Stem Cells Flashcards

Stem Cells: What are they?

  • Cells with the potential to differentiate into other cell types.
  • Possess self-renewal capability, allowing them to bypass senescence.
  • Crucial during development for creating organisms.
  • Serve to repopulate tissues and organs in certain differentiated tissues.
  • Exhibit differing levels of potency:
    • Totipotent: Capable of generating an entire new multicellular organism.
    • Pluripotent: Can give rise to most cell types, but not all.
    • Multipotent/Progenitors: Lack self-renewal capacity but are involved in specific differentiation programs.

Self-Renewal

  • How stem cells make other cells:
    • Asymmetric Division:
    • A stem cell divides into another stem cell and a terminally differentiated cell.
    • Choice determined by asymmetry in the dividing stem cell.
    • Environmental factors play a role in determining cell fate.
    • Independent Choice:
    • A stem cell divides stochastically and/or based on environmental cues.

Stem Cells: Where do we find them?

  • Pluripotent "Adult" Stem Cells:
    • Found in natural reservoirs throughout the body.
    • Can be dormant and activated for tissue repair when needed.
    • Continuously used for tissue maintenance.

Stem Cells: Renewal of Epithelial Tissues

  • Gut Epithelium:
    • Continually renewed and composed of dividing and non-dividing cells.
    • Dividing stem cells located at the base of the crypt.
    • Detected using tritiated thymidine to visualize actively dividing cells.
    • Cell cycle duration: approximately 12 hours when rapidly dividing.
    • Can differentiate into another stem cell or a committed precursor/transit amplifying cell.

Non-Dividing Cells

  • Made by stem cells:
    • Absorptive cells: Brush border and enterocytes.
    • Goblet cells: Secrete protective mucus.
    • Enteroendocrine cells (15 subtypes): Secrete peptide hormones to control neurons, cell proliferation, and growth.

Wnt Signaling

  • Maintains stem cells and their differentiation.
  • FAP (Familial Adenomatous Polyposis) patients: Loss of Apc leads to continuous Wnt signaling, resulting in adenoma.
  • Wnt gradient is important.
    • Normal/High Wnt signaling in the crypt allows division.
    • Less Wnt signaling as cells leave the crypt.
    • Supported by Paneth cells to create a perfect 'niche'.

Notch Signaling

  • Controls diversification through lateral inhibition.
  • Delta expression inhibits notch activation, maintaining the Paneth cell population.
  • Allows other cells to divide.

Stem Cells: Regeneration & Repair

  • Used to repair wounds, limbs, or organisms.
  • Planaria (freshwater flatworms):
    • Can regenerate from even a small piece of tissue.
    • Can selectively "degrow" and grow depending on starvation conditions.
    • 20% of its body is neoblasts, which serve as stem cells.
    • Irradiation stops cell division and causes death.

Vertebrate Regeneration

  • Some vertebrates can regenerate organs.
  • Newt limb regeneration:
    • Starts with the formation of a blastema or embryonic limb bud.
    • Multinucleate cells reenter the cell cycle and provide new growth.
    • Blastema cells are multipotent.

Stem Cells and Differentiation

  • Signals are required to dictate cell fate.

Stem Cells: Connective Tissue

  • Supports epithelial cells, matching their needs.
  • Fibroblasts change character due to chemical and physical signals.
    • Stiff extracellular matrix depositions: lead to strong adhesions and bone cell development.
    • Soft extracellular matrix depositions: lead to rounded cells due to weak adhesions and fat cells.

Stem Cells: Fibroblasts and Connective Tissue

  • Bone Marrow Environment:
    • Mesenchymal stem cells support stromal cells that make fat, cartilage, or bone.
    • Tension of actin-myosin bundles triggers decision-making signaling.
  • Bone:
    • Dense and rigid, growth by deposition of tough type I collagen and calcium phosphate (done by osteoblasts).
    • Osteoblasts become embedded osteocytes.
    • Bone degradation/reabsorption is done by osteoclasts and are controlled by signals from osteoblasts.

Stem Cells: Hemopoietic Cells

  • Hierarchical Process:
    • Lymphocytes: Immune cells (B & T) to make antibodies and kill virus-infected cells.
    • Granulocytes: Grouped based on staining and function.
    • Monocytes: Make macrophages.
    • Red Blood Cells: Carry oxygen.
    • Megakaryocytes: Shed platelets.

Hematopoietic Stem Cells (HSC)

  • Discovered by their ability to replace the entire bone marrow system through a transplant.
  • 1 HSC in every 50,000-100,000 bone marrow cells.

Commitment

  • A stepwise process.
  • Signals from stromal cells maintain potency; loss of contact + other signals lead to differentiation.
  • Progenitors do not require a niche, only a semi-solid matrix for clonal expansion.
  • Colony stimulating factors (CSF) tell cells what to become and are made by various cell types (endothelial cells, fibroblasts, macrophages, lymphocytes).
  • Cells separated after splitting can make different cell types, which suggests epigenetic reprogramming during division.

Stem Cells: Hemopoietic Cells cont.

  • Key factors and markers involved in hematopoietic cell differentiation:
    • LT-HSC (Long-Term Hematopoietic Stem Cell): CD201^+ CD150^+ CD48^- Lin^- c-Kit^+ Sca-1^+ or CD34^- Flk2^- Lin^- c-Kit^+ Sca-1^+
    • ST-HSC (Short-Term Hematopoietic Stem Cell): CD34^+ Flk2^- Lin^- c-Kit^+ Sca-1^+
    • MPP (Multipotent Progenitor): CD34^+ Flk2^+ Lin^- c-Kit^+ Sca-1^+ or CD150^- CD48^- Lin^- c-Kit^+ Sca-1^+
    • CLP (Common Lymphoid Progenitor): Lin^- c-Kit^{lo} Sca-1^{lo} IL7Ra^+ Flk2^+
    • CMP (Common Myeloid Progenitor): CD34^+ FcyRII/III^- Lin^- c-Kit^+ Sca-1^-
    • GMP (Granulocyte-Macrophage Progenitor): CD34^+ FcyRII/III^+ Lin^- c-Kit^+ Sca-1^-
    • MEP (Megakaryocyte-Erythroid Progenitor): CD34^+ FcyRII/III^- Lin^- c-Kit^+ Sca-1^-
  • Various cytokines and factors:
    • IL-3, SCF, GATA, TPO, EPO, IL-11, Gf1, Sall4, Etv6, Sox17, GATA2, Foxo3a, GM-CSF, PU.1, CEBP, G-CSF, M-CSF, IL-5, IFN-Y, Fit3L, TNF-a, IL-10, IL-6, GM-CSF, IL-7, IL-2, IL-4, IL-15

Cellular Reprograming: Somatic Cell Nuclear Transfer (SCNT)

  • Dolly the Sheep (1996):
    • Adult Finn Dorset ewe: Donor of nucleus from mammary gland cells.
    • Unfertilized egg cell from adult Scottish Blackface ewe: Nucleus removed.
    • Donor cells starved (arrested growth cycle).
    • Fusion of donor cell nucleus with enucleated egg cell using electrical pulses.
    • Embryo implanted into surrogate mother.
    • Result: Finn Dorset lamb ("Dolly").

Cellular Reprograming: Embryonic Stem Cells

  • Gathered from the inner cell mass of the blastocyst.
  • Can be used to make any cell type in the organism (except placental cells).
  • Allows development of germ cells.

Cellular Reprograming: Transcription Regulators

  • Define and maintain embryonic stem cell state.
  • High levels of telomerase to avoid senescence.
  • Overexpression of a single transcription factor doesn't change the cell state.
  • Combination of Oct4, Sox2, Klf4, and Myc allows fibroblasts to 'revert' to an embryonic-like state.
  • These factors work together to reprogram the cell transcription profile.
  • The resulting cells are called "induced pluripotent stem cells" (iPSCs).

Reporter Cell Line

  • Used an "embryonic" promoter to drive the expression of a resistance gene.
  • Only when the embryonic promoter is turned on will cells express resistance and survive.

Reprograming Effects

  • Causes massive upheaval of the gene control system.
  • Takes time and early attempts were not very efficient.
  • Modification of chromatin enhances the reprograming process.

Vectors for Cellular Reprograming

  • Integrative viral vectors:
    • Retrovirus
    • Lentivirus
  • Integrative non-viral vectors:
    • Transposons (Piggybac, Sleeping beauty)
  • Non-integrative viral vectors:
    • Adenovirus
    • Sendai virus
  • Non-integrative non-viral vectors:
    • Episomal vectors (plasmids, minicircle)
  • Other methods:
    • Protein
    • RNA
    • miR-302-367

Factors used for iPSC generation

  • Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28

Starting cells for iPSC generation

  • Skin fibroblast
  • Neural stem cell
  • Keratinocytes
  • Melanocytes
  • Adipose tissues-derived cells
  • Immature dental pulp
  • Blood cell

Cellular Reprograming: iPSC Differentiation

  • Need to know the signals required for the pathway of interest.
  • Media and substrate changes as cell types change.
  • Analyze early vs. late protein and gene expression.
  • Use reporters activated only upon late-stage differentiation.

Cellular Reprograming: iPSC Potential

  • Use of patient-specific cells leads to patient-specific medicine.
  • Understand disease progression.
  • Better disease predictions.
  • Drug screening before clinical trials.

Key Concepts

  • Define a stem cell and different “potencies”.
  • Recognize and describe locations where stems cells are used in the human body.
  • How stem cells are supported by their environment.
  • Define stem cell ‘choices’ and what that means for development.
  • Explain the role of cell signaling in cell fate decisions.
  • Identify steps taken to reprogram cells and what they can be used for.