Lecture 21: Kidney Organoids PDF

🥽 Lecture 21: Kidney Organoids Organoids v. other models Direct study of human biology and disease Strengths: Direct study is essential for understanding human biology and...

🥽 Lecture 21: Kidney Organoids Organoids v. other models Direct study of human biology and disease Strengths: Direct study is essential for understanding human biology and disease. Crucial for drug development and therapeutic interventions. Limitations: Limited access to tissue samples. Limited scope of experimentation—testing multiple drug combinations on a single patient is not feasible. Therapies are restricted to well-established options, which may limit individualized treatment. Primary and immortalised cell lines Strengths: Primarily involve 2-dimensional cell culture. Widely used for disease modeling and drug screening. Lecture 21: Kidney Organoids 1  Significant contributions to the understanding of biology. Limitations: Do not replicate the physiological environment. Lack proper modeling of cellular interactions. Model organisms - eg. mice, fruit flies, fish, and worms Strengths: Strong conservation of genetic, cellular, and molecular mechanisms between model organisms and humans. Limitations: Species-specific differences often hinder translational research. Challenges in drug development and metabolism due to: Species-related biological differences. Difficulty distinguishing species-specific discrepancies from individual variations in humans or specific model organisms. How do Human Organoids compare? Strengths: Multicellular human tissues that can be patient-derived. Amenable to genetic manipulation and drug screening. Capable of extensive expansion and experimentation. Recapitulate many aspects of tissue architecture and cellular composition of the modeled organ. Limitations: Lack of physiological context: Limited interaction with immune cell types. Absence of a vascular system or blood supply. Lack of interaction with other organ systems. Lecture 21: Kidney Organoids 2  Aspect Model Organisms Organoids - Strong conservation of Developmental - Easier and faster for developmental Biology genetic manipulation. mechanisms. - Some differences between human and - Do not require full model organism organism development. development. - Superior in capturing - Effective substitute if Physiological whole-organism they replicate key cellular Complexity complexity. processes. - Lacks full systemic physiological context. - Insights into conserved - Directly models human- Human-Specific mechanisms, but species- specific processes, ideal Physiology specific differences exist. for human tissue studies. Establishing organoids 1. Use ES or reprogram to iPSC and direct differentiation Source: derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Process: 1. Pluripotent Cells: Lecture 21: Kidney Organoids 3  Pluripotent stem cells are guided back through developmental signals and processes ⇒ ie. reprogram 2. Developmental Pathways: Cells undergo differentiation following the signals that would naturally guide organ formation. Examples of Derived Tissues: Endoderm-derived tissues: Stomach, lung. Mesoderm-derived tissues: Kidney, blood vessels. Ectoderm-derived tissues: Brain organoids. 2. Isolate and culture adult stem cell Source: derived from adult stem cells or tissue-resident stem cells. Process: 1. Isolation and Culturing: Adult stem cells are isolated and cultured in growth factors and extracellular matrix. This environment promotes the maintenance of the specific cell type. 2. Tailored Factors: A cocktail of specific factors is added to mimic the tissue microenvironment and maintain healthy cell function. 3. Cellular Derivatives: The stem cells produce the cellular derivatives typical for that tissue type. Applications: Successfully used in tissues such as: intestine, stomach, liver, lung, pancreas, and others. 3. Both methods rely on: Understanding the signals needed to regulate stem or progenitor cell maintenance. Lecture 21: Kidney Organoids 4  Knowledge of developmental pathways required for differentiation into specific tissue types. Kidney function and disease Kidney is important - it regulates fluid homeostasis, removal of waste from blood, blood pressure, bone density, and red blood cell count Chronic Kidney Disease (CDK) Definition: gradual loss of renal function, including filtration and hormone production. Causes: genetic mutations, chronic injury - including inflammation or complications like diabetes. The kidney is a major target for toxicity in drug development - often susceptible to drug-induced damage during → often a leading reason for halting further clinical development of drugs. Incidence growing at 6% per annum worldwide Motivation for Kidney Organoids End-stage patients require organ transplantation or life-long dialysis CKD treatment costs Australian economy >$1 billion annually Lecture 21: Kidney Organoids 5  >750 million people affected worldwide Benefits of kidney organoids: Serve as models for kidney disease, including fibrosis, cell defects, cystic diseases, and cancer. Can aid in accelerating drug discovery and development. Potential applications for kidney organoids Human Developmental Biology: Study kidney development and repair/regeneration for the first time using kidney organoids. Toxicology Screening: Assess kidney-specific cell death to evaluate drug toxicity during clinical development. Drug Screening and Discovery: Use organoids to test and discover drugs for specific diseases effectively modeled in kidney organoids. Cellular Therapy and Renal Replacement: Long-term goals include: Generating a functional kidney for patients using their own stem cells or iPSCs. Developing therapies for renal replacement. Lecture 21: Kidney Organoids 6  Knowledge of developmental programs informs understanding of congenital disease and directed differentiation The Argument for Recreating Tissue → Understanding how tissues form during development could enable us to recreate them in vitro Kidney Development: Key Stages and Lineages 1. Primitive Streak Formation: The kidney originates from the posterior primitive streak, which gives rise to: Paraxial mesoderm. Intermediate mesoderm. 2. Intermediate Mesoderm: Anterior Intermediate Mesoderm: Gives rise to the ureteric epithelium (collecting network that drains renal filtrate to the bladder). Posterior Intermediate Mesoderm: Gives rise to the metanephric mesenchyme, a heterogeneous tissue containing progenitors for: Lecture 21: Kidney Organoids 7  Nephrons: Filtration units of the kidney. Stroma: Interstitial tissue between ducts. Vascular progenitors. Understanding mouse development has led to advances in generating and propagating renal progenitors Renal Progenitor Cells: Isolation and culture of nephron progenitors through understanding of self-renewal environments. Cell Reprogramming: Reprogramming progenitors and nephron cells (e.g., podocytes) using transcriptional profiles. Directed Differentiation: Mimicking embryonic progression to generate nephron, stroma, vascular, and ureteric cell types from pluripotent stem cells. Lecture 21: Kidney Organoids 8  Approaches to direct differentiation of renal progenitors Taguchi Protocol: Focused on defining distinct progenitor lineages and their developmental transitions, with a hypothesis that nephron and ureteric bud components could not be generated simultaneously. Correct in Principle: Lineage tracing and mouse developmental studies strongly support the distinct developmental origins of nephron progenitors and ureteric bud cells. Simultaneous induction of both lineages in vivo is unlikely due to these separate origins. Takasato Protocol: Leveraged existing literature to establish a protocol that claimed simultaneous induction of nephron and ureteric bud components. Partially Correct: The protocol showed marker expression consistent with both nephron progenitors and ureteric bud cells. Limitations: Later studies raised questions about whether the induced ureteric bud-like structures were functionally equivalent to true ureteric bud cells in vivo. Comparison of the Taguchi and Takasato Protocols Lecture 21: Kidney Organoids 9  Aspect Taguchi Protocol Takasato Protocol Detailed characterization of Relied on existing literature mouse kidney development for markers and Approach through lineage tracing, gene differentiation drivers without expression profiling, and redoing developmental immunofluorescence. studies. Defined distinct origins of key kidney structures: Claimed simultaneous anterior intermediate induction of both Focus mesoderm (ureteric bud) and metanephric mesenchyme posterior intermediate and ureteric bud in the same mesoderm (metanephric protocol. mesenchyme). - Anterior Intermediate Mesoderm: Ureteric bud and Focused on both ureteric bud Progenitor collecting duct. - Posterior and nephron lineages, relying Lineages Intermediate Mesoderm: on marker expression to Studied Metanephric mesenchyme, validate simultaneous nephron precursors, and differentiation. stromal cells. Identified markers for Used markers derived from progenitor states through Markers Used literature to assess extensive experimental differentiation outcomes. characterization. Investigated signaling Applied known signaling and pathways and transcriptional Drivers of differentiation pathways from regulators to recreate Differentiation literature for directed developmental transitions in differentiation. vitro. Successfully generated iPSC- Successfully generated derived kidney tissue and iPSC-derived kidney tissue Outcomes claimed simultaneous focusing on nephron differentiation of nephron and structures. ureteric bud components. - Lineage tracing in mouse - Marker expression from models. - Gene and protein literature. - Validation expression profiling Immunofluorescence to Techniques (markers). - validate lineage Immunofluorescence to differentiation. confirm differentiation. Lecture 21: Kidney Organoids 10  Aspect Taguchi Protocol Takasato Protocol - Characterized detailed lineage transitions from Showed potential for mouse development. - Distinct simultaneous induction of Hypothesis: simultaneous Contributions nephron and ureteric bud nephron and ureteric bud components in vitro. induction not possible in one protocol. Highlighted importance of Demonstrated feasibility of detailed developmental using literature-based Global Impact understanding for organoid approaches for simultaneous generation. multi-lineage differentiation. Variants developed by Variants developed by Taguchi’s group and others Takasato’s group and others Protocol focused on refining aimed at enhancing dual- Adaptations metanephric mesenchyme lineage differentiation protocols. outcomes. Key Developments in Early Kidney Organoid Protocols 1. Preceding Protocols Early protocols successfully induced intermediate mesoderm (IM) but did not push differentiation toward kidney-specific tissues. 2. Breakthrough by Taguchi and Takasato Achieved Differentiation Beyond Intermediate Mesoderm:Both protocols advanced the field by driving the intermediate mesoderm toward kidney-specific lineages, marking a significant breakthrough in organoid development. Key Factors Used: F9 (Fibroblast Growth Factor 9) WNT signaling activators (e.g., CHIR99021, a GSK3 inhibitor) 3. Common Strategies in Kidney Organoid Protocols Induction of Intermediate Mesoderm (IM): The initial and critical step in generating renal progenitors from pluripotent stem cells. Lecture 21: Kidney Organoids 11  Triggering Nephron Formation: Two distinct approaches: 1. Controlled WNT Signaling Burst: Directs renal progenitors to form nephrons efficiently. 2. Spontaneous Differentiation: Allows nephron structures to develop without precise signaling bursts. Directed differentiation leverages inherent biological programs to yield appropriate cell types and tissue structure Initial Perception of creating organoids → Science Fiction Recreating development in vitro was once seen as impossibly complex due to: The numerous cell fate decisions required from a fertilized egg to fully developed tissue. The need to regionalize tissues in specific time and space, progressing through various progenitor stages. HOWEVER - like other molecular tools (DNA Pol, restriction enzymes), organoids leverage existing capabilities in nature Natural processes like differentiation, self-organisation, and self- patterning Organoid differentiation leverages pre-set developmental programs inherent in cells. Ie. Cells inherently know how to produce specific cell types when properly primed. They do not require exhaustive external instruction. Self-organisation - similar cell types naturally form structures seen in tissues. Inherent patterning - limited patterning capabilities are built into cells, reducing the need for detailed intervention in instructing each step. Lecture 21: Kidney Organoids 12  Characterizing kidney organoids: Takasato Protocol Steps 1. Inducing Posterior Primitive Streak: Used low levels of Wnt signaling via the Wnt agonist CHIR99021 (CHIR). 2. Establishing Intermediate Mesoderm Fate: Added F9 growth factor to stimulate proliferation and control cell identity. 3. 3D Culture Formation: Aggregated the 2D cultured cells into a 3D blob. Placed the blob on a membrane. Administered another pulse of Wnt agonist, enabling nephron formation. 4. Outcome: Formation of self-organizing kidney tissue. Observed distinct patterning with regions corresponding to nephron segments. Results of Characterisation Transcriptional Comparison: Used bulk RNA sequencing to analyze gene expression in kidney organoids. Compared results to human fetal tissues. Key Findings: Kidney organoids closely resembled early human fetal kidney (1st trimester). Organoids displayed relative immaturity but a surprising complement of cell types resembling a developing kidney. Lecture 21: Kidney Organoids 13  Potential applications for kidney organoids Critical factors for success: Cellular composition: The utility of organoids depends on: Presence of relevant cell types (e.g., nephron segments, ureteric epithelium). Lecture 21: Kidney Organoids 14  Accurate representation of the disease process being studied. State of maturation: Mature and functional cell types are essential for: Effectively modeling diseases. Providing reliable results for drug testing. Achieving therapeutic outcomes. Why Continue Research on Kidney Organoids? Challenges: Specificity of Markers in Organoids vs. In Vivo Tissues In embryos, cell types can be identified with confidence as they exist in their natural environment. In organoids, marker expression alone may not definitively confirm cell identity: Example 1: SIX2, a robust marker of nephron progenitor cells in the developing kidney, also shows expression in regions of the nose and brain. Example 2: TCF21, known for marking kidney stroma, is also expressed in other tissues like gut stroma. Challenges in Verifying Cell Identity Starting with pluripotent stem cells, which can differentiate into any cell type, requires rigorous validation to ensure correct differentiation. Simply observing marker expression does not guarantee the generation of the intended cell type: Markers alone cannot distinguish between similar cell types from different tissues (e.g., kidney stroma vs. gut stroma). Limitations of Traditional Analysis Targeted Approaches (e.g., Immunofluorescence or PCR): Lecture 21: Kidney Organoids 15  Only detect markers of interest, potentially missing unexpected cell types or differences. Bulk RNA Sequencing: Averages gene expression across all cells, masking cell- specific profiles. Addressing These Challenges with Single-Cell Sequencing Single-cell RNA sequencing (scRNA-seq) provides a comprehensive view of the cellular composition in kidney organoids: Identifies all cell types present. Allows comparison of organoid-derived cell types to equivalent cell types in the human fetal kidney. This approach helps determine: Conservation of key markers between organoids and in vivo tissues. Unexpected differences or the presence of unintended cell types. Overview: high throughput single cell RNA sequencing What Is Single-Cell Sequencing? A method to study heterogeneous tissues at the level of individual cells. Lecture 21: Kidney Organoids 16  Key Steps: Cell Separation: Isolate individual cells from a tissue. Transcriptional Profiling: Determine the unique set of genes expressed in each cell. Reconstruction of Cell States: Identify cell types or states by clustering cells with similar transcriptional profiles. Transcriptional Profiling and Gene Expression Each cell type expresses a unique set of genes, which defines its identity and state. Single-cell sequencing allows precise mapping of gene expression to specific cell types. Comparison to Bulk RNA Sequencing Bulk RNA Sequencing: Combines RNA from all cells in a sample. Provides an average transcriptional profile of the tissue. Reflects the proportions of contributing cell types but masks cell-type-specific information. Single-Cell Sequencing: Captures the individual profiles of each cell. Provides detailed insights into cell-type-specific gene expression. Application to Kidney Organoids Bulk RNA Sequencing: Historically used for kidney organoids, providing an overview of gene expression but limited resolution. Single-Cell Sequencing: A breakthrough approach to identify and validate the composition of kidney organoids, revealing cell types and their correspondence to fetal kidney cell types. Lecture 21: Kidney Organoids 17  Exploring the cellular composition of human kidney organoids with single cell sequencing Methodology: 1. Preparation: Multiple organoids from different batches were taken. Organoids were dissociated into individual cells. 2. Transcriptional Profiling: The transcriptional profile of each cell was determined. 3. Data Analysis: Clustering algorithms were applied to identify similarities and differences in transcriptional profiles. Clustering allowed the identification of cell identities and states. 4. Clustering Resolution: High-Level Clustering: All cells treated as a single cluster, providing an average blended profile. Detailed Clustering: Divided into smaller clusters based on transcriptional differences. Reflects specific cell types present in the organoid. A clustering tree algorithm was used to define structure, helping to decide the appropriate resolution for clustering. Lecture 21: Kidney Organoids 18  Findings - Kidney organoids contain expected and off-target populations 1. Expected Cell Types: Nephron Cells: Key functional components of the kidney. Endothelial Cells: Essential for vasculature. Stroma Cells: Provide structural support. 2. Off-Target Cell Types: Neural Cell Types: Not typically found in kidney development. Muscle Progenitors: Another cell population not usually present in kidney tissue. Off-Target Differentiation: Likely due to: 1. Variations in cellular response to growth factors. 2. Interactions between cells during differentiation, leading to slight deviations. Comparison to human developing kidney reveals conserved endothelial, nephron, and stromal populations Approach: 1. Data Integration: Lecture 21: Kidney Organoids 19  Single-cell sequencing data from human foetal kidney cells and kidney organoids were integrated. 2. Clustering Analysis: Cells were clustered based on transcriptional profiles to identify cellular identities. Human foetal kidney cells were marked in pink, while kidney organoid cells were marked in blue. 3. Conserved and Divergent Gene Expression: Gene expression profiles within these clusters were analyzed to highlight similarities and differences. Findings: 1. Conserved Cellular Identities: Nephron Clusters: Contributions from both human foetal kidney and kidney organoids were observed across: Nephron progenitor cells. Tubular regions of the nephron. This indicates conservation of cellular identities. 2. Gene Expression Conservation: Key markers for differentiating progenitor cells and other nephron-related states were conserved between the human foetal kidney and organoids. For example, markers of specific nephron states exhibited similar patterns in both datasets. Interpretation: Conservation: Kidney organoids share cellular and gene expression characteristics with the human foetal kidney. Nephron-related cell types are particularly well-represented. Differences: Lecture 21: Kidney Organoids 20  While clustering suggests conservation, subtle differences exist in transcriptional profiles, reflecting inherent variances in the in vitro and in vivo systems. Strengths and limitations of kidney organoids Organoid Strengths: Represent key kidney cell types, including nephron and stromal populations. Useful for developmental studies, especially nephron progenitors and tubule formation. Organoid Limitations: Lack of immune and endothelial cell contributions. Presence of off-target cell types like neural and muscle progenitors. Limited diversity and maturity of nephron subtypes compared to in vivo kidneys. Transcriptional congruence between UE and distal nephron Findings on Ureteric Epithelium Markers in Organoids: Unexpected Marker Specificity: Markers previously believed to be specific to ureteric epithelium (e.g., GATA3 and HOXB7) were observed in cells Lecture 21: Kidney Organoids 21  aligning with the nephron lineage as well. This raises questions about the specificity and reliability of these markers in organoids. Challenges in Defining Lineages: If ureteric epithelium markers are not exclusive, it becomes difficult to confirm that both nephron and ureteric epithelium lineages are produced simultaneously. These findings could suggest: Taguchi's hypothesis: The inability to generate both lineages simultaneously might hold true. Alternatively, the culture conditions may need further refinement to preferentially support one lineage over the other. Implications: For Taguchi and Takasato Protocols: Raises concerns about lineage fidelity in existing protocols. Supports the need for improved methods to selectively promote ureteric epithelium or nephron lineage. Future Directions: Marker re-evaluation: More specific markers need to be identified for ureteric epithelium. Protocol refinement: Adjust culture conditions to better differentiate or isolate the desired lineage. Validation approaches: Use additional lineage-specific functional assays to confirm identity. Refining the cellular components of kidney organoids Marker Definition and Developmental Programmes Taguchi and Professor Ryuichi Nishinakamura revisited the process to: Lecture 21: Kidney Organoids 22  Define the markers that distinguish ureteric epithelium from metanephric mesenchyme. Identify developmental programmes necessary to recreate these lineages in vitro. Key Achievements 1. Ureteric Bud Formation Successfully characterised markers specific to ureteric epithelium. Defined the developmental signals required for ureteric bud formation. 2. Interaction with Nephron Progenitors Combined ureteric bud and nephron progenitors in culture. Achieved interaction between the two populations, mimicking kidney development in vitro. Application: Assessing patterning and lineage relationships in human development Assessing Developmental Relationships in Human Tissues Challenge with Human Embryos: Modifying and studying human embryos directly is not possible. Organoid Advantage: Kidney organoids allow for the manipulation of pluripotent stem cells to track developmental processes in human tissue. Key Research by Sarah Howden and Jess Fensom-Lambrook Genetic Reporters and Labeling: Used genetic reporters to trace nephron markers in organoids. Labelled nephron progenitor populations and different nephron segments. Enabled the study of nephron segment relationships in human tissues. Findings: Lecture 21: Kidney Organoids 23  The labeling successfully identified equivalent nephron segments in kidney organoids. Confirmed that developmental relationships between segments were largely consistent with in vivo observations. Key Insight: Live Imaging of Nephron Development Live Imaging: Utilised fluorescence to visualize podocyte maturation and distal tubule formation. Difference from Embryonic Development: Showed that development in organoids is distinct from that in embryos. Unique Capability: Enabled the study of human nephron development in ways previously impossible. Disease Applications of Kidney Organoids: Modeling Cystic Kidney Disease Study on Autosomal Recessive Polycystic Kidney Disease Objective: Model autosomal recessive polycystic kidney disease (ARPKD) using kidney organoids. Genetic Focus: Mutations in the PKHD1 gene responsible for ARPKD. Methodology Lecture 21: Kidney Organoids 24  Patient-Derived iPSCs: Induced pluripotent stem cell (iPSC) lines were generated from patients with the mutation. A control iPSC line was created by gene correcting the mutation. Stress-Induced Cyst Formation: Cells were stressed using a chemical agent to induce cyst formation. The system was used to test drug efficacy in suppressing cyst formation. Drug Screening Platform Test Example: No Stressor or Drug: Organoids did not form cysts. With Stressor: Cyst formation was observed. With 50 µM Drug: Modest suppression of cyst formation. With 100 µM Drug: Significant suppression of cyst formation. Lecture 21: Kidney Organoids 25  Advantages of Kidney Organoids in Disease Modeling High Throughput Screening: This approach is more amenable to high throughput screening compared to traditional 2D cultures or mouse models. Patient-Specific Modeling: Specific mutations and cell types from individual patients can be studied, providing a personalized therapeutic platform. Limitations of Mice Models: Mice replicate cystic phenotypes but are difficult for high throughput studies. Organoids provide a more scalable and efficient alternative. Application: Automation and High-Throughput Toxicity Screening Using Kidney Organoids Study Overview Lecture 21: Kidney Organoids 26  Objective: Demonstrate proof of principle for high-throughput liquid handling and automated imaging in toxicity screening and developmental modeling using kidney organoids. Protocol: The study utilized a simplified 2D culture format for generating kidney organoids, more amenable to automated screening compared to complex protocols like the Taguchi or Asado protocols. Platform: The organoids were cultured in 384-well plates, making them suitable for use with liquid handling robots and automated microscopes for high-throughput screening. Experimental Approach Growth Factor Variations: Tested the effects of different concentrations of growth factors on kidney organoid development. Nephrotoxicity Testing: Induced toxicity by exposing the organoids to cisplatin, a common chemotherapeutic agent known to cause kidney damage through cell death. Reporter Systems: Built-in reporters assessed markers of individual nephron segments, allowing monitoring of injury, viability, and metabolic activity in response to toxic stimuli. Advantages of the Approach Human-Specific Toxicity Screening: This platform allows for toxicity testing that is specific to human kidney tissue, making it more relevant than traditional animal models. High-Throughput and Automated: The 384-well plate format and automated systems enable screening of multiple cell lines and drug concentrations in a high-throughput manner, greatly enhancing efficiency compared to slower animal model research. Comprehensive Toxicity Assessment: The combination of injury, viability, and metabolic activity assessments provides a multifaceted view of toxicity in kidney organoids. Takeaways This system enables faster and more comprehensive toxicity testing that is specific to human kidney cells, offering a valuable tool for drug screening and nephrotoxicity studies. Lecture 21: Kidney Organoids 27  Knowledge of adult epithelial stem cell regulation underpins patient-derived kidney organoids Key Concept: Stem Cell Regulation: The advancement in adult-derived organoids was based on understanding stem cell regulation in adult tissues. Intestinal Organoid Model: Discovery: Hans Cleaver's lab identified a genetic marker of intestinal stem cells and determined the environmental signals required to maintain their identity. Culturing Approach: By isolating these stem cells and culturing them in a supporting medium, they were able to generate intestinal organoids—the first organoid model, dating back to 2009. Organoid Features: The organoids maintained parts of the stem cell niche and were capable of reproducibly generating different regions of the intestine, mimicking the in vivo environment. Expansion to Other Tissues: Broader Applications: Following the success with intestinal organoids, Cleaver's approach has been adapted for adult epithelial tissues beyond the intestine, including the lung and other adult epithelial systems. Reproducibility: This approach demonstrated that adult epithelium in various tissues is amenable to similar organoid techniques. Application: Modelling virus infection with Adult-derived kidney organoids Key Points: Viral Infections in Kidneys: Kidneys are susceptible to infections from viruses and bacteria, including COVID-19. The BK virus, in particular, causes specific damage to the nephron tubules of the kidney, with no current curative treatment available. Virus Infection Study: The group demonstrated infection with BK virus in kidney organoids to establish a platform for drug screening. Lecture 21: Kidney Organoids 28  Findings: Nuclear Changes: Infection with the BK virus led to an increase in nuclear size, a marker indicative of this infection. Viral Markers: They identified markers of the virus typically used in pathology to confirm the infection. Electron Microscopy: Viral particles were visualized directly using electron microscopy, confirming viral presence. Viral Replication: Polymerase chain reaction (PCR) was used to measure viral replication, showing exponential replication during the first 10 days of growth in the organoid models. New Opportunities: Drug Screening Platform: This study creates a new opportunity to screen for drugs that can prevent viral replication and mitigate infection in kidney tissue, offering potential therapeutic avenues for viral kidney infections. Lecture 21: Kidney Organoids 29  Application: Establishing organoids from Wilms tumors Overview of Wilms Tumor: Wilms Tumor: A common pediatric solid tumor accounting for 5% of childhood malignancies. It is believed to be driven by mutations in developmental regulators, which cause kidney progenitor populations to fail to differentiate and instead develop into cancerous cells. Key Mutations: Mutations have been observed in WT1 (a gene mutation) and Beta-Catenin, which is a Wnt signaling mediator. These tumors express high levels of Six2, a regulator of nephron progenitor cells, which is typically found in the developing kidney. Findings from Organoid Study: Tumor Organoids: Researchers established primary tumor organoids from both healthy tissue adjacent to the tumor and tumor tissue itself. Structural Components: Unlike the tubular organoids, these cancer organoids also replicated structural components of the tumor, consistent with the triphasic histology of Wilms tumors, which contain stromal, nephron progenitor, and epithelial components. Gene Expression: The gene expression of Six2 was maintained in the tumor organoids, but not in the healthy controls, reflecting the tumor-specific features. Genomic Stability: The copy number variations (CNVs), which serve as an indicator of genomic instability or mutations, were found to be equivalent between healthy organoids and healthy tissue, and tumor organoids and tumor tissue. Conclusion: New Avenue for Cancer Research: The organoid models successfully replicated the genetic and molecular features of Wilms tumors, providing a powerful new tool to study this specific type of cancer and potentially for drug screening or therapeutic development. Summary of Kidney Organoid Applications Lecture 21: Kidney Organoids 30  Types of Organoids: 1. iPSC-Derived Kidney Organoids: Generation: Induced pluripotent stem cells (iPSCs) are directed to differentiate towards kidney lineages by recreating developmental signals and pathways. Purpose: These organoids help in modeling kidney development, understanding cellular differentiation, and disease modeling. 2. Primary Kidney Epithelium-Derived Organoids: Generation: Derived from adult kidney epithelium. Purpose: These organoids largely generate tubular structures and are useful for studying kidney repair, regeneration, and potentially more mature cell states compared to iPSC-derived organoids. Key Applications: 1. Development and Regeneration: iPSC-derived and primary kidney epithelium-derived organoids are useful for studying kidney development and regeneration processes. 2. Disease Modeling: Both types are used for modeling kidney diseases, such as cystic kidney diseases, viral infections, and cancers (e.g., Wilms tumor). 3. Biobank and Drug Screening: Tubular organoids offer opportunities to create biobanks of patient samples, enabling drug screening and assessment of drug toxicity. They model the genetic background of the patient, which helps assess the effects of individual genetic variations on phenotypic outcomes. 4. Genetic Manipulation: Lecture 21: Kidney Organoids 31  Organoids are amenable to genetic manipulation, allowing studies of specific gene loci and their effects on kidney function and disease. Advantages of Organoids: 3D Multicellular Human Tissues: Organoids closely resemble human tissues and provide a more accurate model than traditional 2D cultures. Patient-Specific Models: Organoids can represent the genetic background of individual patients, enabling personalized research and therapy. Limitations of Organoids: 1. Lack of Physiological Environment: Organoids cannot replicate full physiological conditions such as immune system interactions, blood supply, and inter-organ communication found in whole organisms. 2. Missing Cell Types: Some organoid protocols lack certain cell types, such as stromal populations and ureteric epithelium. This limits their ability to model diseases involving these cell types. 3. Immaturity of iPSC-Derived Organoids: iPSC-derived organoids tend to have immature cell types, which may not be ideal for studying diseases related to adult cell states or cellular maturation. Lecture 21: Kidney Organoids 32  Kidney Organoids: From Lab to Biotech and Pharma Dream to rebuild kidney from stem cells still requires major obstacles to be overcome Missing cell types, maturation, long term culture methods, tissue patterning, scale Immediate applications in disease modelling, drug screening Proof-of principle shown for cystic diseases, toxicity screening, glomerular disorders Comparison to current models not comprehensive Uptake in biotech → Organovo 3D tissue modelling company Uptake in pharma → Novartis + others Adult-derived kidney organoids likely to address gaps not covered by iPS models Repair and regeneration Infection and cancer Disorders related to ageing and mature cell states Lecture 21: Kidney Organoids 33 Lecture 21: Kidney Organoids 34