Hematopoiesis/Hematopoiesis Physiology

Hematopoiesis/Hematopoiesis Physiology Definitions Hematopoiesis is the process of continuous generation of mature blood cells in the bone marrow (Figure 1). Blood cells represent different kinds of mature circulating cells, their precursors in the bone marrow or the lymphatic organs, and the progenitor and stem cells, residing in the adult marrow, but also circulating in blood in some 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 compromised recipients Probably represent the target for leukemic transformation Quiescence Plasticity (?) These properties, except the last one, are considered essential for the definition of stem cells; plasticity, a recent hypothesis, is currently under investigation. Stem cells are extremely rare. The incidence of reconstituting stem cells is estimated at 1 to 2.5 per 100,000 injected nucleated marrow cells. Progenitor cells When get into active hematopoiesis, they exit the Go phase of the cell cycle, and undergo a series of maturational cell divisions, leading to the generation of progenitor 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 HEMATOPOIESIS assumes that hematopoiesis involves a stepwise process where multipotent stem cells give rise to a hierarchy of progenitor cell populations. During this process proliferative potential and ability for self renewal are gradually lost, whereas differentiation characteristics are acquired (Figure 2). T-lymphocyte platelets megakaryocyte B-lymphocyte NK-cell eosinophil Hematopoietic stem cell basophil neutrophil erhythrocytes monocyte FIGURE 1: Hematopoiesis. 9

self-renewal proliferation Pre-T cell T-lymphocyte self-renewal Common lymphoid progenitor Pre-B cell NK progenitor B-lymphocyte NK-cell BFU-E erhythrocyte Hematopoietic stem cell CFU-Meg megakaryocyte plateles Common myeloid progenitor CFU-Ba CFU-Eo basophil eosinophil CFU-G neutrophil FIGURE 2: Hierarchical model of hematopoiesis. CFU-GM CFU-M monocyte commitment differentiation THE CONTINUUM MODEL OF HEMATOPOIESIS, more recently proposed, suggests that the engraftable stem cell and the progenitor cell population are the same cell population which evidences different phenotypes at different points in the cell cycle, continuously and reversibly changing its gene expression profile and, thus, its surface receptor expression (Figure 3). G 2 /M progenitor 2 G 0 /G 1 stem cell S FIGURE 3: Continuum model of hematopoiesis. progenitor 1 10

H E M A T O P O I E S I S Stemness Stemness is defined as the pattern of gene expression that is common in all stem cells. As self renewal and pluripotency are the main stem cell properties, most of the stemness genes are believed to be related to these functions. In a more extended view, stemness genes may be shared by stem cells from different tissues, although in two studies aiming to prove this, only one gene was common among the 3 purified stem cell populations tested. It is uncertain at this point whether this finding casts doubt on the stemness hypothesis or on the methods used to prove it. Despite these facts, it is already known that certain genes are expressed more often in stem cell populations, permitting investigators to propose a set of stemness genes. Negativity for cell lineage markers, proliferation and self renewal connected genes and markers permitting interaction with specific microenvironments are included. It is most certain that no single marker will emerge to be unique for stemness and at the same time we could also suppose that the absence of a significant number of these genes from a cell population will rather not make these cells fit for the title of stem cells. ARF MDM2 p53 BMI1 p16 INK4A p21 CIP1 p18 INK4C p27 KIP1 mitogenic stimuli CDK 4/6 cyclin CDK 2 D cyclin E prb E2F P P prb E2F P P P P P prb E2F Go/G1 S FIGURE 4: Cell cycle regulation of hematopoietic stem cells. Cell cycle regulation of hematopoietic stem cells Quiescence versus Proliferation or activation Most s are believed to reside in their niches in a quiescent state, at the Go phase of the cell cycle. This state is better described as a state of readiness, as s are able to respond to appropriate signals and proliferate. Various gene products have been implicated in this process of stem cell arousal, which is characterized by a phase of preparation and subsequent phases of early and late proliferation. A re induction of quiescence marks the final stage of activation cyscle. Cell cycle is regulated in s via the actions of specific gene products on cyclin cyclin dependent kinases (cdk) complexes. Of importance among these cell cycle regulating genes seem to be Bmi 1, p21, p27, p18 and arf (Figure 4). Recent experiments have shown that the initial phase of exit from quiescence are mostly stochastic or intrinsic to the, while later phases are increasingly induced by signals from the microenvironment of the hematopoietic niche. As it has been recently reported, constitutive activation of NF κb is not sufficient to disturb normal steady state hematopoiesis. 11

ECM integrins Wnt/frizzled Notch/jagged telomerase stroma cell transcription factors self-renewal HOXB 4 cell cycle regulators ILs and other soluble GFs differentiation apoptosis mobilization FIGURE 5: Molecular basis of hematopoietic stem cell fate decisions. Hematopoietic stem cell fate decisions Self renewal versus Differentiation versus Apoptosis Commitment to terminal differentiation and self renewal represent two opposite outcomes. Normal hematopoiesis requires a balance between these two outcomes. Two models have been proposed to explain the mechanisms that regulate the decision for self renewal or differentiation: the stochastic and the deterministic model. In the stochastic model the decision of an individual stem cell to undergo self renewal or differentiation is thought to be determined by chance. In the deterministic model, the stem cell fate is determined by the action of cytokines and extracellular matrix components (Figure 5). As already stated most stem cells are in a state of quiescence. They, however, do divide and during these divisions some crucial decision is taken between three mainly fates, namely self renewal, differentiation or apoptosis. Intrinsic and extrinsic factors from the BM microenvironment regulate the fate. The non hematopoietic cells from the BM microenvironment can interact with the hematopoietic progenitors through integrins, adhesion molecules, and receptor ligand juxtaposition (i.e. Delta/Notch1, ckit/scf). Additionally these cells can secrete various factors (i.e. chemokines and growth factors) and they can synthesize and display on their cell surface various forms of proteoglycans to enhance cell cell interactions and sequester soluble factors within the BM to. A number of gene products have been implicated in the self renewal versus differentiation process of s. Among these genes Wnt, Notch and HoxB4 seem to play especially important roles. Wnt promotes nuclear translocation of β catenin resulting in increased self renewal of. Notch maintains the pluripotent identity of. The prevailing opinion is that expression of the above genes drives stem cells towards self renewal and preservation of stemness, instead of differentiation. Absence of their collaborative expression, on the other hand, leads as a default choice towards differentiation. Self renewal implies conservation of s numbers and this seems to be mediated via asymmetric divisions. This means that in each division one of the daughter cells maintains stemness and the other is marked for differentiation. Little is understood concerning the basis of this distinction and the signaling pathways involved, although recently substantial progress is made, mostly from studies in lower organisms, where polarity of s is a crucial factor. An important role for the Smad signaling pathway in the regulation of self renewal of s has been reported in an model. Apoptosis is another probable fate of s and its role in normal hematopoiesis is strongly debated. A role for apoptosis and its defects in cancer cells is better understood. 12

H E M A T O P O I E S I S Marrow microenvironment Marrow microenvironment is a term used to denote the specialized milieu of the bone marrow cavity, in which hematopoiesis takes place in the adult (Figure 6). Niche is defined as the specific in vivo regulatory microenvironment where s reside. This structure is composed from different cell types, which contribute via cell receptors and soluble factors to the localization, survival, self renewal and differentiation of s (Figure 7 and 8). This niche concept includes a developmental aspect as well, as different aggregations of cells are supporting hematopoiesis in the AGM region of the embryo, the fetal liver or the marrow. niche bone osteoblasts ECM myofibroblast Endothelial cells Marrow fibroblasts, myofibroblasts and endothelial cells are considered components of the hematopoietic inductive environment; recently the role of endosteal osteoblasts and their precursors has gained considerable recognition, as a prominent player in the process. fibroblast The number of niches is probably determined by PTH stimulated osteoblastic proliferation. Homing of s is their ability to localize and reside at the specialized niches, where hematopoiesis can take place. This is achieved by a dynamic process involving mutual recognition and continuous interaction with stromal cells, via a complex network of surface molecules. BMP FIGURE 6: Bone marrow microenvironment. PTH Mobilization of s may act as a regulator of the hematopoietic stem cell compartment size. Mobilization could be a death pathway, a mechanism that regulates stem cell number. If a replication event occurs and a niche is not available, one offspring mobilizes/dies. After cytokine exposure, many exit the marrow, travel through blood and then repopulate empty marrow niches. FIGURE 7: Regulation of homing, survival, and maintenance by the niche. Endosteal osteoblasts are prominent components of the niche. Osteoblast interactions through cell cell adhesion molecules (N cadherin, β1 integrins, VLA 4 VCAM, LFA ICAM etc), soluble and cell 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 or proliferation and play a key role in the establishment and maintenance of the niche in the bone marrow (BM). Homophilic interaction through N cadherin apears to be critical in keeping s quiescent and may also provide a link to Wnt B catenin signaling pathway that has been shown to regulate self renewal. niche localization and self renewal in the osteoblastic niche is also critically regulated by Jagged 1 (expressed on osteoblasts) Notch signaling. Osteoblasts also secrete Ang 1 which, by activating of Tie2 receptors on s, promotes tight adhesion of stem cells in their niche and stem cell maintenance. osteopontin Cytokines GFs B-integrin osteoblast ICAM-LFA VCAM-VLA ECM N-cadherin B-catenin Homing Quiescence Survival Jagged Notch Tie2 Ang1 M c-kit BMSC These interactions are likely influenced by signals such as PTH and BMPs that regulate osteoblastic function. However, despite the prominent role of osteoblasts, other bone marrow stromal cells (BMSCs) as well as attachment to extracellular matrix (ECM) are also requiered for stem cell maintenance e.g the quiescent is kept in the osteoblastic niche in cooperation with adjacent membrane bound c kit expressing BMSCs. : haematopoietic stem cell, BMPs: Bone morphogenetic proteins, PTH: parathormone, ICAM: intracellular cell adhesion molecule, LFA: lymphocyte function 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. 13

Osteoblastic niche 1 Endothelial cells Osteoblast MMP9 action S c-kit M c-kit BMSC quiescence Vascular niche proliferation 2 Osteoblast differentiation differentiation BMSC Osteoblasts back to osteoblastic niche FIGURE 8: Osteoblastic and vascular niche. Although most s reside in Go within their niches there is always a small number of s that enters the cell cycle to initiate self renewal or differentiation. It is not clear whether proliferation and differentiation occurs into the osteoblastic niche or involves local mobilization to osteogenic stromal cells expressing Jag 1 or vascular niches specialized for proliferation. In one proposed model the activity of matrix metalloproteinase 9 (MMP 9) expressed within the osteoblastic zone results in cleavage of the membrane kit ligant from BM stromal cells (BMSCs). Soluble kit ligant then promotes cell cycle entry and localization of stem cells in the vascular zone where proliferation and differentiation occurs (1). Alternatively assymetric division could occur in the osteoblastic zone and s daugther cells released may return to accessible osteoblastic niches or may be mobilized to the periphery or induced to differentiate (2). Ontogeny of hematopoiesis The developmental origin of adult type hematopoiesis is currently placed at the intrabody portion of the embryo, and especially the aorta gonads mesonephros (AGM) region, rather at the extra embryonic yolk sac, although both sites are rather contributing. Fetal liver is the predominant site at approximately 6 weeks of gestation until about midterm, when bone marrow replaces fetal liver (Figure 9). In some species, as the mouse, spleen is an adult site of hematopoiesis. Primitive and definitive hematopoietic cells in the mouse embryo arise from embryonic endothelial cell marker positive cells. An important difference between fetal and adult hematopoiesis is the inability of the marrow cavity of the fetus to expand in situations of hematopoietic stress. SCL (Stem Cell Ligand)acts not only at the level of erythroid commitment, but also at both multipotent and myeloid committed stages of adult hematopoiesis. Among the molecular mechanisms implicated in early hematopoiesis significant role has been attributed to vascular endothelial growth factor (VEGF) and its receptor flk 1, as well as to bone morphogenetic protein 4 (BMP 4), fibroblast growth factor, and hedgehog proteins. It is of importance that hematopoietic and vascular cells are thought to arise from a common progenitor called the haemangioblast, which is found at highest frequency in the posterior region of the primitive streak, indicating that initial stages of haematopoietic and vascular commitment occur before blood island development in the yolk sac. 14

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