Hematopoiesis: Basic Concepts Blood cell production is highly regulated to maintain circulating cell numbers within relatively constant levels and to respond rapidly to conditions requiring extra cells
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HSC will choose one of 2 pathways: self-renewal (maintains primitive state) or differentiation (driven toward a more mature state)
lymphocytes – terminally diff.: can undergofurther division (i.e. memory cells)
Granulocytes – terminally diff.: no further division
Lineage diagram – outline of how hematopoietic cells are increasingly restricted in the types of progeny to which they can give rise as differentiation proceeds
poiesis is initiated sequentially in different tissues
PRIMITIVE HEMATOPOIESIS occurs in the YOLK SAC at mouse embryonic day 7.5 (E7.5), and probably starts ~ 4 weeks in humans
Primitive hematopoiesis is characterized by the productionof fetal erythrocytes (nucleated) and the lack of lymphocytesand myeloid cells except for macrophages.
For many years the YS was assumed to be the primary siteof formation of the HSCs that migrateto and colonize the fetal liver and subsequently the bone marrow.
1stDEFINITIVE multipotent hematopoietic stem cells are generated within the embryonic AGM region of the para-aortic splanchnopleuric mesoderm (d. 30-37 human, and late d. 10/early d. 11 mice)
DEFINITIVE HEMATOPOIESIS – HSCs can restore long-term multilineage hematopoiesis when transplanted into adult myeloablated recipients, and generates enucleated erythrocytes, various kinds of myeloid andlymphoid cells, and long-term reconstituting hematopoietic stemcells (LTR-HSCs)… Upon transplantationinto NOD-SCID mice, cultured AGM HSC cells showed lymphomyeloidreconstitution; YS cells were only capable of contributingto the myeloid lineage (Tavian et al., 2001).
CD34+ cluster of cells
(also CD45+ hemato-specific)
AGM region –
ventral floor of dorsal aorta
(also umbilical and vitilline arteries @ connection with dorsal aorta)
Shared expression patterns of a number of molecules by both intra-aortic cluster HSCs and underlying endothelial cells supports the existence of a HEMANGIOBLAST or primitive mesenchymal endothelial-like cell with hematogenic potential that lies on the ventral floor of the dorsal aorta… AKA “hemogenic endothelium”
Example: VEGF receptor (VEGF R): neither HSCs nor blood vessel epithelium develop without the ligand, VEGF. Also, angiopoietin: produced by early blood cells to induce blood vessels to grow in the vicinity.
Recent ID of a morphologically distinct layer of cells resembling a stromal layer underlying the ventral floor of the dorsal aorta in the AGM suggests that this region could represent a microenvironment or niche supporting HSC development (Marshall et al., 1999, Dev. Dyn. 215:139)
After about d. 37 (5 weeks), HSCs from the AGM begin to colonize the fetal liver, and at 6 weeks hematopoiesis (definitive) takes place inthe fetal liver until bone marrow isformed
Throughout fetal life, the liver is the chief organ for production of myeloid and erythroid cells.
At about 8 wks, liver HSCs differentiate in the thymus to mature T lymphocytes (~10 wks) which populate fetal lymph nodes, spleen and gut by 12 wks and other peripheral lymphoid tissue by weeks 14-15.
Bone marrow (BM) is seeded by liver HSCs by 8 weeks. B-lymphopoiesis takes place in liver @ 7 wks., then shifts to bone marrow.
Hematopoiesis from fetal BM is mainly myeloid and contributes only minimally to the blood pool throughout fetal life.
After birth, BM becomes main hematopoietic organ.
Many HPC and HSC in circulation during fetal life and immediately after birth; 24-48 hours after birth they disappear due to lodgement in BM.
and adult hematopoiesis
During fetal growth, hematopoiesis takes place in all bony cavities (axial and appendicular skeleton) as well as in liver and spleen.
Prior to birth, splenic and hepatic hematopoiesis disappear, and gradually thereafter hematopoietic tissue (red marrow) is replaced by adipocytes (yellow marrow) beginning in the distal bones and retracting to the adult pattern by age ten. Yellow marrow can be reactivated by an increased demand for blood cells, i.e., blood loss, but does not normally produce blood cells
In the adult, hematopoietic marrow is confined to the axial skeleton (sternum, vertebrae, iliac bones, ribs) and proximal portions of the humerus and femur.
Bone marrow is specially construed to support the proliferation, differentiation, and maintenance of hematopoietic cells
Honeycombed latticework of venous sinuses – large, thin-walled veins
Endothelial cells lining the marrow sinuses are bounded by STROMAL CELLS that generate an EXTRACELLULAR MATRIX that mechanically support hematopoietic cells and vasculature; provide a nurturing microenvironment for hematopoiesis & hematopoietic colonies
Adjacent cells in marrow: endothelial cells, fibroblasts, adipocytes, osteoblasts, and macrophages and reticular connective tissue. Close contact between hematopoietic cells and these cells, especially the stroma facilitates transmission of proliferative signals or diffusion of locally produced cytokines
Maturing blood cells can enter the circulation through openings in the vascular sinuses (megakaryocytes & erythroblasts clustered against sinuses) – usually go first to other hematopoietic tissues for further maturation
Distinct microenvironments per lineage, i.e., erythropoietic, eosinophilic, etc.
From NIH Stem Cell Primer at http://www.nih.gov/news/stemcell/scireport.htm
Functional assay to ID stem cells in vivo: is the cell able to stably generate multiple hematopoietic lineages in irradiated or SCID (immunodeficient) mice?
Sacrifice, take marrow
Short-term reconstituting XX blood cells
Blood is XY
Most strict definition of an HSC: ability to serially
(HSC loses ability to do this after it has
self-renewed several times)
HSC home to marrow
& are quiescent for
up to 48 hrs
to 2ndary host
Expression of new sets of genes
Expression of new sets of genes
•Major component of innate immune system… 1st line of defense against infection
•Surround microorganisms with pseudopodia
•Pseudopod fusion to form phagosome (phagocytosis)
•Granuole release into phagosome
•Secretion of granuole contents
A key concept : The marrow contains a large storage pool of neutrophils which can be reserved for release in a setting of stress, and that the exponential expansion of progenitor cells can be augmented by granulocyte colony stimulating factor (G-CSF) under stress conditions.
2.6 X 109 cells/kg in mitotic pool
Example of differentiation from the precursor stage onward: myeloblast to granulocyte (neutrophil maturation)
Terminal cell division
Increasing development of granuoles (antibacterial and phagocytic)
Increasing phagocytic function – pseudopodia extend around microorganisms and fuse to form a phagosome into which granuole contents are released
Expansion of cell number occurs as cells in the mitotic or proliferative pool replicate
The post-mitotic pool can no longer divide but continues to mature into terminally differentiated cells
In the setting of infection or stress, maturation time may be shortened, divisions may be skipped, and cells may be released into the bloodstream earlier
Mobilization of hematopoietic progenitor cells is a multistep process – common themes in this process (not in chronological order):
1) Adhesion interactions -- must first be disrupted for progenitor cell trafficking. In general adhesion molecules are expressed on hematopoietic progenitor cells in the marrow but are downregulated or degraded to facilitate egress of progenitors from the BM -- selectins, selectin ligands, integrins, CD44 and PECAM
2) Chemotaxis Transendothelial gradients of chemokines (cytokines with chemotactic activity) produced by stromal cells control direction and efficacy of transendothelial migration of hematopoietic progenitors – mature leukocytes respond to a variety of chemokines, but most important so far is SDF-1 stromal cell-derived factor-1 (made by BM stromal cells). Significant chemotactic activity in hematopoietic progenitor and stem cells…significantly enhances retention in BM and homing to BM.
The chemokine receptor CXCR-4 is the receptor for the SDF-1 chemokine similarly upregulated before mobilization and downregulated after transendothelial migration.
3) Paracrine cytokines may support mobilization –
Growth factor stimulated hematopoietic cells produce cytokines such as vascular endothelial growth factor (VEGF) that act on endothelial cells to support migration by increasing endothelial fenestration and permeability.
Mobilization – various molecules administered to donors can mobilize CD34+ stem cells out of marrow into circulation where can harvest from peripheral blood… granulocyte-colony stimulating factor (G-CSF), granuloctye-macrophage colony stimulating factor (GM-CSF), flt-3 ligand, stem cell factor (SCF), and a variety of cytokines (IL-7, IL-3, IL-12) and chemokines (IL-8, SDF-1), as well as chemotherapeutic agents cyclophosphamide and paclitaxel, with varying degrees of efficacy
G-CSF (Neupogen®, Filgrastim®) most commonly used, daily stimulations of healthy donors, sometimes used with the chemotherapeutic agent cyclophosphamide – induce proliferation of hematopoietic cells within the bone marrow
Mobilized PBL cells have a much faster engraftment than do bone marrow cells, due to the increased cell dose of transplanted mobilized cells and increased numbers of committed progenitor cells. BUT – proliferation & differentiation potential of CD34+/CD38- stem cells from mobilized PBL is inferior to that of undifferentiated BM.
Cord blood stem cells: Immediately after birth, relatively high levels of immature CD34 + progenitor & stem cells circulating (for about 48 hours)… umbilical cord blood represents a promising source of stem cells for transplantation, but #s are too low for transplant into adults – need to find a way to expand ex vivo (stromal cells).
Model of mechanisms of stem cell mobilization by G-CSF: disruption of retention in BM & proximity to stromal cells
Lapidot and Petit, 2002 Exp. Hematology 30:973