Lecture 21: Kidney Organoids

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Questions and Answers

What is a significant advantage of using organoids over traditional 2D cultures?

Organoids can be produced faster than 2D cultures.

Organoids can be easily manipulated without losing functionality.

Organoids closely resemble human tissues and allow for better modeling. (correct)

Organoids can replicate full physiological conditions.

Which limitation of organoids is specifically related to the maturation of cells?

Lack of stromal populations in certain organoid protocols.

Inability to replicate inter-organ communication.

Organoids cannot represent the immune system interactions.

iPSC-derived organoids tend to demonstrate immaturity in cell types. (correct)

How do organoids help in the assessment of individual genetic variations?

They provide complete cell type representation.

They model the genetic background of the patient. (correct)

They replicate the entire immune system.

They facilitate quick drug testing.

Which of the following is NOT a limitation of organoids?

Ability to undergo genetic manipulation. Signup and view all the answers

What aspect of organoids limits their ability to model diseases involving specific cell types?

Some protocols do not include all relevant cell types. Signup and view all the answers

What is the primary purpose of using markers derived from progenitor states?

To assess differentiation outcomes Signup and view all the answers

Which signaling pathways were investigated for directed differentiation?

Known signaling and differentiation pathways from literature Signup and view all the answers

What experimental outcome was achieved with iPSC-derived kidney tissue?

Simultaneous differentiation of nephron and ureteric bud components Signup and view all the answers

What technique was employed to validate lineage differentiation?

Immunofluorescence assays Signup and view all the answers

In the context of this research, what does lineage tracing help to establish?

Developmental transitions of different progenitor states Signup and view all the answers

Which outcome is NOT highlighted as a successful methodological approach in this research?

Experimenting with various animal models Signup and view all the answers

Which aspect is emphasized as a focus in marker characterization during differentiation?

Gene and protein expression profiling Signup and view all the answers

What advantage does single-cell sequencing have over bulk RNA sequencing in understanding gene expression?

It provides high resolution insights into individual cell types. Signup and view all the answers

In the context of kidney organoids, what limitation is associated with using bulk RNA sequencing?

It results in an average transcriptional profile that masks individual cell information. Signup and view all the answers

What is the first step in the methodology for exploring cellular composition using single-cell sequencing?

Preparing organoids from different batches. Signup and view all the answers

What role does clustering play in the single-cell transcript profiling methodology?

It allows for identification of similarities and differences in transcriptional profiles. Signup and view all the answers

What is the primary purpose of the newly created drug screening platform discussed in the content?

To find drugs that can prevent viral replication in kidney tissue Signup and view all the answers

How does single-cell sequencing enhance our understanding of kidney organoids compared to bulk sequencing?

It reveals the correspondence of cell types to fetal kidney cell types. Signup and view all the answers

What percentage of childhood malignancies does Wilms Tumor account for?

5% Signup and view all the answers

Which of the following is NOT a benefit of using single-cell sequencing?

Providing an average transcriptional profile of a tissue sample. Signup and view all the answers

Which gene mutations are commonly associated with Wilms Tumor?

WT1 and Beta-Catenin Signup and view all the answers

What can be concluded about high-level clustering in the context of single-cell sequencing?

All cells can be treated as a single cluster to simplify analysis. Signup and view all the answers

What is maintained in the gene expression of tumor organoids from Wilms tumors?

Six2 expression Signup and view all the answers

Why is cell dissociation necessary in the methodology for single-cell sequencing?

To allow for the analysis of individual cell profiles. Signup and view all the answers

What unique structural components were replicated in the tumor organoids related to Wilms tumors?

Nephron progenitor and epithelial components Signup and view all the answers

What is the primary outcome of transcriptional profiling using single-cell sequencing in kidney organoids?

Revealing the diverse cell types within the organoids. Signup and view all the answers

What do the mutations in Wilms tumors cause in kidney progenitor populations?

Development into cancerous cells Signup and view all the answers

What histological component is NOT typically found in Wilms tumors?

Melanocytic components Signup and view all the answers

Which of the following correctly describes Wilms tumor in children?

A common pediatric solid tumor Signup and view all the answers

What is the role of Six2 in relation to nephron progenitor cells?

Regulator of nephron progenitor cells Signup and view all the answers

What is the primary focus of the research conducted by Taguchi and Professor Ryuichi Nishinakamura?

Refining cellular components of kidney organoids Signup and view all the answers

Which key achievement was related to ureteric bud formation?

Characterization of markers specific to ureteric epithelium Signup and view all the answers

What is the significance of the interaction between ureteric bud and nephron progenitors in culture?

It mimics kidney development in vitro Signup and view all the answers

What challenge is associated with direct studies of human embryos?

Inaccessibility of human tissues Signup and view all the answers

Which aspect of kidney organoids allows for studying human developmental processes?

Manipulation of pluripotent stem cells Signup and view all the answers

What approach did Sarah Howden and Jess Fensom-Lambrook use to study nephron markers?

Genetic reporters and labeling Signup and view all the answers

What was the outcome of labeling nephron progenitor populations in kidney organoids?

Identification of nephron segment relationships Signup and view all the answers

What specific markers were traced in the research of kidney organoids?

Nephron and ureteric epithelium markers Signup and view all the answers

How did the researchers overcome the challenge of studying human embryonic development?

Employing kidney organoids as a model Signup and view all the answers

What developmental signals were defined in relation to ureteric bud formation?

Signals critical for ureteric bud development Signup and view all the answers

Study Notes

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).

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Description

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.