Recreating Anatomy for Regenerative Medicine - Notes

Recreating Anatomy for Regenerative Medicine

Key Properties of Stem Cells

  • Cell-cell communication: Niche & Microenvironment
  • Applications of Stem cells in Regenerative Medicine
  • Recreating Anatomy in Regenerative Medicine:
    • Self-organization versus guided engineering approaches
    • Example 1: Intestinal organoids and engineering the niche
    • Example 2: Bioengineering pancreatic cell niche(s)

Overview on Stem Cells

What is a Stem Cell?
  • Stem cells can self-renew (replicating) to maintain the stem cell pool.
  • Stem cells can differentiate (specializing) into specialized cells.
  • Specialized cells:
    • Contribute to all organs.
    • Replace dead or damaged cells.
    • Enable tissue to respond to demand.
Types of Stem Cells
  • Embryonic Stem Cells (ESCs):
    • Originate from the inner cell mass of the blastocyst.
    • Exhibit self-renewal.
    • Are pluripotent (can differentiate into any cell type).
    • Can generate mouse chimeras.
    • Can generate 254 cell types originating adult tissues
  • Adult Stem Cells (ASCs):
    • Created during ontogeny and persist within the niche in most adult animal tissues/organs.
    • Exhibit self-renewal.
    • Are multipotent (can differentiate into a limited range of cell types).
    • Maintain tissue homeostasis in physiological and pathological conditions.
  • Induced Pluripotent Stem Cells (iPSCs):
    • Originate from somatic differentiated cells after transduction with factors like Oct-3/4, Oct4, Myc, Sox2, Klf4.
    • Exhibit self-renewal.
    • Are pluripotent.
    • Can generate mouse chimeras.
    • Can be patient-specific stem cells.
How Cell Identity is Established
  • Cell identity is established through a combination of:
    • Transcriptional & Chromatin program
    • TF (Transcription Factors)
    • Morphogenesis & Cell interactions
    • Extrinsic signals
  • Unraveling control mechanisms of cell identity has direct clinical and translational relevance.
  • Examples of key factors: Oct4, Nanog, LIF.
Induced Pluripotent Stem (iPS) Cells
  • Mature, differentiated cells can be reprogrammed to become pluripotent.
  • Key reprogramming factors: Oct4, Sox2, c-Myc, Klf4.
  • iPS cells can be differentiated into various cell types like neurons, cardiomyocytes, hepatic cells, and pancreatic cells.
  • Applications:
    • Study of development.
    • Pathogenetic study.
    • Stem cell-based regeneration.
Adult Stem Cells
  • An adult stem cell is an undifferentiated cell found in some adult organs, which can self-renew and differentiate to become most or all the specialized cell types within the specific organ.

Niche & Microenvironment

The Stem Cell Niche Concept
  • A Cellular Niche: Involves supporting cells that regulate self-renewal, proliferation, and differentiation of stem cells.
  • B Non-cellular niche: Involves ECM (extracellular matrix) and secreted signals from the niche that regulate self-renewal, proliferation, and differentiation of stem cells.
The Intestinal Stem Cell Niche
  • Structure:
    • Villus: responsible for cell shedding at the tip.
    • Crypt: Contains stem cells and Paneth cells.
    • Crypt-villus junction.
    • Lamina propria.
  • Cell Types:
    • Differentiated cells: Goblet cells, Enteroendocrine cells, Absorptive epithelial cells.
    • Proliferative progenitors: Transit-amplifying cells.
    • Stem cells.
    • Paneth cells.
  • Small intestinal epithelium has an extremely short turnover time of 5 days.
    • Mitotic renewal occurs in 24-36 hours.
    • Differentiation and migration take 24-48 hours.

Stem Cells in Regenerative Medicine

Applications of Pluripotent Stem Cells
  • Cell Therapy:
    • Differentiation of hPSCs (human pluripotent stem cells) into various cell types using specific factors:
      • Hepatocytes (Activin, BMP4, FGF2, HGF, OSM, DEX)
      • Cholangiocytes (Activin, BMP4, NOTCH, FGF10, RA)
      • B-cells (FGF10, RA, KGF, -TGFB)
      • Lung and Airway Epithelial Cells (WNT, SHH, BMP, FGF, RA)
      • Thyroid Cells (FGF, BMP)
    • Examples of cell therapies:
      • Hepatocytes and Cholangiocytes for Liver Disease
      • Pancreatic B-cells for Diabetes
      • Airway cells for Cystic Fibrosis
  • Disease Modeling:
    • Using WT/Corrected iPSCs and Mutated iPSCs to model:
      • Genetic Disorders: Inherited Metabolic Disorders, Cholangiopathies, Cystic Fibrosis, Gastrointestinal Disorders
      • Infectious Diseases: Hepatitis C, Malaria, Bacterial Infections of the Gut
      • Validation of GWAS hits
  • Drug Screening:
    • Using organoids (Intestinal, Gastric) for:
      • Hepatotoxicity
      • Control for cholesterol levels
      • Cystic Fibrosis
      • Diabetes
    • Directed differentiation.
Progress in therapies based on iPSCs
  • New drugs
  • Target validation
  • Autologous therapy
  • Organoids
  • Process:
    • Somatic cells are collected from the patient.
    • Patient-specific iPSCs are generated through reprogramming (KLF4, MYC, OCT4, SOX2)
    • Gene correction can be performed on iPSCs.
    • Directed differentiation of repaired iPSCs into healthy specialized cells.
    • Activity readout is used for drug screening.

Recreating Anatomy in Regenerative Medicine

  • Cells are the building blocks of organs with the ECM (extracellular matrix).
  • Ultimate goal in tissue engineering is the recapitulation of the native organ formation.
  • Key aspects to consider:
    1. Components
    2. Ratios
    3. Spatial distribution-tissue architecture
    4. Assembling
    5. Maturation
    6. Preservation
  • Example components: Cell #1, Cell #2, Vasculature, ECM.
  • Reading: Atlas of the embryonic pancreas (Glorieux et al. 2022).
  • Writing: Bioengineering pancreas.

Organoids

  • Organoids are self-organizing 3D culture systems derived from stem cells.
  • They are miniaturized and simplified versions of an organ.

Self-Organization

  • Self-organization is the process by which local interactions between cells that are initially disordered lead to the emergence of patterns and functions.

Gastrointestinal Organoid Derivation

  • iPSC/ESC are differentiated into organoids through several steps. Factors like Activin, WNT3A, CHIR99021, Noggin, FGF4, RSPO, EGF, IGF-1, FGF-2, and A83-01 are used.
  • These organoids can model different parts of the gastrointestinal tract, including the stomach (gastric organoids) and intestines (intestinal organoids).
  • They can also be used to study diseases like IBD (Inflammatory Bowel Disease), CRC (Colorectal Cancer), and for digestion studies.

Lgr5+ Intestinal Stem Cells

  • Lgr5 is a marker for intestinal stem cells.
  • Lgr5-expressing cells can be tracked using reporters like EGFP.
  • The Lgr5 promoter drives expression of EGFP.

Lgr5+ Organoids Technology

  • Intestinal biopsies are harvested endoscopically.
  • Intestinal crypts are isolated.
  • A culture system is established.
  • LGR5+ stem cells are enriched as organoids.
  • Organoids are formed.
  • Stem cell transplantation

Self-organization versus guided engineering

  • Intestinal organoids grown in patterned tubular matrix organize into crypt-like structures

Engineering the Pancreatic Niche(s)

  • Native Endocrine Pancreas
    • Mesenchymal- cells
    • Tip cells
    • Endocrine cells
    • Trunk cells
    • Blood vessels
    • ECM (Extracellular Matrix)
  • Building blocks to engineered endocrine pancreas
    • Vascular Network
    • Islet/$\$ cells
      *Integration of all the three building blocks

Tissue-Architecture of the Pancreas

  • Exocrine Pancreas comprises ~90% (e.g., Amylase).
  • Endocrine Pancreas comprises ~ 1-2% (e.g., Insulin).

Pancreatic Diseases

  • Diabetes:
    • 422422 million adults have diabetes.
    • 11 in 1111 people.
    • 3.73.7 million deaths due to diabetes and high blood glucose.
    • 1.51.5 million deaths caused by diabetes.
  • Pancreatic Cancer:
    • Pancreatic cancer mortality rates continue to rise throughout Europe.
    • The median survival time for someone diagnosed with pancreatic cancer in Europe is just 4.64.6 months, with patients losing 9898% of their healthy life expectancy at the point of diagnosis.
    • Symptoms:
      • PAIN (ABDOMINAL & BACK)
      • CHANGE IN BOWEL HABITS
      • NAUSEA
      • JAUNDICE
      • WEIGHT LOSS
      • NEW ONSET DIABETES
    • Risk Factors:
      • FAMILY HISTORY
      • SMOKING
      • HEAVY ALCOHOL CONSUMPTION
      • DIABETES
      • OBESITY
      • PANCREATITIS
  • Pancreas lacks regenerative properties and an adult stem cell compartment.
  • Islet transplantation is a therapeutic option to cure diabetes.

Pancreatic organ development

  • Factors involved: FGF10, RA, Notch.
  • Cell types: alpha-cells, beta-cells, blood vessels, mesenchymal cells.
  • ECM (Extracellular Matrix).
  • Adult Islet.

Engineering pancreatic cells using a developmental logic

  • Referenced studies: D’Amour et al. 2006, Rezania et al. 2014, Nair et al. 2019.

Regenerative Cell Therapies for Diabetes

  • Process:
    • Blastocyst --> hESC --> Pluripotent Cell Lines or
    • Somatic cells --> iPSC --> Pluripotent Cell Lines
    • Pluripotent Cell Lines --> Differentiation --> Pancreatic progenitors or B-cells
    • Transplantation --> Reversal of hyperglycemia
  • Pluripotent stem cells --> Definitive endoderm --> Primitive gut tube --> Posterior foregut --> Pancreatic endoderm --> Endocrine precursors --> Immature beta cells --> \-like cells --> Mature B-cells
  • Macro-encapsulation and Micro-encapsulation

Tissue Engineering

  • Engineering functional pancreatic tissue
  • Tissue Development epithelium and mesenchyme
  • 3D bioprinting Integration vasculature
  • Maturation and Functional Analysis

Bioprint pancreatic tissue

  • SC-derived pancreatic cells
  • Organoids mesenchymal cells
  • Self-organization versus guided engineering
  • Bioprinting
  • Endothelial cells
  • Ratio 1:3:2

Summary

  • Fundamental properties of stem cells
  • Potential applications of stem cells in regenerative medicine
  • Self-organization versus guided engineered approaches
  • Intestine: adult stem cells & organoids
  • Pancreas : applying developmental biology lessons to stem cell differentiation & tissue engineering