Blood Flashcards
What are the different functions of blood?
- Transport of O2, nutrients (Glucose) & metabolites (ATP)
- Removal of waste products (urea, CO2, lactic acid)
- Transport of signaling molecules
- innate & adaptive immune responses
- Blood clotting & wound repair
- Thermoregulation
How does blood circulation change in cold vs warm external conditions?
Warm conditions → Blood goes everywhere
Cold conditions → Blood flow is restricted to core organs
*You lose ~ 10-15% of heat by the head
How many blood cells are produced every day?
*Very dynamic process!
> 100 billion blood cells produced/day
Increased ~5-10-fold due to injury or infection
What is the main composition of blood (proportions)
*Centrifuge to visualize
55% Plasma
< 1% Buffy coat (leukocytes and platelets)
45% Erythrocytes
*In leukemic patient, the buffy coat is largely expanded
What are the standard units for blood parameters?
/L
Traditional US units = /mm3
What are the components of plasma?
7% Proteins
91% Water
2% Other solutes (Ions, Nutrients, Waste products, Gases, Regulatory substances)
What are the different plasma proteins?
57% Albumin
38% Globulins
4% Fibrinogen
1% Prothrombin
What are the different leukocytes in blood and their proportion?
60-70% Neutrophils
20-25% Lymphocytes
3-8% Monocytes
2-4% Eosinophils
0.5-1% Basophils
How many RBCs, Platelets and WBCs are found /mm3 of blood?
And main features.
RBCs ~ 5e6/mm3
- O2 transport, Contain Hemoglobin
- Lack nuclei & most organelles
Thrombocytes ~ 2.5e5/mm3
- Blood clotting, Cell fragments (originating from megakaryocytes), 2-3um diameter
- Lack nuclei & most organelles
Leukocytes ~ 7e3/mm3
- Immune and inflammatory response
- Granulocytes (except for lymphocytes?)
What are the physical characteristics of Erythrocytes, Platelets and Lymphocytes?
Erythrocytes:
- Biconcave shape
- 7um diameter
Platelets:
- Cell fragments → 2-3um diameter
Lymphocytes:
- Large round nucleus
- No granules
- 10um diameter
What are the physical characteristics of the different granulocytes?
Neutrophils:
- Multi-lobed nucleus
- Large number of cytosolic granules
- 10um diameter
Monocytes:
- Horshoes-shaped nucleus
- Cytosolic granules
- 10-25um diameter
Eosinophils:
- Nucleus with 2 lobes
- Cytosolic granules
- 10um diameter
Basophils: (appears more purple)
- Nucleus with 2-3 lobes
- Cytosolic granules (less)
- 10um diameter
What is the life span of the different blood cells?
RBCs ~ 120 days
Platelets 7-8 days
Monocytes ~ 3 days
Basophils ~ 60-70h
Neutrophils ~ 5-90h (inactive), 24-48h (active)
Eosinophils ~ 8-18h
T/B-lymphocytes ~ variable
What different cell types are found in bone marrow?
Hematopoietic cells
- Osteoblasts
- Osteoclasts
- Vascular endothelial cells
- Mesenchymal stem cells
- Sympathetic neurons
- Adipocytes (more in older people)
Which embryonic layer is blood derived from?
What is progenitor that gives rise to blood and vascular endothelium?
Mesoderm
Hemangioblasts → Hematopoietic cells + Endothelial cells
Where is blood made during mice gestation period?
Gestation ~ 20 days
Until d7.5 → Yolk sac + blood islands
d7.5-13.5 → AGM (aorta-gonads-mesonephros)
d10.5 until birth → Fetal Liver
d13.5 → Thymus makes T lymphocytes
After birth → bone marrow for all except T lymphocytes, in the thymus
Where is hematopoiesis localized in human embryos at different stages of gestation?
0-2 months fetus → yolk sac
2-7 months fetus → Liver + Spleen
5-9 months fetus → Bone marrow
Infant → Bone marrow (practically all bones)
Adults → Bone marrow (vertebra, ribs, sternum, skull, sacrum and pelvis, proximal ends of femur
*Mostly in the spongy bone at the ends of long bones
What are the main steps of the hierarchical organization of hematopoiesis?
- Stem cells
- Multipotent progenitor cells
- Committed progenitor cells
- Mature cells
*There is plasticity between progenitors
*There are long term and short term repopulating stem cells
Who showed the existence of stem cells?
McCulloch and Till (1961)
What are the 2 key properties of Hematopoietic stem cells? (HPSCs)
*Of all stem cells
- Multipotency → ability to differentiate into all blood cell types
- Self-renewal → maintain stem cell number and funciton throughout life
What lab methods are used to study cell count?
Manual count
Flow cytometry (based on specific expressed markers)
What lab methodologies can be used to study blood cells?
- Blood smear/film (staining)
- Cell count (manual, flow cytometry)
- PCR (DNA)
- Immunoblot, IHC (proteins)
- Erythrocytes sedimentation rate (ESR)
- Gene expression microarrays & next generation sequencing (can distinguish pathologies by looking at gene expression)
What can a high ESR vs a low ESR be indicative of?
*Measure sedimentation over 1h and look how fast RBCs come down
Abnormally high erythrocyte sedimentation rate → infection which causes clumping of RBCs
Abnormally low erythrocyte sedimentation rate → abnormal cells shape
What are the components of a Flow cytometer or FACS machine?
- Forward scatter detector (SIZE)
- Side scatter detector (GRANULARITY)
- Fluorescence detector
- Filters and mirrors
- Charged deflection plates
*FACS = fluorescence activated cell sorting?
Looking at the side scatter of a flow plot, which cells would be the highest vs lowest?
Highest → Lowest:
1. Granulocytes
2. Monocytes
3. Lymphocytes
*RBCs are lysed in tissue prep
How can you assess a single/multiple parameter(s) in Flow Cytometry?
Histogram (single)
*Always compare to isotype control (Ab of the same Ig family, but specific for a protein that is not expressed in you sample)
Dot plot (multiple, 2)
What are the different sources of external donor cells for bone marrow transplantation?
- Bone marrow directly from bones of the donor
- Mobilized peripheral blood
- Donor treated with granulocytes colony stimulating factor (G-CSF); mobilized stem cells are collected from the donor’s blood - Cord blood form umbilical cord stored in public and private cord blood banks
- Limited numbers of stem cells, so mostly for children
What are 3 types sources for bone marrow transplantation?
(And the risks)
Allogeneic (from another donor)
- Risk of graft failure (immune rejection) and GVHD
- Requires immunosuppression
Autologous (self-donation)
- Complete immunological compatibility
- Low risks of failed engraftment & GVHD
- Not always a possible solution depending on problem
Synergeneic (identical twin)
- Complete immunological compatibility
- Low risks of failed engraftment & GVHD
- Still requires immune suppression?
What is myeloablative vs non-myeloablative allogeneic hematopoietic-stem-cell transplantation?
2 types of treatment for leukemia patients
Myeloablative:
1. Total body radiation
2. Chemotherapy
*All cancer cells are dead
3. Transplantation + Immunosuppression
Non-myeloablative:
1. Low dose of chemotherapy
2. Low intensity radiation + transplantation + immunosuppression
*Cancer cells are not really killed, but good for patients that would not survive a full body radiation
What is the case of the Berlin patient?
Cured HIV & Leukemia as he was donated cells without CCR5 (HIV receptor)
What are different possible fates of a stem cells?
What is the purpose of each fate
- Symmetric division into 2 stem cells (stemm cell expansion)
- Asymmetric division into 1 stem & 1 differentiated cells (homeostasis)
- Symmetric division into 2 differentiated cells (meture cell expansion and stem cell depletion)
- Quiescence (most of the time stem cells are in quiescent state)
- Apoptosis (rare, in most cases, stem cells stay your whole life)
What phase of the cell cycle do stem cells spend the most time in?
G0 → quiescent state
Which are the cells with the highest repopulation activity?
HEMATOPOIETIC stem cells
- But very rare and quiescent (G0)
Is differentiation in stem cells reversible in different organisms?
- Unidirectional towards differentiation (mammals 1st hypothesis)
- Balanced, return to stemness from differentiated state is limited, but possible (mammals 2nd hypothesis)
Reversible → Plants and Amphibians
What is a Zygote? What type of cell is it?
Zygote = fertilized egg
Totipotent cells → can give rise to ALL cell types
What are the differences between embryonic stem cells and adult stem cells?
Embryonic stem cells:
- Pluripotent cells
- Derived from blastocyst (embryo)
- More primitive
- Can form most cells types
- Abundant and easy to grow
Adult stem cells:
Multipotent cells → organ/tissue-restricted
ex: Hematopoietic stem cells (blood), satellite cells (muscle), neural stem cells (brain)
- Derived from adult tissue
- More organ specific (can form only few cell types)
- Rare in tissues, more difficult to isolate and grow
What are the different types of stem cells?
- Totipotent
- Pluripotent (Embryonic stem cells)
- Multipotent (tissue/organ restricted, adult stem cells)
- In vitro → induced Pluripotent Stem Cells (reprogramed)
What is plasticity of stem cells?
Plasticity is the ability of an adult stem cell from one tissue to generate the specialized cell type(s) of another tissue
*Requires reprogramming → some studies show clear evidence that some adult stem cells are capable of being genetically reprogrammed
What is a blastocyst?
Blastocyst = 4-5 days in human // 3.5 days in mice
Trophoblast → layer of cells that surround the blastocyst, from trophoectoderm, will become the placenta
Blastocoel → hollow cavity inside the blastocyst
Inner cell mass → group of cells at one end of blastocoel
*ALL embryonic stem cells
In what cases does slow vs fast reprogramming occur?
Differentiated cells → Sow reprogramming/Low efficiency (more steps to go back to iPSC)
*Can’t do with RBCs because they don’t have a nucleus anymore
Adult Stem cells → Fast reprogramming/high efficiency (less steps to iPSC state)
What are the steps from Somatic cells → “mature” iPSCs?
- Somatic cells → inhibit somatic regulators that make up cell signature
- Early intermediates → activation of pluripotency loci → Late intermediates
- Acquisition of factor independence → “immature” iPSCs (4 Yamanaka TFs)
- Complete reprogramming → “mature” iPSCs
- Telomeres kept at proper length/elongated
What are differences between iPSCs and ESCs?
- X chromosome can’t be reactivated in iPSCs
- Some epigenetic modulations are maintained in the iPSCs from their somatic cell type
What are PROS and CONS of ESC?
PROS:
- Low cost
- Established and characterized
- Efficient differentiation
- MHC downregulation is possible
CONS:
- Embryo destruction
- Limited: blastocyst day 5-14
- Difficult to obtain
- Carcinogen risk, mutation rate
- Tissue rejection, incomplete histories
What are PROS and CONS of iPSCs?
PROS:
- No ethical issues
- Any cell types (in theory)
- Easy to obtain
- Blood compatibility, HLA histocompatibility
- Disease modelling possible, drug/toxicity profiles possible
CONS:
- Additional cost
- Retroviral gene delivery → Oncogene activation risk, mutagenesis risk, retention of alterations
What are the 4 reprogramming TFs?
- Oct4
- Sox2
- Klf4
- C-Myc
What are 2 examples of clinical trials using iPSCs
- Macular degeneration:
Skin cell → iPSCs → RPE (retinal precursor sheet) → transplant - Self renewal of satellite cells:
- transplanted satellite cells in an irradiated muscle → these transplanted cells were able to repair a muscle injury
- 3 weeks after single myofiber transplantation, donor-derived cell repopulated the host muscle - Current studies on neural stem cells in mice hippocampi
What are the major clinical uses of HSCs?
- Bone marrow transplantation
- Gene therapy
Which signaling pathway regulates several aspects of the specification of subsquent expansion of HSCs?
Smad signaling pathway:
from
Which transcription factor plays a crucial role in hemangioblasts differentiation?
Ldb1
- Ldb1 KO affects regulation of multiple genes and other TFs
- Ldb1 KO have impaired development from Enbryoid body —> hemangioblast
- Ldb1 KO have impaired endothelial cell differentiation from hemangioblasts
- Ldb1 KO are incapable of differentiating into CD41+ primitive hematopoietic progenitors
What is Flk1? BL-CFC? Ldb1?
Ldb1 = Lim domain binding protein (TF)
Flk1 = receptor for vasuclar endothelial growth factor (VEGF)
BL-CFC = blast colony forming cells (hemangioblasts)
Hemangioblasts are Flk1+BL-CFC (used to isolate them by FACS)
- They require Ldb1 to properly differentiate
What experiment did Till & McCulloch do to prove the existence of stem cells and their clonality?
- Isolated bone marrow cells from femur of a mouse
- Irradiated host mouse (1x250rads)
- Intravenous injection of isolated bone marrow cells into an irradiated host mouse (same inbred strain)
- 2x325 rads in the injected mouse
Total doses:
- Host mouse: 900 rads (cells are dead)
- Injected donor bone marrow cells: 650 rads (cells can still proliferate, but are mutated) - At d10, they seeded the spleen:
- Colonies of variable size, containing differentiated blood cells and a small number of undifferentiated cells (self-renewal)
Results:
- Correlation between the # of cells injected and # spleen colonies
- Abnormal karyotypes among CFU-S from donor bone marrow was used to prove clonality (all cells from 1 colony/1 stem cell had the same mutations/karyotype)
*2 irradiations
*This proved their clonality and the hierachical organization of hematopoiesis
Are the markers for HSC lineage determination in mice and human the same or not?
What marker can be used to differentiate HSCs from multipotential precursor with electron microscopy?
NO, different markers for different species
Mitochondrial staining (Rh123) is only seen in multipotential procursor
What is the main site of hematopoiesis?
Bone Marrow of trabecular bone:
- Quiescent cells are found closer to the cancellous bone niche, farther from blood vessels Endosteal Niche
- More active cells go down closer to blood vessels to be sent into circulation Vascular Niche
What are different regulators of the stem cells niche in the bone marrow?
- Growth factors (soluble, membrane bound)
- Stromal fibroblasts
- Osteoblasts
- Endothelial cells
- ECM components
*They modulate the cytoskeleton, the binding of HSC to the bone
What are the different specific molecules/interactions that regulate HSC proliferation?
Niche - HSC:
SCF - c-kit
*They interact together
What are the different specific molecules/interactions that regulate HSC quiescence and sefl-renewal at the niche?
Niche - HSC:
Ang-1 - Tie2
N-Cad - N-Cad
Jagged-1 - Notch-1
What are the different specific molecules/interactions that regulate HSC homing/adhesion at the niche?
Niche - HSC:
SDF-1 - CXCR4
VCAM - Integrin
What are 3 different molecules that are used to promote HSC mobilization in the blood?
Mobilization = migration from the bone marrow niche → blood stream
G-CSF (most common) = 4-5 days
CXCR4 inhibitor = 6-9 hours
VLA-4 inhibitor = hours
What are 3 types of regulation of stem cell expansion?
- Signaling pathways
- Transcription factors
- Cell cycle regulators
What are 3 main characteristics of quiescent hematopoietic stem cells?
- Protection from stress
- Bone shields from UV light and radiation
- High activity of ABC transporter (ABC-G2)
- High activity of aldehyde dehydrogenase
- Regulation of O2 radical stress - Adhesion to the niche
- Low requirement for growth factors
- Anti-apoptotic machinery
- Stem cell/niche interaction (Cadherin/catenin, integrins)
- Slow cycling (Tie2, p21) - Hypoxic niche
- Osteoblastic zone of trabecular bone
- Low oxidative phosphorylation
- Anaerobic metabolism
What are ethical, legal and social issues with stem cells?
Embryonic, adult stem cells and iPSCs → potential to develop into many different cell types, tissues, organs
Ethical controversy: source of stem cells (in case of embryos)
Cloning:
- Reproductive cloning (ex: Dolly the sheep)
- Therapeutic cloning (for cell transplant into same persone who donated somatic cell nucleus)
Legal issues:
What is our definition of “embryo”, “fertilization”, “conception”??
What is the major federal Canadian funding agency for medical research?
Canadian Institute of Health Reseach (CIHR)
What are different general aspects of hematopoietic differentiation?
Stem Cells → Mature Blood Cell:
- Differentiation
- Lineage commitment
- Cell proliferation
- Cell survival
What did Till & McCulloch’s experiment showed?
- Stem cells & clonality
- Hierarchical organization of hematopoiesis
Why are stem cells from younger donors better?
Stem cell population is more polyclonal → more different types can differentiate
Older donor stem cells have accumulated more mutations
What are the different stages of earliest hematopoietic progenitors?
- Long-Term HSC
- Short-Term HSC
- Multipotent progenitors
- Myeloid (→ common myeloid progenitor) vs Lymphoid (→ common lymphoid progenitor)
*They can both give rise to dendritic cells
Lymphoids → B cells, T cells, NK cells
Myeloids → Erythrocytes, Platelets, Macrophages, Neutrophils, Eosinophils, Basophils
What is the difference between fetal and adult lineage development in the redefined model?
Fetal → HSCs come down as multipotent
- Some differentiate into CMPs, others stay multipotent
- Some differentiate into CLPs, others stay multipotent
*Not one single point in development where everyone choses
Adult → HSCs come down as unipotent
- They are fixed much earlier in development, less plastic
What is the definition of a Growth factor (Cytokine)?
It is a protein that promotes the proliferation of cells
Explain the colony forming assay.
- Isolate cells from bone marrow or fetal liver
- Dilution of cells
- Plating into semisolid medium (methylcellulose) containig growth factors
- Each cell spaced out enough - Incubate → colony differentiation
→ Proliferation, differentiation of hematopoietic progenitor populations - growth colonies (1-2 weeks) and assessment of number of colonies, size and morphology
What are different colonies found in the colony forming assay?
HSC → CMP + CLP
CMP →
CFU-GEMM (colony forming unit that generates myeloid cells) → very large population as they give rise to so many different types of cells
BFU-E (Burst-forming unit-erythroid)
- first progenitor cell stage that is unipotent and committed to the erythroid lineage
CFU-E (colony forming unit erythroid)
CFU-GM
CFU-G (granulocytes)
CFU-M (macrophages)
What are the multiple roles of G-CSF? (of hematopoietic growth factors in general)
- Survival
- Proliferation
- Differentiation/Commitment
- Maturation/Induction
- Functional/Stimulation
*Not just proliferation
What is the structure of the KIT receptor?
What is its ligand?
Ligand = SCF (stem cell factor), KITLG
Ig-like extracellular domains:
D1-3: Ligand binding (Bind SCF)
D4-5: Dimerization (When KIT is bound to SCF, they dimerize)
Intracellular Tyrosine Kinase Domain → transphosphorylate
What are the 2 main isoforms of KIT?
GNNK-
- Anchorage independent growth
- Loss of contact inhibition (focus formation)
- Tumorgenicity
- More rapid and extensive tyrosine autophosphorylation and rapid internalization
GNNK+
- Colony formation but poor focus formation
- No tumorgenicity
*GNNK = sequence between the membrane and D5 (extracellular)
In what cells is the KIT receptor found?
Which intracellular signaling pathways are activated via c-kit?
60-75% of CD34+ cells
- 1-4% of bone marrow cells
Activated intracellular signaling pathways:
PIP-2 → PKC → Alter gene expression
AKT → Survival
SOS/RAS → MAPK → Proliferation
*Negative regulation (termination of signaling) via phosphatase such as SHP-1, SHP-2 and SHIP
What are the different roles of KIT in hematopoiesis?
- HSC and early hematopoietic cells
→ proliferation and survival (in synergy with other growth factors) - Mast cells and dendritic cells
→ Fully differentiated cells maintain high levels of c-Kit expression
→ Proliferation and survival function - Erythroid cells
→ Promoter of erythroid colony formation, but not total dependency on KITLG (SCF) for erythropoiesis
What are other roles of KIT which are not related to hematopoiesis?
Pigmentation → proliferation, survival and migration of melanocytes
Reproduction → protection of germ cells
Gastrointestinal tract → motility
Nervous system → Spatial learning
Cardiovascular system → Cardiac stem cell and cardiomyocyte terminal differentiation
Lung → maintaining lung tissue integrity
What are some pathologies related to different mutations in KIT?
GAIN of function mutation in different c-kit domains:
Ligand binding domain → idiopathic myelofibrosis (bone marrow replaced by fibrous cells)
Extracellular Ig domain → AML
Transmembrane domain → acute leukaemia
Juxtamembrane region → gastrointestinal stromal tumors (GIST)
Phosphotransferase domain → mastocytosis mas cell leukemia (expansion of mast cells)
What is the importance/role of Gleevec/Imatinib?
It is a therapeutics to treat c-kit mutaton pathologies
- Originally developed for inhibition of BCR-ABL oncogenic fusion protein
- Inhibitor of enzymatic (tyrosine kinase) activity of KIT; treatment of chronic myeloid leukemia, GIST
- Mutations of enzymatic site confer resistance to Gleevec/imatinib → solved by using very similar drugs binding to slightly different sites (Midostaurin, Avapritinib)
Mechanism:
It minds to the binding site of ATP on the tyrosine kinase resides → Substrate can’t be phosphorylated using the phosphate from ATP
What does it mean for a growth factor (cytokine) to be permissive vs instructive?
Instructive (inductive) model:
Growth factors transmit specific signals to stem cells or multipotential hematopoietic cells, directing their lineage commitment and differentiation
- Role in lineage commitment
Permissive (stochastic) model:
Lineage commitment and terminal determination are intrinsically determined with cytokines (growth factors) providing only permissive growth and survival
→ increase proliferation potential & survival in stem cells and progenitors
*Till & McCulloch → self-renewal and differentiation occur as probabilistic/stochastic/permissive events
- Follows random distribution
Is Epo a permissive or instructive growth factor/cytokine?
Epo KO animals have committed erythroid progenitors (CFU-E, BFU-E), but no mature erythroid cells → permissive role
*no/minor role in lineage commitment
- Lack of specificity in signaling by cytokine receptor
Epo -/- fetus have not RBCs in liver
What is the difference between RBCs coming from yolk sac and from the fetal liver?
Yolk sac → nucleated precursor
Fetal liver → non-nucleated precursor
What are the stages of erythropoiesis?
Early stage erythropoiesis:
HSC → MEP → BFU-E → CFU-E →
Terminal erythroid differentiation:
Pro-EB → Basophilic-EB → Polychromatic-EB → Orthochromatic-EB → RET → RBC
EPO responsiveness: BFU-E → Baso-EB
Hemoglobin synthesis starts at Basophilic-Erythroblast stage
*BFU-E = burst-forming unit erythroid
MEP = megakaryocyte-erythroid progenitor cell
EB = Erythroblast
RET = Reticulocyte
Is the role of G-CSF and M-CSF permissive or instructive? Why?
Instructive role
They direct cells to favour a specific lineage, not 100% required
What can be the effect of expression of exogenous IL-2bR and GM-CSFR in common lymphoid progenitors + IL-2 and GM-CSF treatment?
Leads to reprogramming into granulocytes & macrophages respectively → instructive
*Does not work for IL-7R or EPOR
What is Ikaros?
A zinc finger TRANSCRIPTION FACTOR
- Promotes differentiation
- Inhibits proliferation
- Controls migration and adhesion
- Regulates gene expression by chromatin remodelling
Ikaros deletions:
- 80% of BCR-ABL-positive ALL patients
- 5% of patients with T cell acute lymphoblastic leukemia
Which organ is missing in Ikaros KO mice?
The thymus
No more B cells or T cells
- Transcription of CD4, CD8, D45, IgM requires Ikaros
How does Ikaros function as a tumor suppressor?
It promotes transcription of factors that inhibit the cell cycle + inhibit PI3K pathway
- Inhibition of Ikaros activity by CK2→ leukemia
*CK2 phosphorylates (multiple times) Ikaros → increases its degradation and prevents it from binding DNA
What is the effect of knocking out C/EBPa in mice?
- Reduction in Monocytes
- Complete loss of Granulocytes
C/EBPa = CCAAT/enhancer-binding protein a
*Role in CMP → GMP differentiation
What is the cross-antagonistic transcription factor model?
Give an example.
- Uncommitted cell expresses both A and B genes which are 2 competing/opposing TFs
- Stochastic/Random event or Environmental factor → A or B to increase in number
- Causes lineage commitment
→ There is even more A to inhibit B and less B to inhibit A so exacerbates the effect
C/EBP & FOG:
1. C/EBP inhibits transcription of FOG (friend of GATA, more like its ennemy)
2. FOG inhibits GATA from transcribing EOS47
*Eosinophilic cell line: transcritpional repression of FOG by C/EBP
Overexpression of FOG: eosinophilic cell line reverts to multipotential precursor phenotype
What is the effect of overexpression of FOG?
It inhibits GATA from transcribing EOS47 → Eosinophilic cell line reverts to multipotential precursor phenotype
Which stages of erythroid differentiation have the following:
- Nuclear DNA
- RNA in the cytoplasm
- Mitochondria
- Occur in the marrow
- Occur in the blood
Nuclear DNA → only in the erythroblast
RNA in the cytoplasm → in erythroblast + reticulocyte
Mitochondria → in erythroblast + reticulocyte
They can all be found in the marrow
Only Reticulocytes and Mature RBC can be found in the blood
What are the main features of EPO? Where is its main site of production?
- 166aa including 27 leader peptide
- Glycoprotein
Mainly produced in the cortex of the kidney (peritubular interstitial cells)
- 2ndary site = liver, brain, spleen, lung, bone marrow
What is NESP?
Novel Erythropoiesis Stimulating Protein:
Hyperglycosylates erythropoietin → increases metabolic stability & half-life
What is the EPO-receptor like?
What are the downstream signaling pathways?
When binds EPO, it dimerizes which brings the 2 JAK2 intracellular domains to close proximity → they can transphosphorylate each other
Downstream pathways:
STAT5 → Survival/Proliferation (CyclinD1, CyclinD2, CyclinG2)
PI3K → AKT → Survival/Differentiation
ERK1/2 → Proliferation
What is the survival ability of the different KO mice in EPO signaling?
EPO KO → embryonic lethal at E13
EPO-R KO → embryonic lethal at E13
JAK2 KO → embryonic lethal at E12.5
STAT5 KO → perinatally lethal, erythroid phenotype (dye not long after birth)
What is HIF?
What is the difference in HIF between hypoxia and normoxia?
Transcription factor:
- basic HLH DNA binding domain
Most studied in mice → HIF-1a
Most common in humans → HIF-2a
*Constantly produced and degraded quickly in normoxia, but if needed, its there
Hypoxia:
- Stabilization
- Heterodimerization
- DNA-binding
- Transcriptional coactivation
Normoxia:
- Rapid degradation via Ubiquitin-proteasome pathway
How is HIF-1a regulated in normoxic conditions?
What happens specifically in hypoxic conditions?
Oxygen activates proplyl-hydroxylases (3) + asparagines-hydroxylase
Prolines 402 and 564 are hydroxylated → recognized by VHL E3 Ligase → inactivation by Ub-mediated proteolysis
Asn 803 is hydroxylated → blocks Asn 803 from being recognized by p300 co-activator
Hypoxic conditions:
*When p300 co-activator can recognize Asn 803 → transcription cascade:
HIF-1a translocates to the nucleus → dimerizes with HIF-1b → with the p300 and HIF-1b, binds to 3’ enhancers of the EPO gene
Other roles in hypoxia:
Erythropoiesis, Angiogenesis, Glucose metabolism, Iron transport, Extracellular matrix remodelling, Apoptosis
Which 2 important gene elements regulate EPO expression?
3’ enhancer (by HIF-1a)
Kidney inducible element: -14kb to -9.5 kb upstream of EPO gene
What are the EPO levels in relationship with Hgb levels?
What are normal Hgb levels?
As Hgb levels decrease → EPO levels increase exponentially
Normal levels of Hgb = 15g/dL
What is the relationship between EPO levels & Hematocrit?
Inverse Linear relationship: When hematocrit % is low, Serum EPO is high and inversely
Non-functional kidney → don’t see that linear relationship because can’t produce EPO when low hematocrit
What stages of Erythropoiesis are EPO responsive?
What is its effect?
BFU-E, CFU-E, Proerythroblasts, Erythroblasts
*NOT all stages
*Not mature RBCs, EPO promotes differentiation
Effect of EPO-EPOR interaction:
- Increase intracellular Ca+
- Increase Hemoglobin synthesis
- Increase transferrin receptor
- Increase in synthesis of integral membrane proteins
- Increase DNA synthesis
*EPO functions as a loop
*EPO production and signaling pathways in tumor growth (often cancer patients are anemic, tried teating them with EPO, but EPO promotes angiogenesis and growth factor VEGF)
At what stage of Erythropoiesis do Hemoglobin starts being expressed?
What about c-kit?
Hemoglobin: Proerythroblasts
c-kit: Pluripotent stem cell up to proerythroblast
What is an important assay for EPO detection?
Radioimmunoassay (RIA)
ELISA (enzyme-linked immunosorbent assay)
*Used Immunoblot to measure natural vs recombinant EPO in cyclists
- Anti-EPO monoclonal capture Ab (immobilized in 96 well plate)
- EPO (in serum, plasma, urine)
- Biotinylated anti-Epo monoclonal detection antibody
- Streptavidin-HRP (horseradish peroxidase) conjugate
- Tetramethylbenzidine substrate reaction (for visualization) → coloration is proportional to EPO concentration
What mutation did Eero Mantyranta have?
XC skier:
Had a mutation making constitutively active EPO receptor → very high hematocrit
