Function and malfunction of hematopoietic stem cells in primary bone marrow failure syndromes

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.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Regenerative cell therapy and pharmacotherapeutic intervention in heart failure

Regenerative medicine represents a promising perspective on therapeutic angiogenesis in patients with cardiovascular disease, including heart failure. However, previous or ongoing clinical trials show ambiguous outcomes with respect to the benefit of regenerative therapy by means of bone marrow stem cell infusion in myocardial infarction patients. Therefore, it is necessary to set up a rational therapeutic strategy in the treatment of congestive heart failure. Chemokines, cytokines and growth factors, as well as pharmaceutical agents, may have an impact on endothelial progenitor cell (EPC) physiology and thus can provide targets for pharmacological intervention. Indeed, EPCs and stem cell niches both in bone marrow and myocardial tissue can be treated as an integral target for recruitment of EPCs from the bone marrow to the cardiac ischaemic niche. In this article, we individually place the signalling factors in their specified context, and explain their roles in the various phases of neovascularisation (see Part 1). (Neth Heart J 2008;16:337-43.)     

Yin and Yang of mesenchymal stem cells and aplastic anemia

Acquired aplastic anemia(AA) is a bone marrow failure syndrome characterized by peripheral cytopenias and bone marrow hypoplasia. It is ultimately fatal without treatment, most commonly from infection or hemorrhage. Current treatments focus on suppressing immune-mediated destruction of bone marrow stem cells or replacing hematopoietic stem cells(HSCs) by transplantation. Our incomplete understanding of the pathogenesis of AA has limited development of targeted treatment options. Mesenchymal stem cells(MSCs) play a vital role in HSC proliferation; they also modulate immune responses and maintain an environment supportive of hematopoiesis. Some of the observed clinical manifestations of AA can be explained by mesenchymal dysfunction. MSC infusions have been shown to be safe and may offer new approaches for the treatment of this disorder. Indeed, infusions of MSCs may help suppress auto-reactive, T-cell mediated HSC destruction and help restore an environment that supports hematopoiesis. Small pilot studies using MSCs as monotherapy or as adjuncts to HSC transplantation have been attempted as treatments for AA. Here we review the current understanding of the pathogenesis of AA and the function of MSCs, and suggest that MSCs should be a target for further research and clinical trials in this disorder.     

Stem cell-conditioned medium does not protect against kidney failure

Paracrine secretion of mediators may be the main route by which stem cells protect against injuries. Stem cells commonly secrete different bioactive molecules. In this study, we examined the hypothesis that administration of conditioned media of stem cells can diminish the burden of kidney injury. A mouse model of cisplatin-induced nephropathy was developed to test the putative renoprotective effects of conditioned media of human umbilical cord blood USSCs (unrestricted somatic stem cells) as well as mouse bone marrow MSCs (mesenchymal stem cells). None of these two types of conditioned medium could protect against kidney failure in terms of serum urea and creatinine, histopathologic examinations and physical activity score. Neither MSC- nor USSC-conditioned media were effective in protecting against kidney injury in our study. Possible explanations for our observations are offered, and related literature is reviewed.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

The therapeutic potential of stem cells in heart disease

Abstract.  Coronary heart disease and chronic heart failure are common and have an increasing frequency. Although interventional and conventional drug therapy may delay ventricular remodelling, there is no basic therapeutic regime available for preventing or even reversing this process. Chronic coronary artery disease and heart failure impairs quality of life and are associated with subsequent worsening of the cardiac pump function. Numerous studies within the past few years have been demonstrated, that the intracoronary stem cell therapy has to be considered as a safe therapeutic procedure in heart disease, when destroyed and/or compromised heart muscle must be regenerated. This kind of cell therapy with autologous bone marrow cells is completely justified ethically, except for the small numbers of patients with direct or indirect bone marrow disease (e.g. myeloma, leukaemic infiltration) in whom there would be lesions of mononuclear cells. Several preclinical as well as clinical trials have shown that transplantation of autologous bone marrow cells or precursor cells improved cardiac function after myocardial infarction and in chronic coronary heart disease. The age of infarction seems to be irrelevant to regenerative potency of stem cells, since stem cells therapy in old infarctions (many years old) is almost equally effective in comparison to previous infarcts. Further indications are non-ischemic cardiomyopathy (dilative cardiomyopathy) and heart failure due to hypertensive heart disease.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Maintenance of rat hepatocytes under inflammation by coculture with human orbital fat-derived stem cells

Preservation of hepatocyte functions in vitro will undoubtedly help the management of acute liver failure. The coculture system may be able to prevent functional decline of hepatocytes. It has already been shown that hepatocytes, when cocultured with bone marrow mesenchymal stem cells, could undergo long-term culture in vitro without loss of functions. In this study, human orbital fat-derived stem cells were isolated and cocultured with rat hepatocytes. When treated with serum from an acute liver failure patient, rat hepatocyte monoculture showed reduction of cell viability and loss of liverspecific functions. However, rat hepatocytes in the coculture system were still able to secret albumin and synthesize urea. IL-6 was significantly elevated in the coculture of rat hepatocyte with orbital fat-derived stem cells, and it might be the key immunoregulator which protects rat hepatocytes against inflammation. Our data confirmed that orbital fat-derived stem cells, or other adipose tissue-derived stem cells, are an ideal candidate to support rat hepatocyte functions in vitro.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Migration, fate and in vivo imaging of adult stem cells in the CNS

Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. One of the most promising cell sources for autologous cell transplantation is bone marrow, containing a heterogenous cell population that can be roughly divided into hematopoietic stem and progenitor cells and mesenchymal stem cells (MSCs). MSCs are multipotent progenitor cells that, in the case of severe tissue ischemia or damage, can be attracted to the lesion site, where they can secrete bioactive molecules, either naturally or through genetic engineering. They can also serve as vehicles for delivering therapeutic agents. Mobilized from the marrow, sorted or expanded in culture, MSCs can be delivered to the damaged site by direct or systemic application. In addition, MSCs can be labeled with superparamagnetic nanoparticles that allow in vivo cell imaging. Magnetic resonance imaging (MRI) is thus a suitable method for in vivo cell tracking of transplanted cells in the host organism. This review will focus on cell labeling for MRI and the use of MSCs in experimental and clinical studies for the treatment of brain and spinal cord injuries.     

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.     

Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria

Within the contemporary multitude of complex methods used in clinical flow cytometry, very few techniques exist which can be described as disease-specific diagnostic tests. Detection of glycophosphatidylinositol (GPI)-linked antigens on hematopoietic cells using monoclonal antibodies and flow cytometry forms the basis of a specific diagnostic test for paroxysmal nocturnal hemoglobinuria (PNH). Absent or markedly diminished expression of GPI-linked antigens is, in the appropriate clinical setting, specific for all patients with PNH. Clinically, PNH is a syndrome characterized by bone marrow failure, acquired hemolytic anemia, and a thrombotic tendency. The molecular genetic lesion responsible for this condition is a somatic mutation of the X-linked pig-a gene within a multipotent hematopoietic stem cell. Due to its rarity, delay in diagnosis is not uncommon for patients with PNH. Once a definitive diagnosis is established, this can make a considerable impact on patient management and prognosis. In this article, we review the complimentary roles that molecular biology and flow cytometry have played in unraveling the genotypic and phenotypic aspects of this unique condition.     

CXCR4 or CXCR7 antagonists treat endometriosis by reducing bone marrow cell trafficking

Adult stem cells have a major role in endometrial physiology, including remodelling and repair. However, they also have a critical role in the development and progression of endometriosis. Bone marrow‐derived stem cells engraft eutopic endometrium and endometriotic lesions, differentiating to both stromal and epithelial cell fates. Using a mouse bone marrow transplantation model, we show that bone marrow‐derived cells engrafting endometriosis express CXCR4 and CXCR7. Targeting either receptor by the administration of small molecule receptor antagonists AMD3100 or CCX771, respectively, reduced BM‐derived stem cell recruitment into endometriosis implants. Endometriosis lesion size was decreased compared to vehicle controls after treatment with each antagonist in both an early growth and established lesion treatment model. Endometriosis lesion size was not effected when the local effects of CXCL12 were abrogated using uterine‐specific CXCL12 null mice, suggesting an effect primarily on bone marrow cell migration rather than a direct endometrial effect. Antagonist treatment also decreased hallmarks of endometriosis physiopathology such as pro‐inflammatory cytokine production and vascularization. CXCR4 and CXCR7 antagonists are potential novel, non‐hormonal therapies for endometriosis.     

Collection, storage and transfusion of blood stem cells for the treatment of hemopoietic failure.

Migration of hemopoietic stem cells via the blood to sites of stem cell need is a principle that becomes established during the embryonic development of hemopoiesis and can be observed in the adult whenever bone marrow transplantations are being performed. The regular presence of stem cells in the peripheral blood lends itself to the study of their collection, storage, and use for transfusion purposes in cases of bone marrow failure. Both in dog and in man, granulocyte-macrophage progenitor cells (CFU-C) can be collected by leukapheresis from the blood in large quantities, particularly if the yield is increased by the administration of mobilizing agents such as dextran sulfate, and appear to be an indicator for the presence of stem cells. For collection and storage, a closed plastic bag system has been developed that allows the safe handling of the cells. The loss of CFU-C from freezing and thawing with DMSO as a cryoprotective agent is only 10%-20%. If frozen and thawed mononuclear leukocytes are transfused into 1200 rad whole-body X-irradiated autologous or allogeneic recipient dogs, a hemopoietic take is observed when 0.2 X 10(5) CFU-C are present among the mononuclear leukocytes (MNC). Graft-versus-host disease can be avoided in the allogeneic situation when a purified CFU-C rich cell fraction is being transfused. In man collection and storage of MNC including CFU-C is feasible and may eventually become a therapeutic tool.     

Sources of adult mesenchymal stem cells and their applicability for musculoskeletal applications

There is significant potential for the use of adult mesenchymal stem cells in regenerating musckuloskeletal tissues. The sources of these stem cells discussed in this review are bone marrow, blood, adipose tissue, synovium, periosteum & cartilage. Adult mesenchymal stem cells of bone marrow origin are the cells which are heavily investigated in many studies and have been shown capable of producing a variety of connective tissues especially cartilage and bone. It has recently been suggested that bone marrow derived mesenchymal stem cells originate from microvascular pericytes, and, indeed, many of the tissues from which stem cells have been isolated have good vascularisation and they may give a varied source of cells for future treatments. Clinical trials have shown that these cells are able to be successfully used to regenerate tissues with good clinical outcome. Other sources are showing promise, however, is yet to be brought to the clinical level in humans.     

脐带血干细胞移植治疗非恶性血液病进展

异基因造血干细胞移植（allo-HSCT）是治愈多种非恶性病的有效方法。脐带血干细胞（UCB）具有免疫原性低、人类白细胞抗原不合耐受性好、移植物抗宿主反应发生率低以及获取相对快捷等特点,可作为非恶性血液疾病患者allo-HSCT的来源。本文简要综述脐血干细胞移植在原发性免疫缺陷病、遗传性骨髓衰竭、遗传代谢病以及自身免疫性疾病等非恶性血液疾病的治疗效果。     

Matrix metalloproteinases in stem cell mobilization

Hematopoietic stem cells (HSCs) have the capability to migrate back and forth between their preferred microenvironment in bone marrow niches and the peripheral blood, but under steady-state conditions only a marginal number of stem cells can be found in the circulation. Different mobilizing agents, however, which create a highly proteolytic milieu in the bone marrow, can drastically increase the number of circulating HSCs. Among other proteases secreted and membrane-bound matrix metalloproteinases (MMPs) are known to be involved in the induced mobilization process and can digest niche-specific extracellular matrix components and cytokines responsible for stem cell retention to the niches. Iatrogenic stem cell mobilization and stem cell homing to their niches are clinically employed on a routine basis, although the exact mechanisms of both processes are still not fully understood. In this review we provide an overview on the various roles of MMPs in the induced release of HSCs from the bone marrow.     

Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord

Background

Human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton''s jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury.

Methodology/Principal Findings

We examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5×10⁵ HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic fibroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 in the host spinal cord, which may help spinal cord repair.

Conclusions/Significance

Transplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.