Neurodegenerative Disease Modeling
Why are some cells more vulnerable to neurodegenerative disorders than others? This is a fundamental question for which research tools are limited. The answer to this question has broad implications for our understanding of pathological mechanisms and the development of pharmacological interventions. Human pluripotent stem cells (hPSCs), which can be differentiated into all cell types, provide an unparalleled tool for studying human neurodegenerative diseases in vitro.
For example, Parkinson’s Disease (PD) is characterized by the almost complete loss of midbrain dopaminergic neurons (DNs) in the substantia nigra pars compacta (SNpc), while their closest relatives in the ventral tegmental area (VTA) are relatively spared. To study cell autonomous and cell non-autonomous contributions to DN vulnerability, we have developed knock-in hPSC reporter lines and differentiation protocols to derive and track not only midbrain DNs but also forebrain striatal medium spiny neurons (MSNs), midbrain astrocytes, and microglia to assemble a simplified nigrostriatal niche.
In another study we compare distinct hPSC-derived midbrain and hypothalamic DN populations against each other to determine autonomous cellular functions that may explain vulnerability.
In previous and ongoing studies we focused on understanding how genetic mutations cause or contribute to the development of PD. In PD the interplay between risk genes and genetic or cellular background is poorly understood. Isogenic hPSC models allow study of disease genes while controlling for genetic background and cell type. Using CRISPR gene editing techniques, we have introduced highly penetrant PD-causing mutations into hPSCs to study PD pathology in its most affected cell type, DNs.
Transcriptomics and proteomics studies revealed how single gene edits can lead to dysregulation of hundreds of genes and we found PD relevant phenotypes including oxidative stress and specific cell death. We observed that PD relevant genes primarily affect various cellular pathways and functions but converge on common pathways such as oxidative stress and impaired lysosomal functions. This involves synergistic effects of PD risk or causative genes, feed-forward loops, and gene networks.
In the most recent studies, we began to explore how PD risk factors contribute to pathology. To achieve this, we are using various molecular tools such as CRISPR technologies including Cas9 knock-ins and misexpression approaches, as well as RNA knockdown using CasRX, to simultaneously and bidirectionally modulate expression of multiple PD relevant genes in a cell-type specific manner and to assess PD phenotypes.
Our long-term goal is to utilize hPSC PD models to understand neurodegeneration and the interactions and pathological contributions of different cell types, familial and risk PD genes, and environmental toxins, and to identify and test the impact of both candidate therapeutics and potential toxins through pharmacological perturbations.
1. Coccia E, Ahfeldt T. Towards physiologically relevant human pluripotent stem cell (hPSC) models of Parkinson’s disease. Stem Cell Res Ther. 2021;12(1). doi:10.1186/s13287-021-02326-5
2. Sarrafha L, Parfitt GM, Reyes R, et al. High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells. STAR Protoc. 2021;2(2). doi:10.1016/j.xpro.2021.100463
3. Fernando MB, Ahfeldt T, Brennand KJ. Modeling the complex genetic architectures of brain disease. Nat Genet. 2020;52(4). doi:10.1038/s41588-020-0596-3
4. Ahfeldt T, Ordureau A, Bell C, et al. Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson’s Disease Discovered Using Isogenic Human Dopaminergic Neurons. Stem Cell Reports. 2020;14(1):75-90. doi:10.1016/j.stemcr.2019.12.005
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2. Ahfeldt T, Litterman NK, Rubin LL. Studying human disease using human neurons. Brain Res. 2017;1656. doi:10.1016/j.brainres.2016.03.051
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5. Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K. A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells. Cell Stem Cell. 2008;3(3). doi:10.1016/j.stem.2008.08.003
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1. Ahfeldt T, Rubin LL. Composition and methods for enhanced knock-in reporter gene expression. Published online December 7, 2018.
2. Cowan C, Schinzel R, Ahfeldt T, Lee Y. Differentiation into brown adipocytes. Published online June 6, 2013.
3. Lum D, Ahfeldt T, Cowan C. Compositions and methods of generating reprogrammed adipocyte cells and methods of use therefore. Published online 2012.
4. Pryor H, Vacanti J, Lum D, Ahfeldt T, Cowan C. Compositions comprising hepatocyte-like cells and uses thereof. Published online April 2, 2010.
Meet the Team
Tim Ahfeldt, PhD
Gustavo Parfitt, PhD
Eveline Gutzwiller, MD