Chapter 1-6: Stem Cells and Regenerative Medicine

Hippo Pathway and Organ Size Control

• Originally identified as a key gene/pathway for controlling organ size.

• Example: A Drosophila larva with a mutations in the Hippo pathway or overexpression of its downstream targets can result in a gigantic fly, showcasing its role in growth regulation.

Hippo Pathway's Role in Trophectoderm and Inner Cell Mass (ICM) Specification

• In the context of the early embryo (morula/blastocyst stage), the Hippo pathway has a distinct role in forming the trophectoderm and ICM.

• Key Proteins Important for Trophectoderm/ICM Specification:

  • aPKC (Atypical Protein Kinase C): Crucial for establishing cell polarity.

  • ECAD (E-cadherin): An adhesion molecule.

  • AMOT (Angiomotin): Initiates activation of the Hippo kinase cascade.

• Mechanism in Trophectoderm Cells:

  • Trophectoderm cells express aPKC and E-cadherin, leading to a visible apical-basal polarity.

  • AMOT is sequestered or repressed at the apical side, preventing the activation of the Hippo pathway.

  • This repression allows the transcription factor TEAD (or sometimes referred to as 'test' in the transcript) to localize to the nucleus and activate the expression of $CDX2$, which is a key transcription factor for trophectoderm specification.

• Mechanism in ICM Cells:

  • ICM cells lack active aPKC or have significantly lower levels.

  • AMOT is active, which allows for the activation of the Hippo kinase cascade.

  • This activation leads to the phosphorylation and nuclear exclusion of TEAD, preventing $CDX2$ expression and promoting ICM development.

Neural Stem Cells (NSCs)

• Niche Definition: Specialized microenvironments that regulate stem cell self-renewal and differentiation.

• Location: In both human and rodent brains, neural stem cell niches are present in two main regions of the cerebrum:

  • Lateral Ventricle: Specifically, the ventricular-subventricular zone.

  • Hippocampus: Located in the subgranular zone.

• Brain Development and Layer Formation:

  • The brain develops from a thin neural tube, which expands by forming distinct layers from inside to out.

  • Ventricular Zone (VZ): The innermost layer.

  • Subventricular Zone (SVZ): Next layer, where most neural stem cells reside.

  • Subgranular Zone (SGZ): A few layers up, containing more granular neurons, particularly in the hippocampus.

• Cell Types in the Ventricular-Subventricular Zone (V-SVZ) Niche:

  • E cells (Ependymal cells): Secondary, supportive cells in the central nervous system.

  • B cells (Type B1 cells): Quiescent neural stem cells (often called Type 1 NSCs) responsible for maintaining the stem cell pool.

  • C cells (Type C cells): Progenitor cells (intermediate level) derived from B cells, capable of generating a few subtypes.

  • A cells (Type A cells): Migrating neuroblasts that are readily differentiate and migrate away from the niche.

• Maintenance of Neural Stem Cell Pool:

  • Structural Adhesion: Maintaining the integrity of the V-SVZ niche is crucial.

    • VCAM-1: An adhesion molecule essential for maintaining the ependymal cell structure. Loss of VCAM-1 disrupts this structure and leads to the loss of E cells.

  • Notch Pathway: Plays a critical role as a "timepiece" for stem cell differentiation decisions.

    • Notch is a cell-surface receptor involved in juxtacrine signaling (cell-to-cell contact).

    • Lateral Inhibition: A mechanism where a cell expressing high levels of Notch ligand inhibits Notch signaling in neighboring cells, influencing differentiation.

    • Mechanism: When Notch ligand binds to the Notch receptor, the intracellular domain of Notch is cleaved and translocates to the nucleus to act as a transcription factor.

    • High Notch Activity: Promotes the stem cell state, preventing differentiation. High Notch levels show low or absent neuronal markers.

    • Decreased Notch Activity: Promotes progenitor proliferation and maturation towards a neuronal fate.

  • Environmental Signals and Paracrine Factors:

    • GABA (gamma-aminobutyric acid): Secreted by migrating neural precursors, it increases proliferation in the niche, thereby generating a gradient that influences different neuronal cell types.

    • GF11 (Growth Differentiation Factor 11): Identified through parabiosis experiments (fusing circulatory systems of old and young mice). A blood-borne molecule from young mice that can enhance neural stem cell proliferation in older mice.

Hematopoietic Stem Cells (HSCs)

• Function: Responsible for the generation of all blood cells.

• High Turnover: More than $100 imes 10^9$ blood cells are replaced daily.

• HSC Niche: Two primary niches exist:

  • Endosteal Niche: Closer to the bone lining, housing long-term dormant (quiescent) HSCs.

  • Perivascular Niche: Located near blood vessels, supports short-term active HSCs that are more actively dividing, proliferating, and differentiating into blood cells that enter the vasculature.

Mesenchymal Stem Cells (MSCs)

• Characteristics: Unlike other stem cells, MSCs are multipotent but typically restricted to forming only a few cell types within a given tissue (e.g., bone, cartilage, fat in connective tissue).

• Self-Renewal and Differentiation: They are able to self-renew and produce different populations of cells.

• Differentiation Influenced by Substrate Stiffness:

  • Softer substrates (mimicking brain tissue) can differentiate MSCs into neurons.

  • Medium stiffness (muscle) promotes muscle cell differentiation.

  • Harder substrates (bone) can differentiate MSCs into osteocytes.

Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs)

• Embryonic Stem Cells (ESCs): Pluripotent cells derived from the inner cell mass of a blastocyst, capable of differentiating into any cell type of the body.

  • Maintenance in culture for indefinite periods.

  • Differentiation can be directed by exposing them to specific combinations, concentrations, and durations of morphogens/factors.

  • Morphogens like $WNT$, $BMP$, $FGF$: In various combinations and gradients, they can differentiate ESCs into ectoderm, mesoderm, or endoderm, which can then be further differentiated into specific cell types.

  • The culture conditions for ESCs often mimic the environment of an actual embryo, guiding their differentiation.

  • Applications: Used in regenerative medicine to replace damaged tissues (e.g., curing motor nerve injuries in rats, generating dopamine-secreting neurons to alleviate Parkinson's-like conditions).

• Induced Pluripotent Stem Cells (iPSCs):

  • Discovery: Identified by Shinya Yamanaka in $2006$. A revolutionary technique that allows differentiated somatic cells to be reprogrammed back into a pluripotent state, circumventing the ethical issues of sacrificing embryos for ESCs.

  • Yamanaka Factors: Four key transcription factors that convert differentiated cells into iPSC-like cells:

    • $OCT3/4$ (same as $OCT4$)

    • $SOX2$

    • $KLF4$

    • $c-MYC$

  • These factors differ slightly from the three major factors for promoting pluripotency in ESCs ($SOX2$, $OCT4$, and $NANOG$).

  • Applications: iPSCs can be differentiated into various cell types, similar to ESCs.

  • Example: Curing Kidney Disease (Sickle Cell Anemia Model):

    1. Create a mouse model for sickle cell anemia (e.g., by introducing a mutant gene).

    2. Harvest somatic cells (e.g., fibroblasts from tail tip).

    3. Culture these cells in the presence of the four Yamanaka factors to induce iPSCs.

    4. Use gene editing techniques (e.g., CRISPR/Cas9) to correct the specific nucleotide mutation in the iPSCs.

    5. Differentiate the corrected iPSCs into embryoid bodies (clusters of cells).

    6. Irradiate the original diseased mouse to eliminate its existing mutant blood precursor cells.

    7. Inject the corrected embryoid bodies back into the mouse, which can then differentiate and replace the diseased cells.

Organoids

• Definition: "Mini-organs" cultured in a dish, rudimentary organs derived from stem cells.

• Sources: Can be generated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells.

• Examples of Organoids Successfully Cultured: Optic cup (eye), gut, kidney, liver, brain.

• Current Status: Organoid technology is still in its early phase. While they can mimic many important embryonic developmental processes and early stages of organ development, creating entire, fully functional organs is not yet possible.

• Challenges: Requires specific 3D culture conditions and takes significant time and research.

• Applications:

  • Disease Modeling: Using iPSCs from patients with specific diseases (e.g., microcephaly) to create organoids that mimic the disease state in vitro. This allows researchers to study disease mechanisms and test potential therapies.

  • Microcephaly Example: Comparing brain organoids from healthy individuals vs. microcephaly patients revealed differences in neural stem cell division angles. In normal individuals, roughly $100$% of neurogenesis involves dividing cells, while in microcephaly patients, this is significantly altered, contributing to smaller brain size.

  • Future (Long-term) Goal: To generate fully functional organs for transplantation, reducing transplant waiting lists.

Summary of Key Stem Cell Concepts

• A stem cell maintains the ability to self-renew (produce a copy of itself) and generate progenitor cells capable of maturing into different cell types.

• Totipotent Stem Cells: Can generate all cell types of both the embryo and extraembryonic tissues.

• The pluripotency of the Inner Cell Mass (ICM) of blastocysts is maintained through E-cadherin (ECAD) interactions with trophectodermal cells, which activate the Hippo kinase cascade and repress the function of the transcriptional regulator $CDX2$ (this statement seems to contain an inversion of the Hippo mechanism regarding $CDX2$ activation/repression in ICM/Trophectoderm, as AMOT represses in trophecto, allowing $CDX2$, and Hippo activated in ICM represses $CDX2$: Correction based on detailed notes above: The immunocell masses of the mast cells are maintained through E-CAD interactions, activating the Hippo kinase cascade in the trophectoderm (where AMOT is repressed, TEAD activates $CDX2$ for trophectoderm) and repressing the function of the transcriptional regulator $CDX2$ in the ICM (where Hippo is active, repressing TEAD and thereby $CDX2$).

• The ventricular-subventricular zone (V-SVZ) of the mammalian brain acts as a niche for neural stem cells (B cells), which generate amplifying C cells, and then into migrating neural precursor A cells.

• Additional signals from neural activity (e.g., GABA) and substances like GF11 (from blood) influence neural stem cell proliferation and differentiation.

• Adhesion to osteoblasts keeps hematopoietic stem cells (HSCs) in the endosteal niche (long-term dormant), while stromal cells are important for multipotent hematopoietic stem cells (short-term active) in the perivascular niche.

• Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs) can be maintained in culture indefinitely and can be coaxed to differentiate into potentially any cell type of the body when exposed to certain combinations of factors or constrained by specific substrates.

• Stem cells are crucial in regenerative medicine for repairing or replacing damaged tissues.

Upcoming: Paper discussion on iPSCs on Monday.