A sensitive quantitative single-platform flow cytometry protocol to measure human platelets in mouse peripheral blood

BACKGROUND: The NOD/SCID mouse is a widely used model for human cord blood (CB) transplantation. Engraftment is generally estimated with semiquantitative methods, measuring the percentage of human cells among mouse cells. To compare protocols aiming to improve hematopoietic recovery, quantitative methods to enumerate human cells would be preferred. This study describes a single-platform protocol to count human platelets (hPLTs) after transfusion and CB transplantation in the peripheral blood (PB) of the mouse. METHODS: With an anti-human CD41 antibody against hPLTs and counting beads, the sensitivity to detect hPLTs in mouse blood by flow cytometry was validated. PLT recovery after hPLT transfusions and PLT kinetics after transplantation with CB CD34+ cells was followed in time in NOD/SCID mice. RESULTS: hPLTs could be reliably detected to a level as low as 1 PLT per microL with this single-platform protocol, what appeared to be at least 10 times more sensitive than detection with the dual-platform protocol. To verify the applicability for mouse studies, hPLTs were measured serially in transfusion and transplantation studies in NOD/SCID mice. The results showed that earlier detection of PLT recovery was feasible with the single-platform protocol. CONCLUSION: A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.     

Application of peripheral blood stem cells (PBSC) mobilized by recombinant human granulocyte colony stimulating factor for allogeneic PBSC transplantation and the comparison of allogeneic PBSC transplantation and bone marrow transplantation.

Recombinant human granulocyte colony stimulating factor (rhG-CSF)-mobilized peripheral blood stem cells (PBSC) are now widely used for allogeneic PBSC transplantation (alloPBSCT). Large numbers of hematopoietic progenitor cells mobilized by rhG-CSF would be considered equivalent or better than bone marrow (BM) cells and would be used as an alternative to BM for allogeneic hematopoietic stem cell transplantation. The complications associated with the administration of rhG-CSF and apheresis in PBSC collection in formal donors are well tolerated and usually acceptable in the short term but some hazardous adverse events such as splenic rupture and cardiac arrest are reported although the incidence is very low. Protective means and stopping rules for safe donation in the collection of PBSC are established. The characteristics of PBSC were clarified; the expression of some adhesion molecules such as CD49d on CD34 positive cells of PBSC have been shown to be low compared to BM stem cells. In alloPBSCT compared with allogeneic BM transplantation (alloBMT), the incidence and frequency of graft versus host disease (GVHD) is of concern because high number of T lymphocytes are infused in alloPBSCT. The incidence and severity of acute GVHD are not increased but chronic GVHD is higher in alloPBSCT compared with alloBMT. The outcome of alloPBSCT and BMT are almost equivalent and conclusive results regarding survival are not yet available.     

Haploidentical stem cell transplantation-bone marrow vs peripheral blood

Hematopoietic stem cells may be obtained by collection of bone marrow, mobilization and collection of peripheral blood stem cells or umbilical cord blood. Transplantation of peripheral blood hematopoietic cells has increased due to faster engraftment and practicability in both the related, unrelated or haploidentical setting. We reviewed the question of which stem cell source - bone marrow (BM) or peripheral blood (PBSC) - is the most suitable for individuals undergoing haploidentical stem cell transplantation. BM or PBSC could be safely used as allograft sources for haploidentical transplantation with good outcomes and acceptable rates of GVHD and graft failure. Prospective randomized studies are needed to evaluate the effect of PB vs BM in haploidentical settings.     

人急性B淋巴细胞白血病-NOD/SCID/IL2rγnull新生小鼠异种移植模型的建立

本研究旨在应用NOD/SCID/IL2rγnull新生小鼠建立人急性B淋巴细胞白血病(B-ALL)的异种移植模型。NOD/SCID/IL2rγnull新生期(出生48 h之内)小鼠经亚致死剂量(100 cGy)137Cs全身照射后,由面静脉输注FACSAria流式细胞仪分选纯化的B-ALL患者骨髓CD3-CD4-CD8-CD34+CD19+细胞,于移植后8-12周用流式细胞术检测受鼠外周血、骨髓和脾脏中人源细胞的植入水平及其免疫表型,并通过HE染色评价人源B-ALL细胞在受鼠各组织器官中的迁移浸润能力。结果表明,在接受B-ALL患者CD34+CD19+细胞移植的受鼠外周血、骨髓和脾脏细胞中,不仅检测到了不同程度的人源B-ALL(huCD45+CD19+)细胞的植入〔(83.36±10.05)%,(93.88±5.05)%,(88.31±5.01)%〕,而且植入细胞具有与B-ALL患者相似的细胞形态和免疫表型特征。此外,人源B-ALL细胞广泛迁移浸润到受鼠的肝脏、肺脏、肾脏和脑等组织器官中。结论:NOD/SCID/IL2rγnull新生小鼠模型能够支持B-ALL患者CD34+CD19+细胞的高水平植入,是对人B-ALL进行体内功能性研究的新型异种移植模型。     

NOD/SCID小鼠脐血单个核细胞骨髓腔内移植的实验研究

目的观察骨髓腔内移植(iBM)人脐血单个核细胞(MNC)对小鼠造血重建和免疫功能恢复的作用。方法NOD/SCID小鼠经137Cs全身照射后,在4h内无菌条件下局部麻醉后从尾静脉或骨髓腔内输注分离脐血MNC。受体小鼠随机分为5组:①对照组:骨髓腔内输注培养液;②阳性对照组(iTV):尾静脉输注脐血MNC3×107/只;③实验Ⅰ组(iBM1):骨髓腔内输注脐血MNC3×106/只;④实验Ⅱ组(iBM2):骨髓腔内输注脐血MNC1×107/只;⑤实验Ⅲ组(iBM3):骨髓腔内输注脐血MNC3×107/只。对照组4只小鼠,实验Ⅱ组7只小鼠,其余每组5只。24h后观察iBM22只小鼠未移植侧胫骨骨髓腔脐血细胞的迁移分布,动态观察移植后小鼠的存活和造血重建情况,7～8周后处死各组小鼠,检测骨髓细胞表面CD分子表达、碳青花(DilCM)染料示踪研究和脐血βactin的DNA标记。结果①照射后骨髓腔内输注脐血MNC,24h后在未输注脐血MNC的一侧胫骨骨髓细胞膜有DilCM标记;②7～8周后小鼠存活14只,其中对照组存活1只,iTV组、iBM1组各存活2只,iBM2组存活4只,iBM3组存活5只;③外周血常规检查结果显示iBM组白细胞的恢复速度比iTV组和对照组快而且稳定;④移植后存活小鼠骨髓细胞表面CD45标记、DilCM染料示踪研究和βactin均显示人源的标记。结论经iBM途径移植脐血MNC至NOD/SCID小鼠骨髓可以重     

Peripheral Blood or Bone Marrow Stem Cells? Practical Considerations in Hematopoietic Stem Cell Transplantation

Although peripheral blood stem cells (PBSC) have worldwide become the predominant source of progenitor cells for hematopoietic stem cell transplantation (HSCT), debate about their role compared with bone marrow (BM) has recently intensified, in large part based on the results of a multicenter Clinical Trials Network study which showed lower incidence of chronic graft-versus-host disease (cGVHD) and improved quality of life in recipients of myeloablative HLA-matched unrelated BM compared with PBSC transplants. However, in certain patient populations, PBSC may lead to improved clinical outcomes due to faster hematologic recovery, a lower risk of graft failure, and possibly a lower probability of relapse. This review will provide a comprehensive summary of studies comparing PBSC with BM as the graft source in terms of acute and chronic GVHD incidence, time to engraftment, and disease-free and overall survival probabilities after HLA-matched related and unrelated donor transplantation and haploidentical donor transplantation. Recommendations based on these studies regarding the use of PBSC versus BM for HSCT are offered.     

无血缘关系供者外周血干细胞移植和骨髓移植治疗白血病的比较

目的比较无血缘关系供者外周血干细胞移植和骨髓移植在造血重建、T 细胞重建、感染、移植物抗宿主病(GVHD)及疗效等的差异。方法 53例白血病患者中,21例接受无血缘关系供者外周血干细胞移植(PBSCT 组),32例接受无血缘关系供者骨髓移植(BMT 组)。统计分析二者移植后白细胞和血小板重建时间、T 细胞重建、感染率、GVHD、白血病复发、无病生存(DFS)情况。结果PBSCT 组和 BMT 组移植后白细胞重建时间分别为(12.43±3.67)天和(16.16±2.99)天(P<0.01),血小板重建时间分别为(14.67±6.19)天和(21.23±8.25)天(P<0.01)。PBSCT 组和 BMT 组移植后1,3,6,9及12个月的 T 细胞重建差异无统计学意义。PBSCT 组和 BMT 组移植后早期感染率分别为42.86%和53.13%(P>0.05)。PBSCT 组与 BMT 组急性 GVHD 的发生率分别为61.90% 和71.885(P>0.05);在可统计的患者中,慢性 GVHD 的发生率在 PBSCT 组和 BMT 组分别为47.06%和43.48%(P>0.05)。PBSCT 组与 BMT 组移植后分别有4例和2例复发,二者复发率差异无统计学意义(P>0.05);PBSCT 组与 BMT 组移植后2年 DFS 率分别为(50.14±12.00)% 和(59.81±8.99)%,二者差异也无统计学意义(P>0.05)。结论无血缘关系供者 PBSCT 后的造血重建比BMT,但二者移植后 T 细胞重建、感染发生率、GVHD 及 DFS 的差异均无统计学意义。     

Validation of human monoclonal HLA Class I antibodies to evaluate the kinetics of donor chimerism in different cell subsets after double‐cord‐blood transplantation in the NOD/SCID model

BACKGROUND: Double‐cord‐blood transplantation (DCBT) in patients is typically accompanied by predominance of a single unit. The causative mechanism, however, is unknown. Identifying the dynamics of mixed donor chimerism in general and in specific subpopulations may help to resolve this question. We conducted studies in a mouse model to develop a new analytic method using anti‐human HLA Class I allele–specific monoclonal antibodies (HLA‐MoAbs) in flow cytometry. STUDY DESIGN AND METHODS: Single‐cord‐blood transplantation or DCBT from HLA‐mismatched donors was performed in NOD/SCID mice. Bone marrow (BM) and peripheral blood were collected from 3 to 20 weeks after transplantation. Donor chimerism was determined quantitatively within human platelets (hPLTs), human CD45+ (hCD45+) cells, and human myeloid and lymphocyte subsets by flow cytometry. RESULTS: Both cord donors stably engrafted in NOD/SCID. The sensitivity to detect chimerism measured with all HLA‐MoAbs was 1% (>10 cells/µL). In mouse BM, the percentage of human cells measured with hCD45+ versus HLA‐MoAbs correlated excellently (r = 0.999). Donor origin could be defined with HLA‐MoAbs for nearly all (>93.6%) human cells in mouse peripheral blood and BM in all lineages. Chimerism of hPLTs in peripheral blood correlated well with hCD45+ cells in BM enabling frequent measurement of chimerism from early after transplantation onward. CONCLUSION: This approach using HLA‐MoAbs enables longitudinal analysis of double‐mixed human chimeric populations despite low absolute concentrations of human hematopoietic cell subsets in peripheral blood and BM in mice. Lacking reactivity with mouse cells, the HLA‐MoAbs are suitable for use in other mouse models and in humans to identify the mechanisms involved in DCBT.     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII(-/-) mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2-12 mU/10(4) cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/10(4) cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34(+) cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/10(4) cells). Following xenotransplantation of transduced CD34(+) into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34(+) progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.     

NoD／SCID小鼠在实验血液学研究中的应用

NOD／SCID（非肥胖糖尿病／重症联合免疫缺陷）小鼠是在SCID（重症联合免疫缺陷）小鼠的基础上与非肥胖性糖尿病小鼠（NOD／Lt）品系回交的免疫缺陷鼠。NOD／SCID小鼠既有先天免疫缺陷，又有T和B淋巴细胞缺乏，各种肿瘤细胞可以植入，且较少发生排斥反应及移植物抗宿主病（GVHD），所以NOD／SCID小鼠逐渐成为血液学实验研究的有用工具。本文从NOD／SCID小鼠的生物学特性及其在建立人类白血病模型、干细胞移植、药物研究及NOD／SCID小鼠应用中存在的不足和改良等方面综合述评。     

Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency

Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.     

Long-term results of allogeneic peripheral blood hematopoietic stem cell transplantation for severe aplastic anemia

ObjectiveAplastic anemia (AA) is a life-threatening disorder and may be associated with significant morbidity and mortality Currently, the first treatment option is allogeneic hematopoietic stem cell transplant (allo-HSCT) for patients younger than 40 years. Bone marrow is recommended as the stem cell source due to less graft versus host disease (GVHD) risk and better outcomes than peripheral blood (PB)-derived stem cell. The aim of this study is to share the data of AA patients who have underwent PB-derived allo-HSCT in our bone marrow transplantation center.MethodsTwenty-seven patients who underwent PB-derived allo-HSCT from human leukocyte antigen matched sibling donors were analyzed retrospectively.ResultsThe median follow-up time was 95.2 months (range, 4.8–235 months). The 10-year survival was 89 %. The median neutrophil and platelet engraftment time was 11 days (range, 9–16 days) and 13 days (range, 11–29 days), respectively. Primary platelet engraftment failure was observed in 1 patient (3.7 %). Acute and chronic GVHD observed in 2 (7.4 %) and 3 (11.1 %) patients, respectively. Neutropenic fever was observed in 13 (44.8 %) of patients until the engraftment after allo-HSCT. One patient died due to CMV infections, two died due to septic shock secondary to fungal infection.ConclusionAlthough there is no prospective data directly comparing BM with PB as stem cell source in AA, observational studies indicates better OS with BM. PB can be used in certain situations such as higher risk for graft failure and donor preference. This study demonstrated that PB-derived stem cell seems to be a reasonable alternative to BM.     

人-NOD/SCID小鼠异种急性移植物抗宿主病模型的建立

目的 建立一种人-NOD/SCID小鼠嵌合的异种急性移植物抗宿主病(aGVHD)模型.方法 NOD/SCID小鼠给予亚致死剂量的60Coγ射线全身照射后经尾静脉输注动员后人外周血单个核细胞(PBMNC),移植后每周检测血常规,采用流式细胞术检测人CD45+细胞的比例,分析人淋巴细胞亚群,取各脏器组织检测人、鼠保守基因序列片段,并进行免疫病理分析.结果 移植后1周起NOD/SCID小鼠的血细胞中有明确的人CD45抗原表达,随人CD45+细胞比例增高逐渐出现以体重减轻和血细胞减少为主要表现的异种急性移植物抗宿主病(X-GVHD),人-NOD/SCID嵌合体的基因组DNA中可扩增出入β-globin及小鼠保守基因HYPSIN序列片段,移植后6周生存率约为14%;人-NOD/SCID嵌合体中以人T淋巴细胞的植人为主,CD4+/CD8+细胞比例倒置;X-GVHD以人的淋巴细胞浸润为主,肝脏与肺脏是主要靶器官.结论 NOD/SCID小鼠预先给予亚致死剂量照射,再经尾静脉输注入PBMNC,能够建立X-GVHD模型.临床表现主要为植入率相关的体重减轻和血细胞减少,病理表现靶器官主要为肝脏和肺脏.该模型建立方法简便,X-GVHD发生时间(2～3周)较早,发生率(94%)高,便于在此基础上进行aGVHD防治的动物实验研究.     

体外转染mFasL-cDNA小鼠骨髓细胞预防移植物抗宿主病的实验研究

为了探讨通过Fas FasL途径清除骨髓移植物中T细胞后 ,预防移植物抗宿主病 (GVHD)的可能性 ,采用脂质体转移法将FasL cDNA转染雌性BALB/c小鼠骨髓细胞 ,证实其获得表达后 ,体外与雄性BAC (BALB/c×C57BL/ 6)小鼠骨髓移植物混合培养后输注给经致死照射的BALB/c鼠 ,然后观察受体鼠GVHD的表现、死亡率 ,进行组织病理学检查 ,骨髓细胞Y染色体及CFU S检测。结果表明 :实验组 10只小鼠 ,60天死亡 2只 ,4只有GVHD改变 ;对照组 10只 ,60天死亡 7只 ,10只均有GVHD改变 ;两组骨髓细胞Y染色体分析显示均有供体鼠来源的骨髓细胞植入 ;CFU S检测表明实验组动物体内 10天即有CFU S形成 ,移植后第 2 0天即达正常水平。结论 :转染mFasL cDNA小鼠骨髓细胞能有效地预防GVHD的发生 ,并能使受体造血功能获得重建。     

In vivo effects of myeloablative alkylator therapy on survival and differentiation of MGMTP140K-transduced human G-CSF-mobilized peripheral blood cells.

High-intensity alkylator-based chemotherapy is required to eradicate tumors expressing high levels of O6-methylguanine DNA methyltransferase (MGMT). This treatment, however, can lead to life-threatening myelosuppression. We investigated a gene therapy strategy to protect human granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells (MPB) from a high-intensity alkylator-based regimen. We transduced MPB with an oncoretroviral vector that coexpresses MGMT(P140K) and the enhanced green fluorescent protein (EGFP) (n = 5 donors). At 4 weeks posttransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, cohorts were not treated or were treated with low- or high-intensity alkylating chemotherapy. In the high-intensity-treated cohort, it was necessary to infuse NOD/SCID bone marrow (BM) to alleviate hematopoietic toxicity. At 8 weeks posttreatment, human CD45+ cells in the BM of mice treated with either regimen were EGFP+ and contained MGMT-specific DNA repair activity. In cohorts receiving low-intensity therapy, both primitive and mature hematopoietic cells were present in the BM. Although B-lymphoid and myeloid cells were resistant to in vivo drug treatment in cohorts that received high-intensity therapy, no human CD34+ cells or B-cell precursors were detected. These data suggest that improved strategies to optimize repair of DNA damage in primitive human hematopoietic cells are needed when using high-intensity anti-cancer therapy.     

Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally

Hematopoietic progenitor cells (HPCs) can home to the bone marrow (BM) after a simple intravenous injection, but the adhesive mechanisms mediating the initial interactions of human HPCs with the BM endothelium have not been evaluated in vivo. Using fluorescence intravital microscopy and homing assays in NOD/SCID mice, we show that endothelial selectins are necessary for human adult CD34(+) cell homing, since rolling on BM endothelium and retention in the BM compartment are drastically reduced (>90%) in endothelial selectin-deficient NOD/SCID mice. Comparative analyses of CD34(+) cells collected from adults and from cord blood (CB) reveal that neonatal cells display reduced rolling fractions compared with adult CD34(+) cells obtained from peripheral blood or BM, suggesting abnormal selectin ligand function on neonatal progenitors. Flow cytometric and intravital microscopy studies suggest that this defect results from nonfunctional P-selectin ligand on a subset ( approximately 30%) of neonatal CD34(+) cells. Further analyses indicate that P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in a nonfunctional form among neonatal CD34(+) cells that do not bind P-selectin and that this subset is enriched in primitive CD34(+)CD38(lo/-) progenitors. These results underscore the potential to improve homing of CB CD34(+) cells to the BM by manipulation of selectins and their ligands.     

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell–dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed ⁵¹Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.     

Efficient oncoretroviral transduction of extended long-term culture-initiating cells and NOD/SCID repopulating cells: enhanced reconstitution with gene-marked cells through an ex vivo expansion approach

Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.     

Establishment of mouse embryonic fibroblast cell lines that promote ex vivo expansion of human cord blood CD34+ hematopoietic progenitors

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic progenitor cells (HPC) is vital to stem cell transplantation. The use of a monolayer of stromal cells on which to grow HPC in direct contact allows high efficiency ex vivo expansion of HPC. Here, we report an establishment of three murine embryonic fibroblast stromal cell lines from adherent cells of day-12 mouse embryos. Among them, HYMEQ-5 was most efficient in supporting long-term maintenance of human umbilical cord blood (CB) CD34(+) cells. Human CB CD34(+) cells cultured on HYMEQ-5 in the presence of stem cell factor (SCF), thrombopoietin, and flk-ligand (FL) showed high expansion of CD34(+)CD38(-) cells and highly proliferative potential-colony forming cells (HPP-CFC). Direct cell-to-cell contact between CD34(+) cells and HYMEQ-5 was important for this expansion. RT-PCR analysis showed that HYMEQ-5 produced FL, SCF, interleukin-6, and macrophage colony-stimulating factor (M-CSF). Expanded CB CD34(+) cells efficiently reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings suggest that HYMEQ-5 provides a milieu that supports long-term human hematopoiesis as well as ex vivo expansion of human CB CD34(+) HPC. This cell line may facilitate elucidation of the mechanism of cellular interactions between HPC and stromal cells.