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ANALYSES OF THE DEVELOPMENT AND FUNCTION OF STEM CELL DERIVED CELLS IN NEURODEGENERATIVE DISEASES.pdf

thesis
posted on 2023-02-03, 13:32 authored by Sailee Sham LavekarSailee Sham Lavekar

Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction.  Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.  

Funding

National Eye Institute (R01EY024984-S1)

National Eye Institute (R01EY033022)

The National Institute for Aging (RF1AG069425)

History

Degree Type

  • Doctor of Philosophy

Department

  • Biological Sciences

Campus location

  • Indianapolis

Advisor/Supervisor/Committee Chair

Dr. Jason Meyer

Additional Committee Member 2

Dr. Scott Canfield

Additional Committee Member 3

Dr. Teresa Mastracci

Additional Committee Member 4

Dr. Teri Belecky- Adams

Additional Committee Member 5

Dr. Benjamin Perrin