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Current Stem Cell Research & Therapy, 2007, 2, 39-52

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Function and Malfunction of Hematopoietic Stem Cells in Primary Bone Marrow Failure Syndromes

Antonio M. Risitano*,1 , Jaroslaw P. Maciejewski2 , Carmine Selleri1 and Bruno Rotoli1

1Division

of Hematology, Federico II University of Naples, Via Pansini 5, 80131 Naples, Italy

2Experimental

Hematology and Hematopoiesis Section, Taussig Cancer Center, The Cleveland Clinic Foundation, 9500 Euclid Avenue, 44195 Cleveland, OH, USA

Abstract: Hematopoietic stem cells (HSCs) are responsible for the production of mature blood cells in bone marrow; peripheral pancytopenia is a common clinical presentation resulting from several different conditions, including hematological or extra-hematological diseases (mostly cancers) affecting the marrow function, as well as primary failure of hematopoiesis. Primary bone marrow failure syndromes are a heterogeneous group of diseases with specific pathogenic mechanisms, which share a profound impairment of the hematopoietic stem cell pool resulting in global or selective marrow aplasia. Constitutional marrow failure syndromes are conditions caused by intrinsic defects of HSCs; they are due to inherited germline mutations accounting for specific phenotypes, and often involve also organs and systems other than hematopoiesis. By contrast, in acquired marrow failure syndromes hematopoietic stem cells are thought to be intrinsically normal, but subjected to an extrinsic damage affecting their hematopoietic function. Direct toxicity by chemicals or radiation, as well as association with viruses and other infectious agents, can be sometimes demonstrated. In idiopathic Aplastic Anemia (AA) immunological mechanisms play a pivotal role in damaging the hematopoietic compartment, resulting in a depletion of the hematopoietic stem cell pool. Clinical and experimental evidences support the presence of a T cell-mediated immune attack, as confirmed by clonally expanded lymphocytes, even if the target antigens are still undefined. However, this simple model has to be integrated with recent data showing that, even in presence of an extrinsic damage, preexisting mutations or polymorphisms of genes may constitute a genetic propensity to develop marrow failure. Other recent data suggest that similar antigen-driven immune mechanisms may be involved in marrow failure associated with lymphoproliferative or autoimmune disorders characterized by clonal expansion of T lymphocytes, such as Large Granular Lymphocyte leukemia. In this wide spectrum, a unique and intriguing condition is Paroxysmal Nocturnal Hemoglobinuria (PNH); even in presence of a somatic mutation of the PIG-A gene carried by one or more HSCs and their progeny, the typical marrow failure in PNH is likely due to pathogenic mechanisms similar to those involved in AA, and not to the intrinsic abnormality conferred to the clonal population by the PIG-A mutation. The study of hematopoietic stem cell function in marrow failure syndromes provides hints for specific molecular pathways disturbed in many diseases of hematopoietic and non-hematopoietic stem cells. Beyond the specific interest of investigators involved in the field of these rare diseases, marrow failure syndromes represent a model that provides intriguing insight into quantity and function of normal hematopoietic stem cells, improving our knowledge on stem cell biology.

Keywords: Hematopoietic stem cells, bone marrow failure, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Fanconi's anemia, dyskeratosis congenita. INTRODUCTION Hematopoietic stem cells (HSC) are responsible for the long-life production of mature blood cells [1,2]; physiologically, they reside inside the bone, where they interact with other cellular types and soluble factors, formally known as microenvironment, to constitute the bone marrow [3]. Hematopoiesis is a hierarchical process which starts from the multipotent HSC and proceeds through more committed and differentiated progenitors [4]; while differentiation and maturation are essential for producing mature circulating cells, self-renewal is the main feature of HSC, ensuring long-term maintenance of hematopoiesis [5]. All these functions are finely regulated by a complex

*Address correspondence to this author at the Division of Hematology, Federico II University of Naples, Via Pansini 5, 80131 Naples, Italy; Tel: +39 081 746 2037; Fax: +39 081 746 2165; E-mail: [email protected] 1574-888X/07 $50.00+.00

network which includes cell-cell interactions between hematopoietic and stromal cells, as well as the action of several soluble cytokines, often working in a paracrine fashion within the so-called hematopoietic niches. Bone marrow is a functional tissue which may be insufficiently performing in several conditions, all clinically presenting as mono- or multi-lineage cytopenia. Bone marrow failure (BMF) may result from various extra-hematological diseases, such as malignancies, infectious diseases and nutritional deficiencies, all of them secondarily affecting HSC function. Similarly, hematopoiesis may be impaired in various hematological diseases, such as lymphoproliferative and myeloproliferative disorders; even when the disease involves HSCs themselves, such as in leukemias, BMF is usually considered secondary to the underlying disease. By contrast, primary BMF syndromes are a heterogeneous group of hematological diseases (Table 1) characterized by the absence of any other disorder potentially affecting marrow

© 2007 Bentham Science Publishers Ltd.

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function; in this paper we review the pathophysiology of these conditions, focusing on HSCs and mechanisms leading to their functional impairment.

Table 1. Bone Marrow Failure Syndromes

Constitutional · · · · · · · Fanconi's anemia Dyskeratosis congenita Shwachman-Diamond syndrome Amegakaryocytic thrombocytopenia Reticular dysgenesis Non-hematological diseases (e.g. Down's syndrome) Familial aplastic anemia o o Acquired Primary Isolated · · Idiopathic aplastic anemia Associated with different medical conditions Hematological diseases o o o · · · Secondary Radiation Chemicals and drugs o o Direct toxicity (cytotoxic agents, benzene) Idiosyncratic reaction (chloramphenicol, nonsteroidal antiinflammatory agents, insecticides, anticonvulsants, antiprotozoals, sulfonamides and some other antibiotics, antithyroid agents, antidiabetes agents, carbonic anydrase inhibitors, sedatives and tranquilizers, allopurinol, gold and other heavy methals) B, C, non-A, non-B, non-C hepatitis (hepatitis-aplastic anemia syndrome) Epstein-Barr virus* Cytomegalovirus* Parvovirus (mostly affecting erythropoiesis)* HIV (rare and multifactorial)* Eosinophilic fasciitis Thymoma and thymic carcinoma Graft versus host disease Hypogammaglobulinemia Paroxysmal nocturnal hemoglobinuria Myelodysplastic syndromes Large granular lymphocyte disorders Human telomerase complex TERT mutations Unknown molecular defect

According to the hierarchical model of hematopoiesis, the desert marrow in AA results from impaired HSCs function. The alteration affecting the HSC may be different depending on the specific form of AA; indeed, even within the AA setting, different entities may be sorted, each one with specific pathophysiologic mechanisms. A first distinction has to be made between constitutional and acquired forms of AA. Constitutional Aplastic Anemias The constitutional forms of AA share the presence of intrinsic defects of the HSC, which are usually inherited through a mendelian pattern of monogenic diseases; since they are germline mutations, the defect usually affects even cells other than hematological, resulting in complex phenotypes which characterize the specific disease. Constitutional AAs are primary diseases of the HSCs; the intrinsic defect within the hematopoietic cells is directly responsible for the marrow failure, through different molecular pathways (Fig. 1). Fanconi's Anemia (FA) is the most frequent inherited bone marrow failure syndrome; it is characterized by a wide range of congenital anomalies, which include short stature and other skeletal abnormalities (mostly of upper limbs and forearms), skin pigmentary changes (cafe'-au-lait spots) and malformations of any other apparatus [7]. The hematological picture is characterized by a moderate mono- or bi-lineage cytopenia, usually presenting within the first decade, which usually proceeds towards a more severe and global marrow failure [8]. FA is also associated with increased risk of developing clonal abnormalities of hematopoiesis, such as myelodysplastic syndromes or overt acute leukemias, as well as solid tumors. FA patients show in vitro findings typical of primary marrow failure, i.e. impaired growth of hematopoietic progenitors; methylcellulose colonies of all lineages (burst-forming unit-erythroid [BFU-E], colonyforming unit-erythroid [CFU-E], colony-forming unitgranulocyte [CFU-G], colony-forming unit-granulocytemacrophage [CFU-GM] and CFU-granulocyte, erythroid, monocyte and megakaryocyte [CFU-GEMM]), are all absent or significantly decreased [9]. Long term hematopoietic cultures demonstrated that marrow cells from FA patients are able to form adherent layers, but fail in producing secondary colonies, as a result of long term culture-initiating cell (LTC-IC) pool contraction [10]. The laboratory and diagnostic hallmark of FA is an increased chromosomal breakage in response to cross-linking agents such as mitomycin C and diepoxybutane (DEB); molecular studies led to the identification of several FA complementation groups and of the corresponding genes. Up to now, eleven FANC proteins have been identified (FANCA, B, C, D1, D2, E, F and G), which may be found altered in FA patients; among their coding genes, the most frequently mutated are FANC-A (70%), FANC-C (10%) and FANC-G (10%) [11]. A number of different mutations of these genes have been identified in individual FA families; the disease phenotype is inherited through an autosomal recessive fashion in all cases, with the exception of the rare FANC-B mutations, which cause a X-linked transmission of the disease. All the FANC proteins are involved in the same

Pregnancy

·

Viruses o o o o o

·

Immune-mediated diseases o o o o

* rare forms, often transient

APLASTIC ANEMIA Aplastic anemia (AA) is the paradigm of bone marrow insufficiency; as other BMF syndromes, it is characterized by peripheral pancytopenia [6]. The hallmark of AA is an empty or fatty marrow as evidenced by bone biopsy, which directly demonstrates the contraction of the hematopoietic cell compartment leading to deficient hematopoiesis.

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DNA repair pathway, functioning as tumor suppressors [12], thus explaining the increased risk of FA patients to develop malignancies. By contrast, the relationship between the FANC proteins and marrow failure is not yet completely understood increased susceptibility to DNA damage by oxydative stress has been hypothesized [13]. FANC genes are highly expressed in HSCs [14], and there their products may be involved in several functions, including cell-cycling, apoptosis, senescence and self-renewal [15,16]. Dyskeratosis congenita (DKC) is a distinct inherited marrow failure syndrome which is clinically marked by the triad of skin pigmentation abnormalities, nail dystrophy and mucosal leukoplakia; other typical features are propensity to develop marrow failure, pulmonary dysfunctions and malignancies [17]. The inheritance pattern is quite heterogeneous: X-linked, autosomal recessive and autosomal dominant forms have been described, in decreasing order of frequency. Even in this setting, intrinsic abnormalities of hematopoiesis have been demonstrated by many experimental data. Hematopoietic progenitor colonies obtained from both unmanipulated as well as stromal layercultured marrow cells (primary and secondary colonies,

respectively) are reduced in all DKC patients; as for FA, DKC stromal cells are normal, whereas the hematopoietic cells grow poorly, thus supporting an intrinsic defect of HSC [18]. In comparison with other BMF syndromes, recent data have led to remarkable insight in understanding the molecular pathophysiology of this disease, even in the context of a considerable genetic heterogeneity. DKC may be linked to mutation of various genes coding for products involved in the functional pathway of telomere length maintenance (Fig. 1); this is confirmed by functional data, demonstrating accelerated telomere shortening in hematological cells from DKC patients [19]. The bestknown X-linked form of DKC, initially shown to be due to abnormalities in Xq28, is caused by a mutation in the DKC1 gene, encoding for dyskerin; dyskerin is a component of small nucleolar ribonucleoprotein particles which cooperates with the stabilization of the telomerase complex [20-22] and is important in ribosomal RNA processing [22] (Fig. 2). Autosomal dominant cases of DKC result from a mutation within the TERC gene [23,24], encoding for the RNA component which constitutes the functional telomerase complex together with the reverse transcriptase TERT (Fig.

Fig. (1). Molecular mechanisms of hematopoietic stem cell damage. HSCs may harbor genetically determined abnormalities affecting pivotal cell functions (in blue), such as DNA repair or telomere length maintenance, leading to reduced self-renewal for cycling arrest or even cell death, both resulting in impairment of normal hematopoiesis. In alternative, HSCs may suffer from extrinsic damage, as a result of direct toxicity from various agents (e.g. drugs, viruses [in green]) or of an aberrant immune response, possibly antigen-specific (depicted in black and white). Typical immune effector mechanisms (depicted in different colors of the red spectrum) include cell-mediated killing via perforine/granzyme or death receptors (Fas, TRAIL-Rs) as well as paracrine production of soluble factors such as the inhibitory cytokines IFN- and TNF-, all leading to the arrest of HSC cycling and eventual apoptosis. Lost of stem cell-ness may be a further mechanism of HSCs exhaustion upon extrinsic injuries. Intracellular pathways of cell damage are in black.

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2). Recently, mutations within the TERC gene have been reported in familial cases of AA, which were initially classified as acquired AA, given the adult onset of pancytopenia [25]. Telomerase function is essential to maintain the appropriate length of telomeres, and is physiologically involved in aging; impairment of this function leads to premature aging resulting in lack of selfrenewal and increased genetic instability [26]. These phenomena respectively translate into exhaustion of the hematopoietic stem cell compartment with eventual marrow failure, and propensity to clonal evolution, both typical of DKC. These observations clearly prove how different genetic lesions affecting the same molecular pathway may account for identical clinical phenotypes (phenocopy).

showing phylogenic relationship and structural homology with known proteins involved in RNA processing and ribosomal-associated functions [31,32], thus linking SDS to other marrow failures defective in nucleolus-associated functions, such as DKC, Diamond-Blackfand anemia and cartilage-hair hypoplasia (see below) [33]. CAMT is a marrow failure characterized by prevalent involvement of megakaryocytopoiesis, which may progress to a more generalized multi-lineage disease; the impairment of hematopoiesis is confirmed by reduced primary and secondary colonies. An intrinsic defect of HSC was initially recognized as faulty response to thrombopoietin (Tpo), which is abnormally elevated in CAMT patients [34]. The genetic defect was subsequently characterized as a mutation in the c-mpl gene, which encodes for the Tpo receptor [35,36]; this type of disease follows a mendelian autosomal recessive transmission. Tpo is essential for megakaryocyte formation but is also involved in early hematopoiesis through survival and anti-apoptotic signals to HSC (Fig. 1); thus, c-mpl mutations causes impaired thrombocytopoiesis and progression to marrow failure, both typical findings in CAMT patients. A number of other inherited marrow failure syndromes are known, which affect selectively only a single hematopoietic lineage; they usually appear as impairment of differentiation and maturation and are not discussed in this review. However, these diseases elucidates the behavior of the hematopoiesis hierarchical pattern in pathological conditions; in fact, unlike in global marrow failure where both HSCs and committed progenitors are affected, in single-lineage failure syndromes the HSC level is at least apparently not involved. Thus, they reflects intrinsic defects either phenotypically appearing in more mature progenitors (e.g., lack of protective mechanisms operating in HSCs but lost during differentiation, or involvement of pathways which are redundant in HSCs but more restricted in progenitors) or affecting cellular functions which become needed upon differentiation. Severe congenital neutropenia (Kostmann disease) is a typical example of marrow failure affecting the granulocytopoiesis [37]; it has been linked to mutations in the ELA2 gene [38], coding for the neutrophil elastase, which has also been found mutated in cyclic neutropenia [38,39]; attempts to explain how the mutated protein causes the disease have been so far elusive. Diamond-Blackfan anemia (DBA) is a constitutional red cell aplasia associated with craniofacial and thumbs malformations (less frequent and less severe than that seen in FA) [40]; in these patients, the marrow failure is usually limited to the red cell progenitors, as confirmed by in vitro colony studies [41,42], even if some of them may subsequently develop neutropenia. The disease has been linked to three loci on 19q [43], 8p [44] and 1q [45]; the first one has been studied in detail, leading to the identification of the causative gene RPS19 [46]. The RPS19 gene encodes for a protein component of the 40S ribosomal subunit which seems to be involved in the maturation of 40S, which is essential for a proper ribosomal function [33]. Interestingly, another form of constitutional red cells hypoplasia, the cartilage-hair hypoplasia (CHH) [47,48], has been linked to mutations in the RMRP gene [49], which encodes for the RNA component of the ribonuclease mitochondrial RNA processing, raising the hypothesis that

Fig. (2). The human telomerase gene complex. Schematic representation of the human telomerase gene complex. The reverse transcriptase of telomerase (TERT), which retains the enzymatic activity, interacts with the RNA component (TERC), which works as a template; to constitute a functional telomerase complex, additional components are needed, such as dyskerin, NOP10, NHP2 and GAR1. TERT, TERC or dyskerin have been found mutated in patients affected by different forms of bone marrow failure.

Two additional forms of inherited marrow failure are the Shwachman-Diamond syndrome (SDS) and congenital amegakaryocytic thrombocytopenia (CAMT). In SDS, hematological abnormalities are associated with exocrine pancreatic insufficiency and skeletal abnormalities such as short stature; the hematological picture is characterized by pancytopenia, usually evolving from initial neutropenia, and propensity to develop myelodysplastic syndromes and leukemia. In vitro findings demonstrate a quantitative and qualitative impairment of the hematopoietic progenitor pool, likely including stromal cells [27], with increased apoptosis playing a pivotal pathogenic role [28]. The syndrome is inherited as autosomal recessive; SBDS on chromosome 7q11, which has been found mutated in 75% of patients, is now recognized as one causative gene [29,30]. The product of the SBDS gene is a highly conserved nucleolar protein

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impairment of ribosome synthesis and functions may be a molecular pathway commonly affected in different constitutional (and possibly some acquired) marrow failure syndromes [33]. Acquired Aplastic Anemia Acquired forms of AA are far more frequent than the constitutional ones; typically they affect young adults or elderly people, who present with peripheral pancytopenia in absence of other hematological diseases. Pancytopenia of AA patients results from the impairment of the hematopoietic progenitor compartment, including HSC and more committed progenitors; the nature of the injury damaging hematopoiesis often remains undetected (Fig. 1). Classically, unlike constitutional forms, acquired AA is thought to result from a damage extrinsic to the HSC compartment (Fig. 3). Cytotoxic drugs and radiation are the best examples of a direct injury to HSC; although stem cells, due to their dormant nature, are more resistant to cytotoxic drugs, for most agents a dose-response relationship with the degree of stem cell damage can be established. However, iatrogenic direct injury by chemotherapy or

radiation is rarely involved in permanent marrow failure syndromes; sometimes, although exposure to a number of drugs may be documented (Table 1), a definitive causative relationship cannot be demonstrated. Indeed, in the rare cases which a putative inciting agent directly attacks the stem cell pool causing a permanent depletion of HSC, the clinical presentation of cytopenia may be delayed for weeks or months, appearing just when a critically low stem cell number is reached. These considerations are applicable to chemical agents as well as to a number of viruses which may infect and destroy hematopoietic progenitors. However, in all these conditions the damaging mechanism may also involve non-direct injury of the HSC. The indirect damage of HSC is mainly sustained by immune effector mechanisms (Fig. 1), which may also be triggered by viruses or by drug metabolites. An appropriate example for such a mechanism is the hepatitis/AA syndrome (HAA), in which AA follows with a delay of months an acute hepatitis caused by HBV, HCV, or more frequently a so far unknown non-B, non-C virus; HAA carries all the characteristics of a viral infection [50,51]. The viral agent responsible for this syndrome has not been identified; it is likely that at the time of overt cytopenia the viral infection has already been cleared, AA

Fig. (3). The spectrum of bone marrow failure syndromes and their pathophysiology. Specific clinical syndromes are positioned according to the contribution to their pathophysiology by intrinsic defect of hematopoietic stem cells (inherited or acquired) and by environmental factors (derangement of the immune system, or, less frequently, direct toxicity). The sizes of segments are indicative (but not exactly proportional) of frequencies of different forms of AA. Constitutional marrow failure (FA, CAMT, SDS, DKC) are caused by intrinsic defect of stem cells, while acquired AA are due to extrinsic injures to the HSCs, mostly through immune mechanisms. However, conditions characterized by both intrinsic abnormality of HSCs and damage by extrinsic factors may exist, as in PNH, in MDS or in some familial forms of AA overlapping to atypical DKC cases (see text). Even in presence of well-defined intrinsic defect or evident extrinsic damage the specific contribution to the development of marrow failure may be argued; for instance, in PNH an intrinsic abnormality of HSCs (the PIG-A mutation, which in contrast with constitutional forms, is acquired), is a hallmark of the disease, but not necessarily causative of the marrow failure.

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being mediated by lymphocytes recognizing a cross-reactive antigen in the marrow. EBV-associated AA has also been described; even this form seems not to be due to direct viral cytoxicity, rather mediated by the immune system [52]. Similar pathogenic mechanisms may be postulated in a majority of cases of idiopathic AA, possibly involving the presence of neo-antigens or cross-reactive triggering antigens which result in a breach of immune tolerance; this would generate an immune-mediated attack toward the hematopoietic progenitors, leading to HSC consumption or functional impairment with subsequent pancytopenia. Regardless the nature of injury, AA patients are characterized by a severe dysfunction of the hematopoietic stem cells; this defect has been deeply characterized on both quantitative and qualitative sides. As those with constitutional AA, patients with acquired AA show a very low number of hematopoietic progenitors, as measured by flow cytometric CD34+ cell assessment or by in vitro colony assays. By flow cytometry, CD34+ cells are reduced in all AA patients, and the contraction affects both committed and immature CD34+/c-kit- or CD34+CD38progenitors [53]. Unlike in murine models, the measurement of more immature progenitors and stem cells is not easily accomplished in humans. Lineage-committed progenitors may be measured in vitro as cells able to originate colonies of blood cells in semi-solid medium (usually methylcellulose), also called colony-forming units (CFU). By in vitro colony assay, hematopoietic progenitors committed to each of the three lineages have been found significantly reduced by several groups [54-57]; in one study, granulocyte colony-stimulating factors (G-CSF) showed at least a partial correcting effect in vitro, suggesting an intrinsic dysfunction of the residual hematopoietic progenitors, too [58]. However, these "primary" colonies do not account for more immature hematopoietic progenitors; consequently, a number of surrogate in vitro stem cell assays have been developed, including long-term culture-initiating cells (LTC-IC) as well as cobblestone forming assay [59,60]. Both methods cultivate hematopoietic progenitors on irradiated (bone marrow-derived) stromal layers; in the latter case, cobblestones are defined as phase-dark areas of 5 or more polygonal cells, corresponding to early hematopoietic colonies. The LTC-IC assay measures the number of cells capable of colony formation after 5 weeks in long-term bone marrow culture; LTC-ICs share the frequency, phenotype, and kinetic properties of true stem cells [60-62]. Several studies indicate a profound deficiency in LTC-IC as well as cobblestone area forming cells in all patients with AA [55,59,63]. At the time of clinical presentation, the number of LTC-IC is usually at least one log below the normal level; combined with a reduction in total marrow cellularity to <10%, the stem cell number in AA is estimated to be reduced of at least two logs compared to healthy individuals [59]. Neither the number of LTC-ICs nor that of colony forming cells correlated with the blood counts, suggesting that in addition to the quantitative defect, a functional impairment may be present [54]; this may be also inferred by the observation that the clonogenic capacity of each individual progenitor is lower than in normals. A reduced clonogenicity was demonstrated both on CD34+ cells (as number of colonies obtained from a purified CD34+ population) and on the putative stem cell LTC-ICs (as

number of "secondary" colonies assayed from LTC-IC in limiting dilution experiments) [54,59,64]. More sophisticated approaches looking at the quiescent HSCs by 5-fluorouracil exposition evidenced that AA LTC-ICs also harbor an abnormal kinetic of growth, with rapid initial proliferation followed by early termination of the cultures, possibly mimicking HSC exhaustion in vivo [65]. These findings suggest that the hematopoietic stem cell compartment is affected by the pathophysiologic process operating in AA, whereas mesenchymal or even more immature pluripotent stem cells may be functionally normal. Indeed, several studies have documented that marrow cells from AA patients are able to generate in vitro perfectly functional stromal layers, as confirmed on cross-over experiments [66-68]. These in vitro data are confirmed by the clinical observation that allogeneic stem cell transplantation is a highly successful therapy for AA, even if most stromal elements may remain of host origin. However, in a recent paper it has been documented that mesenchymal stem cells from AA patients may be defective in suppressing T lymphocyte activity [69]; whether this finding implies an intrinsic defect of mesenchymal stem cells or is related to a more general immune derangement in AA is still to be determined, as well as its specific role in the pathophysiology of AA. Serial studies were conducted to determine the number of stem cells during the course of the disease and the kinetics of decline/recovery in the stem cell number; a profound defect in LTC-IC number may persist for a long time, even in patients successfully treated by immunosuppression [59,63]. In most cases, a residual numerical LTC-IC defect may be permanent, despite a full recovery of the blood counts, while a complete reconstitution is found only in a minority of patients with sustained complete remission [59]. Nevertheless, at least a partial recovery of stem cells is possible, and a highly contracted stem cell pool can sustain a seemingly normal blood cell production, even if the compensatory capacity in response to stress conditions may be diminished. Functional studies of HSCs in AA and result interpretation are complicated by the fact that all the analyses are performed on selected HSCs, i.e. those that have survived the pathogenic injury; thus a qualitative characterization suffers from the bias that such HSCs may harbor substantial biological differences compared to those already disappeared (possibly conferring different susceptibility to the damage), or may themselves have developed phenotypic changes as a result of the pathogenic injury. A qualitative defect of HSC is suggested by the reduced clonogenic potential, as already mentioned above [54]; however, this does not necessarily imply that the dysfunction is intrinsic to the HSC as in constitutional AA. Clearly, the hematopoietic recovery following successful immunosuppression demonstrates that some stem cells must have been spared from the pathologic process; impaired primary and secondary clonogenic capacity concerns a majority of HSCs, which likely are most damaged, but not all individual HSCs. Indeed, it is well known that CD34+ cells from AA patients show a propensity to apoptosis [7072]; it is possible that a few HSCs are able to resist to the apoptotic stimuli in vivo. Several decades of laboratory experiments have suggested that the nature of the stem cell damage in AA involves activation of general immune

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effector mechanisms (Fig. 1) [73-75]. The stem cell damage can be due to direct cell-mediated killing by cytotoxic lymphocytes (CTLs), as well as by cytokine-transduced inhibition; the latter is documented by excess production by circulating and marrow lymphocytes of type I cytokines, especially interferon- and tumor necrosis factor- [76-78]. Additionally, Fas-ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may play an important role as effector cytokines in the hematopoietic inhibition in AA [70,76,77,79]. Such mechanisms may not be restricted to the primary target, but may also attack innocent bystander cells; ultimately, these factors result in apoptosis of all existing cells. Fas, IFN- and TNF- modulate the expression of their receptors through feedback mechanisms; in this way they may enhance each other action [78,80]. Furthermore, the effects of individual factors may be additive or synergistic [80]; chronic exposure to these cytokines in vivo may be more damaging compared with in vitro models, which challenge acute effects of high cytokine concentrations [81]. Apoptosis is the main key mechanism of HSC damage. The apoptotic machinery may be constitutively activated in differentiated cells, which may need appropriate signals to survive; in contrast, in stem cells, apoptosis likely has to be induced by specific stimuli. Indeed, the abundance of trophic signals argues against the lack of survival signals (e.g. growth factors) as a mechanism of apoptosis for AA HSCs [82]. Increased apoptotic rate within the CD34+ cell population from AA patients has been demonstrated [70-72]; in addition, CD34+ cells from AA show increased expression of Fas [71,72,79,83], which may be induced by IFN- and TNF- in the aplastic marrow [70,77,84,85]. Both cytokines can up-modulate Fas expression even in normal CD34+ cells [80,86]; CD34+ cells derived from AA patients appear to undergo apoptosis in response to Fas at a higher rate compared with normal CD34+ cells [71,72,79,83]. Additional pathways of apoptosis may involve nitric oxide or oxygen radical secretion, similarly to what observed in FA; the production of such factors in hematopoietic progenitors may be triggered by classical pro-apoptotic stimuli, such as inhibitory cytokines, leading to apoptosis in a paracrine fashion [87,88]. A more accurate description of HSC in AA has become possible with the oligonucleotide microarray technology, which allows quantitation of the expression levels of a large number of genes. Recently, the gene expression profile in healthy human CD34+ stem/progenitor cells has been reported; the same technology has been applied to assess gene expression in CD34+ marrow cells from AA patients [89]. The study documented that the expression of several genes implicated in apoptosis and cell death was markedly increased in AA CD34+ cells, as well as that of genes involved in the negative control of cell proliferation. Examples of up-regulated genes were those coding for the death receptors Fas, DR3, DR5, TNFRII, and for TRAIL. By contrast, genes coding for products promoting cell cycle progress showed a lower expression compared to CD34+ cells from healthy individuals, possibly explaining the inability of the residual stem cells to compensate the progenitor pool contraction. As anticipated from other evidences of heightened immune activity, several cytokine/chemokine signal transducer genes, stress response

genes, and defense/immune response genes were upregulated. In summary, the transcriptome analysis of HSC in AA is consistent with the presence of stressed, immunologically activated or dying target cells rather than of an intrinsically abnormal population. This support the hypothesis of an organ-specific immune attack on hematopoietic stem/progenitor cells, possibly T-cell mediated. As for most qualitative defects observed in HSCs from AA patients, these abnormalities seem to represent changes resulting from injuries to the HSCs, which become evident in cells experiencing sub-lethal damages or in any case surviving the injury. The presence in the circulation and in marrow of activated CTLs, which inhibit hematopoiesis in vitro, has been reported in AA patients [90,91]; furthermore, we and others have described abnormalities of T cells and of the TCR repertoire [92-96], suggestive of an oligoclonal T cell response. For some patients, CD4+ and CD8+ T cell clones which responded to and destroyed autologous hematopoietic progenitors were characterized after in vitro immortalization [90,96]. More recently, we have identified dominant T cell clonotypes by sequencing the (complementority determining region 3) of the TCR- chain [97]; cells bearing this clonotype were expanded in vivo, likely as a result of an antigen-driven dominant immune response, suggesting their pivotal pathophysiologic role. Indeed, these pathogenic T cell clones appeared to correlate with disease activity, and showed potent cytotoxicity directed against autologous marrow progenitor cells. The observed homology among various patient-specific clonotypes may suggest a semipublic immune response against common epitopes; however, the antigen(s) driving the immune attack on the HSC are still unknown, as well as the possible primary abnormalities of HSC leading to the breach of immune self-tolerance [97]. Other qualitative abnormalities of HSCs and their progeny in AA have been described; the most relevant is telomere shortening. In normal stem cells, self-renewal does not result in telomere shortening, due to the activity of telomerase [98-100]; however, telomerase activity decreases upon commitment and differentiation [101], and telomere length of the progeny reflect the number of doublings of the committed and more mature progenitors. Short telomeres have been reported in constitutional AA, as mentioned; even patients affected by acquired AA show telomere shortening, as measured by various methods [100,102,103]. Apparently paradoxically, patients with chronic moderate AA showed telomeres shorter than those of patients with a more severe disease; actually, in severe cytopenias, telomere shortening may not be evident due to a more extended block of stem cell cycling [100,102,103]. Upon recovery, due to recruitment of intact stem cells, the telomeres of the progeny may show longer measurements again; however, if the stem cell number operating at a given time is small, in order to maintain a normal circulating cell number more divisions are needed and telomere shortening may be more pronounced. Proper function of the telomerase complex requires the presence of an intact RNA primer (TERC), as well as of the functional protein (TERT) (Fig. 2). As for DKC, mutations in the TERC gene have been occasionally reported in patients with acquired AA harboring very short telomeres [104,105]; however, such a defect seems to be restricted to patients with a positive family history, and to certain clinic

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presentation (early onset, chronicity, lack of response to immunosuppression). Thus, these cases likely represent uncommon presentation of forms of constitutional aplasia [25,106]. More recently, AA patients with mutations within the TERT gene have been described [107]. As patients with TERC mutation do, subjects with abnormal TERT show increased telomere shortening and low telomerase activity; this suggests that heterozygous mutations in the TERT gene impair telomerase activity by haploinsufficiency, and may be risk factors for marrow failure, possibly explaining some cases of familial acquired AA. All together, these findings demonstrate how inherited and acquired AA may sometimes converge in a unique terminal damage mechanism, which leads to telomere shortening; this conclusion can be drawn based on the present knowledge of the players involved in the pathway of telomere maintenance. It is to be noted that telomere length and telomerase activity should be studied at the level of stem cells or hematopoietic progenitors, since the telomere shortening is usually found in mature cells of typical acquired forms of AA, and are an epiphenomenon due to the proliferative stress of surviving progenitors rather than the expression of primary lesions of the HSC, as for DKC. Whether the same observations may be extended to other intracellular damage pathways, and more importantly whether that may apply to a majority of AA patients, is still to be determined. Of note, once a critical telomere length is reached, chromosomes become unstable; such a mechanism could be one of the explanations for the possible clonal evolution from AA into myelodysplasia [108,109]. The acquisition of stem cell damage and the expansion of the dysplastic clone is not typical of idiopathic AA, but may represent a clinically relevant complication. Possible mechanisms leading to clonal evolution are: (i). chromosome instability due to telomere shortening; (ii). escape of specific clone(s) from the immune attack; (iii). different susceptibility to the depletive mechanisms between normal and possibly mutated stem cells; (iv). recruitment of a preexisting defective (under normal circumstances quiescent) stem cell, facilitated by the depletion of normal stem cells (oligoclonality theory); (v). iatrogenic effect of exogenous G-CSF interfering with some of these processes [110,111]. LARGE GRANULAR LYMPHOCYTE DISEASES Large granular lymphocyte (LGL) disorders are a group of lymphoproliferative syndromes characterized by the expansion of lymphocytes displaying an effector phenotype [112]. LGL proliferations are a complex spectrum of diseases consisting of different clinical entities, spanning from reactive LGL expansion to aggressive LGL leukemia [112]; transient immune reactions or true malignancies may lead to completely different clinical pictures [113]. LGL diseases are often associated with mono- or multi-lineage cytopenia, often combined with a variable degree of marrow hypoplasia; in some cases, the clinical picture includes some kind of organ-specific immune attack, such as reumathoid arthritis [114,115]. While there is still uncertainty on whether LGL clinical expansions are all true malignancies or may include also some exaggerated selective immune response [113], clues on the pathogenic mechanisms of the associated marrow failure are emerging. HSCs are supposed to be

normal in LGL disorders, since the putative transforming event affects post-thymic lymphocytes; marrow hypoplasia seems not be due to infiltration by the clonal cells, as occurring in other malignancies. It has been hypothesized that the mechanisms of cytopenia include secretion of FasL/soluble Fas receptors that are constitutively expressed by LGL cells and found elevated in patients' serum [116]. It is possible that the TCR specificity of the LGL clone dictates the clinical presentation by highly specific recognition and killing of individual hematopoietic cell lineages. Recent observations have documented non-random selection of dominant and subdominant T-cell clones within the LGL proliferation [117,118]; in fact, molecular characterization of the TCR repertoire in LGL patients showed that significant TCR homologies may be identified in single patients and among different subjects (mostly HLA-related) [118]. This is consistent with the hypothesis that such clones may not evolve randomly; rather, they occur in the context of an immune response specific against identical or highly similar antigens. The initial immune process may provide a signal for the expansion of a clone that through certain, yet not specified, molecular event may possibly acquire autonomous growth and a more aggressive phenotype [113]. According to this theory, "paraneoplastic" marrow failure associated to LGL syndromes would reflect the same pathogenic immune process seen in acquired AA, characterized by an antigendriven expansion of clonal CTLs which damage HSCs. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Paroxysmal nocturnal hemoglobinuria (PNH) is a marrow failure syndrome closely embedded with AA; PNH is a clonal stem cell disorder arising by an acquired somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene [119-121], which is located on the X chromosome (Xp22.1). Affected progeny cells are incapable of synthesizing the glycosylphosphatidylinositol (GPI)-anchor, and present the typical phenotype lacking from their surface all GPI-anchored proteins (GPI-APs). The abnormality affects all hematopoietic lineages; a mutation occurred in a single HSC may sustain hematopoiesis even lifelong [122]. It is well known that PNH red cells have an intrinsic susceptibility to complement-mediated hemolysis, due to the lack from their surface of the GPI-AP complement inactivator CD59 [119]; this leads to the intravascular hemolysis typical of PNH patients. However, other cardinal features of PNH, namely propensity to venous thrombosis and bone marrow failure, remain unclear, as well as the reasons for the PNH clone expansion [119,123]. Thus, PNH is a true disease of the HSC, as marked by the PIG-A mutation, but the HSC defect does not explain entirely the phenotype. In fact, a series of observations suggest that the PIG-A mutation is necessary but not sufficient to cause PNH. (i) The existence of a few (10-50 cells per million) circulating PNH granulocyte may be demonstrated also in healthy individuals by flow cytometry, and confirmed by a nested PCR technique identifying the specific PIG-A mutation [124,125]. Lymphocytes with the PNH phenotype appeared in lymphoma patients during treatment with

(ii)

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alemtuzumab (an antibody that recognizes a GPIanchored protein, CD52) and disappeared at treatment interruption [126]. (iii) Several murine knock-out models were developed to recreate the disease, but the expansion of the aberrant clone seen in PNH patients could not be reproduced [127-129]. Even in experiments employing embryonic stem cells, the PNH clone did not overcome normal hematopoiesis, suggesting that the PIG-A mutation itself did not confer any intrinsic proliferative advantage to PNH HSC [128,129]. This has been confirmed by several in vitro data using PNH and wild-type hemopoietic progenitors obtained by PNH patients [130]. PNH hematopoiesis may be in some cases oligorather than monoclonal, as initially supported by differential susceptibility to complement lysis [131,132] , then by flow cytometry [133] and finally

confirmed by PIG-A sequencing [134]; this observation raised the question whether the expansion of more clones carrying the same functional defect, but molecularly heterogeneous, is compatible with a random process. On these bases, the hypothesis of a dual pathophysiology for PNH has been developed, which is also known as the "relative advantage" [135] or the "escape" theory [136]. According to this theory, a mutation in the PIG-A gene might be a fairly common phenomenon, which has no biological consequences, because the mutated cell has no chance of expanding in presence of a vast majority of normal cells. However, the presence of external conditions may alter this equilibrium, creating an environment permissive for the expansion of PNH clone(s). The nature of such external trigger may be inferred from the close clinical association between PNH and AA. An antigen-driven immune response specifically targeting the marrow tissue may be postulated; if

(iv)

Fig. (4). Models for marrow failure pathophysiology. A. The physiologic hierarchy of hematopoiesis. B. Constitutional bone marrow failure syndromes: inherited mutations cause intrinsic abnormalities of HSCs, with subsequent impairment of pivotal functions (e.g. self-renewal, DNA-repair) leading to exhaustion of progenitors and peripheral pancytopenia. C. Acquired AA: phenotypically normal HSCs may be damaged by a variety of extrinsic factors, which may also injury more mature progenitors, leading to functional impairment of hematopoiesis and subsequent pancytopenia. This model is typical of idiopathic (immune-mediated) aplastic anemia, as well as of marrow failure resulting from viral infections or toxic agents. D, E. The interactive model: marrow failure may result from the interaction between an intrinsic abnormality of HSC and an extrinsic injury. For example, a silent intrinsic genetic abnormality of HSC may cause propensity to develop marrow failure in presence of otherwise inoffensive extrinsic injuries D. This is the case of rare familial AA with mutation in the TERT gene. Finally, in presence of permissive extrinsic conditions acquired mutation may confer a selective advantage to specific HSCs, leading to marrow failure syndromes disclosing intrinsic clonal abnormalities of hematopoiesis E. This model applies to PNH and likely some forms of MDS.

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the target on HSC membrane is a GPI-linked molecule, PNH HSC will escape this injury while normal HSC are killed. Several evidences of immune derangement in PNH have been produced; as for AA, oligoclonality of the T cell pool has been reported [137], and immunodominant pathogenic CTL clones may be detected in most PNH patients [94,97,138]. In rare cases, such expansion may be extremely large, phenotypically resembling a subclinical LGL proliferation [139,140]. It has been recently described that these effector T cells express in excess the activating isoforms of inhibiting superfamily receptors, which elicit a powerful cytolytic activity [141]. However, once again the most striking evidence comes from gene expression profiling: when HSC from PNH patients were separated according to the presence or absence of GPI-APs on their surface, distinct patterns of gene expression were identified. Phenotypically normal (GPI-AP positive) CD34+ cells harbored diffuse abnormalities of their transcriptome, with over-expression of genes involved in apoptosis and immune activity, paralleling the findings seen in CD34+ cells of AA patients. By contrast, PNH CD34+ (GPI-AP negative) showed a gene expression profiling closer to that obtained in CD34+ cells from healthy individuals [142]. This finding, which confirms previous observations on increased susceptibility to Fas-mediated apoptosis of GPI-AP positive CD34+ cells from PNH patients [143], strongly supports the presence of an immune attack to the hematopoietic stem/progenitor cells, which spares the PNH cells. The "escape" of PNH cells may be interpreted in various manners. Contradictory data have been produced on a putative differential sensitivity to inhibitory stimuli between normal and PNH cells; susceptibility to apoptosis has been reported increased, normal or decreased in different models. Recently, it has been shown that human cell lines carrying the PIG-A mutation are less susceptible to NK-mediated killing compared to their normal counterpart [144]. In a more sophisticated model, GPI-deficient cells showed impairment in inducing primary and secondary stimulation of both antigen-specific and alloreactive T cells, providing experimental support for the hypothesis that the PNH clone could inefficiently interact with the immune system [145]. However, the actual mechanisms causing the escape are still elusive. They may include the absence of specific GPI-APs directly targeted by effector immune cells, or a protection due to the absence of important molecules involved in cellcell interaction (e.g., accessory molecules). Alternatively, it may be hypothesized a broader impaired sensitivity to common effector mechanisms, which may be due to the lack of GPI-APs or to non specific structural changes of raft structure in the outer surface [146]. LESSONS FROM HSC IMPAIRMENT IN BMF The study of the stem cell compartment in a variety of marrow failure syndromes may be instructive for the understanding of several aspects of HSC physiology and pathology. In constitutional aplasia specific defects intrinsic to the HSC are genetically determined, and most of them are now molecularly characterized. Different molecular pathways may be affected in specific diseases, such as DNA repair or telomerase function, even if they may lead to similar phenotypes (Fig. 1). All constitutional AA share the

impairment of HSC self renewal. Theoretically, such genetic defects are suitable of correction through gene therapy, when this procedure will become available; however, the engraftment of engineerized stem cells will not influence the extra-hematological clinical phenotype. By contrast, the mechanisms of acquired AA do not necessarily require the assumption of a primary defect of the HSC, since some functional abnormalities may appear as a result of sub-lethal damage by the pathogenic injury (Figs. 3, 4). However, in some cases, the same molecular pathways involved in constitutional forms may be affected, such as the human telomerase gene complex function or enzymes involved in ribosomal functions, or even cell functions pivotal for stem cell-ness maintenance. Although it has to be emphasized that identical molecular findings may derive from completely different mechanisms, converging on the same terminal damage, underlying genetic mutations (either germline or somatic) may participate in the sequence of events leading to AA. Indeed, in acquired AA HSC are thought to be normal; they undergo an extrinsic damage, which is likely caused by the immune system (Fig. 4C). But this simple model needs to be integrated with the recent data suggesting that a genetic background may cooperate with extrinsic factors (Fig. 4D,E); according to this scenario, the contribution to marrow failure pathophysiology of both HSC intrinsic defects and extrinsic damages may allow the appearance of specific phenotypes (Fig. 2). For example, particular HLA genotypes or cytokine polymorphisms are more frequently associated to AA, and may likely represent an environment permissive for autoimmunity. As far as HSCs are concerned, mutations in the TERT gene exemplify the possibility of silent genetic abnormalities of HSC affecting the selfrenewal capability or other important cell functions, which may favor disease development in presence of other otherwise inoffensive injuries. This interaction between intrinsic HSC defect and external factors is well summarized in PNH, where the clonal disease develops only if permissive conditions occur. As an example of non malignant clonal hematopoiesis, PNH demonstrates the complexity of the biology of HSC, exemplifying phenomena that likely occur physiologically in a more subtle fashion. A better understanding of all processes involved in the dynamic of single HSC recruitment, regeneration and clonal expansion may lead to development of new therapeutic approaches for marrow failure syndromes and other hematological diseases. REFERENCES

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Received: January 16, 2006

Revised: March 13, 2006

Accepted: May 10, 2006

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