In vivo expansion of the circulating stem cell pool

Stem cell trafficking between extravascular marrow sites and circulating blood is an essential part of the blood stem cell transplantation technology. Recombinant human G-CSF (rHuG-CSF) is widely used for stem cell peripheralization alone or together with chemopriming mobilizing early and pluripotent CD34+ cell subsets. New cytokine/chemokine mobilization regimens are under investigation such as combined rHuG-CSF and rHu thrombopoietin, rHuG-CSF and interleukin 3, rHuG-CSF and rHu stem cell factor, rHuG-CSF and Flt-3 ligand, human macrophage inflammatory protein, interleukin 1, and interleukin 8. Modifying the adherence of CD34+ cells to extracellular matrix molecules is a new mechanism by which hematopoietic progenitor cells are released into the circulating blood. Blocking the alpha4beta1 integrin receptor on CD34+ progenitor cells by using monoclonal antibodies specific for the heterodimeric complex alpha4beta1 has been shown to further increase the circulating stem cell concentration when given following rHuG-CSF priming. The current clinical research is primarily focused on improving stem cell mobilization efficiency in heavily pretreated and poorly mobilizing patients, and to decrease adverse effects of cytokine treatment.     

Tumor cell purging by ex vivo expansion of hemopoietic stem cells from breast cancer patients combined with targeting ErbB receptors.

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti-ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both--such as trastuzumab or gefitinib--might increase the efficiency of purging.     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血干/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述。     

造血干/祖细胞体外扩增的最新研究进展

造血干/祖细胞体外扩增方法的快速发展为造血于/祖细胞广泛应用于临床开辟了广阔的前景,就造血干/祖细胞体外扩增的方法和培养系统的最新进展做一综述.     

In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse

Summary An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively.     

Pluripotent hemopoietic stem cells give rise to osteoclasts

Osteopetrosis in the ia (incisors absent) rat is the result of reduced bone resorption due to abnormal osteoclasts. The mutant osteoclasts lack a ruffled border—the membrane specialization involved in osteolysis. Studies in the ia mutant have shown that when pluripotent hemopoietic stem cells from normal littermates are transplanted into ia recipients, normal osteoclasts are formed and the skeletal sclerosis is eventually cured. The present study was conducted to provide evidence for the mechanism of the cure. Do the transplanted stem cells provide a helper function, i.e. secrete soluble factor(s) which transform pre-existing osteoclasts, or do they fuse with each other or pre-existing osteoclasts, to form functional osteoclasts? Using the procedures described by Goldschneider and co-workers, and fluorescence-activated cell sorting (FACS), pluripotent hemopoietic stem cells were isolated from normal rat bone marrow, labeled with saturated FITC, and injected intravenously into irradiated ia rats. After 48 hr, the recipients' long bones were removed and split longitudinally, and the endosteal surface was scraped. The resulting cellular suspension containing osteoclasts was examined by phase contrast and fluorescence microscopy. Fluorescing mononuclear cells of donor origin that had homed to the bone marrow demonstrated moderate cytoplasmic fluorescence. Approximately 30% of the osteoclasts observed demonstrated light cytoplasmic fluorescence. When cellular pools incapable of curing osteopetrosis (thymocytes) were labeled and injected into ia recipients, no labeled osteoclasts were observed. These studies indicated that pluripotent hemopoietic stem cells, when transplanted into ia hosts, fuse with each other and differentiate into osteoclasts or fuse with pre-existing osteoclasts.     

Sca1+间充质干细胞支持造血干细胞向树突细胞分化

目的 研究Sca1+间充质干细胞对HSC向DC分化的支持作用,并对获得的DC进行形态、表面标志以及功能的鉴定。方法 取健康Balb/c小鼠骨髓,通过MACS系统分离、纯化CD117＋造血干细胞;以小鼠Sca1+间充质干细胞做饲养层,诱导CD117+造血干细胞定向分化为树突细胞,显微镜观测形态、流式细胞仪检测表面标志,并通过激光共聚焦显微镜和动物实验检测诱生细胞的功能。结果 培养第10天诱生的DC细胞表面树突较短小,呈毛刺状,高表达CD11b,低表达Ia,对外源颗粒具有吞噬功能,在动物器官移植实验中具有免疫抑制功能。结论 Sca1+间充质干细胞对CD117＋HSC向DC分化具有支持作用。     

Effects of Toxocara canis infection on hemopoietic stem cells and hemopoietic factors in mice

The effects of Toxocara canis infection on hemopoietic stem cells and hemopoietic factors were examined in mice. Severe eosinophilia was observed with a peak 14 days after infection. When the numbers of hemopoietic stem cells in peripheral blood, spleen, and bone marrow were examined by spleen colony assay (CFU-S), those in peripheral blood and spleen increased in parallel with peripheral blood eosinophilia. On the other hand, CFU-S in bone marrow did not alter significantly throughout the course of infection. Interleukin (IL)-3, which is known as multi-colony-stimulating factor and is involved in the growth/differentiation of various blood cells including stem cells, was produced by spleen cells of infected mice. The time course study showed that concanavalin A stimulated IL-3 production peaked on day 7 after infection, whereas that with excretory secretory antigen peaked on day 14. Even without stimulation, spleen cells obtained on day 21 after infection produced IL-3 spontaneously. IL-5, which is known to have eosinophil differentiation factor activity, was also produced by spleen cells obtained on day 13 after infection. These results suggest that in response to increased demand for eosinophils, hemopoietic stem cells migrate into various extramedullar hemopoietic organs where they grow/differentiate into mature eosinophils, depending on the hemopoietic factors.     

In search of the in vivo identity of mesenchymal stem cells

In spite of the advances in the knowledge of adult stem cells (ASCs) during the past few years, their natural activities in vivo are still poorly understood. Mesenchymal stem cells (MSCs), one of the most promising types of ASCs for cell-based therapies, are defined mainly by functional assays using cultured cells. Defining MSCs in vitro adds complexity to their study because the artificial conditions may introduce experimental artifacts. Inserting these results in the context of the organism is difficult because the exact location and functions of MSCs in vivo remain elusive; the identification of the MSC niche is necessary to validate results obtained in vitro and to further the knowledge of the physiological functions of this ASC. Here we show an analysis of the evidence suggesting a perivascular location for MSCs, correlating these cells with pericytes, and present a model in which the perivascular zone is the MSC niche in vivo, where local cues coordinate the transition to progenitor and mature cell phenotypes. This model proposes that MSCs stabilize blood vessels and contribute to tissue and immune system homeostasis under physiological conditions and assume a more active role in the repair of focal tissue injury. The establishment of the perivascular compartment as the MSC niche provides a basis for the rational design of additional in vivo therapeutic approaches. This view connects the MSC to the immune and vascular systems, emphasizing its role as a physiological integrator and its importance in tissue repair/regeneration.     

细胞因子在人造血干细胞体外扩增培养中的作用研究进展

自体或异体造血干细胞移植被广泛地应用于临床治疗。因此,将造血干细胞进行体外扩增培养也成为一项极具挑战的实验技术。大量实验证据表明,在体外液体培养体系中加入适当的细胞因子,可以使造血干细胞得以扩增,同时可以调节其生长,即维持静止状态、自我更新、分化、凋亡、在微环境中的迁移等。笔者主要回顾了正向及负向调节细胞因子对造血干细胞体外扩增的作用,总结了几年来使用较多的几种细胞因子组合及造血干细胞体外扩增检测方法。     

Lack of self-renewal capacity in Fancc-/- stem cells after ex vivo expansion

Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc-/-, we tested the ability of CD34- hematopoietic stem cells to support ex vivo expansion. We determined that Fancc-/- CD34- stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.     

Pluripotent hemopoietic stem cells in murine postmortem bone marrow

Although 12-hours postmortem murine bone marrow cells exhibit extensive degeneration, these cells when infused into irradiated mice produce erythrocytic, granulocytic, megakarocytic and mixed colonies in their spleen. These observations clearly demonstrate the presence of pluripotent hemopoietic stem cells and their proliferative capability in 12-hours postmortem, murine bone marrow. These observations suggest that cadaveric bone marrow transplantation may be possible in patients with hematologic disorders.     

In vivo hepatic differentiation potential of human cord blood-derived mesenchymal stem cells

Although recent studies have demonstrated the in vitro hepatic differentiation potential of mesenchymal stem cells (MSCs), the evidence supporting the in vivo engraftment of MSCs, hepatic differentiation and improvement of hepatic function is still lacking. We investigated in vivo hepatic differentiation potential and therapeutic effect of cord blood derived-MSCs (CBMSCs) transplantation in a cirrhotic rat model. CBMSCs (2 x 10(6)) were infused in Wistar rats with thioacetamide-induced chronic liver injury. Biochemical markers, liver fibrosis and engraftment of CBMSCs were assessed. Infused CBMSCs were detected in the perivascular or fibrous region of the liver and did not acquire mature hepatic phenotypes. There was no difference in biochemical markers and in the area of liver fibrosis between the experimental and placebo groups. After infusion of CBMSCs in our experimental cirrhotic rat model we did not observe an improvement of liver function and liver fibrosis. Inversely, CBMSCs could have a pro-fibrogenic potential suggesting that a cautious approach is required in future research.     

In vivo quantum dot labeling of mammalian stem and progenitor cells.

Fluorescent semiconductor nanocrystal quantum dots (QDs) are a class of multifunctional inorganic fluorophores that hold great promise for clinical applications and biomedical research. Because no methods currently exist for directed QD-labeling of mammalian cells in the nervous system in vivo, we developed novel in utero electroporation and ultrasound-guided in vivo delivery techniques to efficiently and directly label neural stem and progenitor cells (NSPCs) of the developing mammalian central nervous system with QDs. Our initial safety and proof of concept studies of one and two-cell QD-labeled mouse embryos reveal that QDs are compatible with early mammalian embryonic development. Our in vivo experiments further show that in utero labeled NSPCs continue to develop in an apparent normal manner. These studies reveal that QDs can be effectively used to label mammalian NSPCs in vivo and will be useful for studies of in vivo fate mapping, cellular migration, and NSPC differentiation during mammalian development.     

Purification of human hemopoietic stem cells for transplantation in Thailand

Stem cell transplantation (SCT) has become the therapy of choice for many hematologic and immunologic disorders. At present, only 25% of patients have suitable HLA-identical donors. In an attempt to increase the donor pool for SCT in Thailand and Southeast Asia, we developed a program whereby parents and mismatched siblings can be used as donors. In this preliminary study, after granulocyte-colony-stimulating factor (G-CSF) was given to adult donors, peripheral blood stem cells (PBSC) were collected and CD34+ cells purified using a CliniMACS immunomagnetic device (Miltenyi Biotec, Germany). In seven experiments, purified CD34+ cells could be obtained from G-CSF-stimulated PBSC in large numbers (1.71 +/- 0.19 x 10(8)), with high purity (93 +/- 2.4%) and excellent recovery (64.28% - 85.62%). Immune reactive T and NK cells were adequately depleted to less than 0.2%. The purification procedure can be completed within 3 hours. In conclusion, a clinical stem cell purification program using this novel device is now established in Thailand and for the first time in Southeast Asia. This should allow further development of advanced SCT therapy including haploidentical and mismatched CD34+ SCT for patients' lacking HLA-identical donors in this region.     

Clonal succession in the hemopoietic system: Number of primitive hemopoietic stem cells and clone lifespan

The fate of individual primitive hemopoietic stem cells is studied by retroviral gene transfer technique. Tens of small hemopoietic clones with a lifespan no longer than 1 month simultaneously function throughout life in lethally irradiated mice reconstituted with bone marrow cells. The disappearing clones are not detected again, which confirms the clonal succession. The number of primitive hemopoietic stem cells in mouse bone marrow has been directly estimated: 1 per 8000 hemopoietic cells, or 30×10³ per mouse, which is at least ten times higher than expected. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 419–423, April, 1999     

In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. Here we quantify dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at Ser 28 as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. We propose that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities.     

In vivo effect of murine recombinant interleukin 3 on early hemopoietic progenitors

Normal and irradiated mice were perfused with recombinant interleukin 3 (rIL3) and the number of early hemopoietic progenitors (CFU-S) was quantified in different organs. Normal mice perfused with rIL3 for 3 or 7 days showed a dramatic increase in the number of CFU-S in the spleen, liver and blood, while the bone marrow CFU-S number was slightly decreased. The total number of CFU-S per animal was only slightly increased; the major effect of rIL3 perfusion was thus on the distribution of CFU-S in the mice. Lethally irradiated mice injected with 5 X 10(4) syngeneic bone marrow cells and perfused with rIL3 were killed after 10 days. A significant increase in both the number and size of the splenic colonies was observed. The CFU-S content of these colonies, determined in a second set of irradiated mice, was increased by more than 25-fold, indicating that in conditions of hemopoietic depletion, rIL3 vastly increased the self-renewal capacity of early progenitors.