Lecture 22: Progenitor Regulation PDF
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This document outlines the differences between pluripotent stem cells, adult stem cells, and transient progenitor cells. It also highlights the role of nephron progenitor cells in kidney health and function. The document provides a detailed explanation on various aspects of kidney development.
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🥽 Lecture 22: Progenitor Regulation Understand the similarities and differences between pluripotent stem cells, issue resident adult stem cells, and transient progenitor cells Pluripotent Stem Cells (ES, iPS):...
🥽 Lecture 22: Progenitor Regulation Understand the similarities and differences between pluripotent stem cells, issue resident adult stem cells, and transient progenitor cells Pluripotent Stem Cells (ES, iPS): Capable of long-term self-renewal in culture. Can differentiate into all three germ layers: endoderm, mesoderm, ectoderm (everything except the placenta). Adult Stem Cells: Maintained for the majority of the lifespan. Capacity to self-renew. Differentiate into tissue-specific cell types. Transient Progenitor Cells: Exist within a limited timeframe during life (e.g., in the developing kidney, present from partway through gestation until birth). Lecture 22: Progenitor Regulation 1 Not maintained long term. Capacity to self-renew. Can differentiate into a limited number of tissue-specific cell types. Recognise that nephron progenitor cells play a critical role in determining kidney function and health Kidney function and disease Kidney role: Regulate fluid homeostasis, removal of waste from blood, acid-base and electrolyte balance Produce hormones that control blood pressure, bone density, and red blood cell count Nephron: Nephrons are specialised filtration units, consisting of blood filtering component, segmented tubular epithelium with distinct functions along different segments Major role in filtration, fluid and solute handling Chronic kidney disease: Affects ~10% of world population Associated with high morbidity and mortality Treatments aim to slow rate of progression or replace function, no current cure Involves progressive nephron loss, which if not slowed or stopped leads to kidney failure. Low nephron number increases risk for kidney disease Nephron Number: Varies tenfold in the human population - ranging: 200,000 to 2,000,000 nephrons per individual. Lecture 22: Progenitor Regulation 2 Low nephron number is a significant risk factor for hypertension, chronic kidney disease (CKD). Premature Birth and Nephron Number: Increasing proportion of premature births (8.6% in Australia). Premature birth, along with genetic and environmental conditions, is consistently associated with low nephron number. Nephron Decline Over Time: No new nephrons form after birth. Existing nephrons have limited capacity for repair. Gradual decline in nephron number over time. Importance of Nephron Endowment: High/Normal Nephron Endowment: Slower progression towards CKD. Low Nephron Endowment: Faster progression towards CKD. Comorbidities (e.g., diabetes) exacerbate the decline. Nephron formation occurs during kidney development All nephrons are formed during kidney development. Lecture 22: Progenitor Regulation 3 1. Formation of Renal Precursors (Progenitor Specification): This is the initial stage where renal progenitor cells are specified. Nephrons are triggered to form at this stage. Kidney organoids model the early phase of development 2. Organogenesis (Nephron Formation): During this phase, the bulk of nephron formation occurs. Involves both progenitor self-renewal and differentiation into nephrons. This phase is not well modeled in organoid systems 3. Maturation: The final phase where the formed nephrons mature and become fully functional. Progenitor depletion can occur during the following stages of kidney development: 1. Organogenesis (Nephron Formation): Self-Renewal and Differentiation Phase: Progenitor cells are actively dividing to both self-renew and differentiate into nephrons. Depletion can occur if the balance tips towards excessive differentiation without sufficient self-renewal, leading to a reduced pool of progenitor cells. 2. End of Organogenesis (Transition to Maturation): As organogenesis progresses, the progenitor cells are gradually used up as they form the final set of nephrons. Near the end of this phase, progenitor depletion becomes complete, as no new progenitors are generated after this stage. Lecture 22: Progenitor Regulation 4 Morphological and molecular evidence of nephron progenitor cell Morphological evidence of progenitor state Observations of progenitors clustering around epithelial tips. Noted mesenchymal-to-epithelial transition forming nephrons, a process still recognized today. Gene Expression Visualization and Knockout Technology → Key Discovery: Transcription Factor Six2 Identified as expressed in the mesenchymal region surrounding epithelial tips. Six2 knockout resulted in loss of progenitor population and halted nephron formation and kidney development. Proposed as both a marker and regulator of nephron progenitor state. Definitive Proof - Lineage Tracing: Six2+ cells give rise to the nephron Lineage Tracing Methodology: Six2 gene used to drive expression of Cre recombinase. Cre recombinase crossed with a reporter line to mark cells expressing Six2 and their descendants. Results: Lineage tracing showed yellow cells (green Cre expression and red lineage marking). Lecture 22: Progenitor Regulation 5 Demonstrated that nephron progenitor populations could self- renew and differentiate into all epithelial cell types of the nephron. Kidney development is driven by interactions between three progenitor populations Components of nephron niche: Nephron progenitor (Six2) Ureteric epithelium Stroma These populations are interdependent, existing in the nephrogenic niche surrounding the developing kidney. Signal Interactions: Nephron Progenitors and Epithelium: Signals from nephron progenitors promote branching and organ growth in the underlying epithelium. Signals from the epithelium promote nephron progenitor self- renewal. Self-Renewal and Differentiation Signals: Wnt9b, Wnt11, FGF (Fibroblast Growth Factor), BMP ligands promote nephron progenitor self-renewal. Spatially Restricted Signals: Lecture 22: Progenitor Regulation 6 Wnt9b: Higher levels of Wnt9b trigger nephron progenitors to undergo mesenchymal-to-epithelial transition. This transition leads to the formation of early-committed nephrons. Early nephrons rapidly patent, segment, and mature into fully formed nephrons. Cessation of nephrogenesis Nephron formation ceases after birth due to loss of progenitor populations Loss of nephron progenitor identity occurs first before the loss of ureteric tip identity Suggests that nephron progenitor cells play a crucial role in maintaining progenitor populations. Imaging development in 3D Challenges in 3D Imaging Wide Field Imaging: Provides surface images of the sample but lacks accurate quantification of 3D structures without additional processing. Sectioning Approach: Cutting tissue slices allows analysis but loses important 3D relationships between cells (e.g., epithelial tips). Lecture 22: Progenitor Regulation 7 3D imaging +/- clearing: Primary Antibody: Binds a specific protein of interest (usually a regulator or cell-type marker). Secondary Antibody: Binds to the primary antibody and is tagged with a fluorescent molecule for visualization under a microscope. Tissue Clearing: Need for Transparency: Large tissues are often opaque, making it difficult for light to pass through (similar to the example with your hand). Advancements in Tissue Clearing: Chemical or electrical treatments make tissues more transparent, allowing light to penetrate and improve 3D imaging quality. Benefits of Tissue Clearing: Enables better quality 3D imaging by enhancing tissue transparency, which is essential for understanding the spatial relationships in developing kidneys. Lecture 22: Progenitor Regulation 8 Multiscale imaging approach to analyse kidney development across time Objective: Address questions about kidney development, progenitor populations, and final organ formation in 3D. Methods Used: Whole Mount Immunofluorescence: Allows visualization of specific proteins across the entire organ. Tissue Clearing: Enhances tissue transparency for better imaging. Imaging Modalities: Capture kidney development at both the organ and cellular scale in 3D. Custom Analysis Approaches: Identify and segment individual niches and structures to quantify cells or proteins within specific volumes. Lecture 22: Progenitor Regulation 9 New insights into kidney development & precision phenotyping Growth Patterns: Kidney growth is not continuous; there is higher growth rate early in kidney development than late. The number of progenitor cells and their reciprocal signaling changes dramatically over time. Progenitor and Epithelial Tip Dynamics: Early Development (Bottom Left Image): Large number of nephron progenitor cells and epithelial tips. Late Development: The number of progenitors decreases in both states, likely due to changes in the signaling environment. Lecture 22: Progenitor Regulation 10 Nephron Formation and Timing: 50% of nephrons are formed shortly after birth (mice) → this aligns with the hypothesis that this period is critical for regulating nephron number. Precise Phenotypic Analysis - Quantitative Kidney Growth Analysis: Instead of subjective descriptions (e.g., slightly smaller/larger), precise measurements are taken to quantify kidney growth. Quantification reveals how these differences relate to progenitor populations and nephron formation. Lecture 22: Progenitor Regulation 11 Nephron progenitor regulation influences kidney size and nephron number Decreased Proliferation or Self-Renewal of Nephron Progenitors: Results in decrease in kidney size and reduced nephron number. Example 1: Reducing FGF signaling (which regulates proliferation) results in fewer nephron progenitors and fewer nephrons. Example 2: Ablating a proportion of nephron progenitors (e.g., using inducible diphtheria toxin) reduces nephron number, although kidney size compensates to match normal levels. Increased Proliferation of Nephron Progenitors: Positive Effect on Kidney Size and Nephron Number. Surprising result: Increasing nephron progenitor proliferation can result in more nephrons but potentially cause pathological consequences Unrestrained nephron progenitor proliferation delays cessation but leads to pediatric cancer Unrestrained Nephron Progenitor Proliferation is linked to Wilms tumor (pediatric cancer). Lin28 Overexpression: Lecture 22: Progenitor Regulation 12 LIN28 highly expressed in progenitor cells and inhibits Let7 - its expression decreases with differentiation Global verexpression of Lin28 led to increased progenitor pool, delayed cessation, and formation of Wilms tumor. Transient overexpression of Lin28b expands nephron progenitors, increasing nephron number and kidney function To avoid pathological consequences, Lin28 overexpression was restricted to a narrow window during kidney development. Results of experiment ^ Increased nephron progenitor population, which extended beyond typical exhaustion time (i.e., 2 weeks post-birth). Doubled nephron number compared to controls. Increased glomerular filtration rate (GFR), indicating better kidney function despite increased nephron number. Significant increase in kidney size, appearing normal initially (2 weeks post-birth). Around 3 weeks or 3 months, kidney distension occurred (fluid buildup). Nephrogenic rests were observed, which are precursors to Wilms tumor. Lecture 22: Progenitor Regulation 13 Lin28b and Let7 have opposing effects on nephron progenitor expansion and kidney function Knockout of Lin28: Reduced kidney size and function. Knockout of Let-7: Increased nephron number and kidney function. Extended cessation by 1 day. Nephron progenitor expansion improves kidney function but must be regulated to avoid disease Lecture 22: Progenitor Regulation 14 Decreasing progenitor proliferation results in a decrease in nephron number. Failure of kidney development Smaller kidneys, increased risk of CKD Increasing progenitor expansion leads to increased nephron number Modest increase in nephrons, normal cessation, no pathology BUT dramatic increase in nephrons, extended cessation, hydronephrosis and cancer SO nephron Progenitor Expansion improves kidney function but must be tightly regulated. Ongoing Efforts: The goal is to find mechanisms (preferably non-genetic) to augment nephron number and improve kidney function. Safe intervention is essential to avoid pathological consequences, such as pediatric cancer, while enhancing kidney health. Lecture 22: Progenitor Regulation 15 Nephron Progenitor Commitment Previous belief of progenitor commitment Progenitor cells reside in the nephrogenic niche traditionally believed to exist in segregated, static subdomains. Classic model suggest spatial segregation and signal Exposure: Top of the niche: Exposure to signals promoting self-renewal. Bottom of the niche: Exposure to signals promoting differentiation. Cells progressively commit to differentiation as they move downward. Live Imaging showed that nephron progenitor cells are not confined to fixed niches but are dynamically moving, influencing their fate based on their position and exposure to different signals. Method: 1. Live Imaging & Lineage Tracing Approach: Researchers used fluorescent reporters to visualize nephron progenitor cells in real-time. They used a Cre driver that activates a reporter gene in cells committed to the early nephron formation. This reporter labels cells red, allowing researchers to track these cells' movements and fates over time. Results: 1. Dynamic Movement: Progenitor cells were observed moving within the niche and between niches. Cells didn't stay fixed in their expected locations (i.e., top or bottom of the niche) but instead moved between domains traditionally thought to be static. 2. Reintegration into Progenitor Population: Some cells originally marked as committing to nephron formation (early nephron cells) migrated back to the Lecture 22: Progenitor Regulation 16 progenitor population. Upon returning to the progenitor domain, these cells re- expressed progenitor markers (like Six2), effectively reverting to a progenitor state. Implications: Impact of Migration on Cell Fate: The movement of cells between niches suggests that their fate isn't fixed and can be influenced by their location and the signals they encounter. This dynamic behavior challenges the static model and suggests that migration could play a role in regulating when and how progenitors commit to differentiation. Potential Effect on Nephron Cessation: Consideration as to whether this back-and-forth movement might impact the overall self-renewal capacity of the progenitor pool. Specifically, it suggests that the reintegration of near- committed cells could contribute to the gradual decline in self-renewal capacity (potentially bringing some level of differentiation programming), potentially influencing reducing the overall ability of the progenitor pool to renew itself and accelerating the timing of nephron cessation. Be able to describe how single cell RNA sequencing can be used to identify candidate signaling molecules to maintain progenitor populations or direct their differentiation to specific cell identities Lineage Tracing and Cell Isolation Challenge: Isolate marked cells in nephron and unlabelled cells in adjacent progenitor population. The objective is to compare cells marked for early nephron commitment and unmarked progenitor cells. Lecture 22: Progenitor Regulation 17 Solution: Single cell sequencing of dissociated kidney cells. Identified nephron progenitors using markers Six2 and Cited1. Tracked lineage with TdTomato RNA to distinguish between previously committed and uncommitted cells. Findings Unexpected Result: Previously committed cells can reprogram back to progenitor state, displaying greater plasticity. Hypothesis: Cell migration modulates environmental exposure, influencing self-renewal and differentiation. Single Cell Sequencing (scRNA-seq) scRNA-seq examines the nucleic acid sequence information from individual cells Process Overview: 1. Sample Preparation: Tissue dissociation or nuclei extraction isolates individual cells. RNA from each cell is captured and barcoded, allowing the identification of which RNA belongs to which cell. 2. Sequencing and Bioinformatics: Pooled RNA is sequenced, and bioinformatics analyses align gene expression, remove duplicates, and perform quality control. This results in a detailed transcriptional profile for each cell, identifying active genes and pathways. Lecture 22: Progenitor Regulation 18 All expected cell types accounted for, cluster profiles cof scRNA-seq is comparable to bulk RNA-seq Initially, there were concerns about the sensitivity of scRNA-seq due to issues like sparse expression and dropout - BUT when scRNA-seq is performed successfully, it can detect all expected cell types and identify known ligands and receptors effectively, similar to what bulk RNA sequencing can achieve. Identification of Candidate Signaling Molecules: Marker-Based Identification: Progenitor populations are identified based on the expression of key markers (e.g., Six2, Cited1 for nephron progenitors). Ligand-Receptor Interaction Analysis: Using databases of known and inferred ligand-receptor interactions, scRNA-seq data helps map potential communication pathways between cells. This identifies candidate molecules involved in maintaining progenitor states or directing differentiation. Example: Discovery of Aspn1 and Aspn3 as ligands involved in nephron progenitor maintenance, which was later validated by knockout studies. Pathway Activity Analysis: Lecture 22: Progenitor Regulation 19 Differential expression within each cell type is analyzed to determine pathway activity. Active pathways are identified based on the expression of pathway targets or components. Example: Pathways like Wnt, Hippo, TGF-beta, and Notch were identified as crucial for nephron progenitor maintenance and differentiation. Trajectory and Spatial Analysis: Cells are ordered by transcriptional similarity, suggesting developmental trajectories. Spatial transcriptomics resolves transcriptional activity within tissue sections, providing context for signaling molecule activity in maintaining progenitor populations or guiding differentiation. Applications: Progenitor Maintenance: By identifying signaling molecules and pathways active in progenitor populations, researchers can pinpoint factors necessary for maintaining progenitor states. Directed Differentiation: Understanding the pathways active in differentiating cells allows for the identification of signaling molecules that can be manipulated to direct progenitor cells toward specific cell fates. Compare response to perturbation between conditions Lecture 22: Progenitor Regulation 20