Induced Pluripotent Stem Cell Derived Human Lung Organoids for Disease Modeling

Abstract

The rise of novel respiratory viruses, chronic lung diseases from metabolic anomalies, and pulmonary genetic disorders has created an unmet demand for biologically relevant models of lung disease. Human-based in vitro models, such as those derived from induced pluripotent stem cells (iPSCs), have become a cornerstone for disease modeling. iPSC-derived organoids are a rapidly emerging system because their 3-D culture and self-organizing nature recapitulate biological phenomena of disease responses in ways 2-D and animal models cannot. Organoids can be tailored into a multitude of organ systems, including the lungs.The emergence of the COVID-19 pandemic, caused by the Severe Acute Respiratory Distress Syndrome Coronavirus-2 (SARS-CoV-2), urged researchers to rapidly develop models that could uncover mechanisms behind virus entry, severe infection mortality, and screen novel therapeutics. We addressed this need by developing an iPSC-derived lung organoid (LO) model. LOs develop in four stages: embryoid body, anterior foregut, progenitor lung, and mature lung, similar to in utero development. Each of these phases of LO development display key lung phenotypes at each stage, such as endoderm upregulation, progenitor lung cells, Alveolar Type I and II cells, and more. This LO system can also be derived from multiple iPSC lines, including CRISPR-Cas9 knockout and patient-derived mutant lines, and cultured for long-term, highlighting their potential to study lung disease development at cellular, long-term, and genetic levels. Once lung phenotypes were confirmed, we tested the feasibility of SARS-CoV-2 modeling in our LO system. LOs display robust infection of SARS-CoV-2, and the utilization of CRISPR-Cas9 iPSC knockout lines reveals ACE2 as the viral entry mechanism. Infected LOs also demonstrate pyknotic nuclei and release of Cleaved Capase-3, hallmark phenotypes of cellular injury and apoptosis. Further examination of tight junctions utilizing a ZO1-GFP-tagged iPSC LO elucidate this cell death pathway by exhibiting tight junction barrier disruption and direct attachment of SARS-CoV-2 to the nuclei in early infection stages. Lastly, we demonstrate the potential to screen therapeutics for SARS-CoV-2 treatment in LOs utilizing an FDA approved drug and novel de novo proteins. Together, these data validate our iPSC-derived LOs as a robust modelling system for SARS-CoV-2 infection and therapeutic screening. The prevalence of comorbidities in SARS-CoV-2 infection severity is severely understudied, especially in the context of diabetes. Diabetes is often looked at as a kidney disease; however, systemic microvascular damage from diabetic complications leads to the development of pulmonary diabetic dysfunction. To understand how diabetes can lead to severe SARS-CoV-2 outcomes, we exposed LOs to glucose-dosed conditions at biologically relevant levels of diabetes. These LOs display differences in organoid morphology and metabolism at different glucose levels, with diabetic conditions exhibiting darkened morphologies and acidic supernatants. LOs cultured in diabetic conditions show elevated levels of ACE2 compared to their non-diabetic counterparts. This elevation in receptor sites could lead to higher levels of infection exposure to SARS-CoV-2 virion particles. Although we were unable to test this during this project, we have established a baseline system to examine the effects of diabetes and other comorbidities related to SARS-CoV-2 infection. Lastly, we sought to demonstrate our LOs potential to model genetic disorders by deriving a cystic fibrosis (CF) LO model. CF is an autosomal genetic disorder due to a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) with a current life expectancy of 35-40 years old. High variance in mutations of CF makes therapeutic development for CF patients extremely limited, highlighting the need for personalized platforms, such as iPSC-derived LOs. Our LO model showed positive expression of CFTR, indicating feasibility of developing a CF LO. We demonstrate the development of LOs from patient-derived CF lines and their mutation corrected controls. These CF lines show morphological and phenotypical differences to non-mutated lines during LO development and successful derivation of mature lung phenotypes. However, CF controls do not express CFTR proteins across multiple analyses and were missing pertinent cell types, such as goblet cells, at the end of LO culture. To fully establish this model, we would develop multiple CF mutant lines or derive CRISPR-Cas9 CF lines with genetically functional controls. The development of an iPSC-derived LO through the derivation of three diseases models, SARS-CoV-2 infection, diabetic pulmonary dysfunction, and Cystic Fibrosis, has reinforced the great potential that iPSCs-derived organoids harness. Overall, this LO system has proven to be an invaluable tool to investigate mechanisms of viruses, chronic illnesses, and genetic diseases that deeply affect pulmonary health.