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.