1
Q
What are the functions of blood (4TIB)?
A
- Transport of O2, nutrients and metabolites
- Transport (removal) of wate products (CO2, lactic acid, urea)
- Transport of signaing molecules (eg. hormones)
- Thermoregulation
- Innate & adaptive response
- Blood clotting & wound repair
2
Q
How many lobes does each granulocyte have?
A
Monocyte: 1 horseshoe-shaped lobe
Basophil: Multiple lobes (2-3)
Eosinophil: 2 lobes
Neutrophil: 3 lobes
3
Q
- Which layer of the embryo gives rise to blood?
2. What progenitor cell is there and what cells does it give rise to?
A
- Mesoderm
- Hemangioblast, gives rise to hematopoietic stem cell and angioblast (which will give rise to vascular endothelial cells).
4
Q
What are the sites of hematopoiesis in the fetus, infant and adult?
A
Fetus:
- Ventral mesoderm
- Yolk sac
- Placenta
- Fetal liver
Infant:
- Thymus
- Bone marrow
Adult:
- Thyme (atrophies with age)
- Bone marrow (not all bones)
5
Q
What are the 2 key properties of hematopoietic stem cells (with definitions)?
A
- Multipotency: Ability to differentiate into all blood cell types
- Self-renewal: Maintain stem cell # and function throughout life
6
Q
What technique of analysis allows visualization of blood cells?
A
Blood smear
7
Q
What are the 3 sources of donor cells in bone marrow transplantation?
A
- Bone marrow
- Mobilized peripheral blood (donor must be treated with G-CSF before collecting mobilized stem cells from blood)
- Blood preservation from umbilical chord
8
Q
What is the difference between myeloablative and non-myeloablative allogeneic hematopoietic stem-cell transplantation and when is each treatment used?
A
Myeloablative: Strong radiation + harsh chemotherapy prior to bone marrow transplantation. Leukemia treated with all 3. Pancytopaenia, infection, organ toxicity.
Non-myeloablative: Used when patient would not be able to survive strong radiation and harsh chemotherapy. Only low-dose chemotherapy + bone marrow transplantation. Leukemia treated mostly with bone marrow transplantation. Mild pancytopaenia.
*In both cases, immunosuppression is required to minimize risk of graft rejection or graft vs host disease. Risk with both: GVHD and infection
9
Q
What are the 5 possible fates of stem cells?
A
- Apoptosis
- Quiescence (sit in G0 phase of cell cycle)
- Symmetric division into 2 stem cells (stem cell expansion)
- Symmetric division into 2 differentiated cells (differentiated cell expansion & stem cell depletion)
- Asymmetric division (one stem cell and one differentiated cell, hematopoiesis)
10
Q
What is the potential of the zygote, embryonic and adult stem cells?
A
Zygote: Totipotent (can differentiate into all cell types)
Embryo: Pluripotent (can differentiate into almost all cell types)
Adult: Multipotent (can differentiate into all cell types of a specific organ or tissue, unless plasticity)
11
Q
What is the potential of the zygote, embryonic and adult stem cells?
A
Zygote: Totipotent (can differentiate into all cell types)
Embryo: Pluripotent (can differentiate into almost all cell types)
Adult: Multipotent (can differentiate into all cell types of a specific organ or tissue, unless plasticity)
12
Q
What is plasticity?
A
The ability for an adult stem cell of a specific tissue/organ to give rise to specialized cell types of another tissue/organ,
13
Q
Where are embryonic stem cells located (describe that location)?
A
In the inner cell mass (ICM) of the blastocyst, which consists of:
- Blastocoel (hollow cavity inside the blastocyst)
- Trophoblast (cell layer surrounding the blastocyst, will become the placenta)
- ICM (group of cells at one end of the blastocoel)
14
Q
What are the 4 differences between the embryonic and the adult stem cell?
A
Embryonic VS adult:
- Rises from the embryo VS rises from adult tissue
- More primitive VS organ/tissue specific
- Pluripotent (can form most cell types) VS multipotent (can for cell types of a specific tissue/organ except if plasticity)
- Abundant, easy to grow VS rare in tissue, difficult to isolate
15
Q
Differentiated cells or adult stem/progenitor cells can be reprogrammed to form induced pluripotent stem cells (iPSCs). What are the 7 steps involved in cellular reprogramming?
A
- Inhibition of somatic cell regulators
- Activation of pluripotent loci
- Acquisition of transcription factor independency
- Complete reprogramming
- Induction of cell proliferation
- Inhibition of senescence and apoptosis
- Immortalization
- iPSC formation from differentiated cells is slower and less efficient compared to formation from stem/progenitor cells
16
Q
What are 3 differences in the use of ESCs and iPSCs?
A
ESCs vs iPSCs:
- ESCs: low cost VS iPSCs: additional cost
- ESCs: difficult obtention VS iPSCs: easy to obtain
- ESCs: embryo destruction VS iPSCs: no ethical issue
17
Q
How can iPSCs be used in therapy and research?
A
- Take somatic cells from a patient (e.g. leukemia patient)
- Cell reprogramming > iPSCs
- iPSCs cell cultivation
- Directed differentiation OR in situ repair of disease-causing mutation prior to differentiation > suitable differentiated cells
- Drug screening, disease model or mechanism, cell therapy (pre-clinical animal model prior to treatment of patient)
- Example of cell therapy: Take patient’s skin cells, cell reprogramming to form iPSCs, directed differentiation of iPSCs into RPE sheet cells, autologous RPE transplant
18
Q
How do we know that satellite cells (muscle stem cells) give rise to myofiber nuclei?
A
One can transplant a single myofiber (which itself has several satellite cells and myonuclei) into a host muscle and damage that host muscle. As a result, numerous donor-derived cells repopulate the host muscle, suggesting that donor satellite cells give rise to myonuclei.
Visualization of cells with fluorescently-tagged Abs against cell-specific markers.
19
Q
What is the role of Lbd1 (lim binding domain 1) in hemangioblasts?
A
Lbd1 interacts with a series of TFs and is therefore involved in stem cell development. When KO Lbd1, reduced number of hemangioblasts and no HSCs, but still angioblasts.
20
Q
What is the role of Lbd1 (lim binding domain 1) in hemangioblasts?
A
Lbd1 interacts with a series of TFs and is therefore involved in stem cell development. When KO Lbd1, reduced number of hemangioblasts and no HSCs, but still angioblasts.
21
Q
Describe the experiments conducted by Till & McCulloch (1960s) in their investigation of stem cells, clonality and hierarchical organization of hematopoiesis.
A
- Took bone marrow cells form the femur of a mouse
- Injected IV those cells in an irradiated mouse (meaning that mouse wasn’t able to generate its own cells)
- Cell colonies of variable size grew in the spleen, most of the cells being differentiated cells and some of them being undifferentiated
- Observed a linear and + correlation btwn the number of bone marrow cells administered and the # of cell colonies
- Abnormal karyotypes among CFU-S from donor bone marrow was used to prove clonality
22
Q
What are the steps from quiescent HSC to differentiated cell?
A
- HSC is activated and either divides symmetrically into 2 multipotential progenitor ( = multipotent precursor) cells or 2 quiescent HSCs or asymmetrically into 1 multipotent progenitor cell and one quiescent HSC.
- Multipotential progenitor cells divide into lineage-restricted progenitors.
- Lineage restricted progenitors divide into differentiated stem cells.
23
Q
What are the steps from quiescent HSC to differentiated cell?
A
- HSC is activated and either divides symmetrically into 2 multipotential progenitor ( = multipotent precursor) cells or 2 quiescent HSCs or asymmetrically into 1 multipotent progenitor cell and one quiescent HSC.
- Multipotential progenitor cells divide into lineage-restricted progenitors.
- Lineage restricted progenitors divide into differentiated stem cells.
24
Q
What are the 2 HSCs’ niches and what are regulatory entities of those niches? What are regulators of stem cell expansion?
A
2 niches:
- Endosteal niche: At extremity of bone marrow, further away form blood vessels, hypoxic environment, maintained quiescent by other players
- Vascular niche: Closer to blood vessels (in the bone marrow), less hypoxic environment, activated and differentiated by other players
Regulatory entities:
- Growth factors
- Endothelial cells
- Osteoblasts
- Stromal fibroblasts
- ECM components
Regulators of stem cell expansion:
- Cell cycle regulators
- Signaling pathways
- TFs
25
What are the 2 HSCs' niches and how are HSCs regulated?
2 niches:
1. Endosteal niche: At extremity of bone marrow, further away form blood vessels, hypoxic environment, maintained quiescent by other players
2. Vascular niche: Closer to blood vessels (in the bone marrow), less hypoxic environment, activated and differentiated by other players
Regulators of HSCs:
- Growth factors
- Endothelial cells
- Osteoblasts
- Stromal fibroblasts
- ECM components
26
What are 3 characteristics of quiescent HSCs?
1. Hypoxic niche
2. Adhesion to niche
3, Protection from stress:
- High levels of ROS scavengers
- Glycolytic metabolism under hypoxia
- High efflux ability
27
Explain the process of therapeutic cloning and its advantages.
1. Take somatic cell from patient and isolate nucleus.
2. Take a donor oocyte and remove the nucleus.
3. Insert the somatic cell's nucleus into the enucleated donor oocyte.
4. Activation to generate an embryo.
5. Isolate ICM from blastocyst to establish embryonic stem (ES) cells with patient immuno-compability
6. Differentiate the ESCs into specific tissue lines (e.g. pancreas, neurons, etc.)
7. Inject back into the patient
Advantages:
Circumvent donor-host immunorejection and disease transmission complications
28
How is a colony forming assay done?
1. Isolate cells from the bone marrow or the fetal liver and dilute them.
2. Plate the cells onto a semisolid medium containing growth factors.
3. Let the cells grow and assess the number of colonies, their size and their morphology.
* You can identify different colonies depending on the growth factor that was added to the mix.
29
What are the 5 roles of hematopoietic growth factors (e.g. G-CSF)?
1. Proliferation
2. Survival
3. Maturation induction
4. Differentiation commitment
5. Function stimulation
30
Explain the action of KITLG (a GF) upon binding to its receptor KIT.
- KIT: Extracellular domain, transmembrane domain, intracellular domain w/ thyrosine kinase activity
- Extracellular domain has 5 domains, 3 of which are involved in binding KITLG, causing dimerization of the last 2
- When KITLG binds KIT, it causes KIT to dimerize, activating intracellular tyrosine kinases, resulting in downstream signalling for survival, proliferation and alteration of gene expression.
31
What do gain-of-function mutations of KIT cause and how can that be treated?
Gain-of-function mutations of KIT result in different blood cancers. Treatment can be done with an inhibitor of KIT's enzymatic activity (tyrosine kinase activity). However, the enzymatic site can mutate to develop a resistance to the drug.
32
What are the roles of KIT in hematopoiesis?
HSCs and early hematopoietic cells:
Survival and proliferation
Mast cells and dendritic cells:
Fully differentiated mast cells and dendritic cells express high levels of c-KIT for proliferation and survival
Erythroid cells:
Promotes erythroid colony formation, but no total dependancy on KIT for erythropoiesis
33
What is the difference between the instructive and the permissive (stochastic) roles of GFs?
Instructive role: GFs transmit specific signals to stem cells or multipotential hematopoietic cells, instructing their lineage commitment and differentiation.
Permissive role: Lineage commitment and terminal differentiation are determined intrinsically and GFs only provide permissive growth and survival signals.
34
Describe the study using Epo to prove the permissive role of GFs.
Method: They mutagenized mice to make Epo KO mice and used WT mice as a control.
Results: Less fetal liver cells in KO mice, but more B-FUE and C-FUE in KO mice than in WT mice.
Conclusions: B-FUE and C-FUE are erythroid committed progenitor cells. Lineage commitment does not require Epo since erythroid committed progenitor cells are still present in KO mice. Epo is only required for these cells to grow into mature RBCs. Therefore, Epo has a permissive role, with lineage commitment and terminal differentiation determined intrinsically in HSCs and Epo only allowing permissive growth and survival of cells.
35
Describe the study using GM (granulocyte/macrophage) progenitors to prove the instructive role of G-CSF and M-CSF (Lecture 2, S.22).
Method:
- Counted the % of granulocyte and monocyte colonies in a mix of cells.
- Treated GM progenitors with M-CSF and counted the % of macrophage colonies.
- Treated GM progenitors with G-CSF and counted the % of granulocyte colonies.
Results: When treating GM progenitors with M-CSF or G-CSF, 90% of the resulting colonies where macrophages and granulocytes, respectively.
Conclusion: The small % of macrophage and granulocyte colonies counted initially = maximal % of colonies rising from lineage restricted progenitors
Difference between the two % values obtained for macrophages corresponds to the minimal instructive M-CSF effect. Same for granulocytes.
G-CSF and M-CSF have an instructive role.
36
What other experiments support the permissive and instructive roles of GFs, respectively?
Permissive: Lack of specificity in signalling by cytokine receptors (e.g. prolactin receptor can replace EPOR).
Instructive: If induce expression of exogenous IL-2BR or GM-CSFR in lymphoid progenitors and treat them with IL-2 and GM-CSF, induce reprogramming of lymphoid progenitors into granulocytes and macrophages, respectively.
37
What is the role of Ikaros (sync finger progenitor) in hematopoiesis and cancer?
Hematoopoiesis:
Ikaros is a zing finger TF required for the differentiation of HSCs into lymphoid progenitors.
- Promotes differentiation
- Inhibits proliferation
- Controls migration and adhesion
- Regulates gene expression via chromatin remodelling
Method: If knock out Ikaros, no thymus. Flow cytometry using CD4 and CD8 as markers for T cells and C45R and IgM as markers for B cells.
Result: No B nor T cells
Conclusion: Ikaros is required for HSC to become lymphoid progenitor cells.
Cancer:
Tumor suppressor. When inhibited via phosphorylation by CK2, cancer (leukemia). Treat with an inhibitor of CK2 such that Ikaros can function as a tumor suppressor. Loss-of-function mutation in Ikaros associated with cancers.
38
Explain the cross-antagonism TF model using C/EBP and FOG as an example.
Cross-antagonism TF model:
2 TFs are present in equal amounts in an uncommitted cell. When one takes over by a stochastic event, the cell commits to a specific lineage (different from the cell lineage specified by the other TF).
C/EBP and FOG:
FOG is a co-repressor of the TF GATA, which is responsible for the commitment of progenitor cells to the eosinophil lineage, and C/EBP is a repressor of FOG.
When C/EBP take over, it represses FOG and the uncommited cell commits to the eosinophil lineage.
When FOG is over-expressed, it represses GATA and the cell returns to a multipotential precursor phenotype.
C/EBP KO: No granulocytes and only half monocytes
39
What characterizes anemia?
- Decreased number of RBCs > Low hematocrit
| - Decreased Hb concentration > Low Hb concentration
40
Define primitive versus definitive erythropoiesis.
Primitive: 1st transient embryonic wave of hematopoiesis that produces mostly RBCs, presumably to provide maximal O2 to the embryo. Occurs in the YS.
Definitive: Independent waves of hematopoiesis that produce all types of blood cells, occurs first in YS, then in fetal liver and finally, in bone marrow.
41
Name 7 differences btwn primitive (EryP) and definitive (EryD) erythropoiesis.
1. Erythrocytes from EryP are macrocytic.
2. EryP occurs in YS only.
3. In EryP, erythropoiesis occurs in YS up to ProE stage. Maturation from ProE to mature RBC occurs in circulation. In EryD, erythroid precursors exit bone marrow only at reticulocyte stage.
4. In EryP, enucleation of reticulocytes doesn't occur before several days. In EryD, enucleation occurs before reticulocytes exit the bone marrow.
5. Erythrocytes from EryP express mostly embryonic globins (higher O2-carrying capacity)
6. When cultured, erythroblasts from EryD are not able to self-renew.
7. Different requirements in cytokines, TFs and signalling pathays.
42
1. Do reticulocytes have mitochondria?
| 2. Are reticulocytes and mature RBCs present in the bone marrow?
1. Yes
| 2. Yes
43
How does the EPO signalling pathway work?
EPO binds to its receptor EpoR, causing its dimerization, activating its intracellular tyrosine kinase activity, causing phosphorylation of kinases like JAK2 and STAT5, leading to downstream signalling resulting in activation of TFs that promote proliferation, survival and differentiation (although Epo has a permissive role).
44
How does HIF-1a regulate EPO production and therefore, erythropoiesis, under hypoxic conditions? What happens under normoxic conditions?
Hypoxia: HIF-1a expression increases, it binds the promoter of the EPO gene, inducing its expression. EPO circulating level rises, it bind EpoR in the bone marrow, stimulating erythropoiesis. Oxygen-carrying capacity of the blood increases, normoxia, HIF-1a level drops and so does EPO level, erythropoiesis no longer stimulated.
Normoxic conditions: O2 activates prolyl-hydroxylase and asparginyl-hydroxylase which respectively hydroxylate proline and asparginine residues of HIF-1a, respectively targeting the TF for degradation via the ubiquitin-proteasome pathway and preventing it from binding its p300-coactivator.
45
How does EPO promote erythropoiesis upon binding EpoR?
1. Increased intracellular Ca2+
2. Increased DNA synthesis
3. Increased transferrin expression (increased iron transport)
4. Increased hemoglobin synthesis
5. Increased synthesis of integral membrane proteins
46
How can EPO be detected? How can it be detected in athletes?
EPO detection via ELISA or RIA (radioimmunoassay):
1. Immobilize a capture antibody against EPO in 96-well plate
2. EPO (from urine sample for instance) binds capture Ab.
3. Detection Ab binds EPO.
4. Horseradish peroxidase binds detection Ab.
5. Tetramethybenzidine substrate binds horseradish peroxidase and turns dark blue.
6. Spectrophotometry
The more EPO, the darker the color.
In athletes:
Urine sample
Electrophoresis
Immunoblot
47
What happens to the oxyHb dissociation curve when exercising?
Shifts to the right, increased p50, lowered affinity for O2.
That is because:
- Increased CO2 in tissues, CO2 diffuses into RBCs, combines with H2O to form H2CO3 (catalyzed by carbonc anhydrase) which dissociates into bicarbonate and H+, H+ binds to Hb and decreases its affinity for O2, releases O2 to tissues.
- Acid lactic production, lowered pH.
- Increased T.
48
What is 2,3-BPG with regard to Hb and how/when does it shift the oxyHb dissociation curve?
2,3-BPG is a heterotropic allosteric regulator of Hb, meaning it modulates the activity of Hb by binding it (although not at the enzyme active site) and since it is not also a substrate of Hb like O2 (homotropic ligand), it is a heterotropic regulator.
2,3-BPG is produced under hypoxia (e.g. high altitude) and shift the oxyHb dissociation curve to the right, higher p50, lower O2 affinity, greater O2 release to tissues.
49
What happens inside RBCs regarding CO2 and O2 at the level of the lungs?
In the lungs, low CO2. CO2 diffuses out of the RBCS (instead of into the RBCs like in the tissues), favouring H2CO3 dissociation into CO2 and H2O (carbonic anhydrase), in turn favouring association of H+ and HCO3- into H2CO3. H+ dissociates from Hb and O2 associates with Hb, driving O2 into the RBC.
50
What is methemoglobin, what gives rise to it and what is its effect?
Methemoglobin: Hemoglobin undergoes auto-oxidation that oxidizes F2+ to Fe3+. It is usually reduced back to Fe2+ by normal RBC metabolism and Fe2+ is often protected form oxidation by globin chains.
However, mutations can cause MethHb to be > 1%:
- Mutation in histidine residue in globin chain involved in binding Fe that stabilized Fe3+
- Mutation in cytochrome b
- Mutation in NADH-cytochrome b15 reductase
Effect: Fe3+ cannot bind O2, but it increases O2-binding affinity in other Hb subunits of the same Hb molecules.
Because of the Fe3+ monomer that cannot bind O2 in MetHb, results in cyanosis (like low-affinity Hb).
51
What is the effect of CO inhalation (smoking) on oxyHb dissociation curve?
Hb has a much higher affinity for CO than O2 (200 fold). Upon binding Hb. CO increases Hb affinity for O2, lower p50, shifts curve to the left.
52
What are quantitative and qualitative Hb defects?
Quantitative defects: a- or B-thalassemia
Qualitative defects:
- High-affinity variants: Comes with compensatory erythropoiesis, leading to erythrocytosis (increased RBC production)
- Low-affinity variants: Cyanosis
- Methemoglobin: Cyanosis
- Polymerizing Hb: sickle cell disease
PHGY 313
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