R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal

Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/β-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.     

Modeling of asymmetric cell division in hematopoietic stem cells--regulation of self-renewal is essential for efficient repopulation

Hematopoietic stem cells (HSCs) are characterized by their ability of self-renewal to replenish the stem cell pool and differentiation to more mature cells. The subsequent stages of progenitor cells also share some of this dual ability. It is yet unknown whether external signals modulate proliferation rate or rather the fraction of self-renewal. We propose three multicompartment models, which rely on a single external feedback mechanism. In Model 1 the signal enhances proliferation, whereas the self-renewal rates in all compartments are fixed. In Model 2 the signal regulates the rate of self-renewal, whereas the proliferation rate is unchanged. In Model 3, the signal regulates both proliferation and self-renewal rates. This study demonstrates that a unique strictly positive stable steady state can only be achieved by regulation of the rate of self-renewal. Furthermore, it requires a lower number of effective cell doublings. In order to maintain the stem cell pool, the self-renewal ratio of the HSC has to be > or =50% and it has to be higher than the self-renewal ratios of all downstream compartments. Interestingly, the equilibrium level of mature cells depends only on the parameters of self-renewal of HSC and it is independent of the parameters of dynamics of all upstream compartments. The model is compatible with the increase of leukocyte numbers following HSC transplantation. This study demonstrates that extrinsic regulation of the self-renewal rate of HSC is most essential in the process of hematopoiesis.     

Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSC) from loss-of-function analysis. Here, we demonstrate that increased expression of Bmi-1 promotes HSC self-renewal. Forced expression of Bmi-1 enhanced symmetrical cell division of HSCs and mediated a higher probability of inheritance of stemness through cell division. Correspondingly, forced expression of Bmi-1, but not the other PcG genes, led to a striking ex vivo expansion of multipotential progenitors and marked augmentation of HSC repopulating capacity in vivo. Loss-of-function analyses revealed that among PcG genes, absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal. Our findings define Bmi-1 as a central player in HSC self-renewal and demonstrate that Bmi-1 is a target for therapeutic manipulation of HSCs.     

The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.     

Evidence for an active hydrophobic form of factor H that is able to induce secretion of interleukin 1-beta or by human monocytes.

We studied the capacity of human factor H to promote the secretion of a lymphocyte-activating factor (LAF) by human monocytes cultured under serum-free conditions. Presence of LAF in the culture supernatants was assessed with the mouse thymocyte assay. Highly purified factor H alone had no effect on thymocyte proliferation. When monocytes were cultured with factor H for 24 h, a significant secretion of LAF was observed. The effect was dose dependent over a range of factor H concentrations from 1 to 15 micrograms/ml. Polymyxin B did not abrogate the capacity of factor H to induce LAF secretion. Adsorption of factor H preparations onto anti-factor H-Sepharose completely suppressed the phenomenon. Conversely, the activity was recovered in the acidic eluate. Furthermore, factor H subpopulation phi 2, that was able to bind to phenyl-Sepharose, was a stronger inducer of LAF secretion by monocytes than the subpopulation phi 1 (which did not bind to phenyl-Sepharose). Using a specific radioimmunoassay for interleukin 1-beta (IL 1 beta), we observed a good correlation between the LAF activity and the amount of IL 1 beta secreted by human monocytes stimulated with factor H. We have shown previously that factor H (phi 2) bound specifically on Raji cells whereas factor H (phi 1) did not. These results argue for the participation of the interaction of factor H with its receptor to stimulate the secretion of IL 1 by monocytes and that the phi 2 form of factor H is a ligand for the human factor H receptor.     

载阿霉素聚合物微泡影响多发性骨髓瘤癌干细胞增殖凋亡的研究

制备载阿霉素(DOX)的PLGA微泡(PLGA-MB),观察载药微泡联合超声(US)辐照体外作用于多发性骨髓瘤(MM)癌干细胞(CSC)的效应。采用声振法与乳液法制备载DOX的PLGA微泡,镜下观察其形态,检测其粒径、电位、载药量和包封率。采用免疫磁珠分选法从MM JJN3细胞株分选获得CD138ˉCD34ˉCSC。以不同声强的超声联合空白微泡作用于MM CD138ˉCD34ˉCSC 30s,筛选最佳超声辐照条件。设对照组(I组)、DOX组(II组)、DOX+US组(III组)、DOX-MB组(IV组)、DOX-MB+US组(V组),细胞计数试剂盒(CCK-8)法检测细胞的存活率,流式细胞术检测细胞的凋亡,透射电镜观察细胞超微结构,Western blotting法检测Bcl-2的表达。DOX-MB粒径均一,分散性好,平均粒径461.6±85.6nm,zeta电位-11.4±2.35mv,包封率(56.51±9.82)%,载药率(7.10±1.04)%。1.0 W/cm2的超声作用30s能大量破泡而对细胞增殖无明显影响。各实验组中,V组的细胞存活率[(41.89±1.11)%]较其他组[I组(98.71±1.16)%,IV组(92.56±3.49)%,II组(73.69±3.24)%,III组(71.13±4.03)%]明显降低(P0.05),V组的细胞凋亡率[(29.05±3.21)%]较其他组[I组(2.33±1.09)%,IV组(3.72±1.58)%,II组(15.14±1.11)%,III组(15.03±3.09)%]明显增强(P0.05)。V组的Bcl-2蛋白表达(灰度值比率0.23±0.06)较其他各组(I组0.82±0.11,II组0.51±0.08,III组0.58±0.12,IV组0.77±0.05)显著降低(P0.05)。PLGA微泡是良好的DOX传递载体,联合超声辐照体外作用于多发性骨髓瘤癌干细胞可通过诱导凋亡产生明显的细胞增殖抑制作用。     

Changes in the cytokine regulation of stem cell self-renewal during ontogeny

The last 10 years have seen the development of a quantitative assay that is specific for transplantable totipotent murine hematopoietic cells with durable in vivo blood-forming ability. Recently, this assay has been successfully adapted to allow the detection and enumeration of an analogous population of human hematopoietic stem cells using myelosuppressed immunodeficient (nonobese diabetic/severe-combined immunodeficiency) mice as recipients. Characterization of the cells detected by this assay indicates their close relationship in both mice and humans with cells detected in vitro as long-term culture-initiating cells (LTC-IC). Culture conditions have now been identified that support a significant net expansion of these cells from both species. More detailed analyses of the cytokine requirements for this response indicate that the viability, mitogenesis and maintenance of LTC-IC function by human CD34+ CD38- cells can be independently regulated by exogenous factors. Superimposed on this uncoupling of hematopoietic stem cell "self-renewal" and proliferation control is a change during ontogeny in the particular cytokines that regulate their responses. These findings unite stochastic and deterministic models of hematopoietic stem cell control through the concept of a molecular mechanism that actively blocks stem cell differentiation and must be maintained when these cells are stimulated to divide by exposure to certain types and concentrations of cytokines.     

Using the galactomannan antigen assay in the diagnosis of invasive aspergillosis after hematopoietic stem cell transplantation

Invasive aspergillosis (IA) is the most common life-threatening infections after hematopoietic stem cell transplant (HSCT). The serum galactomannan (GM) is recognized as an indirect mycological criteria for an early diagnosis of IA. Starting January 2011, we implementing in Fundeni Clinical Institute, Bucharest, for the first time in Romania, the detection of GM antigen (Platelia Aspergillus EIA, Bio-Rad). In 2011, patients undergoing HSCT were screened with the galactomannan ELISA; we performed a retrospective chart review of 162 SCT patients who underwent galactomannan testing. Thirteen of the patients (8.02%) had at least one positive galactomannan ELISA, and four had multiple positive tests. When calculated in reference to a proved or probable diagnosis of aspergillosis, the galactomannan ELISA had a sensitivity of 0.857 and a specificity of 0.913. The positive predictive value was 0.46, and the negative predictive value was 0.993. The Platelia Aspergillus galactomannan antigenemia assay may assist physicians in making an early diagnosis of IA, in correlation with clinical and radiological criteria. The test has a high sensitivity and specificity and a very good negative predictive value. We found the screening of GM ELISA to be a highly specific diagnostic tool in detecting IA manifested in patients undergoing HSCT.     

S-adenosil-l-methionine is able to reverse the immunosuppressive effects of chenodeoxycholic acid in vitro

The study was conceived to evaluate if S-adenosil-L-methionine, a substance commonly used in the treatment of cholestasis in patients with cirrhosis and chronic hepatitis, exerts any immunological effect and if it is able to counterbalance bile acid-mediated immunosuppression. Proliferation and interleukin 2 and interferon-gamma secretion of human lymphocytes, collected from healthy subjects and exposed to mitogenic stimuli (phytohemagglutinin, pokeweed and anti-CD3 monoclonal antibodies), were analysed in the basal condition or after exposure to S-adenosil-L-methionine and/or chenodeoxycholic acid. Chenodeoxycholic acid inhibited phytohemagglutinin-induced lymphocyte proliferation and interferon-gamma secretion, and phytohemagglutinin and pokeweed-mediated interleukin 2 secretion. S-adenosil-L-methionine did not affect lymphocyte proliferation while it reduced interleukin 2 secretion upon phytohemagglutinin and pokeweed stimulation and interferon-gamma secretion upon all stimuli tested. Moreover, S-adenosil-L-methionine counteracted chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion. The results of our study confirm the immunosuppressive role of chenodeoxycholic acid on both secretive and proliferative lymphocyte functions and provide evidence of immunomodulatory activities of S-adenosil-L-methionine and its capacity to antagonize chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion.     

Immunology of hematopoietic stem cell transplantation: a brief review of its history

Summary: The history of clinical marrow transplantation since 1968 is reviewed with an emphasis on immunological and immunogenetic aspects. The events leading to the creation of an international network of volunteer donor registries and the implementation of unrelated allogeneic marrow transplantation as a routine procedure are discussed. Current major issues which need to be resolved are addressed in order to set the stage for the topics covered in this volume of immunological Reviews.     

Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.     

Absolute lymphocyte count on day 30 is a surrogate for robust hematopoietic recovery and strongly predicts outcome after T cell-depleted allogeneic stem cell transplantation.

Several studies have shown that a higher lymphocyte count 3-4 weeks after allogeneic stem cell transplantation (SCT) is associated with better transplant outcome. However, the factors determining early lymphocyte recovery are not defined. To further explore the relationship between lymphocyte recovery and outcome we analyzed lymphocyte counts and other engraftment parameters in 157 patients with leukemia (48 acute myelogenous leukemia, 80 chronic myelogenous leukemia, and 29 acute lymphoblastic leukemia [ALL]) receiving T cell-depleted myeloablative SCT from an HLA-identical sibling. In multivariate analysis the day 30 absolute lymphocyte count (LC30) above the median of 450/muL was associated with improved survival (71% +/- 5% versus 38% +/- 6%, P < .0001), less relapse (21% +/- 5% versus 44% +/- 7%, P = .009), less nonrelapse mortality (NRM; 9 +/- 3 versus 36% +/- 6%, P < .0001) and less acute graft-versus-host disease (aGVHD) (34% +/- 5% versus 51% +/- 6%, P = .025). The beneficial effect of a higher LC30 influenced outcome in patients with both standard and high-risk disease but did not affect survival and relapse in ALL. We found that a higher LC30 correlated with higher lymphocyte counts at all time points between 30 and 90 days post-SCT and also with more rapid neutrophil and platelet engraftment. These results indicate that LC30 is a surrogate for robust engraftment and identifies an "at-risk" population of patients after T cell-depleted SCT.     

Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay

The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferation has hampered the simultaneous tracking of multiple cell populations; furthermore, CFSE is not compatible with green fluorescent protein (GFP), used to identify T cells in various transgenic mice. We have therefore validated the use of the far red dye seminaphthorhodafluor-1 (SNARF)-1 - originally developed to measure intracellular pH - to track murine T cell proliferation in vitro, demonstrating its ability to distinguish multiple cycles of proliferation over three days in a similar fashion to CFSE. The small changes in fluorescence emission attributed to intracellular alkalinisation of proliferating T cells have minimal impact on the ability of SNARF-1 to track cell division and this dye induces minimal cell death at the concentration used in this application. On the basis of these results, we have developed a novel in vitro murine T cell suppression assay, in which the proliferation of both conventional T cells (Tcons) stained with SNARF-1 and regulatory T cells (Tregs) stained with CFSE can be measured simultaneously. We have also demonstrated that SNARF-1 may be used to stain Tcons in assays of suppression involving 'designer' Tregs, generated by the transduction of CD4(+) T cells with constructs encoding the Foxp3(gfp) fusion protein.     

人IL-3基因cDNA的克隆及其在造血干细胞的表达

目的 克隆人IL-3基因cDNA并转导人脐带血造血干细胞，然后研究这一细胞因子的表达情况，从而为脐血扩增及移植打下理论基础。方法 从人外周血单个核细胞中提取IL-3mRNA，用逆转录-聚合酶链反应（RT-PCR）扩增IL-3 cDNA，通过T／A克隆法，构建真核表达型载体pcDNA3／IL-3。将pcDNA3／IL-3质粒转导脐血造血干细胞，并于1—7d检测IL-3表达情况。结果 pcDNA3／IL-3重组质粒的目的基因与ID3基因序列相同；pcDNA3／IL-3转导脐血造血干细胞后，经检测能够有效表达。结论 成功克隆了hIL-3并构建重组质粒pcDNA3／IL-3，该质粒转导脐血造血干细胞后，能在短期内有效表达。