Evaluation of Human Stem Cell-Derived Neurons in 2D and 3D Cultures for Neurodevelopmental Studies, Epilepsy Disease Modeling and Drug Discovery

Author

Hamed Salmanzadeh-Dozdabi, University of the PacificFollow

Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Robert Halliwell

First Committee Member

Atefeh Rabiee

Second Committee Member

David W. Thomas

Third Committee Member

Raj Sevak

Fourth Committee Member

Benjamin Zeitlin

Abstract

Human stem cells have revolutionized the study of early neural development, offering a powerful tool to investigate the complex processes involved in the formation of the nervous system. In this study, the human pluripotent stem cell line, TERA2.cl.SP12, was utilized to derive neuroglial cells and to determine their differentiation and functional maturation up to one year in culture; this model was then investigated as a new tool for the study of neurodevelopmental toxicity, for seizure modeling and as a screening method for the investigation of new anticonvulsants. Immunocytochemistry experiments determined that non-differentiated cells expressed the stem cell marker Oct-3/4 and, when exposed to retinoic acid, differentiated into neurons that expressed the neural marker proteins, β-III tubulin, MAP2 and the glial cells expressed GFAP. Immunoblotting analysis of the neural protein βIII-tubulin expression revealed robust expression from 2 months to 12 months of culture, indicating that the neurons remained viable and stable for an extended period. GFAP expression significantly increased at 4 and 8 months of culture, indicating that gliogenesis followed neurogenesis in vitro. These findings support the concept that the neural differentiation of human embryonal carcinoma (EC) stem cells follows a developmental program that closely mimics the intrinsic timescale observed in vivo, as has been observed in human ES and iPS cells.Multi-electrode array (MEA) studies indicated that these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized firing patterns which all increased significantly over one year in culture and indicated complex network formation and neural maturation. Neural activity in this 2D neuron-glia culture was modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and indicated the presence of inhibitory and excitatory synapses and integrated neural circuits.

To test these stem cell derived neurons as a model for epilepsy disease modeling and drug screening, epileptiform activity was induced by the proconvulsant agent 4-aminopyridine (4-AP) in the neural circuits and five antiseizure drugs, representing a diverse group of clinically important agents for epilepsy, were examined. MEA data showed that 4-AP evoked epileptiform-like activity and increased spike frequency, single cell burst firing, and synchronized network bursting in the neuroglial cultures. In addition, spontaneous neural network activity and 4-AP-evoked epileptiform activity were inhibited by first, second and third generation antiseizure agents, consistent with animal and human studies. Pluripotent stem cell-based systems have proven useful in the study of developmental neurotoxicity. Here the impact of 2 widely used anti-epileptic drugs (AEDs), topiramate and gabapentin, on neural development was determined using TERA2.cl.SP-12 stem cell neurons. Transient exposure to gabapentin (3-300µM) or topiramate (3-300µM) did not affect cell viability, proliferation, or differentiation after a 30-day exposure period, except at concentrations of topiramate above the therapeutic range, and which led to a significant reduction in neural differentiation. The impact of chronic exposure to topiramate and on functional activity of neurons was then determined over ultra-long-term cell cultures. Results showed that topiramate decreased firing rate and synchrony index at a concentration of 30µM compared to control. Notably, these effects did not appear until 36 to 52 weeks, implying ‘silent neurotoxicity’ of developmental processes and functional deficits that do not surface until much later. These findings also suggest that human pluripotent stem cells and their neuronal derivatives can advance toxicological research by reducing the time and costs involved, whilst also minimizing the need for animal testing. Brain organoids offer a highly advanced model for the study of early embryonic and fetal brain development as well as human-specific disorders. In the next phase of this study, TERA2.cl.SP-12 stem cells were differentiated into 3D culture (neural organoids) by utilizing Stemcell Technologies STEMdiff cerebral organoid protocol. After 100 days differentiation, immunolabeling of the neural marker protein (βIII-tubulin) and glial cells (GFAP) revealed strong expression and presence of neuroglial cells differentiated from stem cells in the organoid cultures. Interestingly, immunocytochemistry labeling showed GFAP-positive cells surrounding ventricular-like zone (VZ) areas, suggesting VZ formation in the organoids. Multi-electrode array recordings demonstrated spontaneous spiking, single cell bursts and synchronized which all increased significantly over 30 weeks of differentiation. Neural activity in the organoids was modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and revealed the presence of inhibitory and excitatory synapses and integrated neural circuits. In addition, the convulsant agent 4-AP, evoked epileptiform like activity and increased spike frequency, single cell burst firing, and synchronized network bursting in the organoids, consistent with seizure like activity in these cultures. In further experiments the anticonvulsants, carbamazepine, ethosuximide and diazepam, reduced or blocked 4-AP induced epileptiform-like activity in the organoids at concentrations close to, or within the range of therapeutic dosing for seizure control. Together, the findings reported in this dissertation strongly support the potential value of human pluripotent stem cells in modeling early neural development, neurological disorder and drug investigations in both 2D and 3D cultures.

Pages

170

Recommended Citation

Salmanzadeh-Dozdabi, Hamed. (2023). Evaluation of Human Stem Cell-Derived Neurons in 2D and 3D Cultures for Neurodevelopmental Studies, Epilepsy Disease Modeling and Drug Discovery. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/4175