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Hematopoiesis/Hematopoiesis Physiology

Definitions

Hematopoiesis is the process of continuous generationof mature blood cells in the bone marrow (Figure 1).

Blood cells represent different kinds of mature circulat-ing cells, their precursors in the bone marrow or the lym-phatic organs, and the progenitor and stem cells, residingin the adult marrow, but also circulating in blood insome instances.

Hematopoietic stem cells are specialized, rare cells,sharing some very important basic properties, namely:

• Self−renewal• Extensive proliferative capacity• Multipotent differentiation• Ability to reconstitute hematopoiesis in compro-

mised recipients• Probably represent the target for leukemic trans-

formation• Quiescence• Plasticity (?)

These properties, except the last one, are consideredessential for the definition of stem cells; plasticity, a re-cent hypothesis, is currently under investigation.

T-lymphocyte

monocyte

neutrophil

B-lymphocyte

platelets

megakaryocyte

NK-cell

eosinophil

basophil

erhythrocytes

Hematopoieticstem cell

FIGURE 1: Hematopoiesis.

Stem cells are extremely rare. The incidence of recon-stituting stem cells is estimated at 1 to 2.5 per 100,000injected nucleated marrow cells.

Progenitor cells When HSC get into active hematopoiesis, they exit theGo phase of the cell cycle, and undergo a series of mat-urational cell divisions, leading to the generation ofprogenitor cells. Progenitor cells are characterized by: • Inability to reconstitute hematopoiesis in vivo• Limited proliferative capacity• Decreased or no self−renewal capacity• Active cycling• Irreversible lineage commitment

THE HIERARCHICAL MODEL OF HEMATOPOIESISassumes that hematopoiesis involves a stepwiseprocess where multipotent stem cells give rise to a hi-erarchy of progenitor cell populations. During thisprocess proliferative potential and ability for self−re-newal are gradually lost, whereas differentiation char-acteristics are acquired (Figure 2).

C H A P T E R 1

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THE CONTINUUM MODEL OF HEMATOPOIESIS,more recently proposed, suggests that the engraftablestem cell and the progenitor cell population are thesame cell population which evidences different phe-

notypes at different points in the cell cycle, continu-ously and reversibly changing its gene expressionprofile and, thus, its surface receptor expression (Fig-ure 3).

self-renewal

Hematopoieticstem cell

Common myeloidprogenitor

Common lymphoidprogenitor

BFU-E

CFU-Meg

CFU-Ba

CFU-Eo

CFU-GM

monocyte

neutrophil

eosinophil

basophil

plateles

erhythrocyte

NK-cell

B-lymphocyte

T-lymphocytePre-T cell

Pre-B cell

NK progenitor

megakaryocyte

CFU-G

CFU-M

self-renewalproliferation

commitmentdifferentiation

G2/M

S

G0/G1

progenitor 2

progenitor 1

stem cell

FIGURE 2: Hierarchical model of hematopoiesis.

FIGURE 3: Continuum model of hematopoiesis.

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Stemness

“Stemness” is defined as the pattern of gene expres-sion that is common in all stem cells. As self−renewaland pluripotency are the main stem cell properties,most of the stemness genes are believed to be related tothese functions.

In a more extended view, stemness genes may beshared by stem cells from different tissues, although intwo studies aiming to prove this, only one gene wascommon among the 3 purified stem cell populationstested. It is uncertain at this point whether this findingcasts doubt on the stemness hypothesis or on themethods used to prove it.

pRb

ARF

BMI1

MDM2 p53

p18INK4C

p16INK4A p21CIP1

p27KIP1

E2F

pRb

E2F

Go/G1 S

cyclinE

cyclinD

CDK 4/6 CDK 2

P PpRb

E2F

P P PP P

mitogenicstimuli

FIGURE 4: Cell cycle regulation of hematopoietic stem cells.

Despite these facts, it is already known that certaingenes are expressed more often in stem cell popula-tions, permitting investigators to propose a set of“stemness genes”. Negativity for cell lineage markers,proliferation and self−renewal connected genes andmarkers permitting interaction with specific microen-vironments are included. It is most certain that no sin-gle marker will emerge to be unique for stemness andat the same time we could also suppose that the absenceof a significant number of these genes from a cell pop-ulation will rather not make these cells fit for the title ofstem cells.

Cell cycle regulation of hematopoietic stem cells

Quiescence versus Proliferation or HSC activationMost HSCs are believed to reside in their niches in aquiescent state, at the Go phase of the cell cycle. Thisstate is better described as a “state of readiness”, as HSCsare able to respond to appropriate signals and prolifer-ate. Various gene products have been implicated in thisprocess of “stem cell arousal”, which is characterized bya phase of preparation and subsequent phases of earlyand late proliferation. A re−induction of quiescencemarks the final stage of HSC activation cyscle.

Cell cycle is regulated in HSCs via the actions of spe-cific gene products on cyclin−cyclin dependent ki-

nases (cdk) complexes. Of importance among thesecell cycle regulating genes seem to be Bmi−1, p21, p27,p18 and arf (Figure 4).

Recent experiments have shown that the initial phase ofexit from quiescence are mostly “stochastic” or intrin-sic to the HSC, while later phases are increasingly “in-duced” by signals from the microenvironment of thehematopoietic niche.

As it has been recently reported, constitutive activationof NF−κB is not sufficient to disturb normal steady−statehematopoiesis.

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self-renewal

differentiation apoptosis mobilization

ILs and othersoluble GFs

Notch/jagged

Wnt/frizzledECMintegrins

telomerase

cell cycleregulators

transcriptionfactors

HOXB4

HSC

stroma cell

Hematopoietic stem cell fate decisions

Self−renewal versus Differentiation versus Apoptosis

Commitment to terminal differentiation and self–re-newal represent two opposite outcomes. Normalhematopoiesis requires a balance between these twooutcomes. Two models have been proposed to explainthe mechanisms that regulate the decision for self–re-newal or differentiation: the stochastic and the deter-ministic model. In the stochastic model the decisionof an individual stem cell to undergo self renewal ordifferentiation is thought to be determined by chance.In the deterministic model, the stem cell fate is deter-mined by the action of cytokines and extracellular ma-trix components (Figure 5).

As already stated most stem cells are in a state of quies-cence. They, however, do divide and during these divi-sions some crucial “decision” is taken between threemainly fates, namely self–renewal, differentiation orapoptosis. Intrinsic and extrinsic factors from the BMmicroenvironment regulate the HSC fate. Thenon–hematopoietic cells from the BM microenviron-ment can interact with the hematopoietic progenitorsthrough integrins, adhesion molecules, and receptor–lig-and juxtaposition (i.e. Delta/Notch1, ckit/SCF). Addi-tionally these cells can secrete various factors (i.e.chemokines and growth factors) and they can synthe-size and display on their cell surface various forms ofproteoglycans to enhance cell–cell interactions and se-quester soluble factors within the BM to HSC.

A number of gene products have been implicated in theself–renewal versus differentiation process of HSCs.Among these genes Wnt, Notch and HoxB4 seem toplay especially important roles. Wnt promotes nucleartranslocation of β–catenin resulting in increased self re-newal of HSC. Notch maintains the pluripotent identityof HSC. The prevailing opinion is that expression of theabove genes drives stem cells towards self–renewal andpreservation of “stemness”, instead of differentiation. Ab-sence of their collaborative expression, on the otherhand, leads as a default choice towards differentiation.

Self–renewal implies conservation of HSCs numbersand this seems to be mediated via asymmetric divi-sions. This means that in each HSC division one of thedaughter cells maintains stemness and the other ismarked for differentiation. Little is understood con-cerning the basis of this distinction and the signalingpathways involved, although recently substantialprogress is made, mostly from studies in lower organ-isms, where polarity of HSCs is a crucial factor.

An important role for the Smad−signaling pathway inthe regulation of self−renewal of HSCs has been re-ported in an model.

Apoptosis is another probable fate of HSCs and its role innormal hematopoiesis is strongly debated. A role for apop-tosis and its defects in cancer cells is better understood.

FIGURE 5:Molecular basis of hematopoietic stem cell fate decisions.

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HSC nichebone

Endothelialcells

myofibroblast

osteoblasts

HSC ECM

HSC

fibroblast

Marrow microenvironmentMarrow microenvironment is a term used to denotethe specialized milieu of the bone marrow cavity, inwhich hematopoiesis takes place in the adult (Figure 6).Niche is defined as the specific in vivo regulatory mi-croenvironment where HSCs reside. This “structure” iscomposed from different cell−types, which contribute viacell receptors and soluble factors to the localization, sur-vival, self−renewal and differentiation of HSCs (Figure7 and 8). This “niche” concept includes a developmentalaspect as well, as different aggregations of cells are sup-porting hematopoiesis in the AGM region of the embryo,the fetal liver or the marrow.Marrow fibroblasts, myofibroblasts and endothelialcells are considered components of the hematopoieticinductive environment; recently the role of endostealosteoblasts and their precursors has gained consider-able recognition, as a prominent player in the process.The number of HSC niches is probably determined byPTH−stimulated osteoblastic proliferation.Homing of HSCs is their ability to localize and reside atthe specialized “niches”, where hematopoiesis can takeplace. This is achieved by a dynamic process involvingmutual recognition and continuous interaction with stro-mal cells, via a complex network of surface molecules.Mobilization of HSCs may act as a regulator of thehematopoietic stem cell compartment size. Mobiliza-tion could be a death pathway, a mechanism that regu-lates stem cell number. If a replication event occurs anda niche is not available, one offspring mobilizes/dies.After cytokine exposure, many HSC exit the marrow,travel through blood and then repopulate empty mar-row niches.

FIGURE 6: Bone marrow microenvironment.

osteoblast

HSC

B-catenin

CytokinesGFs

B-integrin

ECMHoming

QuiescenceSurvival

BMSC

N-cadherinosteopontin

Jagged Notch

M c-Kit

ICAM-LFA

BMP PTH

VCAM-VLA

Tie2

Ang1

FIGURE 7: Regulation of HSC homing, survival, and maintenance

by the HSC niche.

Endosteal osteoblasts are prominent components of the HSC niche.Osteoblast−HSC interactions through cell−cell adhesion molecules(N−cadherin, β1−integrins, VLA−4−VCAM, LFA−ICAM etc), soluble andcell−surface associated molecules (Ang−1, Jagged−1, osteopontin),cytokines and growth factors (G−CSF, GM−CSF, M−CSF, IL−1, SDF−1,TGF−β etc) regulate stem cell niche localization, survival, quiesence orproliferation and play a key role in the establishment and maintenanceof the HSC niche in the bone marrow (BM). Homophilic interactionthrough N−cadherin apears to be critical in keeping HSCs quiescent andmay also provide a link to Wnt−B−catenin signaling pathway that hasbeen shown to regulate HSC self−renewal. HSC niche localization andself renewal in the osteoblastic niche is also critically regulated byJagged−1 (expressed on osteoblasts)−Notch signaling. Osteoblasts alsosecrete Ang−1 which, by activating of Tie2 receptors on HSCs, promotestight adhesion of stem cells in their niche and stem cell maintenance.

These interactions are likely influenced by signals such as PTH and BMPsthat regulate osteoblastic function. However, despite the prominent roleof osteoblasts, other bone marrow stromal cells (BMSCs) as well asattachment to extracellular matrix (ECM) are also requiered for stem cellmaintenance e.g the quiescent HSC is kept in the osteoblastic niche incooperation with adjacent membrane− bound c−kit−expressing BMSCs.HSC: haematopoietic stem cell, BMPs: Bone morphogenetic proteins, PTH:parathormone, ICAM: intracellular cell−adhesion molecule, LFA: lymphocytefunction−associated antigen, VCAM: vascular cell adhesion molecule,Ang−1: angiopoietin−1, GFs: growth factors, ECM: extracellular matrix,BMSCs: bone marrow stromal cells, M c−Kit: membrane− bound c−kit.

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FIGURE 8: Osteoblastic and vascular niche.

Although most HSCs reside in Go within their niches there isalways a small number of HSCs that enters the cell cycle toinitiate self−renewal or differentiation. It is not clear whetherHSC proliferation and differentiation occurs into the osteoblasticniche or involves local mobilization to osteogenic stromal cellsexpressing Jag 1 or vascular niches specialized for HSCproliferation.In one proposed model the activity of matrix metalloproteinase 9

(MMP−9) expressed within the osteoblastic zone results in cleavageof the membrane kit ligant from BM stromal cells (BMSCs). Solublekit ligant then promotes cell cycle entry and localization of stemcells in the vascular zone where proliferation and differentiationoccurs (1). Alternatively assymetric HSC division could occur in theosteoblastic zone and HSCs daugther cells released may return toaccessible osteoblastic niches or may be mobilized to the peripheryor induced to differentiate (2).

Osteoblastic niche

Vascular nichequiescence

proliferation

differentiation

differentiation

back to osteoblastic niche

Osteoblast

Endothelial cells

Osteoblast

OsteoblastsHSC

1

2

HSC

HSC

MMP9action

BMSC

BMSC

S c-kit

M c-kit

Ontogeny of hematopoiesis

The developmental origin of adult−type hematopoiesisis currently placed at the intrabody portion of the em-bryo, and especially the aorta−gonads−mesonephros(AGM) region, rather at the extra−embryonic yolk sac,although both sites are rather contributing. Fetal liver isthe predominant site at approximately 6 weeks of gesta-tion until about midterm, when bone marrow replacesfetal liver (Figure 9). In some species, as the mouse,spleen is an adult site of hematopoiesis. Primitive anddefinitive hematopoietic cells in the mouse embryo arisefrom embryonic endothelial cell−marker–positive cells.

An important difference between fetal and adulthematopoiesis is the inability of the marrow cavity of thefetus to expand in situations of hematopoietic stress.

SCL (Stem Cell Ligand)acts not only at the level of ery-

throid commitment, but also at both multipotent andmyeloid committed stages of adult hematopoiesis.

Among the molecular mechanisms implicated in earlyhematopoiesis significant role has been attributed tovascular endothelial growth factor (VEGF) and its re-ceptor flk−1, as well as to bone morphogenetic pro-tein−4 (BMP−4), fibroblast growth factor, andhedgehog proteins.

It is of importance that hematopoietic and vascularcells are thought to arise from a common progenitorcalled the haemangioblast, which is found at highestfrequency in the posterior region of the primitivestreak, indicating that initial stages of haematopoieticand vascular commitment occur before blood islanddevelopment in the yolk sac.

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6 weeks

Midtrimester

SpleenBone Marrow

liver

Yolk sac AGM

FIGURE 9: Ontogeny of hematopoiesis.

Selected references:

Arai, F., A. Hirao, et al. (2004). “Tie2/angiopoietin−1 sig-naling regulates hematopoietic stem cell quiescence in thebone marrow niche”. Cell 118(2): 149−61

Beslu, N., J. Krosl, et al. (2004). “Molecular interactions in-volved in HOXB4−induced activation of HSC self−re-newal”. Blood 104(8): 2307−2314

Blank, U., G. Karlsson, et al. (2006). “Smad7 promotes self−re-newal of hematopoietic stem cells”. Blood 108(13): 4246−54

de la Grange, P. B., F. Armstrong, et al. (2006). “LowSCL/TAL1 expression reveals its major role in adulthematopoietic myeloid progenitors and stem cells”. Blood108(9): 2998−3004

Duncan, A. W., F. M. Rattis, et al. (2005). “Integration of

Notch and Wnt signaling in hematopoietic stem cell main-tenance”. Nat Immunol 6(3): 314

Ema, M., T. Yokomizo, et al. (2006). “Primitive erythropoiesisfrom mesodermal precursors expressing VE−cadherin,PECAM−1, Tie2, endoglin, and CD34 in the mouse embryo”.Blood 108(13): 4018−24

Faubert, A., J. Lessard, et al. (2004). “Are genetic determi-nants of asymmetric stem cell division active inhematopoietic stem cells”? Oncogene 23(43): 7247−55

Fortunel, N. O., H. H. Otu, et al. (2003). “Comment onStemness’: transcriptional profiling of embryonic andadult stem cells” and “a stem cell molecular signature”. Sci-ence 302 (5644): 393; author reply 393

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Ivanova, N. B., J. T. Dimos, et al. (2002). “A stem cell mo-lecular signature”. Science 298(5593): 601−4

Iwama, A., H. Oguro, et al. (2004). “Enhanced self−re-newal of hematopoietic stem cells mediated by the poly-comb gene product Bmi−1”. Immunity 21(6): 843−51

Lessard, J., A. Faubert, et al. (2004). “Genetic programs regulat-ing HSC specification, maintenance and expansion”. Oncogene23(43): 7199−209

Pyle, A. D., P.J. Donovan, et al. (2004). “Chipping away at'stemness”.Genome Biol 5(8): 235

Quesenberry, P. J., G. A. Colvin, et al. (2002). “Thechiaroscuro stem cell: a unified stem cell theory”. Blood100(13): 4266−71

Ramalho−Santos, M., S. Yoon, et al. (2002). “Stemness:transcriptional profiling of embryonic and adult stemcells”. Science 298(5593): 597−600

Reya, T., A. W. Duncan, et al. (2003). “A role for Wnt sig-nalling in self−renewal of haematopoietic stem cells”. Nature423(6938): 409

Schepers, H., B. J. Eggen, et al. (2006). “Constitutive acti-vation of NF−kappaB is not sufficient to disturb normalsteady−state hematopoiesis”. Haematologica 91(12):

1710−1

Srour, E. F., X. Tong, et al. (2005). “Modulation of in vitroproliferation kinetics and primitive hematopoietic poten-tial of individual human CD34+CD38−/lo cells in G0”.Blood 105(8): 3109−3116

Steinman, R. A. (2002). “Cell cycle regulators andhematopoiesis”. Oncogene 21(21): 3403−13

Taichman, R. S. (2005). “Blood and bone: two tissueswhose fates are intertwined to create the hematopoieticstem−cell niche”. Blood 105(7): 2631−9

Venezia, T. A., A. A. Merchant, et al. (2004). “MolecularSignatures of Proliferation and Quiescence in Hematopoi-etic Stem Cells”. PLoS Biology 2(10): e301

Wagner, W., A. Ansorge, et al. (2004). “Molecular evidencefor stem cell function of the slow−dividing fraction amonghuman hematopoietic progenitor cells by genome−wideanalysis”. Blood 104(3): 675−686

Wallenfang, M. R. and E. Matunis (2003). “Developmentalbiology. Orienting stem cells”. Science 301(5639): 1490−1

Zhu, J. and S. G. Emerson (2004). “A new bone to pick: os-teoblasts and the haematopoietic stem−cell niche”. Bioes-says 26(6): 595−9