Retroviral src gene expression in continuous marrow culture increases the self-renewal capacity of multilineage hematopoietic stem cells

To define the action of the retroviral src gene on hematopoietic stem cells, C57BL/6 x DBA/2 (B6D2F1) mouse long-term marrow cultures were infected at initiation with Moloney murine leukemia virus (MuLV) pseudotypes of src-recombinant retroviruses with the src gene inserted in the env region of an amphotropic MuLV (src-Ampho), or in the gag region of Moloney MuLV (src-Mo). Other cultures were infected with Friend spleen focus-forming virus polycythemia-inducing strain (SFFVp), Moloney MuLV, or amphotropic MuLV, or were uninfected controls. Harvested nonadherent cells were tested weekly for multilineage, granulocyte-erythroid-megakaryocyte macrophage (CFU-GEMM) colony formation in vitro in recombinant murine IL-3 and erythropoietin, and individual colonies were removed, split 1:2, with half of each replated for in vitro self-renewal and the other half examined morphologically for number of hematopoietic cellular lineages, or tested for release of MuLV and src virus. Cultures infected with src-Ampho, src-Mo, or SFFVp demonstrated a significant increase in cumulative nonadherent cell and CFU-GEMM production. There was prolonged self-renewal over seven serial transfers of individual CFU-GEMM from src virus-infected cultures over seven serial transfers, and five of 61 individual colonies from the second or third generations contained detectable v-src gene sequences, but none released detectable src virus. Self-renewal of CFU-GEMM was similar to that with permanent IL-3-dependent cell line B6SUtA. In contrast, MuLV-infected or control uninfected cultures produced fewer cells, and self-renewal of CFU-GEMM did not exceed three generations. IL-3-dependent clonal hematopoietic progenitor cell lines, derived from each culture group, formed no detectable tumors in vivo; however, each released the original helper and/or transforming virus. Adherent cell lines, derived from src-Ampho-infected cultures released src virus and formed fibro-sarcomas in vivo. The data support the conclusion that src-recombinant virus expression in long-term marrow cultures increases the self-renewal capacity of multilineage hematopoietic stem cells.     

Induced transgene expression for the treatment of solid tumors by hematopoietic stem cell-based gene therapy

Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-β receptor II (TβRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TβRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.     

Pediatric hematopoietic stem cell transplantation.

The successful use of allogeneic HSCT for children with malignant and nonmalignant diseases continues to be limited by the development of acute and chronic GVHD, infectious complications, delayed recovery of the immune system, acute and long-term toxicity, and relapse of disease. Significant advances have been made, particularly in the ability to identify suitable sources of HSC. Future advances will depend on a better understanding of the biology of HSC sources, GVHD, immune reconstitution, and common complications. Improved therapies are dependent on participation of children in well-designed, translational and clinical transplant studies.     

Regulation of hematopoietic stem cell growth.

Hematopoietic stem cells (HSC) must balance self-renewal and differentiation to provide sufficient primitive cells to sustain hematopoiesis, while generating more mature cells with specialized capabilities. The enhanced self-renewal capacity of primitive HSCs enables their ability to sustain hematopoiesis throughout decades of life and their ability to repopulate a host when used therapeutically in bone marrow transplantation. However, hematopoietic cell perturbations resulting in unchecked self-renewal participate in leukemogenesis. While mechanisms governing self-renewal are still being uncovered, they are thought to bear relationship to the malignant process in a variety of tumor types and may therefore provide useful therapeutic targets in putative cancer stem cells. This review discusses molecular mechanisms recently defined to participate in HSC governance and highlights features of stem cell interactions with the microenvironment that may help guide therapies directed at HSCs.     

造血干细胞动员的进展

使用动员的外周血干细胞(PBSC)已经在很大程度上取代了骨髓作为干细胞源用于异基因和自体干细胞移植.粒细胞集落刺激因子(G-CSF)加或不加化疗是最常用的干细胞动员方案.一些供者或患者,特别是动员前经高强度治疗的患者,用这一方案不能动员到干细胞目标值.新动员剂和新疗法的产生能够改进造血干细胞的动员.     

T细胞受体重排删除环检测在异基因造血干细胞移植中的应用

 异基因造血干细胞移植是治疗血液肿瘤和免疫疾病的重要手段,免疫功能重建是评估移植疗效的重要因素,T细胞受体重排删除环作为胸腺近期输出功能的标志广泛用于评价健康人和造血干细胞移植、血液肿瘤、HIV感染和自身免疫性疾病等的胸腺输出功能。     

Evidence for a stem cell common to hematopoiesis and its in vitro microenvironment: studies of patients with clonal hematopoietic neoplasia

The origin and nature of cells forming the in vitro microenvironment in long-term cultures of human marrow were studied in five patients with clonal myeloproliferative disorders who were heterozygous for glucose-6-phosphatase dehydrogenase (G6PD). The results showed that cells in the adherent stromal layer forming the in vitro microenvironment were derived from the same clonal progenitors involved by the neoplasm in the four patients whose diseases originated in multipotent stem cells. In contrast, stromal cells were derived from normal progenitors in a patient with acute non-lymphocytic leukemia whose clone showed differentiative expression confined to cells in the granulocytic lineage. Mixing experiments demonstrated that the G6PD type displayed by the adherent marrow stromal cells was not obscured by contaminating non-adherent hematopoietic cells or marrow fibroblasts. The data suggest the existence of a pluripotent cell in normal hematopoiesis that gives rise to hematopoietic cells and to their micro-environment.     

间充质干细胞在造血干细胞移植中的应用

 间充质干细胞是一种能够从各种人成体组织分离出来的非造血多能干细胞,近年来,许多研究表明间充质干细胞具有免疫调节能力及促进组织重建等功能。就其在造血干细胞移植中的应用,如急慢性移植物抗宿主病（GVHD）、GVHD造成的移植失败、纯红细胞再生障碍性贫血及免疫性血小板减少性紫癜、出血性膀胱炎作以综述。     

Advances in pediatric hematopoietic stem cell transplantation

Allogeneic and autologous hematopoietic stem cell transplantation (HSCT) has become a therapeutic option for an increasing number of patients with otherwise incurable leukemias, solid tumors, immunodeficiencies, hemoglobinopathies and metabolic diseases. For patients requiring an allogeneic transplant, the addition of unrelated cord blood units and partially matched family member donors as alternate stem cell sources has increased the chances that an appropriate donor can be identified. In addition, new approaches to stem cell graft engineering are yielding insights into potential cellular immune therapies, which may decrease the adverse effects of HSCT such as graft-versus-host disease (GVHD) and harness the alloimmune graft-versus-leukemia effect. Novel conditioning regimens, primarily reduced intensity and non-myeloablative regimens, allow patients with significant co-morbidities to undergo transplantation with reduced morbidity and mortality. Combinations of immune-modulatory cytokines and monoclonal antibodies with autologous and allogeneic transplantation are among the advances being explored in contemporary HSCT.     

Cytomegalovirus in hematopoietic stem cell transplant recipients

This article examines the clinical manifestations of and risk factors for cytomegalovirus (CMV). Prevention of CMV infection and disease are also explored. Antiviral resistance and management of CMV are examined.     

Recent progress in hematopoietic stem cell transplantation

The indication for allogeneic stem cell transplantation (allo-SCT) have been expanded nowadays because many stem cell sources became available and new conditioning procedures such as reduced intensity stem cell transplantation (RIST) have been developed. Stem cell sources can be classified into bone marrow cells, peripheral blood stem cells, cord blood cells and every source derived from related or unrelated donors. Also, HLA mismatched transplantation has been studied especially in haploidentical donors. Now we must select the most compatible stem cell source for the recipient condition and disease status. RIST has expanded the indication of allo-SCT because of low regimen related toxicity. However, evaluation of graft versus leukemia (GVL) effect and control of graft versus host disease (GVHD) are still unresolved problems. Further investigations of the therapy of chronic GVHD and other posttransplant problems are warranted to improve the outcome and quality of life of the patients.     

异基因造血干细胞移植相关研究进展

近年来,单倍体相合造血干细胞移植的发展使“人人都有移植供者”成为现实.因此,选择合适的供者以及解决移植相关并发症,如促进植入、降低移植后移植物抗宿主病的发生率和复发率成为改善移植预后的关键问题.文章介绍了异基因造血干细胞移植的相关研究进展.     

Allogeneic hematopoietic stem cell transplantation in elderly

Most of patients with hematological malignancies are elderly (more than 60 years). Allogeneic stem cell transplantation is an important and effective treatment for most of these diseases. However, the toxicity and the supposed frailty of elderly patients, have limited the applicability of allogeneic transplantation for these patients. Elderly patients are at high risk to develop life-threatening complications, if allogeneic transplantation is performed with myeloablative conditioning regimens and using bone marrow stem source. Since more than 10 years, reduced intensity conditioning regimen have been developed, allowing to overcome the age as contra-indication for allogeneic transplantation. On the other hand, it is the presence of comorbidities which identify frail patients. For these subjects, allogeneic transplantation should be not indicated. Furthermore, advances in the supportive care and the development of new molecules could allow to reduce the toxicity of myeloablative conditioning regimens and thus to offer more intensive regimens before transplantation also in elderly population.     

Managing the toxicity of hematopoietic stem cell transplant

Hematopoietic stem cell transplant is an established treatment modality for a variety of neoplastic, hematologic, and immunologic disorders. Fueled in part by remarkable technologic advances, the number of both autologous and allogeneic transplants has increased dramatically over the past decade. Peripheral blood stem cells have largely replaced bone marrow as the source of hematopoietic progenitors in autologous transplants, and their use in the allogeneic setting has increased substantially. Less toxic transplants, in the form of non-myeloablative conditioning regimens, are being actively investigated, with the promise of expanding indications and age limits for allogeneic transplant. A successful global infrastructure allowing sharing of HLA-typing information has led to increased availability of non-sibling, HLA-matched, unrelated donor transplants for many patients who lack a suitable sibling donor. Finally, umbilical cord blood transplants are being investigated in both children and adult patients. The ability to transplant more individuals with broader indications owes much to a concurrent improvement in supportive care agents and techniques. Although regimen-related mortality and morbidity have decreased, stem cell transplants continue to pose multiple potential complications. A careful proactive assessment to identify, treat, and, hopefully, prevent adverse events is essential to a successful transplant. This review is intended to summarize some of the toxicities of hematopoietic stem cell transplant in a systematic, organ-based fashion and to review the treatment options available for each of these side effects.     

骨髓间充质干细胞在造血干细胞移植中的应用研究

造血干细胞移植(HSCT)后,干细胞的成活、分化、增殖及造血功能的恢复,影响治疗的效果,移植过程中难免会遇到诸多问题。如造血微环境的破坏,干细胞归巢问题及移植后的移植物抗宿主反应(GVHR)均影响移植成败。近年研究发现骨髓间充质干细胞(MSC)为造血微环境的重要组成成分,可分泌多种细胞因子,能够促进造血,加速干细胞归巢,还能参与免疫反应,降低T淋巴细胞反应。而MSC的这些特性恰好可以减少HSC中的以上问题。因此,国内外已将HSC与MSC共移植治疗恶性肿瘤应用于临床,并取得了良好的临床效果。     

Usefulness of hematopoietic cell transplantation-specific comorbidity index after allogeneic hematopoietic stem cell transplantation

We retrospectively investigated the hematopoietic cell transplantation-specific comorbidity index(HCT-CI)to predict non relapse mortality. Of 127 patients who underwent transplantation between January 2000 and December 2003 with conditioning consisting of total body irradiation, cyclophosphamide and thiotepa, HCT-CI scores were obtained for 83 patients. Median age was 42 years. The sources of stem cells included HLA-identical bone marrow or peripheral blood from sibling(30), HLA-matched bone marrow from unrelated donors(45), and HLA-mismatched bone marrow or peripheral blood from family donors(8). Hematological disease was divided into two groups, standard risk(47)and high risk(36). Standard risk indicates acute leukemia in first or second remission and chronic myelocytic leukemia in first chronic phase, while high risk indicates all other diagnoses. There were 45 patients with moderate or severe pulmonary comorbidities. 55 patients with HCT-CI scores of 2 or less had higher 2-year overall survival than 28 patients with HCT-CI scores of 3 or more(65% vs. 36%, p=0.0009). Although the non relapse mortality rate was not different, HCT-CI scores were a more useful indicator to predict survival in high risk patients than in standard risk patients. Prospective evaluation is warranted to clarify the usefulness of HCT-CI.