Twenty-five years of peripheral blood stem cell transplantation.

Peripheral blood stem cell transplantation (PBSCT) is the most common transplantation procedure performed in medicine. Its clinical introduction in 1986 replaced BM as a stem-cell source to approximately 100% in the autologous and to approximately 75% in the allogeneic transplantation setting. This historical overview provides a brief insight into the discovery of circulating hematopoietic stem cells in the early 1960s, the development of apheresis technology, the discovery of hematopoietic growth factors and small molecule CXCR4 antagonist for stem- cell mobilization, and in vivo experimental transplantation studies that eventually led to clinical PBSCT. Also mentioned are the controversies surrounding the engraftment potential of circulating stem cells before acceptance as a clinical modality. Clinical trials comparing the outcome of PBSCT with BM transplantation, registry data analyses, and the role of the National Marrow Donor Program (NMDP) in promoting unrelated blood stem-cell donation are addressed.

Invertebrate hematopoiesis: an astakine-dependent novel hematopoietic factor.

A novel factor, named crustacean hematopoietic factor (CHF), was identified from a library of suppression subtractive hybridization with the aim to find downstream genes of an invertebrate cytokine, astakine 1, in the freshwater crayfish Pacifastacus leniusculus. CHF is a small cysteine-rich protein (∼9 kDa) with high similarity to the N-terminal region of vertebrate CRIM1 in containing an insulin growth factor binding protein variant motif with unknown function. CHF was found to be induced in primary cell cultures of crayfish hematopoietic tissue (Hpt) cells (precursors of crayfish blood cells) after treatment with astakine 1. Silencing of CHF did not affect the renewal of Hpt cells in vitro, but induced apoptosis of Hpt cells. CHF is exclusively expressed in the blood cell lineage of crayfish (Hpt cells and blood cells), and in vivo RNA interference experiments show that knockdown of this gene results in severe loss of blood cells and a higher apoptotic rate in Hpt. Our data further suggest that crayfish CHF is critical for the survival of hemocytes and Hpt cells by preventing their apoptosis, thus it plays an important role in hemocyte homeostasis in crayfish. Our study of CHF may also shed light on the function of this untypical insulin growth factor binding protein motif located in the N-terminal of vertebrate CRIM1.

HIF-dependent hematopoietic factors regulate the development of the embryonic vasculature.

Hypoxia inducible factors (HIFs) regulate adaptive responses to changes in oxygen (O(2)) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF-deficient embryos exhibit a number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures, we show that deletion of the HIF-beta subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild-type Sca-1(+) hematopoietic cells or recombinant VEGF. Arnt(-/-) embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.

Hematopoietic growth factors and the regulation of differentiative decisions.

Hematopoietic growth factors control the growth and differentiation of hematopoietic progenitor cells and bind to specific receptors that are expressed on the surface of immature hematopoietic cells found in the bone marrow. Many studies have demonstrated that these growth factors stimulate cellular growth and division by receptor activation. More recently, it has become apparent that they also influence, either directly or indirectly, the process of cellular differentiation.

Juxtacrine cell signaling molecules.

Cell adhesion molecules and diffusible growth factors have long been studied as two separate forms of intercellular communication. However, biologists working in these two areas are seeing their fields converge. This merge has been promoted by the identification of membrane-anchored growth factors that activate receptors on adjacent cells through intimate cell-cell contacts, and cell adhesion molecules that act as signaling receptors. Juxtacrine stimulation mediated by these two classes of molecules is critical in various aspects of tissue development and maintenance. Our increasing appreciation of juxtacrine interactions should foster rapid progress in this field.

Influence of growth factors and substrates on differentiation of haemopoietic stem cells.

During the last few years there has been major progress in our understanding of the mechanisms underlying the regulation of haemopoietic stem cell proliferation and development. In the past 12 months, advances have been made in identifying how growth factor receptor expression is regulated in primitive haemopoietic cells, in determining the transcription factors that are associated with development, and in recognizing some of the specific molecular interactions that occur between the bone marrow stromal cells and the haemopoietic progenitor cells. These and previous studies clearly demonstrate that the environment can influence the survival, the proliferation and the differentiation of haemopoietic cells at every stage of development.

High-dose therapy and autologous peripheral blood stem cell transplantation in patients with multiple myeloma.

Since its introduction in 1983, high-dose therapy followed by autologous peripheral blood stem cell transplantation is a pillar of the treatment of patients with multiple myeloma. In the last decades, a multitude of clinical trials helped to improve strategies based on high-dose therapy and autologous stem cell transplantation resulting in a continuously prolongation of overall survival of patients. In this chapter we will review the progress, which has been made in order to enhance the mobilisation of autologous stem cells and increase the effectiveness of this treatment.

Early-acting hematopoietic growth factors: biology and clinical experience.

Secreted protein growth factors that stimulate the self-renewal, proliferation, and differentiation of the most primitive stem cells are among the most biologically interesting molecules and at least theoretically have diverse applications in the evolving field of regenerative medicine. Among this class of regulators, the early-acting hematopoietic growth factors and their cellular targets are perhaps the best characterized and serve as a paradigm for manipulating other stem cell based tissues. This chapter reviews the preclinical knowledge accumulated over ~40 years, since the discovery of the first such growth factor, and the clinical applications of those that, upon testing in humans, ultimately gained regulatory approval for the treatment of various hematological diseases.

Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium.

An important goal for the investigation of the proliferation of mammalian cells is to establish a fully defined condition for culturing them in vitro. Here, we report establishment of a fully defined culture condition that supports the primary culture of normal c-kit+IL-7 receptor (IL-7R)+ B precursor cells without the aid of stromal cell lines. This defined culture condition contains IL-7, the ligand for c-kit, transferrin, insulin, and bovine serum albumin as protein components. By using the cell lines derived from RAG2(-/-) mice, which do not differentiate into c-kit- stage, we have evaluated the role of each protein in the cell cycle progression of c-kit+IL-7R+ B precursor cells. Since B precursor cells can grow without insulin, c-kit remains a sole functional receptor tyrosine kinase for their growth. While both c-kit ligand (KL) and IL-7 are the requisite molecules for sustained proliferation of B precursor cells, each molecule plays distinct roles. IL-7 starvation results in prompt arrest of the cells at G1. An accumulation of the cells in the mitotic phase was also detected. Thus, the major role of IL-7 is to regulate the G1/S transition and the process of cytokinesis of B precursor cells. Although prolonged KL starvation over 48 h resulted in accumulation of G1 cells, its effect could not be detected within 24 h, which is long enough for all the cells to complete one cell cycle. This suggests that KL might be involved in the cell cycle progression of B precursor cells in a manner that its signal could still be effective in the one or two cell cycles that follow. Although molecular nature of the signals underlying the present observation awaits future investigation, the method described in this report would provide a useful model system for investigating the signaling pathways that are involved in the cell cycle progression of B precursor cells.

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.

Suppression of a novel hematopoietic mediator in children with severe malarial anemia.

In areas of holoendemic Plasmodium falciparum transmission, severe malarial anemia (SMA) is a leading cause of pediatric morbidity and mortality. Although many soluble mediators regulate erythropoiesis, it is unclear how these factors contribute to development of SMA. Investigation of novel genes dysregulated in response to malarial pigment (hemozoin [PfHz]) revealed that stem cell growth factor (SCGF; also called C-type lectin domain family member 11A [CLEC11A]), a hematopoietic growth factor important for development of erythroid and myeloid progenitors, was one of the most differentially expressed genes. Additional experiments with cultured peripheral blood mononuclear cells (PBMCs) demonstrated that PfHz decreased SCGF/CLEC11A transcriptional expression in a time-dependent manner. Circulating SCGF levels were then determined for Kenyan children (n = 90; aged 3 to 36 months) presenting at a rural hospital with various severities of malarial anemia. SCGF levels in circulation (P = 0.001) and in cultured PBMCs (P = 0.004) were suppressed in children with SMA. Circulating SCGF also correlated positively with hemoglobin levels (r = 0.241; P = 0.022) and the reticulocyte production index (RPI) (r = 0.280; P = 0.029). In addition, SCGF was decreased in children with reduced erythropoiesis (RPI of <2) (P < 0.001) and in children with elevated levels of naturally acquired monocytic PfHz (P = 0.019). Thus, phagocytosis of PfHz promotes a decrease in SCGF gene products, which may contribute to reduced erythropoiesis in children with SMA.

Homing and mobilization in the stem cell niche.

All mature blood cells are derived from the haemopoietic stem cell (HSC). In common with all other haemopoietic cells, stem cells are mobile, and it is this property of mobility that has allowed bone marrow transplantation to become a routine clinical option. Successful transplantation requires haemopoietic stem cells to home to the bone marrow, leave the peripheral circulation and become stabilized in regulatory niches in the extravascular space of the bone marrow cavity. This homing and tethering process is reversible - haemopoietic stem cells can be released from their bone marrow tethering through changes in molecular interactions, which are also important in homing following transplantation. The molecular mechanisms regulating this two-way flow of stem cells are beginning to be elucidated, and much recent data has emerged that sheds light on the processes and molecules involved in these complex physiological events. This article reviews current knowledge of the adhesive, homing and proliferative influences acting on HSCs and progenitor cells.

Hematopoietic growth factors, signaling and the chronic myeloproliferative disorders.

The chronic myeloproliferative diseases (CMDs) are a group of conditions characterized by unregulated blood cell production, that due either to excessive numbers of erythrocytes, leukocytes or platelets, or their defective function cause symptoms and signs of fatigue, headache, ruddy cyanosis, hemorrhage, abdominal distension, and the complications of vascular thrombosis. In the late 19th century Vaquez provided the first description of polycythemia vera (PV) and Hueck defined idiopathic myelofibrosis (IMF). In 1920, di Guglielmo established criteria for patients with essential thrombocythemia (ET). In 1951, Dameshek argued that these disorders, along with chronic myelogenous leukemia (CML) display many similar clinical and laboratory features [Dameshek W. Some speculations on the myeloproliferative syndromes. Blood 1951;6:372-5], and grouped them. In 2002, the World Health Organization expanded the definition of CMDs to also include chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES) and systemic mast cell disorder (SMCD) [Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100:2292-302]. While the molecular pathogenesis of CML is well known [Melo JV, Deininger MW. Biology of chronic myelogenous leukemia-signaling pathways of initiation and transformation. Hematol Oncol Clin North Am 2004;18:545-68], and the causes of CEL/HES and SMCD have been identified in about half of all cases [Gotlib J, Cools J, Malone III JM, Schrier SL, Gilliland DG, Coutre SE. The FIP1L1-PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management. Blood 2004; 103:2879-91; Valent P, Akin C, Sperr WR, Horny HP, Metcalfe DD. Mast cell proliferative disorders: current view on variants recognized by the World Health Organization. Hematol Oncol Clin North Am 2003; 17:1227-41], until very recently the etiologies of the three classically defined CMDs, PV, IMF and ET, were poorly understood. Each of these disorders is characterized by excessive hematopoiesis, a process usually dependent on one or more hematopoietic growth factors (HGFs). This review will focus on how our knowledge of the molecular mechanisms by which HGFs are produced, bind cell surface receptors and transduce survival and proliferative signals have provided the platform on which the multiple origins of CMDs can be understood and novel therapeutic interventions designed.

Hemopoietic regulators.

The production and maturation of blood cells from the eight major blood cell lineages is a complex and continuous process, which is largely controlled by specific glycoprotein hemopoietic regulators. These regulators also control the functional activity of the blood cells through eliciting a diverse set of intracellular responses initiated by a regulator-specific membrane receptor. Twenty of these regulators have now been characterized, and their mass production has led to four already being licensed for clinical use in disease states involving subnormal blood cell formation.

From white spots to stem cells: the role of the Kit receptor in mammalian development.

The Kit tyrosine kinase membrane receptor is essential for melanogenesis, gametogenesis and hematopoiesis during embryonic development and postnatal life. This review summarizes the genetic evidence implicating Kit and its ligand, Steel factor, in the control of stem cell proliferation, migration and survival, with emphasis on mutations in the human and mouse genes.

c-KIT expression enhances the leukemogenic potential of 32D cells.

The growth of human leukemic cells in culture and in vivo is dependent upon the presence of hematopoietic growth factors. Most populations of human leukemic acute myeloblastic leukemia (AML) cells express c-Kit on their surface and respond to Kit ligand (KL) in culture. To determine if this interaction was of potential significance in vivo we used a mouse model system. 32D cells, a murine IL-3-dependent myeloid cell line, were rendered KL responsive by transfection of the murine c-Kit. After injection of 32D or 32D-Kit cells into syngeneic hosts, animals bearing 32D-Kit cells, but not 32D cells, became moribund and were killed. These animals had circulating leukemic blast cells, infiltration of bone marrow, spleen, brain, liver, lung, and kidney. Cells recovered from some of the animals continued to be dependent upon IL-3 or KL for growth while in other cases the cells were factor independent. This model illustrates that the constitutive expression of c-Kit enhances the leukemic potential of 32D cells. The model will be useful for studying the progression of leukemia in vivo and testing whether interruption of the interaction of Kit and KL can affect the growth of leukemic cells.

Lymphohematopoietic cytokines in the female reproductive tract.

The past year has seen an avalanche in publications describing the synthesis of lymphohematopoietic cytokines by the uterus and placenta. Dissection of the particular contributions of these factors will require genetic as well as biochemical approaches. The major events of the year therefore, were the identification and creation of mouse mutants that have provided novel insights into the roles of various cells and cytokines during gestation.

To be or not to be a pro-T?

Recent studies have begun to unravel some of the molecular pathways that appear to control the processes of T cell determination in the earliest thymocyte precursors. In addition, the analyses of mouse mutants with an entirely alymphoid thymus have shed light on the developmental relationship of pro-T cells and thymic dendritic cells, revealing that development of thymocytes and thymic dendritic cells can be dissociated.

Activation of hematopoietic growth factor signal transduction pathways by the human oncogene BCR/ABL.

BCR/ABL is a human chimeric oncogene that causes chronic myelogenous leukemia (CML). The BCR/ABL oncogene is generated from the Philadelphia chromosome (Ph) translocation, t(9;22)(q34;q11), and creates a constitutively active tyrosine kinase. There is clonal expansion of hematopoietic stem cells of several different lineages in CML. CML patients in stable phase usually have high white blood counts and immature cells of granulocytic lineages. Stable phase CML evolves to a more aggressive phase typically within 3.5-5 years, where differentiation is blocked and acute leukemia ensues. The transition of CML stable phase to blast phase is reflected in the loss of growth factor requirement of CML cells and correlates with additional cytogenetic alterations. Some biological effects reported in primary CML cells include reduced apoptosis and altered adhesion to fibronectin; however, the cells are dependent on hematopoietic growth factors. On a molecular level, the BCR/ABL translocation is well characterized. However, the actual mechanism of transformation by the BCR/ABL oncogene of hematopoietic cells is largely unknown. Enhancement of the c-ABL tyrosine kinase activity in BCR/ABL appears to be crucial for transformation. This tyrosine kinase activity leads to activation of several signal transduction pathways that are also utilized by hematopoietic growth factors, including steel factor, thrombopoietin, interleukin-3, and granulocyte/macrophage-colony stimulating factor. In several model systems, BCR/ABL has overlapping biological effects with hematopoietic growth factors, and transformation of hematopoietic growth factor-dependent cell lines leads to growth factor independence. In this review, we will describe the molecular and biological abnormalities in CML and several signal transduction mechanisms utilized by BCR/ABL as compared to hematopoietic growth factors.

Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.