Advancing drug discovery for glomerulopathies using stem-cell-derived kidney models.

Chronic kidney disease (CKD) is an epidemic that affects millions worldwide. The glomerulus, a specialized unit of the nephron, is highly susceptible to injury. Human induced pluripotent stem cells (iPSCs) have emerged as an attractive resource for modeling kidney disease and therapeutic discovery.

SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which has resulted in over 5.9 million deaths worldwide. While cells in the respiratory system are the initial target of SARS-CoV-2, there is mounting evidence that COVID-19 is a multi-organ disease. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often targeted in severe COVID-19, remains poorly understood. We employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes, and examined the expression of host factors for binding and processing of the virus. We studied cellular uptake of the live SARS-CoV-2 virus as well as a pseudotyped virus. Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed cellular uptake even at low multiplicity of infection (MOI) of 0.01. We found that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. We identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

Adriamycin-Induced Podocyte Injury Disrupts the YAP-TEAD1 Axis and Downregulates Cyr61 and CTGF Expression.

The most severe forms of kidney diseases are often associated with irreversible damage to the glomerular podocytes, the highly specialized epithelial cells that encase glomerular capillaries and regulate the removal of toxins and waste from the blood. Several studies revealed significant changes to podocyte cytoskeletal structure during disease onset, suggesting possible roles of cellular mechanosensing in podocyte responses to injury. Still, this topic remains underexplored partly due to the lack of appropriate in vitro models that closely recapitulate human podocyte biology. Here, we leveraged our previously established method for the derivation of mature podocytes from human induced pluripotent stem cells (hiPSCs) to help uncover the roles of yes-associated protein (YAP), a transcriptional coactivator and mechanosensor, in podocyte injury response. We found that while the total expression levels of YAP remain relatively unchanged during Adriamycin (ADR)-induced podocyte injury, the YAP target genes connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61) are significantly downregulated. Intriguingly, TEAD1 is significantly downregulated in podocytes injured with ADR. By examining multiple independent modes of cellular injury, we found that CTGF and Cyr61 expression are downregulated only when podocytes were exposed to molecules known to disrupt the cell's mechanical integrity or cytoskeletal structure. To our knowledge, this is the first report that the YAP-TEAD1 signaling axis is disrupted when stem cell-derived human podocytes experience biomechanical injury. Together, these results could help improve the understanding of kidney disease mechanisms and highlight CTGF and Cyr61 as potential therapeutic targets or biomarkers for patient stratification.

BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood. METHOD: In this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing. RESULT: Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. CONCLUSION: These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro . These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism. SIGNIFICANT STATEMENT: Many patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

A Personalized Glomerulus Chip Engineered from Stem Cell-Derived Epithelium and Vascular Endothelium.

Progress in understanding kidney disease mechanisms and the development of targeted therapeutics have been limited by the lack of functional in vitro models that can closely recapitulate human physiological responses. Organ Chip (or organ-on-a-chip) microfluidic devices provide unique opportunities to overcome some of these challenges given their ability to model the structure and function of tissues and organs in vitro. Previously established organ chip models typically consist of heterogenous cell populations sourced from multiple donors, limiting their applications in patient-specific disease modeling and personalized medicine. In this study, we engineered a personalized glomerulus chip system reconstituted from human induced pluripotent stem (iPS) cell-derived vascular endothelial cells (ECs) and podocytes from a single patient. Our stem cell-derived kidney glomerulus chip successfully mimics the structure and some essential functions of the glomerular filtration barrier. We further modeled glomerular injury in our tissue chips by administering a clinically relevant dose of the chemotherapy drug Adriamycin. The drug disrupts the structural integrity of the endothelium and the podocyte tissue layers, leading to significant albuminuria as observed in patients with glomerulopathies. We anticipate that the personalized glomerulus chip model established in this report could help advance future studies of kidney disease mechanisms and the discovery of personalized therapies. Given the remarkable ability of human iPS cells to differentiate into almost any cell type, this work also provides a blueprint for the establishment of more personalized organ chip and 'body-on-a-chip' models in the future.

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney's filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm's Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Robotic fluidic coupling and interrogation of multiple vascularized organ chips.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking.

G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and here we focus on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in ∼90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, we mutagenized the conserved tryptophan residue in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function.

A 3D bioprinted complex structure for engineering the muscle-tendon unit.

Three-dimensional integrated organ printing (IOP) technology seeks to fabricate tissue constructs that can mimic the structural and functional properties of native tissues. This technology is particularly useful for complex tissues such as those in the musculoskeletal system, which possess regional differences in cell types and mechanical properties. Here, we present the use of our IOP system for the processing and deposition of four different components for the fabrication of a single integrated muscle-tendon unit (MTU) construct. Thermoplastic polyurethane (PU) was co-printed with C2C12 cell-laden hydrogel-based bioink for elasticity and muscle development on one side, while poly(ϵ-caprolactone) (PCL) was co-printed with NIH/3T3 cell-laden hydrogel-based bioink for stiffness and tendon development on the other. The final construct was elastic on the PU-C2C12 muscle side (E = 0.39 ± 0.05 MPa), stiff on the PCL-NIH/3T3 tendon side (E = 46.67 ± 2.67 MPa) and intermediate in the interface region (E = 1.03 ± 0.14 MPa). These constructs exhibited >80% cell viability at 1 and 7 d after printing, as well as initial tissue development and differentiation. This study demonstrates the versatility of the IOP system to create integrated tissue constructs with region-specific biological and mechanical characteristics for MTU engineering.