Detailed Notes on Stem Cells, Cell Asymmetry, and Cell Death

Stem Cells, Cell Asymmetry, and Cell Death

• Pluripotent Stem Cells:
  • Can regenerate planaria tissue.
  • Example: Neoblasts in flatworms.
  • Neoblasts are dividing small mesenchymal cells.
• Cell Growth, Division, and Death:
  • Symmetric Division: Produces two daughter cells with equivalent fates.
  • Asymmetric Division: Produces two daughter cells with different cellular fates.
  • Cell Lineage: A series of cell divisions akin to a family tree.

Gamete Fusion During Fertilization

• Oocyte Structure:
  • Zona pellucida: Translucent material that provides binding material for the sperm.
  • Polar body: A nonfunctional product of meiosis.
• Fertilization Steps:
  • Binding of sperm to zona pellucida: Sperm surface protein GalT recognizes ZP3 (zona pellucida protein).
  • Acrosomal reaction: Release of enzymes from the acrosomal vesicle upon interactions between GalT protein on the sperm surface, and ZP3. Acrosome is a cap-like structure over the anterior half of the head of spermatozoa.
  • Penetration through zona pellucida: Degradation of the zona pellucida allows the sperm to begin entering the egg.
  • Release of cortical granules: Fusion of plasma membranes.
  • Sperm nucleus enters egg cytoplasm.
  • Entry of the sperm nucleus triggers Ca^{2+} release within the oocyte. Cortical granules respond to Ca^{2+} surge by fusing with the oocyte membrane and releasing enzymes that act on zona pellucida to prevent binding of additional sperm.

Cleavage Divisions in the Mouse Embryo

• Cleavage Division: The rapid mitotic division of a zygote, forming a 16-cell morula.
• Morula: Cells are fairly spherical and loosely attached.
• Compaction: Morula cells undergo compaction at the 32-cell stage when E-cadherin appears.
• Late Blastocyst: The later stage is the 128 cells.

Cell Fate Determination in Early Embryo

• Experimental Setup:
  • Morula stage embryos are used.
  • Transplanting a single cell into a mouse morula.
• Blastocyst Structure:
  • Inner cell mass.
  • Trophectoderm (outer layer of blastocyst).
  • Blastocoel: Fluid-filled cavity.
• Embryonic Stem (ES) Cells:
  • Can be maintained in culture; can form differentiated cell types.
  • Can be cultured with or without feeder cells (if specific cytokines are added).
  • Can be maintained for many generations and stored frozen.
• Trophoblasts: Cells forming the outer layer (trophectoderm) of a blastocyst.
  • Provide nutrients to the embryo.

Transcriptional Network Regulating Pluripotency of ES Cells

• Master Transcription Factors: Oct4, Sox2, Nanog
  • Activate genes for self-renewal and pluripotency.
  • Repress genes that induce specific differentiation pathways.
  • Each factor binds to its own promoters and those of the other two, forming a positive auto-regulatory loop.
  • Also bind to promoter/enhancer regions of other genes encoding proteins and micro-RNAs important for proliferation and self-renewal.

Cloning Mice by Somatic-Cell Nuclear Transplantation

• Process:
  • Remove ovum nucleus.
  • Add nucleus from (GFP) olfactory neuron.
  • Add ES cells.
  • Tetraploid blastocyst.
  • GFP mouse.
• Tetraploid Blastocysts (4n):
  • Obtained by fusing two cells at the two-cell stage using an electrical current.
  • Can develop normally to the blastocyst stage and implant in the uterus.
  • 4n cells can form extra-embryonic tissue, but a proper fetus rarely develops.
• Significance: Demonstrates how the genome of a differentiated cell can be reprogrammed completely.

Cell Polarity and Asymmetric Cell Division

• Stem Cell Division: Stem cells divide to give rise to one stem cell and one differentiated cell.
• Cell Polarity: Asymmetric organization of cellular components (plasma membrane, cytoskeleton, organelles).
  • If a polarized cell divides, it undergoes asymmetric cell division.
• Steps in Generating Polarized Cell:
  1. Localized cue.
  2. Sensing cue.
  3. Signal transduction leading to cytoskeletal reorganization.
  4. Cytoskeleton recognition in a polarized manner.
  5. Transport of membrane trafficking organelles and macromolecules.
  6. Reinforcement of polarity determinants.

General Features of Cell Polarity and Asymmetric Cell Division

• Cell Polarity Requirement: Specific determinants (mRNA, proteins, lipids) must be asymmetrically localized.
• Mitotic Spindle: Positioned so that determinants are segregated appropriately.
• External Cue: In response to an external cue, the cell polarizes, and fate determinants become segregated at cell division, leaving one stem cell and one differentiating cell.
• (a) Stem cell interacting in a stem-cell niche (red curved object) orient the mitotic spindle to give rise to a stem cell associated with the niche and a differentiating cell distant from it.

Patterns of Stem-Cell Differentiation

• Stem Cell Differentiation: Stem cells give rise to both stem cells and differentiating cells.
  • (a) Maintain stem cell population. Stem cells undergoing asymmet-ric divisions do not increase the population of stem cells.
  • (b) Increase stem cells. Some stem cells can divide symmetrically to increase their population.
  • (c) Increase differentiating cells. Some stem cells can produce two differentiating progeny, while others differentiating as in ‘b’ .
• Germ-Line Stem Cells (GSCs): Produce sperm and oocytes.
• Germ-Line: The cell lineage that produces oocytes and sperm with steam cells.
• Stem Cell Niches: Well-defined in GSCs in Drosophila.
  • In fly ovary, oocyte precursors form and begin to differentiate next to the tip of the germarium.
• Oogenesis: Takes place inside female ovaries.
  • Female Drosophila have two ovaries, each containing 12-16 ovarioles.
• Germarium: The most anterior portion of the ovariole.
  • Location of terminal filaments, cap cells, germ line stem cells (GSCs), and intergermarial sheath cells.
• Germinal Niche: Located next to the tip of the germanium.

Drosophila Germarium

• Germarium Composition:
  • Stem cells and niches.
  • Signals that create germ-line stem-cell niche.
  • Signals that create somatic stem-cell niche.
• Follicle Cells
• Cap Cells: Secrete Hh (Hedgehog); Secrete two TGFB signals, Dpp and Gbb; Produce Arm (homolog of B-catenin) and Zpg surface proteins
• Inner Sheath Cells: Secrete two signals, Wg and Hh; Produce arm protein (homolog of β-catenin).
• Ovariole: A string of 6 or 7 sequentially developing egg chambers.
• Egg Chamber: Comprises 16 germ-line cells (one oocyte and 15 nurse cells) surrounded by a thin layer of somatic follicle cells.

Germ-Line Stem Cells (GSCs)

• Unipotent: GSCs are normally unipotent, producing only differentiating gametes (cystoblast).
  • A unipotent stem cell refers to a cell that can differentiate along only one lineage.
• Hedgehog (Hh): GSC receives Hh (Hedgehog) through Ptc receptor, promoting its self renewal.
• Bam: Promotes differentiation of GSC to cystoblast. So repression of Bam promotes self renewal.
• TGF Signals (Dpp and Gbb): Interact with TGF receptors on GSC and cause activation of Mad and Med (Medea).
  • Repress bam transcription and allow self renewal.
  • When GSCs move away from the niche (cap cell), they escape TGFB-mediated repression, up-regulate bam expression, and differentiate into cystoblasts.

Somatic Stem Cells (SSCs)

• Wg Signals: SSC receive Wg signals through the Fz receptors promoting self renewal. Wnt signaling pathway
• Arm Protein: SSC produce Arm, which interacts with Arm on inner sheath cells.
• Hh Signal: SSC receive Hh (Hedgehog) signal through the Ptc receptor, promoting self renewal.
• Wg Regulation: Wg regulates proliferation and differentiation of SSC into follicle cells.
• Differentiation: When a newly divided SSC is pushed away from the inner sheath cell, it is deprived of the niche; it then differentiates into follicle cells.

Follicle Cells

• Drosophila Egg Chamber Development:
  • Begins as a single cell, the cystoblast, which undergoes four rounds of incomplete division to produce a cyst of 16 cells.
  • Only one of the 16 cells in the cyst goes on to produce an oocyte and remains arrested in meiotic prophase I; The other 15 cells develop as polyploid nurse cells.
• GSC Markers: Can be recognized by their intracellular spectrosome, which is an ER-derived organelle, having binding affinity to actin and microtubules.
• Spectrosome: A cytoplasmic organelle rich in spectrin (an actin cross-linking protein).
• Drosophila females lay ~400-1000 eggs

Germ-Line Stem Cells in C. elegans

• Distal Tip Cell: Creates and maintains the niche.
• Delta/Notch Signaling:
  • Transmembrane protein delta produced by the distal tip cell binds to the Notch receptor on the adjacent germ-line stem cells.
  • Represses the induction of differentiation and prevents onset of meiosis.
  • Meiosis is blocked by the Delta signal until the cell moves beyond the range of the signal from the distal tip cell.

Intestinal Stem Cells and Their Niche

• Intestinal Epithelium Turnover: Every 3–5 days.
• Lgr5-expressing (Lgr5+) Intestinal Stem Cells: Divide to produce transient amplifying cells.
• Transient Amplifying Cells Divide to provide differentiating cells
• Paneth Cells:
  • Regenerated from Lgr5+ cells and also from transient amplifying cells.
  • Located at base of the crypt.
  • Provide a major part of the stem-cell niche.
  • Secrete antimicrobial defense proteins.
• +4 “Reserve” Stem Cells:
  • Occupy the fourth position from the crypt base.
  • Generated from Lgr5+ stem cells.
  • Can restore the Lgr5+ stem-cell compartment following injury.
• Masanchymal cells surround the base of the crypt secrete Wnt that promote self renewal, offsetting signals for differentiation.

Further Details on Intestinal Stem Cells

• Transient Amplifying Cells
• +4 Stem Cell
• LGR5+ Stem Cell
• Paneth Cell
• Mesenchymal Cells: Surround the base of the crypt and secrete proteins such as Wnt that promote self renewal.

Formation of the Neural Tube and Division of Embryonic Neural Stem Cells

• Neural Tube Formation:
  • A part of the ectoderm rolls up and separates from the rest of the cells.
  • Neural crest cells form from the embryonic ectoderm cell layer and then migrate to contribute to skin pigmentation, nerve formation, craniofacial skeleton, heart valves, peripheral neurons and other structures.
• Notochord: A rod of mesoderm that provides signals affecting cell fates in the nerve tube.
• SVZ Neural Stem Cells:
  • Can divide symmetrically to give rise to two stem cells.
  • Can divide to produce one stem cell and one transiently amplifying (TA) cell, which begins to migrate and differentiate.
• Neural Crest Cells: Specified at the border of the neural plate and the non-neural ectoderm.
• The ectoderm of the embryo differentiates to form the nervous system.

Neural Stem Cell Niche in the Adult Brain

• Adult Neurogenesis: Some cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus continue to act as stem cells.
• Adult Neural Stem Cells (ANSCs) / Type B Neural Stem Cells:
  • Can differentiate to replace lost or injured neurons or glial cells.
  • Have abilities for self renewal and differentiate into neuron, astrocytes and oligodendrocytes (glial cells).
• Transiently amplifying (TA) cells
• Neuroblasts
• Germinal Niche: Provided by cerebrospinal fluid in the ventricle and the ependyma.
  • ANSCs are stimulated to begin differentiation via exogenous cues from the microenvironment, or stem cell niche.

Formation of Blood Cells from Hematopoietic Stem Cells in the Bone Marrow

• Hematopoietic Stem Cells (HSCs): Give rise to lymphoid and myeloid progenitor cells.
  • Multipotent HSC
  • Lymphoid progenitor cell
  • Myeloid progenitor cell
• Blood Cell Lineages:
  • T cells, B cells, NK cells.
  • Monocytes, granulocytes, macrophages, dendritic cells, neutrophil, basophil, eosinophils, mast cell, platelets.
• Supportive Bone Marrow Stromal Cells: Regulate the hematopoietic stem cell niche.
• FACS: Fluorescence Activated Cell Sorting.
• Edward Donnall Thomas received the Nobel prize in 1990

The Hematopoietic Stem Cell Niche in the Bone Marrow

• Niche Components: Osteoblast, osteoclast, fibroblast, endothelial cell, stromal cell, HSC.
• Sinusoids: Bone marrow is permeated by small blood vessels.
• Stromal Cells:
  • Express SCF that binds to and activates a receptor tyrosine kinase c-Kit (CD117) on HSCs.
  • Secrete CXCL12 (or SDF1), a chemoattractant (chemokine) for HSCs.
  • Secrete thrombopoietin, which activates a thrombopoietin receptor that functions as an erythropoietin receptor in HSCs.
  • stromal cells are responsive to cytokine leptine.
• Stromal cells express SCF, CXCL12, leptin receptor
• HSCs express CD150, c-Kit leptin receptor, SCF, CXCL12, thrombopoietin
• Stromal cell CD150, c-Kit, erythropoietin R, CXCL12 R (GPCR)
• In Fetus: Hepatic progenitor cells provide the niche for HSCs.

Meristems as Niches for Stem Cells in Plants

• Plant Stem Cells: Reside in specialized micro environments called meristems.
• Plant Cell Fate: Depends on the cell’s position, not on lineage.
• Cell Identity: Reinforced by intercellular signals (hormones, mobile signaling peptides, miRNAs).
• Shoot Stem Cells: Displaced to the periphery of the meristem and are recruited to form primordia of new organs.
• WUSCHEL (WUS) Gene: Expressed in the Organizing Center underneath the stem cells.
• Quiescent Center (QC): A group of cells in the apical meristem of a root where cell division proceeds very slowly.

Regulatory Network in the Arabidopsis Shoot Meristem Stem-Cell Niche

• WUSCHEL (WUS) Gene: Encodes a homeodomain transcription factor required for maintenance of stem cells in the shoot apical meristem
  • Synthesized in the organizing-center cells.
  • Moves via plasmodesmata into stem cells.
  • Induces CLAVATA3 (CLV3) hormone expression.
• Secreted CLV3 Protein: Activates CLV1, the CLV3 receptor protein kinase on the surface of organizing center cells.
  • Activates signal that represses WUS transcription. The WUSCHEL (WUS) gene encodes a homeodomain transcription factor required for maintenance of stem cells in the shoot apical meristem.
  • A negative feedback loop maintains the size of the apical stem cell population.
• CLV1 is a LRR-RK kind of receptor kinase, which is composed of an extracellular domain containing the consensus hydrophobic leucine residues, a single membrane-spanning domain, and a cytoplasmic kinase domain with serine/threonine specificity.

Apoptosis (Programmed Cell Death)

• Apoptosis: A normal occurrence in which an orchestrated sequence of events leads to the death of a cell.
  • Shrinkage in volume of the cell and its nucleus.
  • Loss of adhesion to neighboring cells.
  • Formation of blebs at the cell surface.
  • Dissection of the chromatin into small fragments.
  • Rapid engulfment of the corpse by phagocytosis.

Role of Apoptosis

• Cell Turnover: ~10^{10}–10^{11} cells in the human body die every day by apoptosis.
• Organ Development: Often requires pruning into the correct form by apoptosis.
• Cancer Prevention: Apoptosis prevents tumor development. If a cell is unable to undergo apoptosis because of mutation or biochemical inhibition, it continues to divide and develop into a tumor.
• Alzheimer's disease, Parkinson's disease, and Huntington's disease: Apoptosis appears to be involved in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

The Discovery of Apoptosis

• Coined Term: The term apoptosis was coined in 1972 by John Kerr, Andrew Wyllie, and A. R. Currie of the University of Aberdeen, Scotland.
• C. elegans Studies: Insight into the molecular basis of apoptosis was first revealed in studies on C. elegans, whose cells can be followed with absolute precision during embryonic development.
  • Of the 1091 cells produced during the development male worms and 959 cells in case of hermaphrodites; 131 cells are normally destined to die by apoptosis.
• CED-3 Gene: In 1986, Robert Horvitz and his colleagues at MIT discovered that worms carrying a mutation in the CED-3 gene proceed through development without losing any of their cells to apoptosis. CED-3 is a cell death gene that encodes an effector caspase (or executioner caspase).

The Targets of Executioner Caspases

• Caspase Cleavage: A cysteine in its active site of caspase nucleophilically attacks and cleaves a target protein at the C-terminal of an aspartic acid amino acid within proteins.
• Target Proteins:
  • Protein kinases (FAK, PKB, PKC, Raf1).
  • Lamins.
  • Proteins of the cytoskeleton (intermediate filaments, actin, tubulin, gelsolin).
  • An endonuclease called caspase-activated DNase (CAD), which is activated following caspase cleavage of an inhibitory protein (iCAD). Once activated, CAD translocates from the cytoplasm to the nucleus where it attacks DNA, severing it into fragments.

Activation of CED-3 Protease in C. elegans

• EGL-1 protein, which is produced in response to environmental signals that trigger cell death, displaces an asymmetric CED-4 dimer from its association with CED-9 on the surface of mitochondria.
• The free CED-4 dimer combines with 3 others to form octamer.
• The octamer binds two molecules of CED-3 zymogen and triggers its conversion into active CED-3 protease.
• The effector then begins to destroy cell components and initiate apoptosis, leading to cell death.

Apoptosis in C. elegans: Key Proteins

• CED-4: Required for activation of CED-3.
  • Forms an octameric apoptosome, which binds the CED-3 zymogen and facilitates its autocatalytic maturation.
• CED-9: A pro-survival protein that inhibits/represses apoptosis.
  • Prevents the release of cytochrome c in the membrane of mitochondria.
• EGL-1: Promotes apoptosis by disrupting the suppressive interaction of CED-9 on CED-4.
  • This disruption promotes activation of the effector caspase, CED-3.

Apoptosis-Related Proteins: Human vs. Worm

• Caspase-8 and caspase-10 are involved in the extrinsic pathway, whereas caspase-9 is involved in the intrinsic pathway

Apoptosis Regulation by Death Signals

• TNFα and FasL: Promote apoptosis; TNFα produced by macrophages triggers cell death
• FADD: Activated trimeric Fas receptor binds to a cytosolic protein FADD; death-inducing signaling complex (DISC) is formed.

Cell Murder: The Extrinsic Apoptosis Pathway

• Death Receptors: Fas and TNF-receptor 1 are examples.
• FADD: An adapter protein containing a DD and a death effector domain (DED).
• Procaspase-8 or Procaspase-10 (Initiator caspase)
• Binding of a FasL to on the surface of one cell to the death receptor Fas on the adjacent cell leads to recruitment of the adapter protein FADD (Fas-associated death domain) and the dimerization and activation of caspase-8 or its homolog caspase 10
• Active caspase-8 (or -10), can also activate the intrinsic apoptosis pathway by cleaving BID and thus producing t-BID.

The Link Between Extrinsic and Intrinsic Apoptosis Pathways

• t-BID: A ‘BH3-only protein’ that binds to Bcl-2 on the mitochondrial outer membrane, leading to release of cytochrome c into the cytosol.
• Active caspase-8 (or -10), cleaves BID to produce t-BID.
• t-BID or Bad or Bim, or Puma binds to Bcl-2 (or Bcl-xL, or Bcl-W) and inactivate it.
• Bcl-2 (or Bcl-xL, or Bcl-W) is an inhibitor of the monomeric channel protein Bak, Bax, or Bok on the mitochondrial outer membrane.
• Apoptosome (Heptamer)

Mitochondrial Apoptosis Pathway: Triggers and Inhibitors

• Internal Cellular Signals lead to the release of cytochrome c from mitochondria and activation of caspases, ultimately resulting in cell death
• Trophic factors e.g., NGF or nerve growth factor) inhibits apoptosis by intrinsic pathway.
• DNA damage: DNA damage induces apoptosis by intrinsic pathway.
• Integrin signaling disruption: Disruption of integrin signaling induces apoptosis by intrinsic pathway

The Mitochondrial Apoptosis-Induced Channel (MAC)

• If the stimulus is prosurvival or antiapoptotic (such as Bcl-2, Bcl-xL, Bcl-W), then apoptosis will not take place
• Inhibition of Bcl-2 by t-Bid, Bad, Bim or Puma will allow formation of MAC channel by oligomerization of Bax or Bak.
• tBid, Bim, Puma, and Bad are a proapoptotic protein that counter the effects of Bcl-2 by binding to it and thus allow Bax oligomerization for pore formation.

Pro-survival proteins: Anti-apoptosis proteins

Pro-apoptosis proteins: Form channels in mitochondrial outer membrane

BH3-only proteins: Regulate activity of Bcl-2 and Bax/Bak proteins

Stimuli and Repressors of Apoptosis

• Multiple signaling pathways regulate intrinsic apoptosis pathway
• Trophic Factor: The presence of specific trophic factors inhibits apoptosis
• DNA Damage: or UV irradiation leads to induction of ‘BH3-only’ Puma protein
• Removal from Substratum: Removal of a cell from its substratum disrupts integrin signaling leading to release of ‘BH3-only’ Bim protein from the cytoskeleton.

Structure of CED-4 and Apaf-1 apoptosomes

• Apoptosome Structure: Forms a heptameric wheel-like complex to activate caspase-9
  CED-4 and Apaf-1 apoptosomes
  During apoptosis, caspases cleave key intracellular target proteins to promote the death of the cell and disposal of the cell corpse. The first caspases activated during this process are the initiator caspases, which in turn cleave and activate the executioner caspases
• (2)CED-9 Complex : In healthy worm cells, CED-4 is sequestered by CED-9 into a (CED-4) :CED-9 complex, in which the CED-4 molecules exist as an asymmetric dimer. In the presence of a death signal, an upstream protein EGL-1 (analogous to mammalian BH3-only proteins) interacts with CED-9 and releases the CED-4 dimer to become activated and form CED-4 octamer

Evolutionary Conservation of Apoptosis Pathways

• IAPs and SMAC/DIABLO: Mammals and flies use IAPs and SMAC/DIABLO to control caspase activity, whereas nematodes do not.
• Mammals and flies, but not nematodes, have IAPs (Inhibitor of apoptosis proteins .Assembly of Bax/Bak channels leads to release of SMAC/DIABLO, which binds to IAPs in the cytosol.