Lecture 22: Progenitor Regulation

Progenitor Stem Cell Regulation

• Pluripotent Stem Cells (ES, IPS): Capable of long-term self-renewal in culture. Can differentiate into all three germ layers (endoderm, mesoderm, ectoderm) except the placenta.
• Adult Stem Cells: Maintained for the majority of the lifespan. Can differentiate into tissue-specific cell types. Have the capacity to self-renew.
• Transient Progenitor Cells: Exist within a limited timeframe during development (e.g., in the developing kidney), present from partway through gestation until birth. Not maintained long term. Can differentiate into a limited number of tissue-specific cell types.

Kidney Function and Disease

• Kidney Role: Regulates fluid homeostasis, removes waste from blood, regulates acid-base and electrolyte balance. Produces hormones that control blood pressure, bone density, and red blood cell count.
• Nephron: Specialized filtration units, composed of segmented tubular epithelium with different functions (filtration and handling of fluid and solute).
• Chronic Kidney Disease: Affects about 10% of the world's population, associated with high morbidity and mortality. Involves progressive nephron loss.

Nephron Number and Development

• Low nephron number increases kidney disease risk. Nephron numbers vary substantially (200,000- 2,000,000 per individual).
• Premature birth is often associated with lower nephron number.
• Existing nephrons have limited capacity for repair; gradual decline in nephron number occurs over time.
• Importance of nephron endowment: high/normal endowment leads to slower progression towards CKD, while low endowment leads to faster progression exacerbated by comorbidities.
• Nephron formation occurs during kidney development. All nephrons are formed during kidney development.
• 50% of nephrons are formed shortly after birth. This period is critical for regulating nephron number.

Formation of Renal Precursors (Progenitor Specification)

• Initial stage where renal progenitor cells are specified.
• Nephrons form at this stage.
• Kidney organoids model the early kidney development phase.

Organogenesis (Nephron Formation)

• Bulk of nephron formation occurs during this stage.
• Involves progenitor self-renewal and differentiation into nephrons.

Maturation

• Final stage where formed nephrons mature and become fully functional.
• Progenitor depletion can occur during kidney development stages.

Self-Renewal and Differentiation Phase

• Progenitor cells are actively dividing to both self-renew and differentiate into nephrons.
• Depletion can occur from excessive differentiation without sufficient self-renewal, resulting in reduced progenitor cells.

End of Organogenesis (Transition to Maturation)

• Progenitor cells are gradually used up as they form the final set of nephrons.
• Progenitor depletion becomes complete as no new progenitors are formed.

Morphological and Molecular Evidence of Nephron Progenitor Cell

• Observations of progenitors clustering around epithelial tips.
• Mesenchymal-to-epithelial transition noted in nephron formation..
• Six2 expressed in mesenchymal regions surrounding epithelial tips. Six2 knockout halts nephron formation and kidney development; proposed as a marker and regulator.

Lineage tracing

• Six2 gene used to drive Cre recombinase expression.
• Cre recombinase crossed with a reporter line to mark cells expressing Six2 and their descendants.
• Lineage tracing showed yellow cells showing green Cre expression and red lineage marking.

Kidney Development

• Interactions between three progenitor populations drive kidney development.
• Components of the nephron niche include: nephron progenitor, ureteric epithelium, and stroma. These populations are interdependent.
• Signal interactions between nephron progenitors and epithelium promote branching and organ growth and signals from the epithelium promote nephron progenitors' self-renewal.
• Wnt9b, Wnt11, FGF, and BMP ligands promote nephron progenitor self-renewal.

Cessation of nephrogenesis

• Nephron formation stops after birth due to loss of progenitor populations.
• Loss of nephron progenitor identity occurs first.
• Nephron progenitor cells play a crucial role in maintaining progenitor populations.

Imaging Development in 3D

• Widefield imaging provides surface images but lacks accurate quantification of 3D structures.
• Sectioning approach allows for analysis but loses important 3D relationships between cells, such as epithelial tips.

3D Imaging +/- Clearing

• Primary antibody binds to a specific protein of interest.
• Secondary antibody binds to primary antibody for fluorescent molecule tagging for visualization.
• Chemical or electrical treatments enhance tissue transparency.

Multiscale Imaging

• Addresses questions about kidney development, progenitor populations, and final organ formation in 3D.
• Whole Mount Immunofluorescence visualizes proteins across the entire organ.

Growth Patterns

• Kidney growth is not continuous; higher growth rates occur early.
• Number of progenitor cells and reciprocal signaling changes dramatically across time.
• Large number of progenitor cells and epithelial tips exist during early development.
• Progenitor numbers decrease during late stages, potentially due to changes in signaling environment.

Nephron Formation and Timing

• 50% of nephrons are formed shortly after birth (mice).

Precise Phenotypic Analysis

• Instead of subjective descriptions, precise measurements are taken to quantify kidney growth.
• This analysis reveals differences related to progenitor populations and nephron formation.

Nephron Progenitor Regulation

• Decreased progenitor proliferation results in decreased nephron number (smaller kidneys, increased risk of CKD).
• Increased progenitor expansion leads to increased nephrons.
• Nephron progenitor expansion improves kidney function, but regulation is needed to avoid disease.

Potential Effects of Migration on Cell Fate

• Progenitor fate is not fixed but influenced by location and the encountered signals; challenging the static model.
• Back-and-forth movement of cells between niches may impact self-renewal capacity of the progenitor pool.

Single Cell RNA Sequencing (scRNA-seq)

• Examines nucleic acid sequence information from individual cells.
• Enables detailed transcriptional profiles for each cell and identification of active genes and pathways.
• Dissociation or nuclei extraction isolates individual cells
• RNA from each cell is captured and barcoded
• Pooled RNA is sequenced, replicates removed, and bioinformatics performs quality control
• Results are detailed transcriptional profiles identifying active genes and pathways.

High Throughput Single Cell RNA-seq

• Enables comparisons to bulk RNA-seq.
• Achieves identification of expected cell types, including clusters.

Pathway Activity Analysis

• Differential expression within cell types is analyzed for pathway activity.
• Pathways are identified by the expression of pathway targets or components.

Explore the intricate processes of nephron development, focusing on the role of progenitor cells and the factors that influence their differentiation and self-renewal. This quiz covers key transcription factors, methodologies used in research, and the significance of cellular dynamics in organogenesis.