Efficient transduction and engraftment of G-CSF-mobilized peripheral blood CD34+ cells in nonhuman primates using GALV-pseudotyped gammaretroviral vectors.

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype. Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient transduction and engraftment of G-PBSC using Phoenix-GALV-pseudotyped vectors. We transplanted three baboons with G/only mobilized CD34(+) cells transduced with GALV-pseudotyped retroviral vectors. We observed high-level, persistent engraftment of gene-modified G-PBSC in all animals with gene marking levels in granulocytes up to 60%. We analyzed amphotropic (PIT2) and GALV (PIT1) receptor expression in G/only cells and found preferential expression of PIT1 after G/only, which may explain the inferior results with amphotropic pseudotypes. These findings demonstrate that high stem cell gene transfer levels can be achieved using G-CSF-mobilized PBSC with Phoenix-GALV-pseudotyped vectors.     

Hematopoietic recovery in cancer patients after transplantation of autologous peripheral blood CD34+ cells or unmanipulated peripheral blood stem and progenitor cells

BACKGROUND: A study of CD34+ cell selection and transplantation was carried out with particular emphasis on characteristics of short- and long-term hematopoietic recovery. STUDY DESIGN AND METHODS: Peripheral blood stem and progenitor cells (PBPCs) were collected from 32 patients, and 17 CD34+ cell-selection procedures were carried out in 15 of the 32. One patient in whom two procedures failed to provide 1 × 10(6) CD34+ cells per kg was excluded from further analysis. After conditioning, patients received CD34+ cells (n = 10, CD34 group) or unmanipulated (n = 17, PBPC group) PBPCs containing equivalent amounts of CD34+ cells or progenitors. RESULTS: The yield of CD34+ cells was 53 percent (18–100) with a purity of 63 percent (49–82). The CD34+ fraction contained 66 percent of colony-forming units-granulocyte- macrophage (CFU-GM) and 58 percent of CFU of mixed lineages, but only 33 percent of burst-forming units-erythroid (BFU-E) (p < 0.05). Early recovery of neutrophils and reticulocytes was identical in the two groups, although a slight delay in platelet recovery may be seen with CD34+ cell selection. Late hematopoietic reconstitution, up to 1.5 years after transplant, was also similar. The two groups were thus combined for analyses of dose effects. A dose of 40 × 10(4) CFU-GM per kg ensured recovery of neutrophils to a level of 1 × 10(9) per L within 11 days, 15 × 10(4) CFU of mixed lineages per kg was associated with platelet independence within 11 days, and 100 × 10(4) BFU-E per kg predicted red cell independence within 13 days. However, a continuous effect of cell dose well beyond these thresholds was apparent, at least for neutrophil recovery. CONCLUSION: CD34+ cell selection, despite lower efficiency in collecting BFU-E, provides a suitable graft with hematopoietic capacity comparable to that of unmanipulated PBPCs. In both groups, all patients will eventually show hematopoietic recovery of all three lineages with 1 × 10(6) CD34+ cells per kg or 5 × 10(4) CFU-GM per kg, but a dose of 5 × 10(6) CD34+ cells or 40 × 10(4) CFU-GM per kg is critical to ensure rapid recovery.     

Kinetic study of CD34+ cells during peripheral blood stem cell collections

The impact of the separated volume on the yield of CD34+ cells during peripheral blood stem cell collections (PBSCC) remains controversial. We therefore studied the CD34+ cell concentration in the peripheral blood of patients (pts) during PBSCC as well as the total amount of CD34+ cells collected after each blood volume (BV) processed and engraftment data for each cycle of high dose chemotherapy (HD Ctx). A total of 21 PBSCC from 20 patients with different malignancies were analyzed. Stem cells were mobilized by chemotherapy and G-CSF (14 pts) or GM-CSF (6 pts). Samples from the pts peripheral blood and the collection bag were taken after each BV processed and analyzed for CD34+ cells, WBC, platelets (plt), and hemoglobin (Hb). The total volume processed was two to five times the pts calculated BV (mean value 17.4 L, range 9.0–24.0 L). Sixteen pts could be evaluated for engraftment. The mean peripheral blood CD34+ cell count was 116±103.5/μl at the start of PBSCC and decreased to 57±61.6/μl after processing of four times the pts BV. The mean number of CD34+ cells collected after each BV was 2.3±2.4, 5.8±5.2, 8.5±7.2, and 11.8±10.3×10⁶ per kg body weight, respectively. The mean plt count decreased by 53±40.2/nl, Hb by 1.±0.5 g/dl and WBC by 0.±6.1/nl after separation of 4 BV. All but two pts reached the target value of 1.5 × 10⁶ CD34+ cells/kg body weight and planned cycle of HD Ctx with 1 PBSCC. All pts engrafted and reached neutrophils>500/μl and plt>20,000/μl at a median of 11 and 13 days, respectively. We could demonstrate, that the yield of CD34+ cells during PBSCC increased continuously with the volume of the separated BV and that up to 5× the patients' BV could be processed safely without serious side effects. Most pts had to undergo only 1 PBSCC to collect sufficient numbers of CD34+ cells to support sequential courses of HD Ctx without delayed engraftment. J. Clin. Apheresis 14:18–25, 1999. © 1999 Wiley-Liss, Inc.     

Assessment of stability of CD34+ cell products enriched by immunoselection from peripheral blood mononuclear cells during refrigerated storage

Durable engraftment of transplanted CD34+ cells largely depends on the quality of the cell product. Limited data are currently available about extended storage of immunoselected CD34+ cells. The aim of our study was to assess the stability of CD34+ cell product with the cells stored in high concentration (80 × 10⁶ in 6 mL) in small bags intended for cell implantation. Cell products were prepared by leukapheresis and immunoselection (Clinimacs^plus procedure) from 13 patients with chronic dilated cardiomyopathy. CD34+ cell products were stored at 2–8 °C and analyzed at time 0 (fresh products), 24, 48 h, 4 and 6 days. Product viability, absolute number of viable CD34+ cells and apoptosis were determined by flow cytometry. Microbiological contamination of the cell products was tested by BACTEC system. The mean viability of CD34+ cells decreased by 2.7% within 24 h, by 13.4% within 48 h and by 37.5% within 6 days. The mean recovery of viable CD34+ cells was 91.1%, 74.8%, 66.3% and 56.2% at 24, 48 h, 4 and 6 days, respectively. The mean fraction of early apoptotic cells in fresh and stored products was 4.9 ± 3.5% at 0 h, 5.9 ± 3.8% at 24 h, 4.2 ± 3.1% at 48 h, 6.3 ± 2.6% at 4 days and 9.3 ± 4.6% at 6 days. All products were negative for microbial contamination.     

外周血CD34+细胞绝对计数可靠预示自体外周血干细胞采集效果

目的　为确定外周血CD34+细胞绝对计数能否可靠预示自体外周血干细胞的采集效果。方法　用流式细胞仪ProCOUNT方法对采集的 2 5份次移植物和采集当天外周血行CD34+细胞绝对计数 ,同时做外周血常规检查和移植物集落形成单位 (CFU)计数 ,每份次移植物以CD34+/kg ,单个核细胞 (MNC) /kg,粒 巨噬细胞集落形成单位 (CFU GM) /kg ,红细胞集落形成单位 (CFU E) /kg等为指标 ,与患者采集当天的外周血CD34+细胞绝对计数、CD34+细胞百分比、WBC ,MNC ,中性粒细胞(NEU)或血小板 (PLT)等各项指标进行相关分析和逐步回归分析。结果　 ( 1)Spearman相关分析结果 :外周血CD34+细胞绝对计数与移植物CD34+/kg高度相关 (r=0 790 ,P <0 0 0 1) ,外周血CD34+细胞百分比与移植物CD34+/kg相关 (r=0 6 17,P <0 0 5 )。外周血WBC、MNC、NEU、PLT或RBC与移植物CD34+/kg无关。外周血CD34+细胞绝对计数与移植物CFU E相关 ,而与CFU GM无关。外周血MNC与移植物MNC/kg相关。 ( 2 )逐步回归分析结果 :移植物CD34+/kg只与外周血CD34+细胞绝对计数高度相关 (P <0 0 0 1) ,而与外周血CD34+细胞百分比无关。结论　移植物CD34+/kg只与外周血CD34+细胞绝对计数高度相关 ,外周血CD34+细胞绝对计数能够可靠预示自体外周血干细胞的采集效果     

Autologous graft-versus-host disease after CD34+-purified autologous peripheral blood progenitor cell transplantation

Autologous graft-versus-host disease (GVHD) has been frequently reported after cyclosporine A (CsA) administration in the autologous setting. This complication is related to the disruption of self-tolerance mechanisms induced by CsA and may exert an antitumor effect. We report the spontaneous occurrence of autologous GVHD after CD34+-purified peripheral blood progenitor cell transplantation (PBPCT) in 5 out of 24 consecutive patients (20.8%). The syndrome was characterized by skin rash (5/5), pruritus (5/5), eosinophilia (5/5), and fever (2/5) occurring at a median of 37 days (range 22-60) after transplantation. Diagnosis was confirmed by skin biopsy in all patients. The syndrome was self-limiting, lasted a median of 25 days, and did not require treatment. The rate of autologous GVHD was high after CD34+-purified autologous PBPCT. In fact, no autologous GVHD was documented in an historical control of 100 consecutive patients submitted to unmanipulated PBPCT at the same institution. The manipulation of the graft by the purging procedure causes a profound T lymphocyte depletion, thus possibly perturbing the equilibrium between autoregulatory cells and autocytotoxic T cells. These observations add new interest to the antitumor efficacy of autologous GVHD and suggest new questions regarding the role of transplantation for autoimmune diseases.     

影响外周血CD34+细胞纯化的相关因素

背景:造血干细胞具有良好的自我复制和更新的能力,CD34+细胞具备有造血干细胞的标志.目的:分析影响外周血CD34+细胞纯化的因素.方法:90例患者,经粒细胞集落刺激因子5 μg/(kg·d)动员1～3 d后,应用COBE血细胞分离机采集外周血单个核细胞液80～100 mL,经Clini MACS免疫磁珠分选技术纯化CD34+细胞.结果与结论:90例CD34+细胞平均数为(1.73±1.15)×107,经流式细胞仪分析,CD34+细胞阳性率大于80%.COBE血细胞分离机单次收集的循环血量在980～1 100 mL时,利于CD34+细胞收集(P=0.005);动员后白细胞浓度在(16～21)×109 L-1时,利于CD34+细胞收集(P < 0.05);中间细胞和淋巴细胞总比例超11%时,利于CD34+细胞收集(P < 0.05);单个核细胞液血小板小于2 100×109 L-1时,利于CD34+细胞的收集(P < 0.05);年龄小于16岁,CD34+细胞数高(P=0.003);CD34+细胞抗体的温度、磁性标记及细胞处理时离心力的大小,均有影响.结果提示,经Clini MACS免疫磁珠细胞分选技术纯化的CD34+细胞能满足临床需要,实验稳定性好,重复性好;注重相关因素的影响,可提高纯化的CD34+细胞数量.     

Prolonged storage of red cells at 4 degrees C

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.     

脐血CD34^＋细胞和外周血单核细胞两种来源的树突状细胞特性？…

目的 体外大量扩增和纯化具有典型表型、形态和功能的树突状细胞（ＤＣ）、以进行相关基础研究和临床应用。方法 采用免疫磁珠江分离脐血ＣＤ３４＾＋细胞及外周血去Ｂ、去Ｔ淋巴细胞的单个核细胞（单核细胞）,然后以ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦα、Ｆｌｔ３配基（ＦＬ）、ＳＣＦ等不同的细胞因子配伍分别诱生ＤＣ,通过流式细胞仪、电镜、光镜分析其特性,同时检测其刺激同种Ｔ细胞增殖的能力。结果 脐民外周血诱生ＤＣ的方     

CD133+CD34+ and CD133+CD38+ blood progenitor cells as predictors of platelet engraftment in patients undergoing autologous peripheral blood stem cell transplantation

BackgroundHematopoietic stem cells (HSC) have been characterized by CD34+ expression and an adequate dose of CD34+ cells is associated with a complete engraftment. CD133 is a more specific marker of HSC.Materials and methodsWe studied the relationship between graft content of CD34+, CD133+, and CD38+ cells and trilineage engraftment after autologous stem cell transplantation in patients with different hematological disorders. Blood samples were obtained before and after mobilization with recombinant granulocyte-colony stimulating factor (G-CSF, 16 μg/kg), from apheresis collections, and after transplantation.ResultsCell subsets were quantified by flow cytometry, and the dose of each population infused was correlated with success of engraftment. G-CSF induced mobilization of CD133+CD38+ cells (12.6-fold) and CD133+CD34+ cells (14.7-fold). A correlation was observed between the infused dose of CD133+CD34+ and CD133+CD38+ cells and platelet engraftment.ConclusionCD133+CD34+ and CD133+CD38+ cells were mobilized with G-CSF and these cell subsets were correlated with platelet engraftment.     

Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.     

MARCH-I expression in cord blood CD34+KDR+ cells

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%-0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR- counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties.     

The quality of fresh-frozen plasma produced from whole blood stored at 4 degrees C overnight

BACKGROUND: The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose. STUDY DESIGN AND METHODS: Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (18-24 hr from donation, Day 1 FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method). RESULTS: In more than 95 percent of Day 1 FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 92 and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by 12, 15, 23, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or 24 hours, but were below the reference range in an additional 2 of 6 units held for 24 hours. The activities of protein S, protein C, antithrombin III, and alpha(2)-antiplasmin were reduced by less than 10 percent in Day 1 FFP (n = 20), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for 18 to 24 hours, but levels of prothrombin fragment 1 + 2 were slightly increased (0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr). CONCLUSION: These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for 18 to 24 hours and that this would be an acceptable product for most patients requiring FFP.     

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.     

Umbilical cord blood progeny cells that retain a CD34+ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34+ cells

BACKGROUND: Because of the limitation of cell numbers associated with cord blood harvests, there is a need to determine the efficacy of using ex vivo-expanded cord blood cells in a transplantation setting. In this study, limiting-dilution analysis was used in nonobese diabetic mice with severe combined immunodeficiency (NOD/SCID) to compare the engraftment potential of progeny cells expressing the CD34+ phenotype after expansion with that of uncultured CD34+ cells. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured in Iscove's modified Dulbecco medium supplemented with 10-percent fetal calf serum (FCS) and IL-6, SCF, megakaryocyte growth and development factor, and Flt3 ligand. The resulting ex vivo-expanded products were assessed for total numbers of nucleated cells, CD34+ cells, and CFUs and long-term culture-initiating cell activity. The engraftment potentials of cultured progeny CD34+ cells and uncultured CD34+ cells were determined by using NOD/SCID mice. RESULTS: After 14 days of culture, total nucleated cell counts increased over input values by 180 +/- 59-fold, CD34+ cell numbers by 44 +/- 13-fold, CFU activity by 23 +/- 5-fold, and long-term culture-initiating cell activity by 20 +/- 6-fold (mean +/- SD; n = 6). The frequency of SCID-repopulating cells (SRC) in mice transplanted with uncultured products was 1 per 20,000 CD34+ cells (95% CI, 1:10,000-1:38,000) and that in mice receiving ex vivo-expanded products was 1 per 418,000 progeny CD34+ cells (95% CI, 1:158,000-1:1,100,000). Taken together, these data indicated that, after 2 weeks of culture, there was a modest twofold increase in the total number of SRCs. However, the levels of human CD45 cell engraftment in NOD/SCID recipients of progeny CD34+ cells were significantly lower than those in mice receiving equivalent numbers of uncultured CD34+ cells (p<0.05). CONCLUSION: Umbilical cord blood progeny cells retaining a CD34+ phenotype after ex vivo expansion have less engraftment potential than do unexpanded CD34+ cells.