CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Cytokine-pretreatment of CD34(+) cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice

Stem cell homing, engraftment and organ regeneration are controlled by cytokines, chemokines and cell-cell interactions. In this paper, cytokine effects on homing- and engraftment-related characteristics of CD34(+) cord blood cells were examined. Untreated CD34(+) cells were mainly in the G(0)/G(1) cell cycle phase, expressed adhesion receptors on a low level, were positive for vimentin, and negative for the epithelial marker cytokeratin 8/18. Treatment with stem cell factor (SCF) stimulated cell proliferation, increased the number of cells in S and G(2)/M cell cycle phase as well as the expression of adhesion receptors. The expression of cytokeratin 8/18 was increased and that of vimentin remained unchanged. Hepatocyte growth factor (HGF) did not stimulate cell proliferation and expression of adhesion receptors, but increased expression of cytokeratin 8/18. In NOD/SCID mice, kinetics of stem cell distribution revealed a fast elimination of human cells from blood. An increase in the number of engrafted cells was observed in different mouse organs in a time-dependent manner, preferentially in bone marrow, spleen and liver. Pretreatment with SCF resulted in reduction of long-term engraftment in bone marrow. HGF pretreatment of cord blood cells showed no significant effects on long-term engraftment capacity in mouse organs compared to untreated cells. Our data provide in vivo evidence that pretreatment of CD34(+) cells with SCF reduces long-term cell engraftment in NOD/SCID mice.     

Hematopoietic reconstitution of CD34<Superscript>+</Superscript> cells grown in static and stirred culture systems in NOD/SCID mice

The hematopoietic reconstitution of cord blood (CB) CD34⁺ cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.     

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells

BACKGROUND: Retrovirus-mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest. METHODS: We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material. RESULTS: Formulation A, consisting of stem cell factor, Flt-3 ligand, thrombopoietin, and IL-3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood. CONCLUSIONS: Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.     

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.     

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.     

High-level gene transfer to the cornea using electroporation

Background

Methods for gene transfer to the cornea that yield high‐level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.

Methods

To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square‐wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin‐6 (IL‐6) production.

Results

The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000‐fold more gene product was produced using electroporation. Expression was detected as early as 6 h post‐electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.

Conclusions

These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer. Copyright © 2001 John Wiley & Sons, Ltd.

     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.