UC San Diego

Human pluripotent stem cells (hPSCs) serve as an excellent starting material for tissue engineering due to their renewability, capacity to differentiate into various tissue types, and remarkable self-organizing capabilities. However, a crucial challenge lies in establishing techniques for cultivating lab-grown organ constructs from hPSCs that accurately mimic the multi-lineage and vascularized nature of authentic human organs. In my thesis, I concentrated on innovating methods to produce multi-lineage and vascularized tissue, employing both in vitro and in vivo tissue engineering strategies.

In my first aim, I introduce an in vitro engineering approach to construct multi-lineage vascularized human tissue. This involves merging cell reprogramming with chemically directed organoid differentiation. Unlike standard organoid protocols, which often result in single-lineage tissue, this method combines transcription factor overexpression and blood vessel organoid- directed differentiation within a single organoid construct. The overexpression of transcription factors serves as an intrinsic reprogramming signal, overriding extrinsic media cues. This synergy drives parenchymal cell fate within the vascular organoid, essentially allowing for the creation of multi-lineage vascularized tissue constructs. In conclusion, in my first aim I developed a modular tissue engineering platform which allows for the programmable introduction of parenchymal cell types into a vascular organoid scaffold, contributing to the bottom-up engineering of complete human organs in vitro.

In my second aim, I present an in vivo engineering approach for building multi-lineage, vascularized human tissue by molecularly sculpting teratomas. Teratomas are often used in stem cell research to examine the differentiation potential of hPSCs and are usually formed via the subcutaneous injection of hPSCs in immunodeficient mice. Considering teratomas are a multi- lineage and large-scale source of vascularized human tissue, I chose to leverage them as a launching pad for multi-lineage, vascularized tissue engineering. Although teratoma tissue contains a panoply of self-organized, fetal-like structures from all three germ layers, the inherent randomness of global differentiation patterns precludes its utility in tissue engineering. However, by implementing microRNA (miRNA)-mediated suicide gene circuits, in this aim I demonstrate a unique teratoma sculpting approach, in which one can modularly select for desired tissue types in vivo based on the endogenous expression of a lineage-specific miRNA. All in all, this method of multi-lineage, vascular tissue engineering leverages the teratoma as powerful starting material for tissue sculpting and eventual top-down in vivo organ engineering.

In my third and final aim, I utilize the teratoma, along with time-course fetal-to-adult human organ tissue data, to conduct a biological investigation into the role of the RNA editing proteins adenosine deaminases acting on RNA (ADARs) in human development and cell-fate specification. ADARs impact diverse cellular processes and pathological conditions, and while we possess important insights into their roles in adult tissues, their functions in early cell fate specification remain less understood. To address this, we devised a comprehensive framework to investigate ADARs in human development, utilizing time-course human organ tissue, time-course teratoma tissue, and ADAR family CRISPR-KO screens in teratomas. After first establishing the teratoma as a faithful model of human development, we conducted pooled ADAR-KO (ADAR1- KO, ADARB1-KO, and ADARB2-KO) CRISPR-Cas9 screening in teratomas to uncover the functional roles of ADAR proteins in developing tissue, across all three germ layers. Via multi- lineage screening, we discovered that knocking out ADAR1 led to a significant fitness defect in mesodermal tissues, and an enrichment of adipogenic cells, revealing a novel role for ADAR1 in mesenchymal differentiation and obesity phenotypes. In brief, in my third and final arm, I demonstrate a modeling application for the multi-lineage and vascularized teratoma tissue, towards investigating the developmental role of ADARs and RNA editing across all germ layers.Collectively, my thesis work seeks to develop novel techniques for generating multi- lineage vascularized lab-grown organ constructs and demonstrating their utility in biological investigation.