Lecture 21: Kidney Organoids

Kidney Organoids

• Organoids offer a direct study of human biology and disease, crucial for drug development and therapeutic interventions
• Strengths: Direct study of human biology & disease; crucial for drug development & interventions
• Limitations: Limited access to tissue samples; limited scope of experimentation (e.g., 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 immortalized cell lines have strengths: Primarily involve 2-dimensional cell culture; widely used for disease modeling and drug screening
• Model organisms (e.g., mice, fruit flies, fish, and worms) have strengths: Strong conservation of genetic, cellular, and molecular mechanisms between model organisms and humans.
• Model organisms limitations: Species-specific differences often hinder translational research. Challenges in drug development and metabolism (species-related biological differences); difficulty distinguishing species-specific discrepancies from individual variations in humans
• Human organoids are multicellular human tissues that can be patient-derived. They are amenable to genetic manipulation, drug screening, and experimentation; capable of recapitulating many aspects of tissue architecture and cellular composition of the modelled organ. 
• Limitations of human organoids: Lack of physiological context (limited interaction with immune cell types, lack of a vascular system or blood supply, lack of interaction with other organ systems).
• Establishing organoids can use ES or reprogram to iPSCs and direct differentiation. Source is derived from ESCs or iPSCs
• Processes for establishing organoids involve pluripotent cells

Organoids vs Model Organisms

• Organoids: Easier and faster for genetic manipulation. Do not require full organism development. Effective substitute if they replicate key cellular processes, but lack full systemic physiological context. Direct models of human-specific processes.
• Model Organisms: Strong conservation of developmental mechanisms. Differences between human and model organisms. Superior in capturing whole-organism complexity, but insights into conserved mechanisms, but species-specific differences exist.

Kidney Function and Disease

• Kidneys regulate fluid homeostasis, remove waste from blood, regulate blood pressure, bone density, and red blood cell count
• Chronic kidney disease (CKD) is a gradual loss of renal function; includes filtration and hormone production.
• Many causes including genetic mutations, chronic injury (inflammation), and complications like diabetes.
• Kidneys are a major target for toxicity in drug development; often susceptible to drug-induced damage.
• Incidence is growing at 6% per annum worldwide
• CKD treatment costs are a burden on the Australian economy (>$1 billion annually)
• Patients with end-stage disease require organ transplantation or dialysis.

Potential Applications of Kidney Organoids

• Human Developmental Biology: Study kidney development, repair, and regeneration.
• Toxicology Screening: Assess kidney-specific cell death to evaluate drug toxicity.
• Drug Screening and Discovery: Test and discover drugs for specific diseases effectively modeled in kidney organoids
• Cellular Therapy and Renal Replacement: Generate functional kidneys for patients, develop therapies for renal replacement.

Potential Applications for Kidney Organoids (cont'd)

• 750 million people worldwide affected by kidney problems.

• Kidney organoids can serve as models for kidney disease (fibrosis, cell defects, cystic diseases, and cancers).
• Aids in accelerating drug discovery and development.

Establishing Organoids

• Use ES or reprogram to iPSC and direct differentiation
• Source = derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)
• Process starts with pluripotent cells

Kidney Organoid Protocols

• Taguchi Protocol: Focused on defining distinct progenitor lineages. Hypothesis that nephron and ureteric bud components cannot be generated simultaneously
• Takasato Protocol: Leveraged existing literature for markers and differentiation drivers to claim simultaneous induction
• Key developments in early protocols involve the successful induction of intermediate mesoderm, but not necessarily kidney-specific tissues. Subsequent breakthroughs involve differentiation beyond intermediate mesoderm and toward kidney-specific lineages (marked by F9 and Wnt signaling activators)

Kidney Organoid Protocols (cont'd)

• Correct in Principle: Lineage tracing and mouse developmental studies strongly support the distinct origins of nephron progenitors and ureteric bud cells.
• Limitations: Later studies raised questions about the functional equivalence of induced ureteric bud-like structures to true ureteric bud cells in vivo.
• Partially Correct (The Takasato protocol) : The protocol showed marker expression consistent with both nephron progenitors and ureteric bud cells.

Approaches to Direct Differentiation of Renal Progenitors

• Taguchi Protocol: Focused on defining distinct progenitor lineages and their developmental transitions. Hypothesis that nephron and ureteric bud components couldn´t be generated simultaneously; lineage tracing, gene expression profiling, and immunofluorescence
• Takasato Protocol: Relied on existing literature for markers and differentiation drivers without repeating developmental studies

Characterizing Kidney Organoids: Takasato Protocol

• Inducing Posterior Primitive Streak: Used low levels of Wnt signaling via the Wnt agonist CHIR99021 (CHIR).
• Establishing Intermediate Mesoderm Fate: Added F9 growth factor to stimulate proliferation
• 3D Culture Formation: Aggregated 2D cells into a 3D blob and placed on a membrane
• Outcome: Formation of self-organizing kidney tissue. Observed distinct patterning.

Key Steps for Kidney Organoids

• Cell Separation: Isolate individual cells from a tissue.
• Transcriptional Profiling: Determine the unique set of genes expressed in each cell.
• Reconstruction of Cell States: Classify/group cells with similar transcriptional profiles
• Single-cell sequencing: Precise mapping of gene expression to specific cell types
• Bulk RNA sequencing: Combines RNA; provides an average transcriptional profile. Masks cell-type specific information.
• Single-cell sequencing: Captures the individual profiles of each cell, offering detailed insights.

Exploring Cellular Composition in Human Kidney Organoids with Single-Cell Sequencing

• Methodology: Preparation (multiple organoids taken and dissociated into individual cells), Transcriptional Profiling, Data Analysis (Clustering algorithms), Clustering Resolution (High-level vs Detailed)

Kidney Organoids: Expected and Off-Target Populations

• Expected: Nephron cells, endothelial cells, stroma cells
• Off-Target: Neural cell types, muscle progenitors, potential variations in cellular response to growth factors, interactions during differentiation.

Single-Cell Sequencing for Kidney Organoids

• Addresses (challenges): Issues with using bulk RNA sequencing to mask cell type-specific information.
• Comprehensive view of cellular composition; identifies all cell types; compares organoid-derived cells to equivalent in vivo cells
• Helps determine conservation of key markers between organoids and vivo, revealing unintended cell types.

Why Continue Research on Kidney Organoids?

• Need for accurate representation of disease processes in studies
• Requirement for functional, mature cell types essential in modelling diseases, performing drug testing, achieving therapeutic outcomes
• Specificity of markers in organoids vs in vivo tissues; in embryos cells are easier to identify, but in organoids marker expression alone isn't sufficient.

Challenges in Verifying Cell Identity

• Stem cell differentiation (crucial validation needed); simply observing marker expression doesn't automatically guarantee intended cell type.
• Similar cell types from different tissues can have the same markers (e.g. kidney vs gut stroma).

Limitations of Traditional Analysis

• Focuses on targeted approaches (e.g., Immunofluorescence, PCR).

Explore the roles of kidney organoids in studying human biology and disease. Understand their strengths and limitations, along with the comparisons to primary cell lines and model organisms for drug development. This quiz will deepen your knowledge of therapeutic interventions and experimental challenges in this field.