Hematopoiesis 4 Learning Objectives—Level l - PowerPoint Presentation

Slide1

Hematopoiesis

4

Slide2

Learning Objectives—Level lAt the end of this unit of study, the student should be able to:Describe the basic concepts of cell differentiation and maturation.Compare and contrast the categories of hematopoietic precursor cells: hematopoietic stem cells, hematopoietic progenitor cells, and maturing cells, including proliferation and differentiation potential, morphology, and population size.

continued on next slide

Slide3

Learning Objectives—Level lAt the end of this unit of study, the student should be able to:Describe the hierarchy of hematopoietic precursor cells and the relationships of the various blood cell lineages to each other (including the concept of colony-forming units/CFUs).

continued on next slide

Slide4

Learning Objectives—Level lAt the end of this unit of study, the student should be able to:List the general characteristics of growth factors and identify the major examples of early acting (multilineage), later acting (lineage restricted), and indirect acting growth factors.Compare and contrast paracrine, autocrine, and juxtacrine regulation.

continued on next slide

Slide5

Learning Objectives—Level lAt the end of this unit of study, the student should be able to:List examples of negative regulators of hematopoiesis.Define hematopoietic microenvironment.

Slide6

Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Compare and contrast the phenotypic characteristics differentiating the hematopoietic stem cells and progenitor cells.Identify the key cytokines required for lineage-specific regulation.Describe the structure and role of growth factor receptors.

continued on next slide

Slide7

Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Summarize the concept of signal transduction pathways.Explain the roles of transcription factors in the regulation of hematopoiesis and differentiation.Outline current clinical uses of cytokines.

continued on next slide

Slide8

Learning Objectives—Level llAt the end of this unit of study, the student should be able to:Identify and describe the cellular and extracellular components of the hematopoietic microenvironment.List and explain the proposed mechanisms used to regulate hematopoietic stem/progenitor cell proliferation/differentiation.

Slide9

HematopoiesisTissue homeostasisMaintenance of an adequate number of cells to carry out functions of an organismCareful balance between:Cellular proliferationCellular differentiationCell death (apoptosis)

Slide10

HematopoiesisHematopoiesisThe process responsible for the replacement of circulating cellsDepends on the proliferation of precursor cells that still retain mitotic capabilityGoverned by multiple cytokinesTakes place in a specialized microenvironment

Slide11

HematopoiesisDifferentiationProcess that generates the diverse cell populationsAppearance of different properties in cells which were initially equivalentCommitmentWhen two cells derived from the same precursor take a separate route of development

Slide12

HematopoiesisMaturationThe entire process from commitment to when the cell has all of its characteristics

Slide13

HematopoiesisTwo primary characteristics:The variety of distinct blood cell types producedThe relatively brief life span of the individual cells

Slide14

HematopoiesisCirculating cells are:MatureIncapable of mitosisException: lymphocytes"Limited life span" or terminally differentiatedMust be replaced by less differentiated, mitotically active precursor cells

Slide15

HematopoiesisHematopoietic precursor cells Located primarily in the bone marrow in adultsConsist of a hierarchy of cells

continued on next slide

Slide16

HematopoiesisHematopoietic precursor cells Enormous proliferation potentialDaily production of:~ 2 × 1011 RBCs~ 1 × 1011 WBCs~ 1 × 1011 plateletsCan increase production of cells rapidly and efficiently when necessary

Slide17

Hematopoietic Precursor Cells

Stem Cells

↓

Progenitor Cells

↓

Maturing Cells

Slide18

Stem CellsUndifferentiated cells (HSCs)Give rise to all of the bone marrow cells by the process of proliferation and differentiationMultipotential precursorsHigh self-renewal capacityGive rise to daughter cells that are exact replicas of the parent cellGive rise to all lineages of blood cells

Slide19

Stem CellsStem cell population is sustained throughout the individual's lifespan.

Slide20

Stem CellsHumans contain ~ 2 × 104 HSCsNot morphologically distinguishableSimilar to small lymphocytesDefined functionally by their ability to reconstitute lymphoid and myeloid cells when transplanted into recipient

Slide21

Proposed Stem Cell PhenotypeCD34+Thy-1+CD49f+CD38–Lin–HLADR–Rh123LoCD 34+: glycoprotein on stem cells, early progenitor cells and vascular progenitor cells

continued on next slide

Slide22

Proposed Stem Cell PhenotypeCD34+Thy-1+CD49f+CD38–Lin–HLADR–Rh123LoThy-1(CD90): membrane glycoprotein that participates in T-lymphocyte adhesion to stromal cellsImportant marker in conjunction with CD34 for HSC identification

continued on next slide

Slide23

Proposed Stem Cell PhenotypeCD34+Thy-1+CD49f+CD38–Lin–HLADR–Rh123LoCD38–: early myeloid differentiation antigenCD49f+: important in cell adhesion

Lin

–

: absence of known differentiation markers or antigens present on lineage-restricted progenitors

continued on next slide

Slide24

Proposed Stem Cell PhenotypeCD34+Thy-1+CD49f+CD38–Lin–HLADR–Rh123LoHLADR–: Human major histocompatibility complex antigensRH123Lo: low intensity staining of rhodamine

123

, a supravital dye

Slide25

Stem Cells"Age hierarchy" in stem cell compartmentLong-term repopulating cells (LTRs)Short-term repopulating cells (STRs)More likely to be proliferatingDecreased self-renewal potentialMore important to blood formation first few months following HSC transplantation

Slide26

Figure 4-1 Derivation and fates of hematopoietic stem cells (HSCs). Hemangioblasts are precursor cells giving rise to both HSCs and vascular endothelium during embryonic development. LTR (long-term repopulating) HSC and STR (short-term repopulating) HSC refer to the length of time these HSC subpopulations take to repopulate depleted hematopoietic tissue and the duration of hematopoiesis arising from each. LTR cells are developmentally more primitive than STR cells. HSCs have three possible fates: self-renewal, commitment to differentiation (becoming common lymphoid progenitors [CLP] or common myeloid progenitors (CMP]), or apoptosis. This cell-fate decision is highly regulated and involves specific transcription factors.

Slide27

Stem CellsHSC pool must balance simultaneous process of expansion (self-renewal) and differentiation.Process of asymmetric cell division where one daughter cell self renews (retaining parent cell properties) and other daughter cell undergoes differentiation

Slide28

Stem CellsReside in unique "stem cell niches" in bone marrowOsteoblastic niche—supports and maintains HSC quiescence and/or self-renewalVascular niche—provides signals for proliferation and differentiation

Slide29

Stem CellsStem cells have four possible fates:QuiescenceSelf-renewalCommitment to differentiationApoptosis (cell death)

Slide30

Stem CellsRegulation of HSCs is by cell-intrinsic functions and regulatory signals provided by nicheSCL (stem cell leukemia gene)LM02 (LIM-only protein 2)GATA2, AML1, MYB transcription factorsOsteoblasts regulate HSC number and function

Slide31

Progenitor CellsSome stem cells will initiate differentiation.Transition correlates with:Downregulation of HSC-associated genesUpregulation or activation of lineage-specific genes

Slide32

Progenitor CellsUpon commitment to differentiation, HSC enters the hematopoietic progenitor cell compartment (HPC).

Slide33

Progenitor CellsDaughter cell of the HSC Initially retains the potential to generate cells of all hematopoietic lineagesMultipotential progenitor cells (MPPs)Gradually become restricted in differential potential to one cell lineUnilineage or committed progenitor cell

Slide34

Progenitor CellsHematopoietic progenitor cell compartment (HPC)Contains all precursor cells located between HSCs and morphologically recognized precursor cellsConstitutes ~ 3% of total nucleated hematopoietic cellsTransit cells without self-renewal

continued on next slide

Slide35

Progenitor CellsHematopoietic progenitor cell compartment (HPC)Not morphologically identifiable, but functionally defined by mature progeny they produceGrowth-regulatory glycoproteins or cytokines influence survival and differentiation of precursor cells

Slide36

Progenitor CellsAble to produce colonies of cells in semisolid media in vitroColony-forming units (CFUs)CFU-GEMM—progenitor cell giving rise to granulocytes, erythrocytes, monocytes, megakaryocytesCFU-Mk—progenitor cell giving rise to megakaryocytes

Slide37

Maturing CellsConstitute >95% of total hematopoietic precursor cell poolCommitted (unipotential) transit populationPopulation can be numerically amplified by proliferationProliferative sequence complete before full maturity

Slide38

Maturing CellsMaturing cells are morphologically recognizableMeasured by nuclear and cytoplasmic characteristicsClassify the lineageStage of developmentEarliest recognizable cell—"Blast"Lymphoblast, myeloblast, megakaryoblast

Slide39

Table 4-1

Comparison of Hematopoietic Precursor Cells

Slide40

Precursor Cell ModelPluripotential hematopoietic stem cellSelf-renewal abilitiesGive rise to all hematopoietic elementsEarliest differentiating daughter cells give rise to Common lymphoid progenitor cell (CLP)Common myeloid progenitor cell (CMP)

Slide41

Precursor Cell ModelUnder additional differentiation steps the:CLP gives rise to: T and B cells, natural killer cells, lymphoid dendritic cellsCMP gives rise to: Neutrophils, monocytes, eosinophils, basophils, erythrocytes, megakaryocytes

Slide42

Table 4-3 Phenotype of Hematopoietic Precursor Cells

Slide43

Figure 4-4 The pluripotential hematopoietic stem cell (HSC) gives rise to erythrocytes, platelets, monocytes, macrophages, granulocytes, and lymphoid cells. Under stimulation from selective growth factors, stem cell factor (SCF), Flt ligand (FL), and interleukins (IL), the HSC in quiescence (G0) enters the cell cycle (G1) and differentiates to the common myeloid progenitor cell (CMP) and, subsequently, to the colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte (CFU-GEMM). The CFU-GEMM then differentiates into granulocytes, erythrocytes, monocytes, and megakaryocytes under the influence of specific growth factors, erythropoietin (EPO), thrombopoietin (TPO), granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-5 (IL-5). Different combinations of hematopoietic cytokines regulate the differentiation of HSCs into the common lymphoid progenitor cell (CLP) and subsequently into B and T lymphocytes, natural killer (NK) cells, and lymphoid dendritic cells. SDF-1 = stromal cell derived factor-1

Slide44

Cytokines (Growth Factors)Govern hematopoietic precursor cell survival, self-renewal, proliferation, and differentiationColony-stimulating factors (CSFs)Cytokines = Interleukins

Slide45

Cytokines (Growth Factors)Produced by many cellsMonocytes, macrophages, activated T lymphocytes, fibroblasts, endothelial cells, osteoblasts, adipocyte (bone marrow stromal cells)

Slide46

Cytokines (Growth Factors)Most are produced by stromal cells in the hematopoietic microenvironmentException is erythropoietin (EPO)Produced mainly in the kidneyMost are not lineage specific Each has multiple functions acting on more than one cell type (pleiotrophy)

Slide47

Cytokines (Growth Factors)Interact with surface receptors on the target cellCreate complex cell-to-cell communication systemHematopoietic cell growth requires the continuous presence and interplay of GFs.Remove GF and the cell will dieAct synergistically

Slide48

Table 4-4 Characteristics of Hematopoietic Growth Factors (GFs)

Slide49

Cytokine CommunicationCytokine network has signal amplification circuitsAutocrineSignal produced by and act on the same cellParacrineSignals produced by one cell and act on nearby cell

continued on next slide

Slide50

Cytokine CommunicationCytokine network has signal amplification circuitsJuxtacrine Signals are not secreted by the cell that produced it.Remains membrane boundRequires direct producer cell–target cell contact for desired effect

Slide51

Figure 4-5 Mechanisms of cytokine regulation. Autocrine signals are produced by and act on the same cell. Paracrine signals are produced by one cell and act on an adjacent cell, typically over short distances. A juxtacrine signal is a specialized type of paracrine signaling in which the cytokine is not secreted by the producing cell but remains membrane bound, necessitating direct cell–cell contact to achieve the desired effect. In contrast, endocrine signals (classic hormones) typically act over fairly long distances.

Slide52

Table 4-5 (continued) Hematopoietic Growth Factors (GFs)

Slide53

Cytokines (Growth Factors)GF requirements change during the differentiation process.Early-acting (multilineage) GFsLater-acting (lineage-restricted) GFs

Slide54

Early-Acting Growth FactorsDirect effect on multipotential precursor cellsAffect proliferation of noncommitted progenitor cellsSCF, FL, IL-3, GM-CSF, IL-6, IL-11

Slide55

Early-Acting Growth FactorsSCF—Stem cell factorAlso called mast cell growth factor (MCGF) or kit ligand (KL)

continued on next slide

Slide56

Early-Acting Growth FactorsSCF—Stem cell factorPromotes proliferation and differentiation of:Stem cells, multilineage progenitor cells, committed progenitor cells (CFU-GEMM, CFU-GM, CFU-Mk, BFU-E), mast cell precursorsAlso functions outside of hematopoietic system in melanocyte development and gametogenesis

Slide57

Early-Acting Growth FactorsFL—Flt3 LigandIncreases recruitment of stem cells and progenitor cells into the cell cycleInhibits apoptosisPotent stimulator of proliferation and differentiation of:Granulocytic/monocytic cellsB cells Dendritic cells

Slide58

Early-Acting Growth FactorsIL-3—Interleukin 3Directly affects: Multipotential progenitor cellsEarly committed progenitor cells (BFU-E)

Slide59

Early-Acting Growth FactorsGM-CSFStimulates clonal growth of all lineages except basophilsActivates functional activity of mature phagocytes (neutrophils, macrophages, eosinophils)

Slide60

Early-Acting Growth FactorsIL-6 and IL-11 (Interleukins 6 and 11)Overlapping growth stimulatory effects on Myeloid cells, lymphoid cells, primitive multilineage cellsBoth work with IL3, SCF, and other cytokines

continued on next slide

Slide61

Early-Acting Growth FactorsIL-6 and IL-11 (Interleukins 6 and 11)Have significant effects on: Megakaryocytopoiesis and platelet productionIL-6 stimulates production of hepcidin, a regulator of iron absorption

Slide62

Later-Acting Growth FactorsHave a narrower spectrum of influence Function to induce maturation along a specific lineageMost not lineage specific, but demonstrate predominant effect on committed progenitor cells of single lineage

Slide63

Later-Acting Growth FactorsInduces differentiation of more mature cellsGranulocyte stimulating factor (G-CSF)Monocyte stimulating factor (M-CSF)Erythropoietin (EPO)—erythrocytes

continued on next slide

Slide64

Later-Acting Growth FactorsInduces differentiation of more mature cellsThrombopoietin (TPO)—megakaryocytes and plateletsInterleukin 5 (IL-5)—eosinophilsIL-2, -4, -7, -10, -12, -13, -14, -15—lymphocytes

Slide65

Indirect Acting Growth FactorsRegulate hematopoiesis by inducing accessory cells to release direct-acting factorsIL-1 induces neutrophilic leukocytosis

Slide66

Lineage-Specific RegulationErythropoiesisProgenitor cells give rise to: BFU-E regulated by IL-3, GM-CSFCFU-E which depends primarily on EPOGives rise to the first recognizable erythrocyte precursor, the pronomoblast EPO is pivotal in preventing apoptosis and inducing proliferation/differentiation of erythroid cells.

Slide67

Lineage-Specific RegulationGranulocytopoiesis and monopoiesisDerived from GMP, which is derived from CFU-GEMMGrowth factorsGM-CSF and IL-3M-CSF supports monocyte differentiationG-CSF supports neutrophil differentiation

continued on next slide

Slide68

Lineage-Specific RegulationGranulocytopoiesis and monopoiesisEosinophils and basophils come from CFU-GEMMEosinophils—IL-5Basophils—IL-3/IL-4

Slide69

Lineage-Specific RegulationMegakaryocytopoiesis/ThromobopoiesisPlatelets are derived from megakaryocytesCome from MEPCFU-MksGrowth factors that influence the proliferation and differentiation of megakaryocytesIL-11 and TPO induce greatest increase in platelet production

Slide70

Lineage-Specific RegulationLymphopoiesisOccurs in multiple anatomic locationsBone marrow, thymus, lymph nodes, spleenMultiple GFs, acting synergistically, play a role in lymphopoiesis

Slide71

Negative RegulatorsNegative regulators of hematopoiesisLimit the production of hematopoietic precursor cellsHematopoiesis may be inhibited by:Decreasing production of stimulating factorsIncreasing factors that inhibit cell growth

Slide72

Table 4-7 Negative Regulators of Hematopoiesis

Slide73

Cytokine PathwaysCytokines Must bind to surface membrane receptors to express their activity

continued on next slide

Slide74

Cytokine PathwaysCytokines Signaling pathways The cytokine (external stimuli) binds to its specific receptor causing an intracellular signal.The intracellular signaling molecules translocate to the nucleus.Recruit transcription factors Activate or silence genes

Slide75

Figure 4-6 A model for the transfer of signals from extracellular stimuli (cytokines) into appropriate intracellular responses. The binding of a cytokine or ligand (L) to its cognate receptor generally induces receptor dimerization, the activation of a cascade of downstream-signaling molecules (A-, B-, C-signal transduction pathways) that converge on the nucleus to induce or repress cytokine-specific genes. The result is an alteration of transcription, RNA processing, translation, or the cellular metabolic machinery.

Slide76

Cytokine ReceptorsCytoplasmic tyrosine kinase domainsAre transmembrane proteinsHave cytoplamsic "tails" that contain a tyrosine catalytic site or domainGF binds to the receptorReceptor chains dimerize enhancing catalytic activity of the kinase domainActivates intracellular signaling pathwaysIncludes M-CSF, SCF, FL

Slide77

Cytokine ReceptorsReceptor superfamilyMultichain transmembrane proteinsCytokine binding and receptor activation leads to:Recruitment of cytoplasmic kinasesPhosphorylation of cellular substratesServes as a docking site for the adaptor molecules

continued on next slide

Slide78

Cytokine ReceptorsReceptor superfamilyReceptors for these cytokines are divided into three groups based on shared peptide subunitsβc4 family: IL-3, IL-5, GM-CSFGP-130 family: IL-6, IL-11γ-chain gene family: IL-2, IL-4, IL-7, IL-9, IL-15, IL-21

Slide79

Signaling PathwaysTransfer of signals requires "signal transduction pathways"Initiated by cytokine binding to receptor, then activation of downstream signaling moleculesCausing the nucleus to modulateTranscriptionRNA processing

continued on next slide

Slide80

Signaling PathwaysTransfer of signals requires "signal transduction pathways"Initiated by cytokine binding to receptor, then activation of downstream signaling moleculesCausing the nucleus to modulateProtein synthetic machinery (translation)Cellular metabolic machineryCytoskeletal-dependent functions

Slide81

Signaling PathwaysMost pathways involve protein phosphorylation that occurs by activating either: An intrinsic kinase domain within the receptorA cytoplasmic kinase

Slide82

Signaling PathwaysReceptors that have intrinsic domains are:Receptor tyrosine kinases (RTKs)Receptor serine kinases (RSKs)Receptor protein tyrosine phosphatases (PTPs)Cytokine binding to the receptor promotes oligomerization resulting in activation of their cytoplamic kinase domains.

Slide83

Signaling PathwaysReceptors without instrinsic kinase domainsUse their intracellular "tails"Induce association and assembly of multisubunit protein complexesResults in enzymatic activity

continued on next slide

Slide84

Signaling PathwaysReceptors without instrinsic kinase domainsCalled protein tyrosine kinases (PTKs)Most hematopoietic receptors signal throughThe Janus family (JAKS)

Slide85

Signaling PathwaysJAK-STAT signaling pathwayActivated JAK kinases recruit Signal relay molecules that include:The STAT (signal transducers and activators of transcription) family of transcription factors STAT proteins are phosphorylated, dimerize, translocate to the nucleus, bind to DNA sequences, and activate or inhibit specific gene expression

Slide86

Figure 4-7 Cytokine receptor-JAK-STAT model of signal transduction. Cytokine (e.g., EPO) interaction with its specific receptor (EPO-R) leads to receptor dimerization and activation of JAK kinases associated with the activated receptor. Activated JAK kinases mediate autophosphorylation as well as phosphorylation of the receptor, which then serves as a docking site for signal transducers and activators of transcription (STATs). These STATs are phosphorylated, dissociate from the receptor, dimerize, and translocate to the nucleus where they activate gene transcription.P = phosphorylated protein

Slide87

Transcription FactorsTFs are DNA binding proteins that interact with regulatory promoter regions of their target genesEffect of a particular TF can be either:Gene expression or gene silencingTFs in hematopoiesis allow for:Cell viabilityCell proliferation

Slide88

Transcription FactorsDifferent TFs are restricted to particular lineages and differentiation.Many have been shown to be involved in chromosomal abnormalities leading to leukemias.

Slide89

Clinical Use of Hematopoietic Growth FactorsCloning and characterization of genes encoding hematopoietic GFs enable production of cytokines through recombinant DNA technology.Used in therapeutic regimens for hematopoietic disorders

Slide90

Table 4-9 Clinical Applications of Hematopoietic Growth Factors

Slide91

Hematopoietic MicroenvironmentNormally confined to certain organs and tissuesCrucial for: Proliferation and maturation of precursor cellsMaintains the hematopoietic system throughout an individual's lifeContains cellular elements and extracellular components

Slide92

Hematopoietic MicroenvironmentCellular componentsStromal cellsAdipocytes (fat cells)Endothelial cellsFibroblasts (reticular cells)Osteoblasts

continued on next slide

Slide93

Hematopoietic MicroenvironmentCellular componentsAccessory cellsT LymphocytesMacrophagesMonocytes

Slide94

Hematopoietic MicroenvironmentExtracellular matrix (ECM)Produced and secreted by the stromal cellsComposed of: CollagenGlycoproteinsGlycosaminoglycansCytoadhesion molecules

Slide95

Hematopoietic MicroenvironmentExtracellular matrix (ECM)Provides adhesive interactions needed for: Stem cells, progenitor cells, and growth regulatory proteins

Slide96

Table 4-10 Hematopoietic Microenvironment

Slide97

Hematopoietic MicroenvironmentPrecursor cells (PCs) of different lineages and different stages can be found in distinct "niches" of the marrowPCs can interact only with some types of ECM components

Slide98

Hematopoietic MicroenvironmentImportant molecular determinant for geographic location of hematopoiesisPresence of membrane receptors for ECM proteins and stromal cells

Slide99

Hematopoietic MicroenvironmentAdhesive interactions among the stem cell, progenitor cell, and the ECM function to:Hold hematopoietic cells in microenvironment nichesAllow for close proximity with growth-regulatory cytokines

Slide100

Figure 4-8 A model for regulation of hematopoietic precursor cells in the bone marrow microenvironment. The hematopoietic stem cell (HSC) attaches to bone marrow stromal cells via specific receptors and ligands. The HSC is then influenced by both positive and negative regulatory growth factors.CAM = cell adhesion molecule; SCF = stem cell factor; FL = Flt3-ligand; IFN = interferon; TNF = tumor necrosis factor; TGF- β transforming growth factor β; MIP- 1α = macrophage inflammatory protein- 1α [stem cell inhibitor]; G-CSF = granulocyte colony-stimulating factor; GM-CSF = granulocyte-monocyte CSF; IL = interleukin

Slide101

Hematopoietic MicroenvironmentStem cell nichesLocalized in osteoblastic niches that block response to differentiation-inducing signalsHypoxic envirornment to protect HSC from oxidative stress

Slide102

Hematopoietic MicroenvironmentLymphoid nichesB cells located close to endosteal surfaceT cells close contact with IL-7 secreting stromal cells in bone marrow

Slide103

Hematopoietic MicroenvironmentErythroid nichesErythroblastic islands in marrow sinusoids or scattered throughout bone marrowMegakaryocytic nichesNear marrow sinusoidal endothelial cellsPositioned to release platelets