Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.     

Quiescent subpopulations of human CD34-positive hematopoietic stem cells are preferred targets for stable recombinant adeno-associated virus type 2 transduction

We have previously demonstrated recombinant adeno-associated viral (rAAV) transduction of human CD34(+) hematopoietic stem cells (HSCs) capable of serial engraftment in vivo. Here we evaluated the capacity of rAAV2 to mediate gene transfer into nondividing, quiescent, primitive CD34(+) cells subdivided on the basis of metabolic, mitotic, and phenotypic properties. Results revealed that CD34(+)CD38() marrow cells are the most quiescent, exist primarily in G(0) at isolation and are the only population to remain nondividing during the entire exposure to free rAAV. Despite significant differences in the extended clonogenic capacities of CD34(+) subsets in stromal cultures, the frequency of rAAV marking of colonies derived from primitive progenitors was similar in all three populations, suggesting that both primitive and more differentiated progenitors were initially transduced at equal levels. After transduction, episomal and integrated rAAV genomes were detected in all CD34(+) subsets. However, the more quiescent cells displayed higher levels of integrated rAAV than did rapidly dividing cells. Importantly, stable long-term integration was observed only in the most primitive, quiescent CD34(+)CD38(-) subset, indicating that this HSC compartment comprises the preferred substrate for stable rAAV2 transduction. Previously described rate limitations to transgene expression were observed in transduced CD34(+) cells and could be overcome by tyrphostin pretreatment, which resulted in augmented second-strand synthesis. These results represent the first demonstration of rAAV-mediated gene transfer to primitive, quiescent human CD34(+)CD38(-) stem cells and reveal that nondividing CD34(+)CD38(-) HSCs are the optimal CD34(+) targets for rAAV transduction.     

Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector.

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells.     

In vitro selection of lentivirus vector-transduced human CD34+ cells

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.     

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34(+) cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.     

2型重组腺相关病毒对人脐血CD34+造血干/祖细胞的转导效率研究

为探讨 2型重组腺相关病毒 (rAAV 2 )载体能否有效地转导脐血CD34+造血干 /祖细胞 ,采用rAAV 2 /GFP感染经免疫磁珠法分离的脐血CD34+造血干 /祖细胞 ,在荧光显微镜下观察GFP的表达。结果显示 ,转导 19小时后感染复数 (MOI)为 2× 10 5时 ,CD34+细胞GFP基因的表达率为 4 3%。结论 :rAAV 2能有效地转导脐血CD34+造血干 /祖细胞。     

Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virus-based gene transfer vectors

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.     

Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34+ cell-derived human dendritic cells

The efficient genetic modification of CD34+ cell-derived dendritic cells (DC) will provide a significant advancement towards the development of immunotherapy protocols for cancer, autoimmune disorders and infectious diseases. Recent reports have described the transduction of CD34+ cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC. Since there is significant apprehension regarding the clinical uses of HIV-based vectors, in this report, we compare a murine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bicistronic vector for gene transfer into human CD34+ cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dominant selectable marker DHFR(L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood CD34+ cells. However, in vitro expansion and differentiation in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resulting in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced cells expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols.     

Stable transduction with lentiviral vectors and amplification of immature hematopoietic progenitors from cord blood of preterm human fetuses

Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells. To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34(+) cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34(+) cells was 42 +/- 9%, resulting in GFP expression in 23 +/- 4% of colonies derived from colony-forming units (CFUs) and 11 +/- 1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated transduction and GFP expression in cells in the G(0) phase, which contains immature hematopoietic progenitors. Transduced fetal CD34(+) cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with Flt-3 ligand. At week 10, expression of GFP was observed in 40.5 +/- 11.7% of CFU-derived colonies. While prestimulation of CD34(+) cells with cytokines prior to transduction increased the efficiency of GFP transfer 2- to 3-fold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation.     

Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction

Human mesenchymal stem cells (hMSC)-nonhematopoietic cells within the bone marrow microenvironment that can be culture expanded to a uniform population of fibroblastic cells-have been shown to support long-term hematopoiesis of CD34+ cells. Because direct contact between stromal elements and CD34+ cells enhances long-term engraftment, we postulated that hMSC would be a good alternative to the more heterogeneous stroma currently used in gene transfer studies. We used hMSC to support retroviral gene transfer of the G156A MGMT (deltaMGMT) gene encoding an alkyltransferase (AGT), which confers drug resistance to a combination of O6-benzylguanine (BG) plus the alkylating agents BCNU and temozolomide (TMZ) in human hematopoietic progenitors. In the presence of IL-3, IL-6, SCF, or leukemia inhibitory factor (LIF) and Flt-3 ligand, hMSC facilitated expansion and retroviral transduction of human peripheral blood-mobilized CD34+ cells. Furthermore, the transduced cells expressed AGT in 29% of hematopoietic cells and were 5-fold more resistant to BCNU and TMZ than were untransduced cells. Unirradiated hMSC present as support cells were simultaneously transduced and expressed AGT in 26% of the cells. Thus, the homogeneous nature of hMSC, and their ability to support gene transfer and be transduced themselves suggest they may be useful in clinical gene transfer protocols and have broad therapeutic applications.     

mdr-1和DHFR双基因共转染人CD34+细胞拓宽造血细胞耐药谱的体外研究

目的　探讨将多药耐药基因 (mdr 1)和二氢叶酸还原酶基因 (DHFR)同时导入人CD3 4 细胞 ,以拓宽造血细胞耐药谱 ,改善骨髓耐受联合化疗的可行性。方法　将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架 ,通过引入IRES序列构建获得含mdr 1和DHFR(L2 2Y)双耐药基因的逆转录病毒载体pSF DIM ,通过脂质体介导包装 ,单嗜性和双嗜性包装细胞上清交叉感染提高病毒滴度。低温离心病毒上清转染人脐血CD3 4 细胞 ,用流式细胞仪检测P gp的表达 ,基因组PCR检测外源性耐药基因的整合 ,CFU GM培养观察耐药性变化。结果　逆转录病毒载体pSF DIM转导人脐血CD3 4 细胞后 ,P gp的表达较未转基因组增加了 10 .98% ;基因组PCR同时检测到两种外源性耐药基因的整合 ;与未转基因组比较 ,在 48nmol/L甲氨蝶呤和 10ng/ml及 12ng/ml紫杉醇 (商品名Taxol)浓度水平 ,CFU GM集落形成显著增加 (P <0 .0 5 )。结论　重组双耐药基因逆转录病毒载体pSF DIM可以有效介导mdr 1和DHFR双耐药基因进入人脐血CD3 4 细胞并且获得共表达 ,拓宽了造血细胞耐药谱     

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.     

Potent inhibition of simian immunodeficiency virus (SIV) replication by an SIV-based lentiviral vector expressing antisense Env

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.     

Tpo、IL-11基因修饰的基质细胞对CD34+CD38-早期造血祖细胞扩增的影响

目的　观察经Tpo、IL 11基因修饰的基质细胞对脐血CD3 4 + CD3 8-细胞体外扩增的影响。方法　用载有Tpo、IL 11基因的重组逆转录病毒感染成纤维样基质细胞HFCL ,通过Northernblot法检测基因修饰的HFCL细胞Tpo、IL 11基因的表达。以未经基因修饰的HFCL细胞作为对照 ,将脐血CD3 4 + 造血干 /祖细胞在这种基因修饰的HFCL细胞支持下 ,进行 7d体外扩增后 ,用锥虫蓝拒染法计数活细胞总数 ,并用流式细胞仪分析扩增细胞中CD3 4 + 细胞以及CD3 4 + CD3 8-细胞的比例。结果　在Tpo基因修饰的HFCL细胞、IL 11基因修饰的HFCL细胞和Tpo +IL 11基因共同修饰的HFCL细胞的支持下 ,扩增后CD3 4 + CD3 8-早期造血祖细胞的比例分别为 (1.8± 0 .2 4 ) %、(1.6 2± 0 .2 3) %、(2 .4 5±0 2 8) % ,细胞扩增倍数为 4 .2倍、3.6倍、6 .9倍 ,高于对照组的 (0 .80± 0 .2 3) %和 1.5倍 ,同时扩增细胞总数和CD3 4 + 细胞比例亦高于未经基因修饰的HFCL细胞所支持的扩增体系。结论　Tpo、IL 11基因修饰的基质细胞可有效促进脐血CD3 4 + 造血干 /祖细胞体外扩增 ,同时能有效维持扩增体系中的CD3 4 + CD3 8-细胞以及促进其扩增。     

mdr—1和DHFR双基因共转染入CD^＋34细胞拓宽造血细胞耐药谱的体外研究

目的：探讨将多药耐药基因（mdr-1)和二氢叶酸还原酶基因（DHFR)同时导入人CD^ 34细胞,以拓宽造血细胞耐药谱,改善骨髓耐受联合化疗的可行性,方法：将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架,通过引入IRES序列构建获得含mdr-1和DHFR（L22Y）双耐药基因的塑转录病毒载体pSF－DIM,通过脂质体介导包装,单嗜性和双 包装细胞上清交叉感染提高病毒滴度,低温离心病毒上清转染人脐血CD^ 34细胞,用流式细胞仪检测P－gp的表达,基因组PCR检测外源性耐药基因的整合,CFU－GM培养药性变化,结果：逆转录病毒载体pSF-DIM转人脐血CD^ 34细胞后,P-gp的表达较未转基因组增加了10．98％,基因组PCR同时检测到两种外源性药基因的整合,与未转基因组比较,在48nmol/L甲氢蝶呤和10ng/ml及12ng/ml紫杉醇（商品名Taxol）浓度水平,CFU－GM集落形成显著培养（P＜0．05）。结论：重组双耐药基因逆转录病毒载体pSF-DIM可度水平,CFU－GM集落形成显著增加（P＜0．05）,结论：重组双耐药基因逆转录病毒载体pSF-DIM可以有效介导mdr-1和DHFR双耐药基因进入人脐血CD34^ 细胞并获得共表达,拓宽了造血细胞药谱。     

慢病毒载体介导的基因转导技术对人脐血CD34+细胞基因表达谱的影响

目的 研究慢病毒载体(lentivirus vector)基凶转导技术对脐血CD34+细胞基因表达的影响.方法 将携带绿色荧光蛋白(GFP)基因的第三代自身失活(self-inactivating,SIN)慢病毒载体导入CD34+细胞,提取被病毒感染细胞的总RNA,应用cDNA微阵列技术对慢病毒载体感染的脐血CD34+细胞及正常对照细胞进行差异基因表达谱分析.结果 在23000个被检测基因中,与无基因导人的对照组细胞比较,基因导入细胞中表达的上调基因共2个,分别为IGSF4和HEC基因,下调基因共6个,分别为HNRPAO、ZNF205、FLNA、CLK3、LDB1、ACTA1.进一步对筛选出的差异表达基因进行半定量RT-PCR分析证实基因表达水平变化不明显.结论 本实验中应用的慢病毒载体对脐血CD34+细胞基凶表达无明显影响,有望成为临床基因治疗有效且安全的工具.     

FLT3 ligand preserves the uncommitted CD34+CD38- progenitor cells during cytokine prestimulation for retroviral transduction

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34+ cells. The effect of cytokine prestimulation on transduction efficiency and the population of uncommitted CD34+CD38- cells was determined. CD34+ cells harvested from umbilical cord blood were kept in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL-3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04). However, prestimulation with SCF in combination with IL-3 and IL-6 also reduced the percentage of CD34+ cells (p = 0.02). Then, prestimulation with SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34+CD38- cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytokine combinations increased the transduction efficiency, but reduced the fraction of CD34+ cells. Importantly, the use of FLT3 ligand seemed to preserve the population of uncommitted cells.     

GFP在人脐血CD34~+细胞中的表达及意义

目的探讨慢病毒载体在CD34+脐血细胞(CBCs)中的基因转导效率,为基因治疗的临床应用提供关键材料。方法应用1型人类免疫缺陷病毒(HIV-1)改造而成的第三代自身失活(self-inactivating,SIN)慢病毒载体(lentiviralvector)系统,通过流式细胞术检测基因导入细胞百分比,评价该载体系统在人CBCs中的基因转导效率。结果 HIV载体的转导效率在95%以上。结论基于慢病毒载体基因转导的高效性,该载体系统可作为CD34+CBCs基因转导的极好工具。