Glycosylphosphatidylinositol-anchor-deficient mice: implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis. Abnormal hematopoietic cells from patients with PNH are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins and clonally dominate various hematopoietic lineages in the bone marrow and the peripheral blood. Analysis of many patients with PNH has showed that somatic mutation in the X-linked gene PIG-A is responsible for the GPI-anchor deficiency in PNH. The PIG-A mutation must also be relevant to the clonal dominance of GPI-anchor deficient (GPI-) blood cells because two or more PIG-A mutant clones become dominant in many patients. However, whether the PIG-A mutation alone is sufficient for clonal dominance is not known. To address this question, we generated chimeric mice using Pig-a (the murine homologue of PIG-A) disrupted embryonic stem (ES) cells, in which the animals are chimeric with respect to the surface expression of GPI-anchored proteins. The chimerism of hematopoietic and nonhematopoietic tissues in such mice was always low, suggesting that the higher contribution of Pig-a disrupted GPI- cells had a lethal effect on the chimera. GPI- cells appeared in the peripheral blood of some of the chimeric mice. However, the percentage of GPI- erythrocytes did not increase for 10 months after birth, implying that the Pig-a mutation alone does not immediately cause the clonal dominance of GPI- blood cells; another pathologic or physiologic change(s) in the hematopoietic environments or in the clone itself may be necessary.     

The effect of sperm-mucus interaction test on the outcome of in vitro fertilization and ovulation induction combined with intrauterin insemination

The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow-up of AA. All metaphases in CD59-deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59-deficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single-base deletion mutation in the PIG-A gene of this patient's peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation.     

Paroxysmal nocturnal hemoglobinuria and its association with aplastic anemia

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haematopoiesis. Clinically it is characterized by intravascular haemolysis, venous thrombosis and often by bone marrow hypoplasia. Haemolysis and thrombosis develop as a consequence of deficiency of several proteins on the cell membrane of the affected clone of blood elements. This is caused by somatic mutations in the PIG-A gene, which encodes an enzyme involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchor. Spectrum of mutations in the PIG-A gene is different to that observed in other genes. The mutations are mainly small deletions and insertions causing frameshift; large deletions are rare. Recently, however, a 88 base pairs direct tandem repeat insertion has been reported in a patient with PNH developed on the background of aplastic anaemia (AA). The peculiar pattern of the PIG-A gene mutations and the finding that more than one mutated clone is commonly present in patients with PNH might suggest that some form of hypermutability, caused by decreased DNA stability, deficient repair or increased generation of mutagens, might underline PNH. As most mutations cause cell death, it would explain the hypoplastic nature of the disorder and its association with AA. Other models of pathogenesis of PNH are also discussed.     

Paroxysmal nocturnal hemoglobinuria: molecular pathogenesis and molecular therapeutic approaches

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic stem cell disorder classified as an intravascular hemolytic anemia. Abnormal blood cells are deficient in glycosylphosphatidyl inositol (GPI)-anchored proteins. Deficiencies of GPI-anchored complement regulatory proteins, such as decay accelerating factor (DAF) and CD59, render red cells very sensitive to complement and result in complement-mediated hemolysis and hemoglobinuria. In the affected hematopoietic cells from patients with PNH, the first step in biosynthesis of the GPI anchor is defective. Three genes are involved in this reaction step and one of them, an X-linked gene termed PIG-A, is mutated in affected cells. Granulocytes and lymphocytes from the same patient have the same mutation, indicating that a somatic PIG-A mutation occurs in hematopoietic stem cells. The PIG-A gene is mutated in all patients with PNH reported to date. We review these recent advances in the understanding of the molecular pathogenesis of PNH. Furthermore, we present an hypothesis regarding the predominance of the PNH clone, caused by positive selection by hematopoietic suppressive cytokines, such as transforming growth factor (TGF)-beta. In addition, we discuss the possibility of cure for PNH through molecular therapeutic strategy using gene transfer techniques. (Key words: paroxysmal nocturnal hemoglobinuria, glycosylphosphatidylinositol-anchored proteins, PIG-A, clonal dominance, growth advantage, transforming growth factor-beta, gene therapy, molecular therapeutic approach).     

Paroxysmal nocturnal haemoglobinuria clones in patients with myelodysplastic syndromes

Among acquired stem cell disorders, pathological links between myelodysplastic syndromes (MDS) and aplastic anaemia (AA), and paroxysmal nocturnal haemoglobinuria (PNH) and AA, have been often described, whereas the relationship between MDS and PNH is still unclear. We analysed blood cells of patients with MDS to determine the incidence of the PNH clone, and analysed the PIG-A gene to find mutations characteristic of the PNH clone in MDS. In four (10%) of 40 patients with MDS, flow cytometry showed affected erythrocytes and granulocytes negative for decay-accelerating factor (DAF) and CD59. The population of affected erythrocytes was smaller in MDS patients with PNH clone (MDS/PNH) than in patients with de novo PNH, and haemolysis was milder in the MDS/PNH patients. PIG-A mutations were found in granulocytes of all patients with MDS/PNH. In type and site, the PIG-A mutations were heterogeneous, similar to that observed in de novo PNH; i.e. no mutation specific to MDS/PNH was identified. Of note, three of four patients with MDS/PNH each had two PNH clones with different PIG-A mutations, suggesting that PIG-A is mutable in patients with MDS/PNH. In a MDS/PNH patient with trisomy 8, FISH detected a distinct karyotype in a portion of granulocytes with PNH phenotype, indicating that PNH and MDS partly shared affected cells. Thus, MDS predisposes to PNH by creating conditions favourable to the genesis of PNH clone. Considering the increasing prevalence and incidence of MDS, these disorders could be useful for investigating the mechanism by which PIG-A mutation is induced.     

New somatic mutation in the PIG-A gene emerges at relapse of paroxysmal nocturnal hemoglobinuria

We report a detailed longitudinal study of the first patient to be treated (in 1973) for paroxysmal nocturnal hemoglobinuria (PNH) with syngeneic bone marrow transplantation (BMT). The patient subsequently relapsed with PNH in 1983, and still has PNH to date. Analysis of the PIG-A gene in a recent blood sample showed in exon 6 an insertion-duplication causing a frameshift. Polymerase chain reaction (PCR) amplification of the PIG-A exon 6 from bone marrow (BM) slides obtained before BMT showed that the duplication was not present; instead, we found several single base pair substitutions in exons 2 and 6. Thus, relapse of PNH in this patient was not due to persistence of the original clones; rather, it was associated with the emergence of a new clone. These findings support the notion that the BM environment may create selective conditions favoring the expansion of PNH clones.     

Levels of soluble stem cell factor in serum of patients with aplastic anemia

Aplastic anemia (AA) is a rare bone marrow (BM) disorder characterized by an unexplained failure of hematopoietic precursors to proliferate. In vitro growth of AA BM cells can be improved by the addition of the hematopoietic growth factor SCF (stem cell factor), which suggests that deficiency of SCF may be one of the underlying causes of the disease. In this study, we measured the concentration of SCF in sera of patients with severe AA. One hundred twenty-eight serum samples from 32 patients, at diagnosis and following therapy, were analyzed. Before treatment, SCF levels varied between 0.33 and 6.1 ng/mL; no correlation between hematopoietic function and SCF serum levels was apparent. Therapy with antilymphocyte globulin (ALG) or bone marrow transplantation (BMT) did not result in a recognizable pattern of changes in SCF levels. However, serum concentration of SCF in many patients with AA was at the low range of control serum levels determined in healthy blood donors. Of 128 AA serum samples tested before and after therapy, 107 were below the mean normal value of 3.3 ng/mL, including 26 samples below the minimum normal value of 1.3 ng/mL, as estimated in 267 controls. We also found that SCF levels in peripheral blood serum correlate well with factor concentrations in the BM plasma. Clinical observations suggest that higher SCF serum levels are often associated with a better clinical status of the patients in terms of survival and transfusion requirements. The data indicate that a deficient production of soluble SCF may contribute to AA in some patients; thus, suggesting a potential therapeutic benefit of SCF in this disorder.     

Comparison of baseline-nitrate technetium-99m sestamibi with rest-redistribution thallium-201 tomography in detecting viable hibernating myocardium and predicting postrevascularization recovery

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (> or = 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG-A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.     

Empirically supported comprehensive treatments for young children with autism

Aplastic anemia can be either acquired or congenital. The paradigm for the congenital form is Fanconi's anemia (FA). FA is an autosomal recessive, genetic syndrome characterized by progressive bone marrow failure, developmental abnormalities, and a predisposition to malignancy. The clinical manifestations of FA are heterogeneous, but one common outcome in the majority of patients is the development of life-threatening hematologic disease. FA is thought to affect the hematopoietic stem cell, and the hematologic consequences of FA can be effectively treated by complete replacement of patient stem cells by those from a histocompatible donor. Unfortunately, allogeneic stem cell transplantation is currently limited to patients with an unaffected matched sibling donor. Transplantation from alternative donors, while successful in selected cases, is associated with a high risk of graft failure and must be carefully considered in terms of risk and benefit for each individual. For FA patients lacking an appropriate donor, new therapies need to be devised. This review summarizes both the scientific rationale and the progress of gene therapy strategies aimed at correcting the hematopoietic defect of FA.     

CD34 positive blood cells for allogeneic progenitor and stem cell transplantation

The transplantation of allogeneic peripheral blood progenitor cells (PBPC) provides complete and sustained hematopoietic and lymphopoietic engraftment. In healthy donors, large amounts of PBPC can be mobilized with hematopoietic growth factors. However, the high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic PBPC to an elevated risk of acute and chronic graft-versus-host disease. Thus, the use of appropriate T-cell reduction, but not depletion might reduce this risk. The hazards of graft rejection and a higher relapse rate can be avoided by maintaining a portion of the T-cells in the graft. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1000-fold reduction of T-cells. These purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation and can be used in the following instances: 1. As hematopoietic stem and progenitor cell transplantation instead of bone marrow cells, from HLA-identical family donors; 2. for increasing the stem cell numbers from HLA-mismatched or three HLA-loci different family donors in order to reduce the incidence of rejection but without increasing the T-cell number; 3. boosting of poor marrow graft function with stem cells from the same family donors; 4. transplantation from volunteer matched unrelated donors; 5. split transplantation of CD34+ and T-cells; 6. addition of ex vivo expanded CD34+ cells to blood cell or bone marrow transplantation; 7. generation of antigen specific immune effector cells and antigen presenting cells for cell therapy.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

The mechanisms involved in the impairment of hematopoiesis after autologous bone marrow transplantation

Hematopoiesis after autologous bone marrow transplantation (BMT) is characterized by a prolonged and severe deficiency of marrow progenitors for several years, especially of erythroid and megakaryocyte progenitors, while the peripheral blood cells and marrow cellularity have reached relatively normal values within a few weeks. These anomalies are comparable to those reported for allogeneic BMT, despite the absence of any allo-immune reaction or post-graft immunosuppressive therapy. Post-graft hematopoietic impairment is the consequence of quantitative and qualitative changes involving both stem cell and stromal compartments which are expressed by an impaired capacity of stem cell self-renewal and commitment towards erythroid and megakaryocytic lineages. Besides the toxicity of conditioning regimens, hematopoietic reconstitution using autologous grafts is particularly dependent on a combination of factors related to the patient, such as underlying disease and pre-graft chemotherapy regimens, and to the graft processing itself, such as in vitro purging with chemotherapeutic agents.     

Postnatal somatic growth and insulin contents in moderate or severe intrauterine growth retardation in the rat

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.     

The role of granulocyte colony-stimulating factor in mobilization and transplantation of peripheral blood progenitor and stem cells

The article provides a review of the role of granulocyte colony-stimulating factor (G-CSF) for mobilization and transplantation of peripheral blood progenitor and stem cells. Recombinant gene technology has permitted the production of highly purified material for therapeutic use in humans. Progenitor cells can be assessed using semisolid and liquid culture assays or direct immunofluorescence analysis of cells expressing CD34. This antigen is found on lineage-determined hematopoietic progenitor cells as well as on more primitive stem cells with extensive self-renewal capacity. Administration of G-CSF during steady-state hematopoiesis or following cytotoxic chemotherapy leads to an increase of hematopoietic progenitor cells in the peripheral blood. The level of circulating CD34+ cells post-chemotherapy is greater compared with G-CSF administration during steady state. On the other hand, CD34+ cells harvested post-chemotherapy contain a smaller proportion of more primitive progenitor cells (CD34+/HLA-DR- or CD34+/CD38-) compared with G-CSF treatment alone. Independent of the mobilization modality, the amount of previous cytotoxic chemo- and radiotherapy adversely affects the yield of hematopoietic progenitor cells. While continuous subcutaneous administration of G-CSF between 5 and 16 micrograms/kg bodyweight is preferred, additional dose-finding studies may be helpful to optimize current dose schedules. Adhesion molecules like L-selectin, VLA (very late antigen)-4 and LFA (leukocyte function antigen)-1 are likely to play a role in mobilization, since these antigens are expressed on CD34+ cells from bone marrow in different densities compared with blood-derived CD34+ cells collected following G-CSF-supported cytotoxic chemotherapy. It is also relevant for transplantation that during G-CSF-enhanced recovery post-chemotherapy, peripheral blood is enriched with a greater proportion of CD34+ cells expressing Thy-1 in comparison with CD34+ cells from bone marrow samples obtained on the same day or before the mobilization therapy was started. The early nature of the CD34+/Thy-1+ cells is very likely since this phenotype has been found on stem cells from human fetal liver and bone marrow and on cord blood cells. As a result, G-CSF-mobilized blood stem cells provide rapid and sustained engraftment following high-dose therapy, including myeloablative regimens. Positive selection of CD34+ cells as well as ex vivo expansion using different cytokines are currently being investigated for purging and improvement of short-term recovery post-transplantation. Future developments include the use of blood-derived hematopoietic stem cells for somatic gene therapy. The availability of growth factors has been an important prerequisite for the development of these new avenues for cell therapy.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cells transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho+/++ (dull/bright) and Rho- (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho+/++ cell population contained > 99% of committed progenitor cells with in vitro colony-forming ability. The Rho- cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho- stem cells or purified Rho- stem cells in combination with large numbers of Rho+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho- stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.     

Gene transfer into hematopoietic stem cells

The ability to insert a gene into hematopoietic stem cells and achieve lineage specific expression of the transferred gene within hematopoietic organs following bone marrow transplantation would create the potential to effectively treat many genetic and acquired diseases. The use of retroviral vectors to achieve this purpose has been investigated extensively in animal models and most recently, in humans. In the murine model, about 20-30% of repopulating stem cells can be genetically modified with a retroviral vector. Peripheral blood stem cells, mobilized by cytokine administration in splenectomized animals, are readily transduced and are capable of long-term reconstitution of transplant recipients with genetically modified cells. Similar protocols have been utilized to transduce highly purified stem cells from rhesus monkeys. Although long-term repopulation with cells that persistently express the transferred gene has been achieved, the frequency of cells containing the vector genome is only about 1-2%. Genetic marking of human bone marrow and peripheral blood cells has been utilized to investigate their potential for contributing to long-term reconstitution following autologous transplantation. Future work will focus on improving gene transfer efficiencies for specific therapeutic applications.     

Enhanced maintenance and retroviral transduction of primitive hematopoietic progenitor cells using a novel three-dimensional culture system

Current techniques for the in vitro maintenance of hematopoietic stem cells often lead to loss of pluripotency. Overcoming the technical difficulties that result in alterations in stem cells in vitro has important implications for areas of basic science and clinical medicine such as cell expansion, bone marrow transplantation and gene therapy. Recent insights into hematopoietic stem cell biology have demonstrated that the three-dimensional architecture of the culture environment may influence the maintenance of stem cell pluripotency in vitro. An intriguing hypothesis is that the utilization of three-dimensional culture systems may improve the maintenance and manipulation of these cells in vitro. We report that a novel, three-dimensional, tantalum-coated porous biomaterial (TCPB) may be employed effectively as a hematopoietic progenitor cell culture device that offers distinct advantages over conventional culture systems. Specifically, we demonstrate that the use of TCPB for culturing hematopoietic progenitor cells in the absence of exogenous cytokines results in enhanced hematopoietic progenitor cell survival, improved maintenance of the immature CD34+/38- phenotype, and improved retroviral transduction of CD34+ cells and long-term culture initiating cells (LTCIC), without compromising multipotency, as compared with cultures in plastic dishes or bone marrow stroma. These findings suggest that this three-dimensional culture system may be useful in advancing the in vitro culture and transduction of hematopoietic stem and progenitor cells.     

Myelodysplasia following aplastic anaemia-paroxysmal nocturnal haemoglobinuria syndrome after treatment with immunosuppression and G-CSF: evidence for the emergence of a separate clone

Myelodysplasia (MDS) and aplastic anaemia-paroxysmal nocturnal haemoglobinuria (AA/PNH) syndrome developed in a severe aplastic anaemia (AA) patient after treatment with immunosuppressive (IS) therapy. Glycosylphosphatidyl inositol (GPI)-linked proteins were determined, and during the AA/PNH phase, a high proportion of neutrophils were found to be negative, without clinical evidence of haemolysis. However, MDS developed with cytogenetic abnormalities of monosomy 7,9q- and a rearranged chromosome 6; the GPI-linked protein negative cells were completely replaced by positively expressing cells. This represents the emergence of a GPI-linked protein positive myelodysplasia clone arising separately from an AA/PNH clone.