Stem Cells In Order Research Articles

An Extended Mathematical Model of Pathophysiology and Response to Treatment in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells that results in increase in myeloid, erythroid cells, and platelets in the peripheral blood and marked myeloid hyperplasia in the bone marrow. This disorder is characterized by the specific cytogenetic abnormality, the Philadelphia (Ph) chromosome, which results from a balanced translocation between the long arms of chromosomes 9 and 22, generating the bcr/abl chimeric gene that expresses an abnormal fusion protein with altered tyrosine kinase activity. Imatinib mesylate (IM, Gleevec, Novartis, Basel, Switzerland), is a potent and selective competitive inhibitor of the BCR-ABL protein tyrosine kinase and has shown to induce a high rate of cytogenetic and hematologic response in patients with chronic phase (CP) CML both as initial therapy and as secondary therapy after previous interferon therapy failed. Because the pathophysiology of CML and the mechanism for the clinical effects by IM is relatively uniform among patients, simplification and generalization with mathematical models have been proposed and they have excellently simulated the regression of leukemic cells by IM therapy and the regrowth of CML cells after appearance of IM-resistant clones. These models are based on the assumption that the transition rate of leukemic stem cells or precursor cells to more differentiated fractions are profoundly diminished by the administration of IM. This assumption is sufficient to explain the response as long as the observation period is short. In contrast, the issue regarding the influence of IM on the self-reproduction rate of leukemic stem cells was not focused on in these models because this issue had little effect on short-term outcomes with IM. After a decade since the appearance of IM, accumulated observations of CML patients treated with IM revealed long-term effectiveness; novel transformations to accelerate phase or blastic crisis are rarely observed in patients who continue to receive 400mg/day of IM for five or six years. Our aim is to clarify the effect of IM on leukemic stem cell fractions by extending and modifying the existing models so that they are compatible with actual long-term outcomes of IM therapy. First, we demonstrated that sustained effectiveness of IM for over six years cannot be achieved unless a stem cell fraction of CML is decremented by IM. In order to estimate the degree of stem cell attack by IM, we computed the rate of novel generation of IM-resistant clones before and after IM administration. In this model, we presumed that this rate is proportional to the accumulated number of self-duplication of leukemic stem cells. In order to simulate the actual observation that the clonal evolution decrease annually after IM administration, we illustrated that the rate of self duplication is depleted to at one fourth or less with IM compared to without IM. With this simulation, we show that the tyrosine kinase inhibitors can eradicate malignant cells thus leading to the radical cure of the disease. We also showed that the achievement of major molecular response (MMR; defined as at least three-log reduction of bcr/abl positive clones in the peripheral blood) at the 18th month of IM therapy is roughly associated with the absence of resistant clones at the moment of IM administration, and is obviously linked to successful therapy of CML after IM therapy is launched. This provides the supportive evidence of the previously reported observation that MMR at 18th month is associated with long-term effectiveness. Our model underscores the significance of prompt elimination of leukemic stem cells in order to diminish the generation of novel resistant clones and accomplish complete cure of CML. Development of the evaluation system to quantify residual leukemic stem cells would verify this hypothesis and pursuit to maximal response including early administration of second-generation tyrosine kinase inhibitors would be justified.

High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming

Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15–590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.

A importância do uso das células tronco para a saúde pública

Stem cells were elected 'Breakthrough of the Year' by Science(1) magazine in 1999, having shown that stem cells from adult tissues retained the ability to differentiate into other tissue types. During the previous year, the first human embryo stem cell lines were established. Since then, the number of scientific papers on stem cells has been increasing exponentially, establishing new paradigms that are rapidly challenged by subsequent experiments. This paper reviews the stem cell research field, divided into two groups: embryo and adult stem cells. While the differentiation potential of the former is well characterized in mice and humans, their use in cell therapy and research has been hampered by histocompatibility, safety and ethical issues. In contrast, adult stem cells do not present these problems. However, the extent of their plasticity is still under investigation. Nevertheless, numerous clinical trials in humans are under way, mainly with stem cells derived from bone marrow. This paper discusses discuss the importance of working with both classes of human stem cells in order to fulfill the promise of stem cell therapies.

Characterization of human myoblast cultures for tissue engineering

Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal muscle tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.

Treatment of chronic spinal cord injured patients with autologous bone marrow-derived hematopoietic stem cell transplantation: 1-year follow-up

Transplanted bone marrow (BM) cells have been found to improve neurologic disease in central nervous system (CNS) injury models by generating neural cells or myelin-producing cells. The results in treated patients and animal models suggest that BM cells could potentially be used as a therapy for spinal cord injury (SCI) patients. Nine patients with chronic complete SCI with American Spinal Injury Association (ASIA) Impairment Scale (ASIA) grade A were included in this study. They were treated with autologous BM-derived hematopoietic progenitor stem cell transplantation without any serious complications. All patients completed the protocols successfully. Three weeks after the operation all patients' movements and sensations were improved. All patients had ASIA grade B or C after the operation. We used autologous hematopoietic progenitor stem cells in order to avoid the problems associated with immunologic rejection and graft-versus-host (GvH) reactions, which are frequently caused by allografts. The advantage of this type of cell therapy is that it is not associated with carcinogenesis, which sometimes occurs with embryogenic stem cell therapy. To evaluate the patients we used neurologic impairment scales (ASIA scores), pre- and post-operative Somato Sensorial Evoked Potential (SSEP) assessments and pre- and post-operative Magnetic Resonance Imaging (MRI). All the data showed that BM-derived autologous stem cell therapy is effective and safe for the treatment of chronic SCI.

Stem cells and the Planarian Schmidtea mediterranea

In recent years, stem cells have been heralded as potential therapeutic agents to address a large number of degenerative diseases. Yet, in order to rationally utilize these cells as effective therapeutic agents, and/or improve treatment of stem-cell-associated malignancies such as leukemias and carcinomas, a better understanding of the basic biological properties of stem cells needs to be acquired. A major limitation in the study of stem cells lies in the difficulty of accessing and studying these cells in vivo. This barrier is further compounded by the limitations of in vitro culture systems, which are unable to emulate the microenvironments in which stem cells reside and which are known to provide critical regulatory signals for their proliferation and differentiation. Given the complexity of vertebrate embryonic and adult stem cell populations and their relative inaccessibility to in vivo molecular analyses, the study of stem cells should benefit from analyzing their counterparts in simpler model organisms. In the past, the use of Drosophila or C. elegans has provided invaluable contributions to our understanding of genes and pathways involved in a variety of human diseases. However, stem cells in these organisms are mostly restricted to the gonads, and more importantly neither Drosophila, nor C. elegans are capable of regenerating body parts lost to injury. Therefore, a simple animal with experimentally accessible stem cells playing a role in tissue maintenance and/or regeneration should be very useful in identifying and functionally testing the mechanisms regulating stem cell activities. The planarian Schmidtea mediterranea is poised to fill this experimental gap. S. mediterranea displays robust regenerative properties driven by a stem cell population capable of producing the ∼40 different cell types found in this organism, including the germ cells. Given that all known metazoans depend on stem cells for their survival, it is extremely likely that the molecular events regulating stem cell biology would have been conserved throughout evolution, and that the knowledge derived from studying planarian stem cells could be vertically integrated to the study of vertebrate stem cells. Current efforts, therefore, are aimed at further characterizing the population of planarian stem cells in order to define its suitability as a model system in which to mechanistically dissect the basic biological attributes of metazoans stem cells. To cite this article: A. Sánchez Alvarado, C. R. Biologies 330 (2007).

MR imaging in assessing cardiovascular interventions and myocardial injury

Performing an MR-guided endovascular intervention requires (1) real-time tracking and guidance of catheters/guide wires to the target, (2) high-resolution images of the target and its surroundings in order to define the extent of the target, (3) performing a therapeutic procedure (delivery of stent or injection of gene or cells) and (4) evaluating the outcome of the therapeutic procedure. The combination of X-ray and MR imaging (XMR) in a single suite was designed for new interventional procedures. MR contrast media can be used to delineate myocardial infarcts and microvascular obstruction, thereby defining the target for local delivery of therapeutic agents under MR-guidance. Iron particles, or gadolinium- or dysprosium-chelates are mixed with the soluble injectates or stem cells in order to track intramyocardial delivery and distribution. Preliminary results show that genes encoded for vascular endothelial and fibroblast growth factor and cells are effective in promoting angiogenesis, arteriogenesis, perfusion and LV function. Angiogenic growth factors, genes and cells administered under MR-guided minimally invasive catheter-based procedures will open up new avenues in treating end-stage ischemic heart disease. The optimum dose of the therapeutic agents, delivery devices and real-time imaging techniques to guide the delivery are currently the subject of ongoing research. The aim of this review is to (1) provide an updated review of experiences using MR imaging to guide transcatheter therapy, (2) address the potential of cardiovascular magnetic resonance (MR) imaging and MR contrast media in assessing myocardial injury at a molecular level and labeling cells and (3) illustrate the applicability of the non-invasive MR imaging in the field of angiogenic therapies through recent clinical and experimental publications.

Selective Targeting of Cancer Stem Cells

Although the concept of 'cancer stem cell' was first proposed more then a century ago, it has attracted a great deal of attention recently due to advances in stem cell biology, leading to the identification of these cells in a wide variety of human cancers. There is accumulating evidence that the resistance of cancer stem cells to many conventional therapies may account for the inability of these therapies to cure most metastatic cancers. The recent identification of stem cell markers and advances in stem cell biology have facilitated research in multiple aspects of cancer stem cell behavior. Stem cell subcomponents have now been identified in a number of human malignancies, including hematologic malignancies and tumors of the breast, prostate, brain, pancreas, head and neck, and colon. Furthermore, pathways that regulate self-renewal and cell fate in these systems are beginning to be elucidated. In addition to pathways such as Wnt, Notch and Hedgehog, known to regulate self-renewal of normal stem cells, tumor suppressor genes such as PTEN (phosphatase and tensin homolog on chromosome 10) and TP53 (tumor protein p53) have also been implicated in the regulation of cancer stem cell self-renewal. In cancer stem cells, these pathways are believed to be deregulated, leading to uncontrolled self-renewal of cancer stem cells which generate tumors that are resistant to conventional therapies. Current cancer therapeutics based on tumor regression may target and kill differentiated tumor cells, which compose the bulk of the tumor, while sparing the rare cancer stem cell population. The cancer stem cell model suggests that the design of new cancer therapeutics may require the targeting and elimination of cancer stem cells. Therefore, it is imperative to design new strategies based upon a better understanding of the signaling pathways that control aspects of self-renewal and survival in cancer stem cells in order to identify novel therapeutic targets in these cells.

The Influence of a Human Embryonic Stem Cell–Derived Microenvironment on Targeting of Human Solid Tumor Xenografts

The awareness of the important role that the surrounding tissue microenvironment and stromal response play in the process of tumorigenesis has grown as a result of in vivo models of tumor xenograft growth in immunocompromised mice. In the current study, we used human embryonic stem cells in order to study the interactions of tumor cells with the surrounding microenvironment of differentiated human cell tissues and structures. Several cancer cell types stably expressing an H2A-green fluorescence protein fusion protein, which allowed tracking of tumor cells, were injected into mature teratomas and developed into tumors. The salient findings were: (a) the observation of growth of tumor cells with high proliferative capacity within the differentiated microenvironment of the teratoma, (b) the identification of invasion by tumor cells into surrounding differentiated teratoma structures, and (c) the identification of blood vessels of human teratoma origin, growing adjacent to and within the cancer cell-derived tumor. Mouse embryonic stem cell-derived teratomas also supported cancer cell growth, but provided a less suitable model for human tumorigenesis studies. Anticancer immunotherapy treatment directed against A431 epidermoid carcinoma cell-related epitopes induced the complete regression of A431-derived tumor xenografts following direct i.m. injection in immunocompromised mice, as opposed to corresponding tumors growing within a human embryonic stem cell-derived microenvironment, wherein remnant foci of viable tumor cells were detected and resulted in tumor recurrence. We propose using this novel experimental model as a preclinical platform for investigating and manipulating the stromal response in tumor cell growth as an additional tool in cancer research.

Uptake of Protamine Sulphate Complexed Fluorescent Nano-Particles Is Defined by Cell Cycle Status in Primary Human CD34+ Cells: Use of a Multi-Color p27 kip1 Based Flow Cytometric Assay.

The use of iron based nano-particles for multi-modal imaging is gaining interest, since it allows high resolution non-invasive in vivo imaging of human hematopoietic homing and engraftment events in xenograft models. The uptake of ferridex nano-particles complexed to cationic protamine sulphate is believed to be non-specific through mechanisms like endocytosis, but this has not been well defined for hematopoietic stem cells (HSC). In defining ex vivo cultivation strategies for manipulation of human HSC, a key factor is the responsiveness of the most primitive cells to the in vitro conditions, with the aim of maintaining viability without inducing terminal differentiation. Here, we present a novel flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo protocol, aimed at facilitating labeling of human stem/progenitor cells using protamine sulphate complexed nano-particles for subsequent in vivo imaging. We used intracellular staining for the cell cycle inhibitor p27kip1, which is present in the highest levels in non-cycling cells, as the primary flow cytometric marker in combination with CD34, CD133 and Alexa 488, 647 and 750 conjugated ferridex nano-particles and the membrane dye PKH26. An assay was developed to simultaneously assess the molecular events occurring in individual human cord blood Lin− or CD34+ cells while they were cultured for up to 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates with or without nano-particles. Co-expression of p27kip1, CD34 or CD133 in the cultured cells slowly decreases from 86.1% CD34+p27kip1 (T=0) to 76.7%+/−12.2% (T=72) and from 89.6% CD133+p27kip1+ (T=0) to 54.1%+/−10.4% (T=72). We suggest that this slow decrease represents cells dividing and potentially differentiating over the time course of the ex vivo cultivation period. Assessing uptake of fluorescent conjugated nano-particles over a 72 hr period showed that the uptake of particles in CD34+ and CD133+ cells declined significantly after the first 24 hrs., from 32.5+/−3.7% nano-positive CD34+ cells to 19.2+/−2.9% at 48 hours ex vivo with a more significant decline to only 8.3+/−3.7% nano positive CD34+ cells in the culture after 72 hours ex vivo. The same decline in uptake over time was observed in cultured human CB cells that were positive for CD133. PKH26 co-staining demonstrated that the majority of cells that undergo cell division within the first 24 hours of ex vivo culture are the most likely to uptake the nano-particles. In summary, using a multi color p27kip1 based flow-cytometry assay, we found that human Lin−, CD133+, and CD34+ cells uptake Fe-Pro in a fashion which is not entirely cell cycle independent as previously suggested. These data indicate that cell cycle or metabolic status may influence the ability of human hematopoietic stem and progenitor subsets to uptake the protamine sulphate-complexed nano-particles. These findings emphasize the need to carefully develop ex vivo conditions for nano-particle labeling of primary human stem cells in order to perform accurate in vivo imaging of the most primitive human hematopoietic stem and progenitor cells.

Pancreatic differentiation

Over the last decade, with the advent of new techniques and technologies in modern molecular biology, our understanding of the underlying mechanisms responsible for organ differentiation has developed rapidly. Despite this, our knowledge of these signaling pathways is still far from complete. Some of these advances, such as the creation of transgenic mouse models, have given us new tools to help us understand the interactions of the various transcription factors that are responsible for the creation of various cell types from a single cell type during embryogenesis. This knowledge then gives rise to the concept of creating new ways to manipulate stem cells in order to correct the deficiencies present in various disease processes. Here, we present work that focuses specifically on pancreatic development. The ultimate goal of our research in studying the mechanisms of the basic differentiation of pancreatic precursor cells is to gain the knowledge necessary to be able to engineer stem cells specifically into beta-cells in the treatment of diabetes.

Neural stem cells and cell replacement therapy: making the right cells.

The past few years have seen major advances in the field of NSC (neural stem cell) research with increasing emphasis towards its application in cell-replacement therapy for neurological disorders. However, the clinical application of NSCs will remain largely unfeasible until a comprehensive understanding of the cellular and molecular mechanisms of NSC fate specification is achieved. With this understanding will come an increased possibility to exploit the potential of stem cells in order to manufacture transplantable NSCs able to provide a safe and effective therapy for previously untreatable neurological disorders. Since the pathology of each of these disorders is determined by the loss or damage of a specific neural cell population, it may be necessary to generate a range of NSCs able to replace specific neurons or glia rather than generating a generic NSC population. Currently, a diverse range of strategies is being investigated with this goal in mind. In this review, we focus on the relationship between NSC specification and differentiation and discuss how this information may be used to direct NSCs towards a particular fate.

Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag of marbles, in germline stem cells in the Drosophila ovary.

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters, cystoblasts. Here we show that Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for Med and punt, two essential Bmp signal transducers, bam transcription is also elevated. Finally, we show that Med and Mad directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal.

Early stages of hematopoietic differentiation.

Mouse bone marrow contains hematopoietic stem cells as well as progenitor cells, which are partially differentiated offspring of stem cells. We have utilized several approaches to separate progenitors from stem cells in order to characterize essential differences between these two stages of development. As a first approach, we utilized the supravital fluorescent dye rhodamine-123 (Rh-123) to distinguish quiescent stem cells (Rh-123(low)) from metabolically active progenitor cells (Rh-123(hi)). Analysis of megakaryocyte potential in a tissue culture assay demonstrated that Rh-123(hi) progenitor cells were capable of robust megakaryocyte differentiation, while Rh-123(low) stem cells produced fewer colonies containing megakaryocytes. Transplantation of the two cell populations into irradiated recipients revealed the opposite outcome, suggesting that the tissue culture assay failed to predict behavior in a transplant setting. We also evaluated functional potential of lymphoid progenitors isolated by selecting for differential expression of Thy-1.1 and c-kit. The potential of defined cell populations to differentiate as T or B lymphocytes in vivo was dependent upon the time post transplant at which animals were evaluated. These studies underscore the need for caution in the interpretation of lineage potentials evaluated by both in vitro and in vivo assays.

A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells.

To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.

Prospects for the temporary treatment of acute liver failure.

At present, the most successful treatment of acute liver failure is orthotopic liver transplantation, with survival rates ranging from 70% to 85%. However, mortality rates for liver failure remain high because of the shortage of available donor organs. Therefore, there has been renewed interest in temporary treatment methods for patients with acute liver failure to either allow liver regeneration or await liver transplantation. It is thought that the function of the liver can only be replaced with the biological substrate, e.g. liver cells or a whole liver specimen, which requires the availability of liver tissue from xenogeneic or human sources. In this review, existing temporary liver support techniques are summarized and the potential hazards are described. These include the immunological implications of these techniques, e.g. the host versus graft reaction, which may influence the effectivity of the support system, and in the long run may sensitize the patient to subsequent allogeneic transplantation. The graft versus host reaction is also considered. At present, one of the major concerns is the threat of pig-to-human transmission of activated endogenous retrovirus present in the pig genome. An overview is given of literature concerning the transmission of retrovirus particles in vitro and in vivo. Finally, new solutions for the development of ex vivo systems for temporary treatment of patients with acute liver failure are discussed. These include the use of new immortalized human cell lines and human fetal hepatocytes, and the possibility of isolating, expanding and genetically manipulating stem cells in order to have stable differentiated and committed cells.

Autologous peripheral blood haematopoietic stem cell transplantation for chronic myelogenous leukaemia

In these last four decades there has been extraordinary progress in our understanding of the biology of, and therapeutic approach to, chronic myelogenous leukaemia (CML). During these decades new observations arising from studies of the biological behaviour of diploid and leukaemic stem cells and, recently, from clinical investigations have received the most attention. From a clinical point of view, allografting is still the only procedure which is able to cure CML. For patients without HLA-compatible donors, current therapeutic options include conventional chemotherapy (hydroxyurea), interferon-α (IFN-α) and autografting. While IFN-α (±low-dose ARA-C) must be considered the first-line therapy, autografting, according to our approach, or other procedures, raises the question of an ideal sequential strategy in the management of CML patients (diploid stem cell mobilization, autografting, IFN-α). Because it seems that the diploid haematopoietic reservoir declines with time, it may be desirable to mobilize and collect diploid stem cells in order to store them as soon as diagnosis is possible when the WBC count has been controlled by hydroxyurea.

Collection, Tumor Contamination, and Engraftment Kinetics of Highly Purified Hematopoietic Progenitor Cells to Support High Dose Therapy in Multiple Myeloma

AbstractUnfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.

Collection, Tumor Contamination, and Engraftment Kinetics of Highly Purified Hematopoietic Progenitor Cells to Support High Dose Therapy in Multiple Myeloma

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.

Establishment of the role of IL-6 and TNF receptor 1 using gene knockout mice

Cytokines are known to be key players in host response to infection, immunological disorders, and tissue injury in the attempt of an organism to overcome the insult and restore homeostasis. Another important aspect of cytokines, however, is their normal physiological role during development in the unchallenged organism. The most elegant way to analyze both of these functions is to introduce targeted mutations in embryonic stem cells in order to create new mouse strains deficient for a given cytokine and identify the functions that are consequently impaired or lost. This review summarizes the mutant phenotypes of mice carrying a null mutation in the cytokine IL-6 gene or the tumor necrosis factor receptor 1 (Tnfr1) gene. Results for interferon-- and interferon-gamma receptor-deficient mice are included for comparison.