Discipline: Technology and Engineering
Subcategory: Biomedical Engineering
Room: Exhibit Hall A
Alexxa Cruz-Bonilla - University of Puerto Rico, Mayaguez
Co-Author(s): Shiva Razavi and Ron Weiss, Massachusetts Institute of Technology, MA
Annually approximately 4.5 million are diagnosed with chronic liver disease (CLD). The previous treatments for CLD have proven ineffective, calling for the development of new targeted therapeutics in the form of small molecule drugs. The current platforms for testing the efficacy of existing drugs are either two-dimensional cell culture that lacks the cell type heterogeneity and the three-dimensional aspect of a liver or is low throughput and high risk, such as animal testing and human clinical trials; calling for the design of a novel synthetic biology solution. We propose an innovative high throughput approach to develop human induced pluripotent stem cells (hiPSCs) containing a previously engineered synthetic genetic circuit with the expression of a hepatic regulator (GATA6 protein) into a 3D organotypic culture (organoids). When conglomerating the hiPSCs, this platform will capture the liver cellular heterogeneity and produces both albumin and urea, the markers of a functional liver. The previous organoids that were being developed in the lab were producing the ideal amounts of albumin but seemingly deficient in urea production. We sought to increase urea production from approximately 10ug/million cells/day to the physiological synthesis rate at 40ug/million cells/day. The Wnt pathway has previously been shown to control urea and albumin production in the liver. To test this, we treated the organoids with Wnt ligands and inhibitors at various concentrations and treatment durations to study the role of the pathway in urea production and reach our target. In parallel, we realized the need for a more sensitive urea assay that would allow higher detection rates. When changing the assay, we saw an increase in urea production in non-treated organoids by 3 to 4-folds; putting us close to the desired physiological rates without the need to perturb the Wnt pathway. Even though the Wnt treatments did not cause a significant increase in urea production, morphological phenotypes were observed with the Wnt inhibitor cardamonin. We further explored these apparent differences by immunostaining for confocal microscopy and observed changes in the distribution of hepatocytes. Our work validated that our organotypic platform is now suited for high throughput drug target screening. Moreover, we will continue to explore the vast dosage, timing, and duration landscape of Wnt inhibitors to be able to fine-tune the complexity of our 3D liver organoid model. We aim to further translate these treatments to synthetic circuits that can be activated endogenously without external factors, similar to the natural liver development path. References: Guye, P., Ebrahimkhani, M. R., Kipniss, N., Velazquez, J. J., Schoenfeld, E., Kiani, S., … Weiss, R. (2016). Genetically engineering self-organization of human pluripotent stem cells into Birchmeier, W. (2016). Orchestrating Wnt signalling for metabolic liver zonation. Nature Cell Biology, 18(5), 463–465. doi: 10.1038/ncb3349. References: Guye, P., Ebrahimkhani, M. R., Kipniss, N., Velazquez, J. J., Schoenfeld, E., Kiani, S., ? Weiss, R. (2016). Genetically engineering self-organization of human pluripotent stem cells into Birchmeier, W. (2016). Orchestrating Wnt signalling for metabolic liver zonation. Nature Cell Biology, 18(5), 463?465. doi: 10.1038/ncb3349
Funder Acknowledgement(s): I thank Dr. Caley Allen (EBICS Education & Diversity Programs Manager) for her mentoring, support in this project endeavors, and for having invited me to return to the program as an EBICS Alumni. I also thank Dr. Erez Pery and Dr. Jose Vargas-Asencio for the provided supporting data. Funding was provided by an NSF Grant: CBET-0939511.
Faculty Advisor: Prof. Ron Weiss, firstname.lastname@example.org
Role: In this research, I was responsible for the cell culture of the hiPSCs and the development of the organoids. Just as well, I administered the treatments while researching new possible variables that might affect urea production. I was also responsible for optimizing the past urea assay used in the lab and finding new assays that when optimized would fit our platform. I was also tasked with the immunostaining but received support for the confocal microscopy. Consequently, all of the collected data was analyzed by me and was reviewed by my mentor Dr. Shiva Razavi and PI Dr. Ron Weiss.