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PHGY 351 > Nijnik > Flashcards

Nijnik Flashcards

1
Q

What are the 2 key properties of stem cells?
*Needed to prove you have a stem cell colony

A
  1. Ability to self-renew indefinitely
  2. Differentiation into multiple cell types → can get cells from all 3 germ layers from that one stem cell (mesoderm, endoderm, ectoderm)
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2
Q

What are the different types of stem cells?

A
  1. Embryonic Stem cells:
    - Derived from early stage-embryo
    - pluripotent (differentiate to any cell inthe body of the embryo)
  2. Adult Stem cells:
    - mediate tissue renewal throughout life
    - multipotent (can differentiate into several cell types, more restrictive)
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3
Q

What are embryonic stem cells derived from?

A

Derived from inner cell mass of a day 4.5 embryo / blastocyst (earliest stage where cells are different from one and other)

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4
Q

When were mice vs human Embryonic stem cells 1st derived?

A

Mouse → 1981 by Martin Evans and Matthew Kaufman, Cambirdge

Human → 1998 by James Thomson, University of Wisconsin-Madison

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5
Q

What are the Yamanaka factors (2006) ?

A

They are the 4 TF required for cell reprogramming of induced pluripotent stem cells:
- OCT4
- SOX2
- KLF4
- c-MYC

*Take fibroblast and induce pluripotency (no ethical issue)

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6
Q

What 3 types of cells can we grow in cell culture?

A
  1. Primary somatic cells: (from healthy donors)
    - maintained in vitro for limited duration of time
    - difficultot isolate in high number
  2. Cell lines:
    - self-renew indefinitely
    - transformed cancerous cells, abnormal properties
    - very limited differentiation potential
  3. iPSCs/ESCs:
    - self-renew indefinitely
    - differentiate into ALL cell lineage
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7
Q

What is required for ESC/iPSC culture?

A
  1. Grown on feeder cells or on matrix:
    - gelatin, fibronectin, matrigel
    - STO feeder cell lines (fibroblasts)
    - mouse embryonic fibroblast
  2. Add specific cytokines to repress ESC/iPSC differentiation
    - mouse ESC/iPSC → use leukemia inhibitor factor (LIF)
    - human ESC/iPSC → use fibroblast growth factor (FGF)
  3. Add chemical inhibitors of ESC/iPSC differentiation
    - 2i consiting of speciifc GSK3b inhibitor and Mek1/2 Inhibitor

*The more you inhibit differentiation, the slower they grow (differentiation associated with increased proliferation)

  1. Media composition has to be highly controlled
    - use pre-tested fetal calf serum (variable so test to make sure it matches previous batch)
    - Chemically defined serum replacement also available (mimic fetal calf serum, more consistent)
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8
Q

What are different applications of ESC/iPSC?

A
  1. Study ESC biology → mechanisms of cell differentiation in early embryonic development
  2. In vitro derivation of rare cell types for experimental work → regenerative medicine
  3. Production of transgenic mouse lines → study mammlian gene functions and model human diseases (KO mouse lines)
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9
Q

What are different markers of ESC/iPSCs?

A
  1. Colony morphology
  2. Cell Surface markers → SSEA1 (mouse), SSEA3/4 (human, found in non-differentiated cells)
  3. Transcription factors → Nanog, Oct2, Sox2, KIf4, c-Myc
  4. Telomerase activity (to maintain self-renewal)

*Nanog → maintain balance between self-renewal and pluripotency/differentiation

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10
Q

What tissues are found int he mesoderm, endoderm and ectoderm?

A

Mesoderm: (middle layer)
- Cardiac muscle cells
- Skeletal muscle cells
- Tubule cell of the Kidney
- RBC
- Smooth muscle cells

Endoderm: (internal layer)
- Lung cells
- Thyroid cells
- Pancreatic cells

Ectoderm: (external layer)
- Skin cells of epidermis
- Neuron cells
- Pigment cells

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11
Q

What in vivo and in vitro experiments can prove pluripotency of ESC/iPSCs?

A

In Vitro → 1 single ES cell can form colonies containing progeny cells of all 3 germ layers

In Vivo → when injected into a mouse blastocyst, ES cells can contribute to all different cell types/tissues

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12
Q

How can iPSCs be used to produce rare cells in vitro?

A
  1. Collect blood from the patient
  2. Culturing fo monocytes
  3. Differentiation of monocytes into DC
  4. DC are coltured outside of the body with proteins extracted from tumour lysate or syntehsized peptides
  5. Infusion back into the patient

*Used ESC and iPSC to derive dendritic cells in vitro

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13
Q

What are key points of the protocol for ESC/iPSC differentiation to dendritic cells?

A
  1. Remove inhibitors of ESC/iPSC differentiation (feeders, LIF, 2i inhibition)
  2. Embryoid body formation in non-adherent plates → 3D aggregate of in suspension of pluripotent stem cells
  3. Add cytokines GM-CSF and IL-3 to drive dendritic cell differentiation
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14
Q

What neural cells can be derived from ESC/iPSC differentiation?

A
  • Neurons
  • Astrocytes
  • Microglia
  • Cerebral organdies
  • NSCs/NPCs
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15
Q

What size of air particles are removed by the HEPA filter?

A

0.3 um and above

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16
Q

How does the Acetic Acid with Methylene Blue cell counting method work?
What is it useful for?

A

Useful for selective counting of nucleated cells (when get blood sample for example, richin RBC and platelets, but want to count WBC)
→ Reagent lyses cells → Stains the nucleus Blue

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17
Q

How does the Trypan Blue cell counting method work?
What is it most useful for?

A

Useful for differentiating live vs dead cells → Trypan blue stains dead cells blue (live cells stay white)

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18
Q

What is the composition of physiological blood?

A

55% Plasma (5L) + 45% Erythrocytes + <1% Buffy coat (platelets and leukocytes)

19
Q

What is the definition of the hematocrit?

A

% of Red blood cells/volume

20
Q

What is the concentration and main functions of the different blood cells?

A

RBC ~ 5x10^6 /mm3 → Oxygen transport (contain Hb)
Platelets ~ 2.5x10^5 /mm3 → Blood Clotting
Leukocytes (WBC) ~ 7x10^3 /mm3 → Immune and inflammatory response (neutrophils is the most abundant WBC)

21
Q

What are the different Leukocytes?

A

Granulocytes: → innate immune response, combat infection
1. Neutrophils (60-70%)
3. Eosinophils (2-4%)
4. Basophils (0.5-1%)

Monocytes (3-8%) → phagocytosis + can differentiate into macrophages and DC

Lymphocytes (20-25%) → B and T cells, adaptative immune response

22
Q

What are the characteristics of neutrophils?

A
  • Multi-lobed nucleus
  • Many cytosolic granules
  • Small cells → 10 um
  • 1 - 5 days life-span
23
Q

What are the characteristics of monocytes?

A
  • Horseshoe-shaped nucleus (U)
  • Some (little) Cytosolic granules
  • Large cells → 10-25 um
24
Q

What are the characteristics of lymphocytes?

A
  • Large round nucleus
  • No granules
  • Small cells → 10 um
25
Q

What is the life-span of RBC and platelets?

A

RBC → 120 days
Platelets → 14 days
*Short compared to other tissues

High production rate → > 100 billion blood cells produced/day, can increase 5-10 fold during injury/infection (most are neutrophils)
*Allows to donate a lot of blood without secondary effects

26
Q

What is the primary site of blood cell production in heathly individuals?

A

During development (fetus) → in the fetal liver
After birth → bone marrow (liver and spleen will compensate if problem)

27
Q

What is the hierarchy of progenitor cells for Macrophages and Dendritic cells?

A

HSC → Common Myeloid Progenitor (CMP) → Granulocyte Monocyte Progenitor (GMP) → Monocytes → Macrophages/Dendritic cells (or directly from GMP to dendritic cells)

28
Q

What hierarchy of progenitor cells leads to formation of granulocytes?

A

HCS → Common Myeloid Progenitors (CMP) → Granulocyte Monocyte Progenitor (GMP) → Granulocytes (Basophils, Neutrophils, Eosinophils)

29
Q

What hierarchy of progenitor cells leads to formation of Platelets and RBC?

A

HCS → Common Myeloid Progenitors (CMP) → Megakaryocyte, Erythroid Progenitor (MEP) → Megakaryocyte (→ platelets) + RBC

30
Q

What hierarchy of progenitor cells leads to formation of T and B lymphocytes and NK cells?

A

HSC → Common Lymphoid Progenitor (CLP) → B and T Lymphocytes + NK cells

31
Q

How does Multipotency, Self-renewal and Differentiation change with evolution of HSC → Erythrocytes, platelets, granulocytes and macrophages?

A

HSC = Max multipotency and Max self-renewal
Erythrocytes, platelets, granulocytes and macrophages = max Differentiated state

Differentiated cells don’t undergo self-renewal + are short-life

32
Q

What are cellular components of the Hematopoietic Stem Cell Niche in the bond marrow?

A
  1. Mesenchymal stem cells → Osteoblasts (bone producing cells), Osteoclasts (bonde degrading cells), Adipocytes
  2. Vascular Endothelial cells
  3. Neurons

*Mimiching this microenvironment in culture may be key to promoting HSC expansion!

33
Q

How is hematopoiesis (differentiation) regulated?

A

With different Growth Factors:
In the lab, we used GM-CSF to promot dendritic cells differentiation
GM-CSF = Granulocyte-Macrophage Colony Stimulating Factors

For Platelets → TPO (thrombopoietin)
For Erythrocytes → EPO (erythropoietin)
For Granulocytes → G-CSF (HSC → SCF/IL-3 → CMP → G-CSF → Granulocytes)
B, T Lymphocytes → IL-7 and others (IL-15 for NK cells)

34
Q

What are the 4 mechanisms Hematopoietic Growth factors act on?

A
  1. Stimulate proliferation
  2. Inhibit apoptosis
  3. Differentiation
  4. Maturation Activation

*Many act through the JAK signalling pathway (cytokine receptor)

35
Q

What is the only form of stem cell therapy in routine clinical use?

A

Bone marrow/ hematopoietic stem transplantation

36
Q

What are the 2 possible sources/types of donor stem cells?

A
  1. Allogeneic (from another donor)
    - Donor and recipient need to be immunologically compatible
    - Risk of immune rejection of the donor cells by recipient (graft-failure)
    - Risk of graft-vs-host disease (GVHD)
  2. Autologous (self-donation)
    - Complete immunological compatibility → low risk of failed engraftement and no risk of GVHD
    - Not suitable for genetic diseases (has to be combined with gene therapy)
37
Q

What is the difference between GVHD and graft-failure?

A

Graft-vs-host disease (GVHD) = immune attack of the donor cells against the recipient

Graft-failure → no perfect matching, immune rejection of the donor cells by the recipient

38
Q

What are possible sources of donor stem cells?

A
  1. Bone marrow → direct colleciton of bone marrow from the bones of the donor (rarely used)
  2. Mobilized peripheral blood → donor treated with G-CSF and mobilized stem cells are collected from the dono’r blood (higher concentration)
  3. Cord Blood → Blood from placenta and umbilical cord stored in public and private cord blood banks
    - disadvantage → cells are immunologically immature, 1 unit not enough for an adult recipient (good for kids), less risk of GVHD
39
Q

What are possible future outlooks for Hematopoietic Stem Cell Transplantation?

A
  1. Application for treatment of wider range of immunological disorders
  2. Expanding limited numbers of donor-derived HSC in culture
  3. Combining BM/HSC transplantation with gene therapy (BM = bone marrow)
  4. Deriving new HSC in culture from iPSCs or other cell types
40
Q

Whathy is introducing DNA into cells through homologous recombination highly inefficient?

A

Can’t make KOs or change both alleles because the flanking sequences of the 1st recombination will be prefered to exchange wiht new sequence → will stay Heterozygous

Method:
In vitro → electroporation of of cells → homologous recombination → selection with resistance marker → inject in a blastocyst

41
Q

How does CRISPR-Cas9 work?

A

Mdoern Genome Editing tool:
1. Introduce a dsDNA break at a specific target site → highly efficient
GuideRNA + Cas9 (cleaves)

Can do direclty to the zygote, no need for ESC injected in the blastocyst, can do on both alleles

42
Q

How many HEPA filters?

A

1 for air going into the hood + 1 for air going back to the ambient room

43
Q

What diseases are currently treated by bone marrow transplantation?

A
  1. Hereditary life threatening disorders of the blood and immune system:
    - Immunodeficiency
    - Blood clotting disorders
    - Anemias
    - Bone marro failures

PHGY 351 (3 decks)

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