Medical Cell & Tissue Biology

Hematopoiesis

Dr. Cathleen Pettepher February 12, 2002

Formation of Blood Cells

• Blood cells have a limited life cycle

– Cells are a component of the peripheral blood only in part of the life cycle

– Production and destruction occur constantly

• Bone Marrow

– RBCs, granulocytes, monocytes and platelets are formed here

– Lymphocytes are formed in marrow and in lymphoid tissues

Hematopoiesis in Embryonic and Fetal Life

• Primitive hematopoiesis

– Transient production of blood cells in the “blood islands” of the yolk sac in embryos (days 15-18)

– RBCs are nucleated and expressing embryonic globin chains

• Definitive hematopoiesis

– Hematopoietic centers appear in the liver & lymphoid tissues (days 335-342)

– RBCs are non-nucleated and expressing fetal or adult globins

– Origin of hematopoietic stem cells is the AGM

• (dorsal aorta, gonads and mesonephros)

Hematopoiesis after Birth

• Occurs in the red bone marrow and lymphoid tissues

• Pleuripotential Stem Cell

– Long term repopulating hematopoietic stem cell

• LTR-HSC

• In vivo transplantation into lethally irradiated adults, resulted in long-term multi-lineage repopulation within 4-6 months

– Colony-forming Unit

• Nodular colonies contain all of the hemopoietic cell lines

• All of the cells are progeny of one pleuripotential CFU

Colony Forming Units

• Multipotent progenitors

– CFU-S, spleen

– In vivo transplantation into lethally irradiated adults resulted in macroscopic colonies in the spleen within 8-16 days

• Committed single and multi-lineage progenitors

– CFU-C, culture

– In vitro culture in semi-solid medium in the presence of hematopoietic factors.

• Morphologically indistinguishable from lymphocytes

• Only one in several thousand nucleated bone marrow cells is a CFU

• Less common in peripheral blood

– Only one in a million nucleated cells is a CFU

Erythropoiesis

• 2.5 x 10 ¹¹ erythrocytes are generated everyday

• Two types of unipotential progenitor cells:

– Burst-forming units (BFU-E)

• Erythropoietin is produced by the kidney when RBC count is low

• With IL3 and granulocyte-monocyte CSF, it induces CFU-S to differentiate into BFU-E

• These cells undergo a burst of mitotic activity forming CFU-Es.

– CFU-E

• Require low levels of erythropoietin to survive and to form the first recognizable erythrocyte precursor

– proerythroblast

Erythrocyte Development: Proerythroblast

• First recognizable cell beginning the process of erythropoiesis

• Derived from a CFU

• Relatively large cell (12-15 um)

• Large spherical nucleus

– 1 or 2 nucleoli

• Cytoplasm shows mild basophilia

– Presence of free ribosomes

Erythrocyte Development: Basophilic Erythroblast (N1)

• Smaller than a proerythroblast

• Nucleus

– Becomes smaller

– Progressively more heterochromatic

• Deeply basophilic cytoplasm

– Large number of free ribosomes that are making hemoglobin

Erythrocyte Development: Polychromatophilic Erythroblast (N2)

• Smaller cell (9-12 um)

• Markedly condensed nucleus

– Coarse checkerboard pattern

• Lilac colored cytoplasm

– Presence of increasing amounts of hemoglobin

– May see distinct colored regions (pink or blue)

• Last cell in series capable of mitosis

Erythrocyte Development: Normoblast (N3)

• Orthochromatophilic erythrocyte

• Slightly larger than mature erythrocyte

• Small, compact, intensely stained nucleus

– pyknotic

• Nucleus is extruded at this stage

– Passes into blood sinus of marrow

• Cytoplasm acquires acidophilia

Erythrocyte Development: Reticulocyte

• Polychromatophilic erythrocyte

• Constitute 1-2% of RBCs

• No nucleus!

• Acidophilic cytoplasm with trace of grey

• Special stains demarcates reticular network of polyribosomes

– Still able to synthesize hemoglobin

Kinetics of Erythropoiesis

• Erythroblasts will undergo mitosis

– Proerythroblasts

– Basophilic erythroblasts

– Polychromatophilic erythroblasts

• Nearly all erythrocytes are released into circulation as soon as they are formed

• Bone marrow is not a storage site for RBCs!

• RBC formation and release are under the regulatin of erythropoietin

– Glycoprotein secreted by kidney in response to decreased oxygen tension.

Breakdown of RBCs

• At 4 months (120 days), they become fragile and subject to breakage

• Macrophage system phagocytoses the degrading RBCs

• Iron is separated from the hemoglobin

– Stored as ferritin in spleen

– Reused in hemoglobin synthesis

• Heme moiety binds to albumin

– Transported to liver where it is partially degraded, conjugated and excreted via gallbladder as bilirubin

Granulocyte Development: Myeloblast (M)

• 14-16 um in diameter

• Derived from CFU

• Oval nucleus with finely dispersed chromatin

• Thin rim of basophilic cytoplasm

• Devoid of granules

Granulocyte Development: Promyelocyte (P)

• First recognizable cell in granulopoiesis

• 17-26 um in diameter

– Largest cell in series

• Large oval nucleus

– Muliple nucleoli

• Azurophilic (primary) granules in cytoplasm

– Produced only at this stage!

Granulocyte Development: Myelocyte (M1)

• Spherical nucleus

– Becomes increasingly heterochromatic

• Prominent Golgi apparatus

– Negative image

• Lots of azurophilic granules

• Formation of specific granules

– Emerge from Golgi (cis face) complex

– Characteristic staining reactions for each line

Granulocyte Development: Metamyelocyte (M2)

• First stage that is clearly divided into separate lines

• Few hundred granules present in the cytoplasm

– Specific granules outnumber the azurophlic granules 4:1

• Nucleus

– Heterochromatic

– Indentation deepens to form horse-shoe shape

Granulocyte Development: Band Cell (M3)

• Last immature stage in Neutrophilic series

• Sometimes seen in circulation

– Particularly during states of chronic infection

• Nucleus is elongated and of uniform width

• Nucleus constricts

– 2-5 lobes are formed

– PMNs

Kinetics of Granulopoiesis

• Mitotic stage

– Stops by late myelocyte stage (lasts ~ 1 week)

• Postmitotic stage

– Metamyelocyte to mature granulocyte (~ 1 week)

• Mature granulocytes circulate in peripheral blood for 8-12 hours

• Leave to go into perivascular CT

– Neutrophils live for ~ 1-2 days, then they are destroyed by macrophages

– Unknown exactly how long eosinophils and basophils live in the CT

Megakaryocyte Development: Megakaryoblast

• Derived from Pleuripotential CFU

• ~30um in diameter

• Non-lobulated nucleus

• No evidence of platelet formation is seen at this stage

• Successive endomitoses occurs

– Chromosomes replicate

– No karyokinesis nor cytokinesis

– Ploidy increases to 16-64n, chromosomes cease to replicate >>>> Megakaryocyte

Megakaryocyte

• 50-70um in diameter

• Multi-lobulated nucleus

– Increased in size in proportion to ploidy of cell

• Scattered azurophlic granules

• Clusters of platelets at edge

Lymphopoiesis

• Lymphocytes constitute ~30% of all nucleated cells in the bone marrow

• Progeny of T-cell lymphopoietic stem cells

– Leave marrow and go to the thymus

– Complete their differentiation there

– Enter circulation as long-lived small lymphocytes

• Progeny of B-cell lymphopoietic stem cells

– Originate in several sites

• Bone marrow, gut-associated lymph tissue (GALT) and the spleen

• Precursors to small lymphocytes in the marrow are called “transitional cells”

• Slightly larger than small lymphocytes

• Thin rim of cytoplasm

• Nucleus is filled with fine chromatin

Monocyte Development

• Derived from Pleuripotential CFU

• Promonoctyes represent progenitor cells for this line

– Half are rapidly dividing

– Other half are reserve population of near stem cells

• Stem cell to monocyte transformation takes ~55 hours

• Monocytes remain in circulation only about 16 hours prior to emigrating into tissues

– Differentiate into macrophages

Bone Marrow

• Consists of:

– Blood Vessels

– Specialized Units of blood vessels – sinuses

– Sponge-like network of hemopoietic cells

• Lie in cords between sinuses or between sinuses and bone

Red Bone Marrow

• Active bone marrow

• Cords of hemopoietic cells

– Developing blood cells

– Megakaryocytes

– Macrophages, mast cells & fat cells

• Appears unorganized

– Specific types develop in nests or clusters

• Once mature, cells penetrate the endothelium to enter the circulation

Yellow Bone Marrow

• Non-active

• Found in medullary cavities of bones in adult

• Retains its hemopoietic potential

– When necessary it can revert to red bone marrow to resume hemopoiesis