• origin of hematopoietic cells
• early in development: yolk sac mesenchymal cells Þ hematopoietic cells
• seed liver and other lymphoid organs
• late gestation: seed bone marrow
• red bone marrow is the site of all hematopoiesis in adults

• recent evidence shows that one pluripotential stem cell in bone marrow produces all types of blood cells
• divide at a slow rate to produce more pluripotential stem cells and differentiated cells
• differentiated cells produce different blood cell types
• pluripotential stem cells first form committed myeloid stem cells and committed lymphoid stem cells
• myeloid—produce separate progenitor cells for erythrocytes, eosinophils, basophils, megakaryocytes, and monocytes/neutrophils
• lymphoid—some seed thymus (T-lymphocytes), others stay in bone marrow (B-lymphocytes)—both then seed other lymphoid tissue

Regulation of Hematopoiesis—number of each cell type relatively constant, so production indep. regulated

Blood Cell Development—only late stages of hematopoiesis are recognizable in the bone marrow by LM or EM

• Erythropoiesis
• pronormoblast Þ basophilic normoblast Þ polychromatic normoblast Þ orthochromatic normoblast Þ reticulocyte Þ RBC
• accompanied by several gradual changes:
• decrease in cell size and loss of cellular organelles
• decrease of basophilia in cytoplasm due to loss of polysomes
• increase of acidophilia in cytoplasm due to hemoglobin accumulation
• decrease in nuclear size and increase in chromatin density (nucleus is eventually extruded)
• loss of ability to divide
• Granulopoiesis
• myeloblast Þ promyelocyte Þ myelocyte Þ metamyelocyte Þ band form Þ mature granulocytes
• accompanied by several gradual changes (similar to erythropoiesis):
• decrease in cell size
• decrease in nucleus size and increase in chromatin density—also segmentation of nucleus
• decrease of basophilia in cytoplasm
• decrease in amount of azurophilic granules, appearance and increase of specific granules
• loss of ability to divide
• Lymphocyte Formation
• produced from lymphoblasts and prolymphocytes—both are larger than lymphocytes
• Lymphocytes are also produced in other lymphoid tissues in response to immunological stimulation
• Monocyte Formation
• produced from monoblasts and promonocytes
• circulate in blood for only 1-2 days and then migrate to tissue and differentiate into macrophages
• Platelet Formation
• megakaryoblast Þ megakaryocyte Þ platelets (from fragmentation of megakaryocyte cytoplasm)
• megakaryocytes/-blasts are the largest cells in bone marrow—polyploid with large amount of cytoplasm

Hematopoiesis – process of production of blood cells; occurs in the bone marrow of adults

Development of Blood and Bone Marrow – begins 19^th day of fetal life with RBCs production in the yolk sac

• Yolk Sac: mesoblastic cells in the yolk sac form blood islands Þ forms large erythroblasts which retain their nuclei in the circulation and contain embryonic forms of hemoglobin (Hbg) – Only RBCs are produced
• Fetal Liver (also spleen to a lesser degree): at 6 wks yolk sac production of blood cells begins to decline and fetal liver becomes primary site of hematopoiesis until 6 ½ months Þ forms smaller erythroblasts which contain fetal Hbg
• First production of white cell precursors
• Bone Marrow: medullary hematopoiesis begins at 20 weeks; sole site of blood cell production in normal state
• Extramedullay hematopoiesis occurs in childhood with severe sustained anemia and only occurs in adults with disease of the bone marrow and/or hematopoietic stem cells
• First large scale production of granulocytes and platelets
• At birth and early childhood all marrow spaces is occupied by proliferating hematopoietic cells
• After age 4 or 5, skeletal growth surpasses the need to Ý RBC levels and hematopoietic cells are replaced by fat cells (bone pain may be related to Ý hematopoiesis)
• At age 20 bone marrow hematopoiesis is confined to the vertebrae, pelvis, sternum, ribs and the proximal ends of the long bones (10% of the marrow)

• Vasculature – bone marrow is extremely vascular with blood supply from a nutrient artery and periosteal capillary system; vessels are highly fenestrated
• the arteries form the vascular sinuses which drain to the periphery to form the central veins of the long bones
• Stromal cells – nonhematopoietic cells essential for normal hematopoiesis; controls hematopoietic microenvironment which supports the survival and self renewal of the hematopoietic stem cells
• Endothelial cells – line medullary sinuses and allow for entry of nutrients and the egress of newly formed blood cells
• transport of blood cells into the capillaries is an active process and cells only differentiated cells with specific antigens (Ag) are transported in the normal state
• Adventitial or Reticular cells – large, broad cells which cover the outside surface of the sinus; fibroblast in origin and provide the structural framework by producing reticulin fibers, hematopoietic cell GFs and providing the binding sites for GF and developing cells; assist in the release of mature cells
• Fat cells – represent accumulation of fat in the adventitial cells; maintain space in the marrow for future expansion of hematopoiesis if needed; produce GFs
• Macrophages – only stromal cell of hematopoietic origin; produces GF and maintain phagocytic functions (phagocytize the extruded RBC nucleus); erythropoiesis occurs in clusters around a supporting macrophage (erythroblastic island)
• Extracellular Matrix – contains adhesive proteins such as laminin, fibronectin, hemonectin supported by glucose; bind stem cells and GFs to provide physical support and influence regulation of hematopoiesis; as marrow cells differentiate, they lose adhesion molecules and receptors for matrix that allows mature cells to leave medullary cavity
• Stem cells – undifferentiated cells with the capability of self renewal

• Hematopoiesis Þ process where a small population of undifferentiated stem cells give rise to a large numbers of fully differentiated mature blood cells of many cell lines
• 8 cell lines in total (erythrocytes, platelets, neutrophils, monocytes, eosinohpils, basophils, and T and B lymphocytes)
• involves a series of complex cell divisions associated with the acquisition of differentiated characteristics and amplification of the maturing cell population
• Four cell populations:
• stem cells – extensive capacity for self renewal; capable of differentiation; only 1-2 million cells
• committed progenitor cells – capable of self renewal and further differentiation
• morphologically identifiable immature cells–little or no capacity for self renewal;predominant cell in marrow
• mature blood cells – majority of cells in peripheral blood
• Pluripotent Stem Cell – most primitive stem cell Þ can give rise to all blood cell types
• morphologically indistinct, undifferentiated and have extensive capacity for self renewal – self renewal capacity is not unlimited, but far exceeds needs of normal hematopoiesis; most stem cells are quiescent (G₀)
• capable of self renewal and producing progeny which can differentiate along various cell lines; descendants of the stem cells which begin differentiation lose self renewal capacity
• committed progenitor cells and morphologically identifiable immature cells undergo majority of cell amplification
• Hematopoiesis is a unidirectional process- differentiation is irreversible
• Pluripotent stem cells express CD34 and do not express HLA-DR or Ag that are expressed on lineage-restricted cell lines
• Regulation of differentiation – not well understood
• partly under the control of the mediators of the hematopoietic microenvironment and partly occurs randomly
• differentiation into either the myeloid or lymphoid cell line is the first step of development
• the lymphoid stem cell has not been isolated and less is known about early lymphoiesis
• myeloid cell line: red cells, granulocyte/monocyte (neutrophils, basophils, eosinophils, macrophages) platelets
• Myeloid stem cell: termed CFU-S (Colony forming unit - spleen) and CFU-GEMM (Colony forming unit-granulocyte/erythrocyte/macrophage/megakaryocyte or CFU-Mix) in the human
• CFU-S or CFU-GEMM cells are capable of self renewal or forming differentiated cells

• Growth Factors (GF) – substances that act upon hematopoietic stem cells to promote proliferation and differentiation
• GFs regulate blood cell formation to so that an organism can produce the right type and number of blood cells appropriate to a current condition; GF can have both positive and negative control over hematopoiesis
• Approximately 20 GF have been identified
• Most GF have multiple biological activities and act upon multiple cell types to control hematopoietic cell growth, differentiation, programmed cell death and specialized functions
• some GF are integral membrane proteins with tyrosine kinase activity; interaction of the extracellular GF with their receptors initiates a cascade of intracellular signaling responses leading to modification of nuclear transcription factors and cell cycle components Þ specific gene expression and growth regulation
• Most GF are cytokines Þ produced by one cell to act on another; produced in the marrow by stromal cells
• Only erythropoietin which controls red cell production is a true hormone
• Growth Factors Acting Early in Hematopoiesis – stimulate quiescent stem cells into active the cell cycle and promote commitment to a particular cell lineage
• (1) Stem Cell factor (SCF, kit ligand) – Glycoprotein ligand for kit receptor expressed on hematopoietic stem cells as well as melanocytes, mast cells, Purkinje cells and germ cells
• Produced by fibroblasts, bone marrow microenvironmental cells and other interstitial cells
• promotes cell survival and is mitogenic for mast cells; synergistic with IL-6, IL-3, GM-CSF and G-CSF in promoting the proliferation of stem cells and differentiation into colonies in vitro
• SCF defect (in mice) causes a poor microenvironment and anemia; defect in kit causes anemia, albinism, and sterility
• Uses: produced for clinical use
• (2) FLT3 Ligand (FL, FLT3/FLK2) – essential to early hematopoiesis; FLT3 ligand has been cloned from stromal cells and T cells and is widely expressed in many tissues
• the FLT3 receptor (flt3) is limited primarily to hematopoietic stem cells and early committed progenitor cells particularly of the myeloid lineage
• FLT3 ligand enhances early stem cell survival in vitro, and with other cytokines, promotes stem cells growth and the expansion of colony forming cells; has the potential to be used clinically in bone marrow transplantation
• (3) IL-3 (Interlukin-3) – Produced by: activated T-lymphocytes and helps stimulates the proliferation of early hematopoietic cells including CFU-GEMM and CFU-MEG (megakaryocytes), CFU-GM (granulocyte-macrophage), and BFU-E (erythroid); limited effect on the differentiation and activation of mature cells; modest effect on granulocyte, red cell and platelet production in in vivo studies
• Uses: clinically in investigational trials to aid in bone marrow transplantation
• (4) GM-CSF (Granulocyte/macrophage colony stimulating factor) – Produced by: a number of cell types including monocytes, fibroblasts, endothelial cells and glial cells
• Actions: Ý production during inflammation when cells are stimulated by IL-1, TNF, and other inflammatory mediators
• In vitro Actions: promotes proliferation and differentiation of CFU-GEMM, CFU-GM, CFU-G, CFU-M but has little to no direct effect on lymphocytes, platelets or red cells
• In vivo Actions: GM-CSF Ý release of mature myeloid cells from bone marrow and production of all myeloid cell lineages within marrow Þ Ý marrow cellularity results in Ý circulating granulocytes, macrophages and eosinophils
• Uses: commercially available and used in bone marrow transplantation and treatment of hematologic malignancies
• (5) IL-6 (Interlukin-6) – produced by: primarily endothelial cells and T-lymphocytes after activation by IL1 and TNF during the inflammatory response
• Actions: most promising GF identified to stimulate quiescent pluripotent stem cells
• many biological actions including stimulation of plasma cell proliferation
• In vitro actions: acts synergistically with IL-3 to promote primitive hematopoietic stem cell self proliferation and renewal
• In vivo action: Ý platelet production
• (6) IL-11 (Interlukin-11) – Produced by: bone marrow stromal cells
• Actions: many; induces quiescent stem cells into active cell cycle similar to IL-6 and IL-11
• In vitro Action: stimulates CF-GEMM and the more committed precursors CFU-GM, CFU-MEG and BFU-E
• In vivo Action: stimulates megakaryopoiesis
• Uses: commercially available to treat chemotherapy-induced thrombocytopenia

• BFU-E (Burst forming –erythroid) is the earliest recognizable marrow precursor committed to RBC production
• GF from GM-CSF, IL-3, CSF influence BFU-E proliferation
• BFU-E and immediate progeny are motile Þ causes characteristic "burst" seen in bone marrow cultures
• Each BFU-E forms 10,000 mature RBCs after about 14 days; BFU-E forms CFU-E, which forms 50-200 RBCs
• CFU-E stage – erythropoiesis is totally dependent upon erythropoietin (EPO)
• EPO is a 18-34 Kd glycoprotein produced by the peritubular interstitial cells of the kidney
• Binds to specific high affinity surface receptors on BFU-E (few receptors) and CFU-E (many receptors) and maturing red cell precursors, and is essential for the continued survival and proliferation of these cells
• produced in the kidney in response to cellular hypoxia and circulates to marrow to stimulate red cell production
• chronic renal failure leads to severe anemia due to ß EPO production
• Use: to correct anemia associated with chronic inflammatory or infectious states (Rheumatoid arthritis or AIDS); not as useful in anemias secondary to disease of the marrow itself such as plastic anemia or myelofibrosis
• Maturation of the Erythrocyte
• Stages: proerythroblast (normoblast) Þ basophilic erythroblast Þ polychromatophilic erythroblast Þ orthochromatic erythroblast Þ Reticulocyte Þ Erythrocyte
• 25-30% of marrow cells are maturing red cells which arise from CFU-E
• Proerythroblast – earliest cell; large cell with basophilic cytoplasm and open chromatin; undergoes three cell divisions to produce 8 mature red cells
• Cytoplasm Changes: hemoglobin accumulates in cytoplasm as cell matures and changes color from blue to pink
• reticulocyte contains some remnants of cellular RNA which have removed during passage through the spleen in the first 48 hours in circulation
• Nucleus Changes: nucleus condenses and is eventually extruded prior to release of the reticulocyte into circulation
• Normal life span of the erythrocyte is 120 days.

• Definition – commitment to the specific production of granulocytes and monocytes begins at the CFU-GM stage
• CFU-GM (granulocyte/monocyte) proliferate under the influence of GM-CSF and IL-3
• CFU-G (granulocyte) and CFU-M (monocyte) are well characterized while the progenitors for eosinophils and basophils remain unclear
• Lineage specific GF in Granulocyte production include
• (1) G-CSF (Granulocyte – colony stimulating factor)
• Produced by: monocytes, fibroblasts and endothelial cells; also Ý under conditions of inflammation (stimulated by IL-1, IL-6, TNF and other inflammatory cytokines)
• Action: stimulates the production of granulocytes from CFU-G; is a potent inducer of granulocyte; may also act early in hematopoiesis to stimulate quiescent stem cells; acts on committed granulocyte precursors
• In vivo Action: Ý granulocytes within 12 hours and over 3-5 days causes Ý CFU-G in the marrow and granulocyte release from the marrow
• Uses: to prevent or ameliorate neutropenia following chemotherapy and in patients with AIDs; also used to treat congenital neutropenias
• (2) M-CSF (Macrophage-colony stimulating factor)
• Produced by: fibroblasts and endothelial cells
• In vitro Actions: stimulates CFU-M to produce monocytes and to augment functional acivity of mature macrophages
• (3) IL-5 (Interlukin-5)
• Produced by: eosinophil
• Actions: GF that also stimulates B lymphocytes
• no specific GF has yet been described for basophilic production; IL-3, IL-4 and SCF have activity in vitro

• Myeloblast Þ Promyelocyte Þ Myelocyte Þ Metamyelocyte Þ Band Þ Segmented Þ Mature Neutrophil
• production of mature neutrophils from CFU-G takes approximately 6-10 days
• majority of marrow cells are maturing granulocytes
• monocyte maturation (and likely eosinophil and basophil maturation) proceed along similar lines
• Myeloblast – earliest recognizable cell of the granulocyte lineage
• large immature cell with high nuclear/cytoplasmic ratio and one or more nucleoli
• no granules are present at this stage
• capable of cell division
• Promyelocyte – recognizable by the presence of intensely azurophilic granules Þ the primary granules
• capable of cell division
• Myelocyte – specific granules (neutrophil, eosinophil, basophil) granules begin to appear
• still capable of cell division
• Metamyelocyte – not capable of cell division
• nucleus begins to indent; nuclear indentation progresses to the band stage
• Band Form – only a small percentage of band forms are seen in the circulation
• Ý bands with inflammation, infection or other stresses (Left shift)
• Mature Neutrophil (polymorphonuclear leukocyte (PMN) or Poly) – segmented nucleus allows the cell to be mobile
• circulate for only 6-8 hours before entering the tissues
• only polys and bands are functional as phagocytes
• Monocyte maturation is analogous to neutrophil maturation – mature monocytes are released in to circulation where they enter the tissues and further mature into macrophages

• thrombopoiesis or megakaryopoiesis – production of platelets
• BFU-MEG (Burst forming unit- Megakaryocyte) and CFU-MEG (Colony forming unit-Megakaryocyte, aka. CFU-MK and CFU-Mega) are the progenitors specific for platelet production with analogy to red cell production
• BFU-MEG arise from the CFU-GEMM and give rise to CFU-MEG under the influence of a number of GF including IL-3, SCF, IL-6 and IL-11
• Thrombopoietin (TPO, c-MPL ligand) Þ GF with specificity for CFU-MEG and maturing megakaryocytes
• Stimulates the production of platelets
• CFU-MEGs give rise to megakaryoblasts which present in marrow in small numbers
• megakaryoblasts are formed by fusion of precursor cells, so are multinucleated
• TPO promotes cell growth, DNA replication and nuclear division without cell division; this process (called endoreduplication) forms mature megakaryocytes Þ large multiploid cells with a nuclear content of 8-32 nuclei
• Platelets are fragments of mature megakaryocyte cytoplasm which are released directly into the circulation
• new platelets migrate to the lungs where they undergo further maturation before reentering the circulation
• life span of a mature platelet is 7 days