Finite lifespans of T cell clones derived from CD34+ human haematopoietic stem cells in vitro

Several studies have documented finite lifespans of at least the vast majority of cultured human T cell lines and clones. However, there is a great deal of variation among the different preparations, ranging from < 25 PD up to > 100 PD. The cultured T cells in all these studies originated from mature T cells isolated from peripheral blood of adult donors. It was, therefore, impossible to assess the contribution of differences in in vivo age to the subsequent differences between clones in in vitro aging. In an attempt to circumvent this difficulty, we have developed a culture system that supports the differentiation of highly purified human CD34+ cells into CD3+ T cells in vitro. This features the use of a serum-free medium supplemented with the cytokines flt-3 ligand, IL 3, stem cell factor (c-kit ligand) and IL 2, together with IL 7 or oncostatin M (OM). In this way it is possible to perform "longitudinal" studies on T cells derived de novo in vitro. We show here that T cell clones derived under these circumstances also manifest variable finite life expectancies, for which the only uncontrolled (nonstochastic) effects of aging must already have occurred at the stem cell level.     

The AFT024 stromal cell line improves MDR1 gene transfer to CD34+ cells derived from human umbilical cord blood

Human hematopoietic stem cells (HSCs) are difficult to transfect with retroviral vectors because of their quiescent nature. Based on the theory that the murine fetal stromal cell line AFT024 can recruit significant numbers of HSC into cell cycle without loss of their primitive function, we transduced human umbilical cord blood cells (UCB) derived CD34+ cells with a retroviral vector pHaMDR1/A containing the human multidrug resistant 1 gene (MDR1) during co-culture with the AFT024 feeders. We found that the presence of the AFT024 cells increased the proportion of Rh-123dull cells up to 35.5%+/-11.4% and transduced colony-forming cells (CFCs) up to 15.2%. Six weeks after transplantation of 5x10(4) day 0 uncultured CD34+ HSCs or their equivalents expanded in the presence or absence of the AFT024 cells for 21 days into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, we found that CD34+ cells expanded in the presence of the AFT024 cells engrafted in each receptor mouse and the percentage of CD45+ cells reached 18.8%+/-9.5%, of which 18.1%+/-6.0% were Rh-123dull cells. These results suggest that the AFT024 stromal cells can significantly improve MDR1 gene transfer efficiency and maintain the engrafting ability of the CD34+ HSCs derived from UCB.     

半胱天冬氨酸蛋白酶-12小干扰RNA对小鼠肝细胞凋亡的阻抑

目的观察小鼠半胱天冬氨酸蛋白酶-12（caspase-12）基因特异性小干扰RNA（siRNA）构建的表达载体pRNAT-casp12对caspase-12基因的抑制及其对内质网应激介导的小鼠肝细胞凋亡的影响。方法以小鼠肝细胞株Hepa1-6为靶细胞，利用脂质体与重组质粒pRNAT-casp-2共转染，分别在转染24、48和72h后收集细胞，采用实时荧光定量PCR分析和Western印迹检测caspase-12的表达；利用毒胡萝b素（TG）诱导细胞，建立内质网应激介导的细胞凋亡模型，通过DNA梯带凝胶电泳检测，选出适合的诱导时间；TG诱导已转染了pRNAT—casp12的干扰组细胞后，以空质粒转染组为对照，利用Western印迹检测caspase-12蛋白表达水平的变化，通过DNA梯带凝胶电泳、流式细胞仪、Hoechst33258染色等方法检测细胞凋亡，观察caspase-12siRNA对细胞凋亡的影响。结果pRNAT—casp12转染细胞24、48和72h后，caspase-12mRNA的水平分别下降了45．6％、72．5％和59．5％；caspase-12蛋白表达下降了17．1％、37．3％和60．1％；2μmol／L TG处理细胞30h后，成功诱导细胞凋亡；与对照组相比，干扰组细胞经TG诱导后，caspase-12蛋白表达水平下降了54．6％，流式细胞仪检测发现早期调亡率下调了51．4％（P〈0．01）。结论小鼠caspase-12siRNA对Hepa1—6细胞caspase-12基因的表达具有显著的抑制作用，能够明显阻抑内质网应激介导的凋亡，有望发展成为新一代抗凋亡药物。     

Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells.

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.     

Factors predicting engraftment of autologous blood stem cells: CD34+ subsets inferior to the total CD34+ cell dose.

Data were analyzed on 178 consecutive patients (median age 43 years) who underwent autologous blood stem cell transplantation (ABSCT) at a single institution to determine if CD34+ subsets (CD34+38-, CD34+33-, CD34+33+, CD34+41+) or various clinical factors affect hematopoietic engraftment independent of the total CD34+ cell dose/kg. Using Cox proportional hazards models, the factors independently associated with rapid neutrophil engraftment were higher CD34+ dose/kg, use of G-CSF post-ABSCT, and conditioning regimen (single-agent melphalan +/- TBI slower). Factors independently associated with rapid platelet engraftment were higher CD34+ cell dose/kg, higher ratio of CD34+33-/total CD34+ cells infused, conditioning regimen (mitoxantrone, vinblastine, cyclophosphamide faster), and no CD34+ cell selection of the autograft. The CD34+ cell selection process seemed to deplete CD34+41+ cells to a greater extent than total CD34+ cells which may explain our observation that it resulted in slower platelet engraftment. In conclusion, the total CD34+ dose/kg was a better predictor of hematopoietic engraftment following ABSCT than the dose of any CD34+ subset. Platelet engraftment, however, was also influenced by the ratio of CD34+33-/total CD34+ cells for unmanipulated autografts, and possibly by the CD34+41+ dose for autografts manipulated by CD34+ selection. The use of CD34+ subsets requires further investigation in predicting engraftment of autografts which undergo ex vivo manipulation.     

Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34⁺ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-α, TGF-β and Flt-3 ligand (fold increase of CD1a⁺ cells = 102 ± 32 after 2 weeks of culture). CD34⁺ cells were also grown under continuous flow conditions in an artificial capillary system; after 14 d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 ± 2 v 18.9 ± 4) but the percentage of CD1a⁺/CD83⁺/CD80⁺ cells was considerably higher, whereas the CD14⁺ cells were significantly reduced (8.9 ± 2 v 26 ± 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a⁺/S100⁺/CD83⁺/CD80⁺/CD14^?‘large cells’ with great internal complexity (mature DCs); the second including ‘small cells’ either CD33⁺/CD14⁺, CD33⁺/CD15⁺ or CD33⁺/CD13^±/CD14^?. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.     

RNA干扰对XWLC-05肺癌细胞水通道蛋白3表达的影响

目的构建靶向水通道蛋白3（AQP3）的siRNA表达载体,探讨其对XWLC-05肺癌细胞系AQP3表达的影响。方法构建AQP3特异性siRNA表达载体和对照组表达载体,以脂质体法转染XWLC-05肺癌细胞,用RT—PCR检测AQP3mRNA的表达,用Westernblot方法检测APQ3蛋白的表达。结果成功构建靶向AQP3的siRNA表达载体1和2,分别命名为pAQP3-siRNAl、pAQP3-siRNA2。转染48h后pAQP3-siRNA1和pAQP3．siRNA2转染组AQP3 mRNA的表达量分别是对照组的65％和79％,组间比较有统计学差异（P〈0．05）;pAQP3-siRNAI和pAQP3-siRNA2转染后的蛋白表达量分别是对照组的53％和。73％,pAQP3-siRNA1干扰效果明显高于pAQP3-siR—NA2（P〈0．05）。结论AQP3特异性siRNA能够有效地抑制XWLC-05肺癌细胞系AQP3的表达。     

Expression of the nlsLacz gene in dendritic cells derived from retrovirally transduced peripheral blood CD34+ cells

BACKGROUND AND OBJECTIVE: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs. DESIGN AND METHODS: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a. RESULTS: Addition of IL-4 was found to induce dendritic differentiation preferentially by inhibiting proliferation and differentiation of the macrophage lineage. As assessed by X-Gal staining, LacZ gene expression was observed in cells from both the dendritic lineage (CD1a+/CD14-) which still exhibits the highest immunostimulatory activity in mixed lymphocyte reaction and from the macrophage lineage (CD1a-/ CD14+). INTERPRETATION AND CONCLUSIONS: This study sets out the possibility of transducing dendritic and macrophage progenitors present in the CD34+ cell population and in using a marker gene such as nlsLacZ to study gene expression in antigen presenting cell compartments.     

Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment

Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.     

小干扰RNA对大鼠血管内皮细胞ICAM-1表达的抑制作用

目的:探讨小干扰RNA对大鼠血管内皮细胞细胞间黏附分子-1(ICAM-1)表达的抑制作用.方法:使用 ICAM-1siRNA及错配siRNA转染肿瘤坏死因子-α刺激过的大鼠血管内皮细胞,聚合酶链反应和蛋白印迹检测转染后48 h ICAM-1 mRNA和蛋白表达水平.结果:与空白对照组比较,大鼠血管内皮细胞在转染48 h后ICAM-1 mRNA表达减少90.0％,蛋白表达减少83.0％.结论:使用小干扰RNA能有效抑制大鼠血管内皮细胞ICAM-1表达.     

CD7 expression on CD34+ cells from chronic myeloid leukaemia in chronic phase.

Thirty-seven patients with chronic phase chronic myeloid leukaemia and fourteen healthy controls have been evaluated for lineage differentiation with immunological markers on purified bone marrow CD34 positive cells by multiparameter flow cytometry. The myeloid-associated antigen CD33 and the stem cell factor receptor (CD117, c-kit) was expressed by 82.3% and 73.5% on CP-CML patients and by 57% and 57.5% on healthy donors, respectively (P < 0.005). CD34+/CD19+ or CD34+/CD10+ B-lymphoid cell population represented 9. 1% and 10.7% of the CD34+ cells in CML whereas in normal controls this subpopulation was expressed by 27.9% and 30.4% of the CD34+ cells, respectively (P< 0.005). The T-lineage associated markers (CD7 and CD2) were detected on a minor population of CD34+ BM cells of healthy controls (mean, 3.6% and 4.6%, respectively). The CD2 positive cells represented 1.5% of the CD34+ cells in CML patients. CP-CML patients co-expressed the CD7 antigen on a mean of 32.6% of the CD34+ BM cells. Moreover, 93% of this CD34/CD7 double positive subpopulation co-expressed CD33 antigen in CML patients. Co-expression of CD7 on CD34+ cells was induced to decrease significantly after short-term in vitro culture with the differentiation-inducing agent phorbol ester (PMA) and with a combination of cytokines (stem-cell factor, interleukin-3 and granulocyte colony-stimulating factor). In conclusion, a high co-expression of CD7 antigen is demonstrated on CD34+ cells of chronic phase-chronic myeloid leukaemia patients. The loss of CD7 marker following incubation with PMA and cytokines suggests that this antigen is expressed transiently in early myeloid leukaemic CML haemopoiesis.     

Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.     

Interleukin-6 directly modulates stem cell factor-dependent development of human mast cells derived from CD34(+) cord blood cells.

In the present study, we attempted to clarify the effects of interleukin-6 (IL-6) on the growth and properties of human mast cells using cultured mast cells selectively generated by stem cell factor (SCF) from CD34(+) cord blood cells. The addition of IL-6 to cultures containing mast cells resulted in a substantial reduction of the number of progenies grown by SCF in the liquid culture. This IL-6-mediated inhibition of mast cell growth may be due in part to the suppression at the precursor level, according to the results of a clonal cell culture assay. Moreover, a flow cytometric analysis showed that the cultured mast cells grown in the presence of SCF+IL-6 had decreased c-kit expression. The exposure of cultured mast cells to SCF+IL-6 also caused substantial increases in the cell size, frequency of chymase-positive cells, and intracellular histamine level compared with the values obtained with SCF alone. The flow cytometric analysis showed low but significant levels of expression of IL-6 receptor (IL-6R) and gp130 on the cultured mast cells grown with SCF. The addition of either anti-IL-6R antibody or anti-gp130 antibody abrogated the biological functions of IL-6. Although IL-4 exerted an effect similar to that of IL-6 on the cultured mast cells under stimulation with SCF, the results of comparative experiments suggest that the two cytokines use different regulatory mechanisms. Taken together, the present findings suggest that IL-6 modulates SCF-dependent human mast cell development directly via an IL-6R-gp130 system.     

Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy- 1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor

Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34- allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1- phycoerythrin. These cells were later stained with 4', 6-diamidino-2- phenylindole for simultaneous DNA analysis and immunophenotyping. BM- derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.     

CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells

Malignant cells in haemopoietic autografts can contribute to post-transplant relapse. Engraftment of myeloma patients with CD34⁺ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7–4.5 logs. Residual tumour cells detected in CD34⁺ selected cells may be due to selection impurity or the existence of malignant CD34⁺ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34⁺ progenitors selected with immunomagnetic beads and highly purified CD34⁺ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections. Using allele-specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi-quantitative ASO-PCR was capable of detecting one malignant cell in 10⁴–10⁵ normal white blood cells. Selection of CD34⁺ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two-colour flow-cytometry we observed CD34^?, BB4⁺ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of >99% pure CD34⁺ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO-PCR detected no malignant cells in >99.9% pure CD34⁺ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34⁺ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34-negative cells.     

The relative extent and propensity of CD34+ vs. CD34- cells to undergo apoptosis in myelodysplastic marrows

The paradox of peripheral cytopenias despite cellular bone marrow (BM) observed in myelodysplastic syndromes (MDS) has been associated with excessive intramedullary apoptosis of hematopoietic cells. Since MDS is regarded as a stem cell disorder, the present studies were undertaken to examine the relative susceptibility and propensity of early progenitor CD34+ cells to undergo apoptosis as compared to more maturing/matured CD34- cells. Five serial studies were performed on 4 independent groups of 36 newly diagnosed MDS patients. First, in 2 separate groups of 16 and 8 patients each, measurement of the extent of apoptosis in CD34+ and CD34- fractions of the BM aspirate mononuclear cells was carried out using independent biparametric flow cytometry methods, CD34 labeling/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (n = 16), and CD34 labeling/reduced uptake of nucleic acid staining dye LDS751 (n = 8). The difference in the median degrees of apoptosis in CD34+ vs. CD34- cells was not statistically significant by either technique (P = 0.583 and P = 0.674 for TUNEL and LDS751, respectively). In the next group of 4 MDS patients, a double-labeling was performed on plastic embedded marrow biopsy sections, to detect CD34 antigen with specific monoclonal antibody and apoptosis by in situ end labeling (ISEL) of fragmented DNA. Despite high overall apoptosis (56.2% +/- 18.4%), only an occasional CD34+ cell was found to be simultaneously labeled with ISEL. Finally, in the last group of 8 MDS patients, CD34+ cells were separated from CD34- cells on affinity column and cultured in serum containing medium for 4 hours. At 0- and 4-hour time points, ISEL was carried out to label apoptotic cells. In addition, a fluorometric assay was employed to estimate the activity of a proapoptotic enzyme, Caspase 3. Both the net increase in % ISEL labeled cells (apoptotic index or AI) and Caspase-3 activity were significantly lower in CD34+ cells as compared to CD34- cells (AI, 0.87% +/- 0.5% vs. 3.97% +/- 1.4%, n = 6, P = 0.028 and Caspase-3 Units/mg protein, 46.9 +/- 25.0 vs. 71.7 +/- 23.03, n = 5, P = 0.042, respectively). We conclude that when estimated in a total population of mononuclear cells, CD34+ cells and CD34- cells show comparable degrees of apoptosis. However, once separated the CD34+ fraction demonstrates lower propensity to undergo apoptosis, thereby suggesting the CD34- fraction as being a possible source for proapoptotic signaling.     

Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34⁺ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34⁺ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34⁺ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34⁺ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

A major objective of recent HIV research is to develop a “cure” for this virus infection that avoids lifelong adherence to antiretroviral therapy (ART). One of the approaches toward reaching this objective has been to genetically delete or mutate genes encoding for proteins that promote HIV infection and spread. An attractive candidate for this strategy is the Ccr5 gene, for which a genetic mutation causing a 32-bp deletion has been shown to be associated with natural protection from HIV infection and disease (1, 2). The Ccr5 gene encodes CCR5, a human cell-surface chemokine receptor that is a coreceptor for HIV attachment and infection of cells (3, 4). The Ccr5 allele with its 32-bp deletion results in a truncated isoform of the CCR5 receptor, CCR5Δ32, which is not expressed at the cell surface. Thus, entry of the virus into the cell is blocked (5).Induced pluripotent stem (iPS) cells (6), because of their capacity to differentiate into CD34⁺ hematopoietic stem cells (HSCs) (7), can reconstitute a full immune system (8, 9). These iPS cells are therefore a target of choice for genetic engineering. Our group and others have demonstrated that iPS cells generated from the peripheral blood mononuclear cells (PBMC) of both healthy individuals (10) and HIV-infected patients under ART (11) can have their wild-type allele of the Ccr5 gene genetically edited to carry the Ccr5 Δ32 mutation (12, 13). Notably, using CRISPR/Cas9 technology, the Ccr5 gene can be modified to have the naturally occurring Δ32 variant allele that has been associated with resistance to R5-tropic viruses. Moreover, while it is not present at the cell surface, the truncated CCR5Δ32 protein is still expressed and, as such, could have other important physiological roles (14–17).We have confirmed that the genetically modified Ccr5 Δ32 iPS cells can be differentiated into CD34⁺ HSCs in vitro (10, 18). Under appropriate cell culture conditions, they can give rise to various myeloid and lymphoid cell lineages (10, 11, 18). This result can also be observed with the formation of teratomas following the injection of large quantities of iPS cells into mice. Teratomas are multicellular tumors composed of many different cell types including HSCs. Notably, immune cells with the CCR5Δ32 mutation differentiated in vitro from the genetically modified iPS cell-derived HSCs and inoculated with HIV are resistant to R5-tropic virus infection (10, 18).These results have suggested that editing Ccr5 in iPS cells from HIV-infected subjects can be a promising strategy toward an HIV cure. The pluripotent stem cells can be induced from a small number of PBMC from the patients and genetically modified to become resistant to HIV infection (10, 11, 18). In this case, leukapheresis to obtain large amounts of these cells (19) is not required. The edited HSCs could then be transplanted back to the original patient without concern for immune cell rejection. Therefore, because these experiments were performed in cell culture, an important remaining question is whether in vitro-edited iPS cells can differentiate into HSCs that can be transplanted back into a recipient in vivo (20).To address this question, transplantation of the in vitro-derived CD34⁺ cells was attempted under various conditions in animal models of humanized or immunodeficient mice (21). In approaches to obtain sufficient numbers of CD34⁺ cells for transplantation, our ability to grow them in vitro offered an opportunity. However, although we could expand CD34⁺ cells substantially in culture (18), we observed that engraftment of these cell culture-derived CD34⁺ cells in humanized NSG-BLT mice did not occur. Thus, alternatively, to study the genetically edited cells in vivo, we explored the use of differentiated CD34⁺ cells in vivo via the generation of teratomas from iPS cells. We found that not only did these teratomas successfully yield human CD34⁺ cells, but importantly, these CD34⁺ cells could engraft in recipient immunodeficient NSG mice. This observation has been made by Nakauchi and colleagues (22) with different mouse strains. Finally, we confirmed that the PBMC formed in mice from these teratoma-derived genetically edited CD34⁺ cells are resistant to ex vivo R5-tropic HIV infection when they carry the mutant Δ32 Ccr5 allele.     

Flk1+ cells derived from mouse embryonic stem cells reconstitute hematopoiesis in vivo in SCID mice

OBJECTIVE: Embryonic stem (ES) cells are pluripotent and can differentiate into any cell type, including the hematopoietic lineage. We examined whether hematopoietic progenitor cells derived from ES cells reconstitute hematopoiesis in irradiated SCID mice. MATERIALS AND METHODS: ES cells (E14.1, H2K(b)) were cultured for 4 days in semisolid medium containing methylcellulose. Irradiated SCID mice were used as recipients of hematopoietic progenitor cells. Cell surface antigen expression was analyzed by flow cytometry. The spleens of the recipient mice were studied by hematoxylin and eosin staining and immunohistochemical staining. RESULTS: After cell culture of ES cells in methylcellulose for 4 days, the cells expressing Flk1 (VEGF receptor 2), a tentative marker of hemangioblasts, were increased, whereas cells expressing CD31 (PECAM-1) and E-cadherin (nonmesodermal adhesion molecule) were dramatically reduced. Flk1+ cells expressed c-kit predominantly. Circulating leukocytes and thrombocytes were increased in irradiated SCID (H2K(d)) mice transplanted with ES cell-derived Flk1+ cells compared with vehicle-injected control mice. H2K(b+) and VE-cadherin(+) vascular endothelial cells were prominent in spleens of the recipient mice. Flow cytometric analysis demonstrated that H2K(b+) cells were increased in the bone marrow of recipient mice. In addition, Flk1+ cells accompanying enhanced c-kit expression preferentially repopulated in the bone marrow, and leukopoiesis and thrombopoiesis of the recipient mice were evident. CONCLUSION: The Flk1+ hematopoietic cells derived from ES cells reconstitute hematopoiesis in vivo and may become an alternative donor source for bone marrow transplantation.     

siRNA沉默Annexin-1基因对膀胱癌T24细胞生物学特性的影响

目的观察RNA干扰技术沉默Annexin-1基因表达对膀胱癌T24细胞增殖能力的影响。方法针对Annexin-1基因不同部位设计并化学合成3对不同的靶向小干扰RNA(siRNA),脂质体介导瞬时转染膀胱癌T24细胞,转染后应用半定量RT-PCR和Western印迹法检测Annexin-1mRNA和蛋白的改变,透射电镜观察细胞凋亡情况,用MTT法检测沉默Annexin-1表达对膀胱癌T24细胞增殖的影响。结果转染siRNA后膀胱癌T24细胞Annexin-1mRNA和蛋白水平显著下降(P0.05),转染siRNA沉默Annexin-1基因表达后膀胱癌T24细胞出现典型凋亡细胞,增殖能力显著下降(P0.05)。结论 RNA干扰Annexin-1基因后其mRNA和蛋白水平显著下降,诱导了膀胱癌T24细胞凋亡,并且抑制细胞的增殖。