Control of Haemopoiesis

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  • Haemopoiesis: Prefix of Haem =Blood
    Poiesis =to make
    High level of turnover, from the need to replace mature circulating blood cells at a rapid rate , necessitated by the limited lifespan of mature cells
    The principal cause of blood cell loss is aging.
    Granulocytes few hours, RBC’s 120 days, therefore 1013 cell per day to maintain steady state blood counts
    Equivalent to the Annual number of cells approximating the TOTAL BODY WEIGHT
    However total BM of an adult human contains only on 1012 total cells , 10 fold less than daily needs.
    During 70 years, average 70kg human will produce 7 tons of blood cells
    Blood cells for lifelong haemopoiesis cannot be preformed on the body
    Hence we need a renewable source
    Stem cells
  • Mesenchymal stem cells, or MSCs, are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include
    osteoblasts, greek for bone, osteoblasts are sophistocated fibroblasts,
    A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen[1], the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals
    Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose adipose tissue, specialized in storing energy as fat.
    chondrocytes greek cartilage
    Biological information in a DNA molecule is contained in its base sequence To make this information available is called gene expression. This information goes from DNA to RNA to protein To go from DNA to RNA is transcription to go from RNA to protein is translation The rate at which transcription happens the rate at which the DNA is transcribed into the M RNA is controlled by transcription factors. DNA binding proteins. Don’t forget they can act to promote or to suppress the action. For example they can bind to the TIC transcription initiation complex or they may block the binding site. PU1 and GATA -1 are transcription factors.
    While the terms Mesenchymal Stem Cell and Marrow Stromal Cell have been used interchangeably, neither term is sufficiently descriptive as discussed below:
    Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells.
    Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. While this is an accurate description for one function of MSCs, the term fails to convey the relatively recently-discovered roles of MSCs in repair of tissue.
    Because the cells, called MSCs by many labs today, can encompass multipotent cells derived from other non-marrow tissues, such as adult muscle or the dental pulp of deciduous baby teeth, yet do not have the capacity to reconstitute an entire organ, the term Multipotent Stromal Cell has been proposed as a better replacement.
    Transcription factors commit the stem cells to certain cell lineages which cell is selected for which lineage is selected for differentiation is dependent on chance and external signals rec’d by progenitor cells.
    Several transcription factors have been isolated that regulate differentiation along the major cells lines i.e PU1 commits to myeloid lineage
    GATA-1 essential role in erythropoieitc and megakaryocyte differentiaion
    . Like other cells of connective tissue, fibroblasts are derived from primitive mesenchyme. Thus they express the intermediate filament protein vimentin, a feature used as a marker to distinguish their mesodermal origin. However, this test is not specific as epithelial cells cultured in vitro on adherent substratum may also express vimentin after some time.
  • Stem cells are the most important cells in haemopoietic production.
    They are responsible for regenerating haemopoieisis following damage to the haemopoietic system.
    MOST important cells for haemopoietic production stem cells are capable of both self renewal and differentiation into at least one type of mature cell type.
    Self renewing maintaining their population level by cell division, producing new stem cells to maintain the stem cell pool(stem cell renewal) and cell division to differentiating cells that are the progenitor cells of each of the blood lineages. When increased demand stem cells have enormous proliferative capacity
    Difficult to study they are : 2 reasons
    Scarce :One stem cells per 20 million nucleated cells, Difficult to grow in vitro
  • Haemopoiesis occurs in a suitable microenvironment provided by a stromal matrix on which stem cells grow and divide.
    Bone marrow provides a stromal matrix. The stromal cells adipocytes, fibroblasts endothelil cells macrophages secrete extracellular molecules such as collagen, glycoproteins and glycosamines (hyaluronic acid and chondroitin), to from the extracellulmatrix.
    Mesenchymal cells thought to be critical in stromal cell formation. HSC Transplant with additional mesenchymal cells evidence that takes quicker.
  • Earliest recognizable red cells precursors are large nucleated cells demonstrable at 2 weeks At 2 weeks the embryo is little more than two sacs the amniotic sac and the yolk sac separated by a wedge of tissue called the embryonic plate
    As the embryo develops the amnitic sac expands to fill the uterus and the placenta is formed. The yolk sac becomes compressed into a narrow stalk to form the core of the umbilical cord. The embryo develops from the embryonic plate.
    Major hb Gower delta and epsilom
    Leucopoiesis and Thrombopoieisi do not commence until about 6 weeks when megakaryocytes and granulocytes can be seen in the yolk sac
    Lymphocytes not formed in the yolk sac but in lymphocyte s@7weeks
    Liver is the primary source of blood cells until @week 30 gestation then ceases at 40 wks.
    Major source of HB during hepatic haemopoietic phase is HB F alpha & Gamma
  • Spleen starts producing cells @10 weeks and continues through t 2nd trimestder. However even at the height of its activity it is only of secondary importance as a haemopoietic organ compared to the hepatic haemopoiesis
    Bone cavaties form @20 wks and quickly provide the right environment for the haemopoiesis hence by @40 weeks the BM becomes the sole source of production. This process is accompanied by gradual replacement of the Hb F production with Hb A the normal adult Hb alpha and beta
    Chains
    By birth every available bone cavity is filled with haemopoietically avtive BM RED marrow hence there is no reserve in infants to produce extra cells in time of increased demand. The only response an infant can have if increased haemopeitic activity is required is to expand the marrow volume. This is the cause of skeletal deformities in severe erythropoietic states such as thalassaemia.
  • During childhood marrow volume increases in parallel with increased marrow space made available by growth
    By 3 years 1500 ml of active marrow available enough to meet the needs of an adult therefore as the child grows to adult hood the space available fills with inactive yellow marrow. The process begins peripheral diaphyses of long bone and continues until adult has ¾ of red marrow in pelvis vertebrae and sternum Yellow marrow can be converted into red active marrow therefore an adult has reserves to call upon in times of extra demand. Adult six fold reserve capacity
    Extra medullary haemopoiesis is when the bone marrow is unable to meet the demands for blood cells such as when the bone marrow space is invaded by a metastatic tumour and haemopoiesis may revert to foetal sites of liver and spleen
  • Cell cycle is simply, if you can call any of this simple !!the cell division cycle. It is at the heart of haemopoiesis. If this cycle is disrupted it is also the key to developing malignant disease
    M phase during which the cell physically divides
    Interphase duplication of chromosomes and cell growth prior to division 3 stages to interphase
    G1 cell begins to commit to replication, during the S phase the DNA content doubles and the chromosomes replicate and during G2 phase cell organells copied and the cytoplasmic volume increases
    If the cells rest prior to division they enter the state of G0 where they can remain for long periods
  • .These two checkpoints coordinate the division at the end of G1 and G2
    Is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation).
    Cyclins:A family of proteins that control progression through the cell cycle by activating cyclin dependent kinases (CDK) enzymes like p21 A cyclin dependent kinase is activated by a cyclin forming a cyclin dependent kinase complex. . CDK’s phosphorylate downstream proteins targets on serine and threonine amino acid residues. And the cyclins regulate their activity
    If this phosphorylation does not take place the cycle is disrupted and malignancies can occur. E.g Mantle cell lyphoma has a specific translocation of t(11;14) q13;q32 which leads to a downregulation of Cyclin D1
    Morphologically HSC’s are undifferentiated and resemble small lymphocytes.
    Majority ofHSC’s are in the quiescent phase where they are resting and protected from drugs that are cell cycle dependent
    Drugs in the chemotherapy regimens
    Cell cycle dependent drugs such as 5’Fluorouracil and S phase specific agents such as cytosine arabinoside and hydroxyurea.
  • While the p53 protein normally is short lived and is present at low levels in unstressed mammalian cells, in response to both genotoxic and nongenotoxic stresses it accumulates in the nucleus where it binds to specific DNA sequences
    Genomic approaches have shown that p53 induces or inhibits the expression of more than 150 genes including p21, GADD45, MDM2, IGFBP3, and BAX [4], many of which mediate cell cycle arrest or apoptosis. p53 modulates DNA repair processes [5,6], and the arrest of cell cycle progression may provide time for the repair of DNA damage. In some circumstances, cell cycle arrest is permanent and indistinguishable from senescence [7].
    Alternatively, stress signaling may initiate p53-dependent apoptosis [2]. The biochemical links between p53, G1 arrest, senescence, and apoptosis are cell-and stress-type dependent. These observations suggest that specific posttranslational modifications to the p53 protein, at least in part, determine cellular fate. In turn, these modifications reflect the specific pathways that become activated in response to any particular stress condition. pathways that modulate p53 stability and activity in response to genotoxic and nongenotoxic stresses through covalent posttranslational modifications to p53 including the phosphorylation of serine and threonines and the acetylation of lysines.
    When there is DNA damage p53 signalling is initiated to cause cell cycle arrest, it stops the cell cycle. This allows time to repair DNA damage and can induce apoptosis which is also induced by p53,
    P53 targets p21 a cyclin dependent kinase that regulated the activity of cyclin cdk complexes, as p21 is an inhibitor it inhibits, stops, the kinase from performing its task of phophorylation and the cell cycle is disrupted
    Inj this case it prevents the phophorylation of the retinoblastoma protein
    Don’t forget all many processes act together to give a haemopoietic haemostasis. Part of that process is also apopoptosis the programmed cell death of a cell. It is an induced and ordered process in which the cell actively participates in bringin about its own death. For example leukocytes produced by haemopoiesis have a characteristic lifespan and then dies by programmed cell. E.g neuts have a @24 hour lifespan before the programmed death is initiated. Apoptosis does not lead to necrosis so it does not secrete substances as the cells die. Therefore no inflammatory response as a pose to necrosis unprogrammed cell death where an the dying cell will release it contents and provoke inflammatory response. ecellinflofre e
  • The retinoblastoma protein remains bound to the transcription facto of E2F family and trascription prevent . Information is not passed on
    Therefore the genes required for progression of the cell cycle are not transcribed and the cells remain in the quiescent phase.
  • Control of haemopoiesis is important, therefore control of HSC’s is critically important for regulation of haemopoietic cell production
    Control of stem cell renewal versus differentiation and how it its manipulated is still incomplete.
    Stem cell factor is a cytokine produced by stromal cells and occurs as a transmembrane protein as well as a soluble protein
    It binds its receptor c-kit that is expressed on HSC ‘s essential for normal cell production
    Flt 3 ligand Transmembrane protein expressed on human tissues. It binds haemopoietic cells and is important for cell survival and cytokine responsiveness
    Stem Cell Leukaemia haemopoietic transcription factor and GATA-2are both required for development of haemopoieisis in the yolk sac
  • Control of Haemopoiesis

    1. 1. CONTROL OF HAEMOPOIESIS Denise Pegnall
    2. 2. AIM  Demonstrate an understanding of the haemopoietic process  Where haemopoiesis takes place  When haemopoiesis takes place  Stem cells  Stromal microenvironment  Cell Cycle
    3. 3. HAEMOPOIESIS  The formation of blood cells  High level turnover, need 1013 new cells daily  Bone Marrow contains only 1012  During 70 years, average 70kg human will produce 7 tons of blood cells  Blood cells for lifelong haemopoiesis cannot be preformed on the body  Renewable source
    4. 4. STEM CELLS  Mesenchymal  osteoblasts, chondrocytes , adipocytes  Neural, Muscle, In the crypt of the gut,  Hair follicle stem cells  Haemopoietic stem cells (HSC)  Erythrocytes, platelets, monocytes, neutrophils, eosinophils, basophils, lymphocytes , natural killer cells  Transcription factors commit HSC’s to cell lineages  PU-1  GATA-1
    5. 5. STEM CELLS o Important cells for haemopoietic production o Haemopoietic stem cells, HSC’s o Capable of self renewal o maintain stem cell pool o Differentiation o progenitor cells of each blood lineage o Regulation of Haemopoiesis starts with stem cell division o One to self renew, One to differentiate o Enormous proliferative capacity o One stem cell: 106 mature blood cells after 20 divisions o Rare: One stem cell per 20 million nucleated cells
    6. 6. STROMAL MICROENVIRONMENT o Stem cells require a suitable environment to grow and divide o Stromal Matrix o Stromal cells o Adipocytes, fibroblasts, endothelial cells, macrophages o Microvascular network o Collagen, glycoproteins, glycosamines o Stromal cells also secrete growth factors necessary for stem cell survival o Mesenchymal cells, critical for stromal cell formation o osteoblasts, chondrocytes , adipocytes
    7. 7. BONE MARROW HUMAN STEM CELL
    8. 8. EMBRYONIC HAEMOPOIESIS  Earliest recognizable at 2 weeks  Large nucleated red cell precursors  Haemoglobin Gower 1 (ζ2 ε2 )  Leucopoiesis/Thrombopoiesis @6 wks gestation  Lymphocytes in lymph sacs@7 wks gestation  Primary source foetal cells until @30 wks Liver  Ceases @ 40wks  Haemoglobin F (α2 γ2 )
    9. 9. BONE MARROW  Foetal spleen produces blood cells @10 wks  Continues through second trimester  Bone cavities from @20 wks gestation  Humans Bone marrow sole source of blood cells by 40 wks  Gradual replacement of Hb F with Hb A (α2β2 )  Birth, haemopoietically active marrow fills every available space
    10. 10. THALASSAEMIA PATIENT WITH FACIAL DEFORMITIES Orthodon.CraniafacialRes.10200736-44
    11. 11. HAEMOPOEISIS TO ADULTHOOD o Childhood marrow vol.increases parallel to increased marrow space made available by growth o Average 3 year old, 1500ml active marrow o Entirely active & sufficient for needs of adult o As child grows, further expanding marrow space filled with inactive marrow o Adult ¾ active marrow pelvis, vertebrae, sternum o Adult, six fold reserve capacity o Extramedullary haemopoiesis
    12. 12. CELL CYCLE  Cell division cycle  M. Phase, mitotic phase: division of cell  Interphase: duplication of chromosomes  G1: cell begins to commit to replication  S phase: DNA content doubles, chr. replicate  G2: organelles copied, cytoplasmic vol. Increased  G0 State: resting stage  Controlled by two checkpoints
    13. 13. CELL CYCLE  Co-ordinate division end of G1 & G2  Controlled by  Cyclin dependent protein kinases (CDK)  Cyclins  Majority of HSC’s in quiescent G0 stage  Cell cycle dependent drugs, 5’Fluorouracil  S phase specific agents  Cytosine arabinoside  Hydroxyurea
    14. 14. P53  Quiescent state maintained by Transforming growth factor β (TGFβ) mediated by p53  Tumour suppressor gene  Normally, short lived protein present at low levels in unstressed mammalian cells  Under stress, p53 accumulates in the nucleus & binds to specific DNA sequences
    15. 15. P53  Induces or inhibits expression > 150 genes  p21,GADD45,MDM2 ,IGFBP3 ,BAX4  DNA damage, p53 signalling network activated, induces cell cycle arrest, DNA repair and apoptosis  p53 targets enzyme p21, a cyclin dependent kinase inhibitor  p21 regulates activity of cyclin-CDK complexes  Inhibitors of c-CDK’s prevent phosphorylation of retinoblastoma proteins
    16. 16. CELL CYCLE  Retinoblastoma proteins remain bound to transcription factor of E2F family  Therefore genes required for progression of cell cycle not transcribed  Cells remain in quiescence
    17. 17. MAINTENANCE OF STEM CELL QUIESCENCE PostgraduateHaematology5thEd.2005
    18. 18. CONTROL OF HAEMOPOIESIS o Intrinsic, extrinsic or both? o Extrinsic o Cell-cell interaction in microenvironment o Cytokines o Stem Cell Factor / receptor c-kit o Flt3 ligand/ receptor Flt3 o Intrinsic o SCL, Stem cell leukaemia haemopoietic transcription factor o GATA-2 o Both required for haemopoiesis in the yolk sac
    19. 19. E2F Retinoblastoma

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