A Novel 3-D Culture System for Modeling Autosomal Dominant Polycystic Kidney Disease
Abstract/Contents
- Abstract
- Autosomal dominant polycystic kidney disease (ADPKD) is the world’s most common monogenic disorder. It is characterized by mutations in the PKD1 and PKD2 genes that cause cyst formation in kidney epithelial cells and bilaterally enlarge the kidney, leading to renal failure by age 60. While gene therapy to correct the underlying mutations in PKD1 and PKD2 could someday serve as a cure for ADPKD in the future, it is limited by the lack of a simple and inexpensive model with a clear phenotypic readout. Here, we describe a novel immortalized 3-D cell model for modeling cyst formation in vitro that shows rapid cyst formation in response to inducible Pkd2 knockout (KO). The cell line for this model was created by crossing the temperature-sensitive simian virus 40 (SV40) “Immortomouse” with a homozygous, Tet-On, Pkd2-inducible KO mouse and then isolating kidney tubular epithelial cells. The temperature-sensitivity of the SV40 transgene allows this cell line to renew indefinitely at 33 °C, but revert to a primary-like state upon transfer to 37 °C. Cells in this primary-like state were seeded into a modified 3-D Matrigel culture system to initiate cyst formation and treated with doxycycline (DOX) to induce Pkd2 KO. Although the wild-type (WT) cells also developed cyst-like structures, the Pkd2 KO cysts grew significantly faster after DOX treatment than the WT controls (p < 0.001), demonstrating that cyst growth in our model was Pkd2 KO specific. This is the first immortalized, inducible cell culture system described in the PKD field with a cystic size phenotype that varies between WT and PKD KO, and will be extremely useful for testing various gene therapy cures for ADPKD in a simple, low-cost, in vitro environment.
Description
| Type of resource | text |
|---|---|
| Date created | June 2020 |
Creators/Contributors
| Author | Maxim, Demetrios | |
|---|---|---|
| Primary advisor | Bhalla, Vivek | |
| Degree granting institution | Stanford University, Department of Biology, 2020 | |
| Advisor | Axelrod, Jeff | |
| Advisor | Stearns, Timothy |
Subjects
| Subject | Biology |
|---|---|
| Subject | polycystic kidney disease |
| Subject | 3-D culture |
| Subject | gene therapy |
| Subject | cyst formation |
| Genre | Thesis |
Bibliographic information
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- Use and reproduction
- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Share Alike 3.0 Unported license (CC BY-SA).
Preferred citation
- Preferred Citation
- Maxim, DS, Axelrod JA, Stearns, T, and Bhalla VB (2020). A Novel 3-D Culture System for Modeling Autosomal Dominant Polycystic Kidney Disease. Stanford Digital Repository. Available at: https://purl.stanford.edu/jr760vf1471
Collection
Undergraduate Theses, Department of Biology, 2019-2020
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- Contact
- dmaxim@stanford.edu
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