HEMATOLOGY#3 -- Hematopoietic Organs and Progenitors Definition: Hematopoiesis (hemopoiesis) is the process by which pluripotent stem cells mature into functional mature blood cells

I. HEMATOPOIETIC TISSUE DEVELOPMENT

A. Mesoblastic phase 1. 2-3 weeks after fertilization 3 layer development (ecto, meso, and endoderm) 2. mesoderm gives rise to “blood islands” with basophilic cells 3. central basophilic cells detach as peripheral cells of form vessels allowing primitive

blood cells (look nucleated erythroblasts) to circulate between the yolk sack and embryo 4. formation of blood islands occurs in multiple locations

B. Hepatic phase 1. Liver develops starting at 5weeks 2. Blood cells begin developing in liver from migration of yolk sac blood island cells and

establisment of new colonies 3. Yolk sac production disappears by 10th week 4. Bona fide erythropoiesis begins with the development of mature anucleated erythrocytes 5. Spleen begins developing in 10th week providing new sites for blood cell development 6. Hematopoietic activity starts to pick up in lymph nodes, thymus, and bone marrow

C. Medullary (Myeloid) phase 1. Starts in the 4th month and becomes dominant by 7 months 2. At birth most myeloid hematopoietic activity takes place in the marrow 3. Lymphoid hematopoieisis continues in the thymus, lymph node. GALT, MALT

II. ORGANS OF HEMATOPOIESIS -Bone Marrow -- All bood cells -Spleen--E,MØ,G,M -Lymph nodes--L,MØ -Thymus--L,MØ

B. Bone marrow - Structure, Development, Function

1. Marrow Function -- To supply circulating blood with mature blood cells during steady state conditions and during periods of increased demand; therefore, hemopoiesis must be highly regulated and is controlled @ level of progenitor cells

a. Principal site for blood cell formation - produces: 1) 2.5 x 109 rbc/day/kg 50 kg female has 1.2 x 1011 /day 2) 2.5 x 109 platelets/day/kg 50 kg female has 1.3 x 1011 /day 3) 1 x 109 granulocytes/day/kg 50 kg female has 5 x 1010 /day 4) Production is regulated 0-4x these rates depending on need

b. Production of monocytes c. Production of virgin uncommitted lymphocytes d. Synthesis of B cells e. Recognition and removal of senescent and abnormal cells f. Production of progenitors of osteoblasts/osteoclasts invoved in bone turnover

2. Marrow Development

a. Among largest organs - 3.5-4.5% kg body weight or 50kg x 0.05 = 2.5kg b. Difficult to study - complex cellular composition, soft tissue had to await certain technical advances

-- Cell separation -- Romanovsky stain, Wright's -- pooling knowledge allowed

better understanding -- culture technique

c. BM cavities form during 5th fetal month

- -site of granulocytic and megakaryeytic formation - -prenatal erythropoiesis - starts in liver & spleen moves to marrow in last trimester

d. Birth -- BM cavities energized with hemopoietic cells

-- now the only site of hemopoiesis => spill over of immature forms in peripheral blood -- infant profiles ╪ adult profiles; --lymphocytes: low at birth

>40% @ 1 mo, reduced to

<20% as adult e. 4th year - significant # of fat cells in diaphysis (shaft of bone) expand centripetally until . 18 ys adult;replacement of hemopoietic cells continues f. 18th year - hemopoietic cells found in vertebrae, ribs, skull, pelvis, proximal epiphyses of bone ends, femora and humer.

g. 25-70 years - most activity resides; vertebrae, sternum, rib, fat cells 8 3. Marrow Structure - structure of blood forming tissue, hemopoietic cells are enmeshed in blood vessels and branched fibroblasts

a. Nutrient source – blood b. Nutrient artery through bone and cortex - branches to central artery, branches

to periosteal capillaries in cortex where it joins periosteal arterial flow (periosteum-connective tissue covering bones)

c. Branches out into network supporting hemopoietic cells - vessels have specialized thin walls providing volume compensation

d. Space surrounding these cells is in a highly branched sinus network drawn into a central sinus which drains into venous system.

4. Site of Hematopoiesis

a. Extravascular space between marrow sinuses or marrow cords, wedge shaped in x-section. Sinus wall has a luminal layer of broad, flat, overlapping, interdigitated endothelial cells with very thin "fenestra" - that may provide exit points (migrating loci) for hemopoietic cellse

b. endothelial cells are actively endocytic involved in chemical and particle traffic to marrow sinoids clathrin coated pits, endosomes and on to lysosomes;

c. laterally differential surface carbohydrate distribution, stops @ fenestrae. c. Sinus endothelium

--coated with "adventitial reticular cells" --separated by thin basement laminae

--adventitial reticular cells synthesize reticular fibers w/other cytoplamic process makes up meshwork in which hemopoietic cells reside

--"not" phagocytic as in phagocytes which require specific receptors;but can take up particles and substances -- cannot develop into hemopoietic cells -- develop into adipocytes by lipogenesis

d. Enervation (myelinated and non-myelinated) on perioaterial sheath regulate vessel tone/neurotransmitter release in marrow e. Extracellular matrix -

a. Adhesion "RGD" and other specific motifs lamin; fibronectin; hemonectin; collagen => mediate specific attachment of hemopoietic cells; loss of RGD receptors=> emigration of reticulocytes b. Proteoglycan - chondroitin sulfate; hyaluronic acid; heparin sulfate (bind growth factors)

f. Hemopoietic Cells a. location - chords or wedges between vascular sinus b. erythroblasts - close to vascular sinus in islands or clusters around a central MØ inner are erythroblast, outer - more mature c. Megakaryocytes (are precursors of platelets) - lie in vascular wall release platelets d. Granulocyte precursors - deeper in hemotopoietic chords away from sinuses - can easily migrate e. myeloid stem cells & progenitors in subcortical regions f. lymphocytes & MØ - conc. around arterial vessels in central portion g. Mature cells -- cellular release or egress; --migration between adventitial recticular cells through endothelial channels fenestrae.

--mature cells press against endothelium, pseudopods enter cytoplasm of endothelial sinus cells w/o breaking luminal PM; i.e,. migrating cells make hole in endothelial cytoplasm;

--channel size - much smaller than diameter of migrating cell => substratum deformation

5. Hematopoiesis depends on several important factors a. Production of autocrine, paracrine, & endocrine growth factors;

--activation of cell division & differentation; e.g. endotoxin,TNF --cytokines - growth factor or activators or differentiation factor

produced by cells; - interleukins - cytokines produced by leukocytes - now

understood to be produced by a variety of cells b. Existence of receptors for the above growth factors 1 TMD/TK; 7 TMD/G-protein c. Specific recognition of one cell type by another d. HIM-Hematopoietic Inductive Microenvironment

--Sl locus or steel mutation defective in HIM --Sl/Sld marrow cells correct hematopoiesis in irrad mice --Irradiated Sl/Sld mice not be corrected by normal marrow cells

B. Spleen -- a highly specialized connective tissue organ consisting of a 3D reticular meshwork serving as a filter in the path of circulation

1. Specific functions-- immunoreactive center, filter for blood, iron recapture, blood cell resevoir, blood volume regulator

a. sort, alter, destroy erythrocytes b. retain, modifiy reticulocytes c. retain pool of platelets in exchangeable state with circulating pool d. sequester monocytes e. induce monocyte ====>to MØ f. sort and group B & T cells into specific spleen zones g. trap circulating antigens, microbes h. process Ag through antigen presenting cells to induce AB formation j. concentrate immune components

2. Development/evolution --merger of lymphatic immunologic tissue ----white pulp --merger of hematopoietic tissue red pulp --shares many function with other tissue and is expendable --progressive involution (degeneration) during aging --50% of it 100-200g weight is blood

3. Morphology a. location

--upper abdomen, below thorax, between stomach & diaphragm --freely moveable --no afferent connection to lymphatic system

b. structure --150g in adult human --lobed, i.e. repeating structures developed around vasulature --outer layer covering is peritoneal --trabeculae extend into body of spleen to form arteries, veins, efferent lymphatic ducts --stroma fibroblasts and fiber lining trabaculae and capsule --parenchyma white pulp;B&T cell zones surround periarterial vessels (10-15% exchangeable pool --parenchyma red pulp; reticular meshwork & sinus terminal

branches of central artery interweave or perpendicular plasma

skimmed away from cellular element of blood forcing them through < 3µm meshwork, traps aged, rigid cellular elements

--most arteries end in a marginal zone rich in MØ for phagocytosis and destruction of cells --antigen presenting cells are present

--splenic sinuses lined by advential reticular cells, endothelial cells,and fiberous discontinuous basement membrane arranged as staves and hoops of a barrrel through which blood cells must squeeze; --ennervated to allow muscles and intermediate filament rich endothelial cells to contract and squeeze out reserve blood supply --upon infection barrier cells differentiate from fibroblasts and aid in walling of white pulp thus enhancing immune response, increasing filtration and binding capacity of red pulp

C. Lymph Nodes --encapsulated dense collection of lymphocytes, plasma cells, and MØ organized along the cousrse of large blood vessels through several regions of the body including abdomen,axilla, inguinal, neck

1. Function a. filtration of lymph to remove foreign substances b. passage of "cleaned' lymph back to blood c. facilitate interaction of immune cells to promote immune reactions d. " production of antibody producing plasma cells from B-cells

2. Structure to be covered in Lymphopoiesis D. Thymus -- to produce and export Thymus-dependent lymphocytes (T cells which

recognize processed antigen in the context of MHC molecules) to peripheral lympoid tissues and thus

express cell mediated immune reactivities 1. Function

--Primary site of development of T lymphocytes --capture of T cell progenitors exported by marrow --provide environment for antigen free proliferation --elimination of T cell clones that react with self antigens exposed on thymic cortical epithelial cells --provide source of T cell for further maturation n lymph nodes

2. Structure --lymphoid cells support by epithelial stroma --other cell types fibroblasts, MØ,dendritic cells, myeloid cells, plasma cells basophils and eosinophils --outer cortex rapidly dividing immature T cells and aggregates of

matured T cells --inner medulla matured T cells in throughout --only 1% of maturing T cells survive

E. Blood Vessels -- Confines blood and blood cells -- Allows for the orderly and regulated distribution, entry, and egress of cells and plasma constituents to the tissues

Stacked laminar flow in blood favors leukocyte endothelial interactions, minimizes rbc interactions, Most rapid flow is in center of vessel cavity, intermediate speed flow, between center and edge, slow flow or no flo on edges; causes stratification of cell flow -- rbcs (ρ= 1.069 g/cc) in center; -- dense leuckocytes (mostly PMNS ρ= 1.035) in middles layers; -- light leukocytes (mostly lymphocytes) along edge

2. Plasma skimming --right angle branching in kidney favors more contact with plasma and incresases efficiency of solute transfer as is observed in kidney afferent glomerular arterioles; -- spleen red pulp and white pulp require separation of leukocytes and erythrocytes

3. Regulated escape and recycling from blood vessels and lymphatic ducts

--plasma washing of tissues in the capillaries --recirculating lymphocyte pool --diapedesis --inflammatory egress --trauma

4. Vascular differentiation minimal (hepatic sinusoids, ear lobe lg. veins) Summary -- hematopoietic organs are interactive- lg vasular bed surround by specialized connective tissue that promotes or impedes flow allowing full

differentiation and growth of hematopoietic cells.

III. CELLS OF HEMATOPOIESIS

A. Stem Cells -- give rise to all the differentiated blood cells; 1. undergo continuous proliferation for self renewal and for production; of more

differentiated cells commited to particular cell lineage; 2. Stem cells (& lateer progenitors) look like small mononuclear

lymphocytes; 3. have life long capability for self renewal w/ multipotential capacity.

e.g. can produce lymphoid - T&B cells or myeloid cells ( rbc, platelet, granulocytes, monocyte, MØ)

4. Serial transplantations have a limit; experimentally multipotential stem cells become permanently exhausted => limited renewal capacity, loss of non renewable multipotential stem cells during each regenerative effort. 5. called alpha cells replaces self, gives rise to more differentiated type 6. called CFU-LM (colony forming unit lymphoid, myeloid, found in lymphoid organs irradiation/repopulation ) just being characterized – recently identified, repopulate marrow after marrow injury and grow and differentiate in culture; lethal irradiation==> hematopoietic injury. 7. in mice - lethal irradiation salvaged by isogenic(same species) marrow transplant 1961 Till & McCulloch - discovered distinct colonies of marrow (derived from cells called Colony Forming Units or CFUs) in a spleen 7-10 days after injected small # marrow cells into irradiated mice.

a. require HIM (Hemopoietic Inductive Microenvironment) & cytokines such as IL-3, IL-6, IL-1 to divide, differentiate i.e. lose their differentiation options; b. change to uni-, bi-, or multi potential cells of limited capacity. progenitor cells look alike - small mononuclear lymphocyte-like cells.

B. Myeloid prognitors -- produces Monocytes, MØ, Granulocytes, Erythrocytes, Thrombocytes (platelets) -differentiate and proliferate under stimulation of variety of multipotential colony stimulating factors and HIM;

1. CFU-GEMM (or CFU-S) forms all myeloid cells, other marrow cells(?)

-- require HIM and IL-3 and GM-CSF(Granulocyte,MØ-Colony

Stimulating Factor -- precursor for BFU-E ===> CFU-E accompanied by an increase in size, but nothing else morphologically, CFU-GM - looks the same. --sequestered by spleen after injection single cell subsequent to

irradiation make colonies 5th day-responsive to effectors and microenvironments, yield pure and mixed colonies.

-- surface colonies are erythroid CFU-E; when same cells implanted in marrow cavity produce granulocytes --development of megakaryocytes imply => HIM - hematopoietic

inductive micro-environment modify & influence stem cells and differential path. --development also studied in plastic culture flasks provided with stroma of endothelial cells, MØ, fibroblasts, fat cells, and others still depends on surface and hemopoietic factors.

Signal for differentiation comes from:

1. committed progenitor 2. cellular depletion (anemia) 3. injury (endotoxin) 4. several multi CSF - induce self-renewal, proliferation,differentiation; 5. growth & division depends on continuing presence of growth factors 6. rapid (daily control of blood cell # is possible/myeloid cells

- " " " " " not poss & w/erytho because there are too many numbers of cells in blood

2. Erythroid Precursors - identified with in vitro culture techniques, surface of spleen in Till McCulloch experiment (irradiation transplantation); plasma clot culture

a. BFU-E - (burst forming unit erythroid)

--extensive proliferation potential; --requires high EPO (erythropoietin made in liver) concentration

b. CFU-E - more differentiated closer to a proerythroblast, --requires small amounts of EPO, in addition to IL-3, GM-CSF, IL-4; --forms small erythroid cluster, --proliferation more rapid than BFU

3. Granulocyte precursors - CFU-GM - committed progenitor of granulopoiesis

--precursor for CFU-G, CFU-M, granulocytes & macrophages --NOT precursor for CFU-Eo, CFU-Bas --CFU-Eo, CFU-Bas appear to derive directly from CFU-GEMM

--requires colony stimulating factor for proliferation and growth

4. Platelet (Thrombocyte) precursors - CFU-meg & BFU-meg --proliferation determined by CSF's;

--terminal differentiation determin by HIM, specific factors including: Thrombopoietin & IL-3 or GM-CSF + IL-3 or IL-4, IL-1, EPO

C. Lymphoid Progenitors--produce lymphocytes, B(Bone Marrow-derived) & T(Thymus-derived) cells, plasma cells, NK (natural killer) cells1. Marrow and Thymus provide continuous flow of primary lymphoid progenitors

-- no involvement of antigen stimulation; --enter B or T cell pathway --generation of a large part of antibody diversity; -- " of precursor pool; -- " tolerance of self;

2. Circulating lymphocytes; 3. Lymphopoiesis continues in secondary lympoid organs, spleen, lymph nodes, and other peripheral lymphatic tissue and other organs to produce secondary progenitor cells

--involvement of antigen specific stimulation --immunocompetance --development of regulatory T cell subsets --development of cytotoxic T cell subsets --development of delayed hypersensitivity T cell subsets

3. Differentiated progenitors are produced that become ultimate examples of unipotential stem cells for a particular epitope of immunological memory or helper, killer, or delayed hypersensitivity cells