Person:

Peters, Derek T.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.

A complex interplay of genetic and environmental factors underlies the development of common human diseases such as cardiovascular disease. Despite widespread use of existing medications, coronary heart disease (CHD), including myocardial infarction (MI), remains a significant cause of morbidity and mortality worldwide. The results of recent human genetic studies have provided unprecedented opportunities to elucidate the genes and molecular pathways that underlie CHD and risk factors like plasma lipid concentrations. Translating these findings into mechanistic insight promises to improve our understanding of disease pathogenesis and aid in the development of novel therapies. Precise functional characterization is required to bridge this gap; yet doing so can be challenging using traditional approaches.

The properties of human pluripotent stem cells (hPSCs) make them uniquely suited to address this challenge - they retain a normal human genome in culture, can be genetically modified, and can be differentiated into multiple cell types. The work presented in this thesis demonstrates the use of hPSCs and genome editing technology to generate human disease models in vitro. We developed a genome editing system optimized for hPSCs that can be used to efficiently generate hPSC lines with targeted genetic modifications. Modified and isogenic control hPSCs are then differentiated into a relevant cell type for phenotypic characterization. We applied this approach to investigate the functional effects of human genetic variation underlying plasma lipid concentrations.

We used TALEN genome editing to clarify the role of the SORT1 gene as a mediator of cellular metabolic processes. We targeted the ANGPTL3 gene using TALEN and CRISPR/Cas9 genome editing to create an in vitro model of a monogenic disorder, familial combined hypolipidemia, and investigated the putative role of ANGPTL3 in the regulation of low-density lipoprotein cholesterol metabolism. To facilitate the use of hPSC-models to study subtle phenotypes involving human liver, we developed a method for purifying hepatocyte-like cells following hPSC differentiation. Finally, we combined these approaches to model the tissue-specific effects of a common non-coding genetic variant in the chromosome 1p13 locus that is associated with risk of MI. This result demonstrates the feasibility of using hPSCs to characterize disease-associated common genetic variation.