Session Number
Project ID: MEDH 21
Advisor(s)
Dr. Srinivas D. Narasipura; Rush University Medical Center
Dr. Lena Al-Harthi; Rush University Medical Center
Dr. Tanner Shull; Rush University Medical Center
Dr. Hemil Gonzalez, MD; Rush University Medical Center
Discipline
Medical and Health Sciences
Start Date
20-4-2022 10:05 AM
End Date
20-4-2022 10:20 AM
Abstract
Astrocytes are one of the most abundant cell types in the central nervous system (CNS), playing an important role in regulating the environment by participating in glutamate uptake, immunoinflammatory response of the CNS, and supporting the blood brain barrier (BBB). Sourcing a consistent supply of primary astrocytes for in vitro experiments is vital to understanding their role in CNS health and biology. In the past, astrocytes were mainly sourced from adult deceased brains, gliomas (U87MG, U138MG), aborted fetal tissues and mice/rat brains. However, such sources can be difficult to access and can pose limitations due to lack of biological relevance, costliness, and legal implications. Recently, hiPSCs (human induced pluripotent stem cells) were successfully demonstrated to differentiate into induced astrocytes (iAs). iAs have become an important tool in studying astrocyte biology and function because, compared to other sources, iAs are a consistent and timely supply of proliferative astrocytes.
The canonical Wnt/β-catenin pathway is a pro-survival pathway robustly expressed in human astrocytes such as gliomas and fetal astrocytes. β-catenin is the central mediator of this pathway. The pathway regulates important astrocyte functions, such as glutamate uptake, immunoinflammatory response and HIV transcription. This pathway is not studied in iAs yet. Hence, in this study we seek to determine if Wnt/β-catenin pathway is expressed robustly in iAs. We demonstrated that Topflash, a specific reporter of this pathway, is robustly active when integrated via lentivaral approach. Further, inducing the pathway by using a molecular activator, CHIR99021 resulted in significantly higher induction of the TopFlash reporter activity. Furthermore, we detected robust expression of TCFs/LEFs (transcriptional activators of the pathway) mRNAs through RT-qPCR. Taken together, these results demonstrated that iAs do indeed robustly possess the Wnt/Beta-Catenin pathway.
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Characterization of the Wnt/β-Catenin Pathway in iPSC Induced Human Astrocytes
Astrocytes are one of the most abundant cell types in the central nervous system (CNS), playing an important role in regulating the environment by participating in glutamate uptake, immunoinflammatory response of the CNS, and supporting the blood brain barrier (BBB). Sourcing a consistent supply of primary astrocytes for in vitro experiments is vital to understanding their role in CNS health and biology. In the past, astrocytes were mainly sourced from adult deceased brains, gliomas (U87MG, U138MG), aborted fetal tissues and mice/rat brains. However, such sources can be difficult to access and can pose limitations due to lack of biological relevance, costliness, and legal implications. Recently, hiPSCs (human induced pluripotent stem cells) were successfully demonstrated to differentiate into induced astrocytes (iAs). iAs have become an important tool in studying astrocyte biology and function because, compared to other sources, iAs are a consistent and timely supply of proliferative astrocytes.
The canonical Wnt/β-catenin pathway is a pro-survival pathway robustly expressed in human astrocytes such as gliomas and fetal astrocytes. β-catenin is the central mediator of this pathway. The pathway regulates important astrocyte functions, such as glutamate uptake, immunoinflammatory response and HIV transcription. This pathway is not studied in iAs yet. Hence, in this study we seek to determine if Wnt/β-catenin pathway is expressed robustly in iAs. We demonstrated that Topflash, a specific reporter of this pathway, is robustly active when integrated via lentivaral approach. Further, inducing the pathway by using a molecular activator, CHIR99021 resulted in significantly higher induction of the TopFlash reporter activity. Furthermore, we detected robust expression of TCFs/LEFs (transcriptional activators of the pathway) mRNAs through RT-qPCR. Taken together, these results demonstrated that iAs do indeed robustly possess the Wnt/Beta-Catenin pathway.