input settings to equilibrate with 95% oxygen and 5% carbon dioxide at 34 degrees Celsius. Transfer the brain slices to a chamber filled with artificial cerebrospinal fluid. Load glass pipettes with iced intracellular solution, ensuring electrode resistance is between seven to 10 megohms. Mount slices in a submerged recording chamber superfused at two milliliters per minute with oxygenated artificial cerebrospinal fluid at 34 degrees Celsius. Position pipettes using motorized micromanipulators under infrared differential interference contrast microscopy. Establish whole cell configuration using gentle suction to patch neurons with resistance greater than one gigaohm. Clamp the cells at minus 70 millivolts using a patch clamp amplifier and acquire baseline spontaneous excitatory post-synaptic currents for eight minutes at 10 kilohertz sampling rate. Add 50 micromolar clozapine N-oxide to the recording chamber immediately after the baseline measurement. Continue recording data for 16 minutes and monitor changes in spontaneous excitatory post-synaptic current frequency or amplitude. Finally, perform a washout procedure by replacing the clozapine N-oxide solution with fresh artificial cerebrospinal fluid and continue recording to evaluate recovery in synaptic activity. Hemogenetic modulation using clozapine N-oxide reduced contralateral forelimb movement in hM4Di-transplanted mice and increased it in hM3Dq-transplanted mice compared to saline treatment, demonstrating bidirectional behavioral control. Electrophysiological recordings revealed that hM4Di-transplanted cells had longer intervals and smaller peak amplitudes in spontaneous excitatory post-synaptic currents, while hM3Dq transplanted cells had shorter intervals and larger amplitudes, indicating respective suppression and enhancement of synaptic activity. Immunofluorescence analysis confirmed the in vivo expression of tyrosine hydroxylase and ZsGreen in the transplanted hM4Di and hM3Dq cells, supporting successful graft integration.