Morphological Analysis and Lentiviral Transduction of Fetal Monkey Bone Marrow-Derived Mesenchymal Stem Cells

Lee, Charles C.; Kohn, Donald B.; Ekert, Jason E.; Tarantal, Alice F.

doi:10.1016/j.ymthe.2003.09.019

Cited by 47 publications

(45 citation statements)

References 47 publications

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“…Remarkable progress has been made in the recent past in using retroviral or lentiviral vectors to deliver therapeutic genes into MSCs (Zhang et al, 2002;Lee et al, 2004;Reiser et al, 2005;Barzilay et al, 2009;Jacome et al, 2009) or in deriving iPS cells from adult somatic cells (Welstead et al, 2008;Sommer et al, 2009) Townes, 2008). However, both retroviral and lentiviral vectors undergo random integration into host chromosomal DNA, and there is persistent risk for insertional mutagenesis (Cockrell and Kafri, 2007;Nair, 2008).…”

Section: Discussionmentioning

confidence: 99%

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

Jayandharan

et al. 2010

Human Gene Therapy

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Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is *10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by *130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80-90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors. Overview SummaryFibroblasts and mesenchymal stem cells (MSCs) are promising targets for gene and cell therapy. Recombinant adeno-associated virus 2 (AAV2) vectors are currently in use in several Phase I/II clinical trials for gene therapy. However, the existing data show that AAV2 vector-mediated transduction of fibroblasts and MSCs is inefficient. We observed that AAV2 vectors containing mutations in three surface-exposed tyrosine residues significantly increase transduction efficiency in fibroblasts, as well as in both human and murine primary MSCs. The increased transduction efficiency of these triple-mutant AAV2 vectors correlates well with improved viral intracellular trafficking in fibroblasts. These data provide strong evidence that high-efficiency gene delivery and transgene expression by AAV vectors are indeed feasible, which should facilitate the generation of induced pluripotent stem cells, as well as the use of MSCs in combinatorial gene and cell therapy.

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Section: Discussionmentioning

confidence: 99%

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

Jayandharan

et al. 2010

Human Gene Therapy

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“…A candidate for this minor population could be the still elusive osteoprogenitor cell that maintains the potential to proliferate and to differentiate (42). Progenitors are indubitably present in primary osteoblast cultures, which undergo several population doublings before senescence (43), and previous work has shown that LVs transfer genes in bona fide stem cells (44), including bone precursors (45). Although unidentified, the osteoprogenitor targeted by MET likely belong to the family of mesenchymal stem cells, which are also the precursors of rhabdomyosarcomas.…”

Section: Discussionmentioning

confidence: 99%

MET Overexpression Turns Human Primary Osteoblasts into Osteosarcomas

et al. 2006

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The MET oncogene was causally involved in the pathogenesis of a rare tumor, i.e., the papillary renal cell carcinoma, in which activating mutations, either germline or somatic, were identified. MET activating mutations are rarely found in other human tumors, whereas at higher frequencies, MET is amplified and/or overexpressed in sporadic tumors of specific histotypes, including osteosarcoma. In this work, we provide experimental evidence that overexpression of the MET oncogene causes and sustains the full-blown transformation of osteoblasts. Overexpression of MET, obtained by lentiviral vector-mediated gene transfer, resulted in the conversion of primary human osteoblasts into osteosarcoma cells, displaying the transformed phenotype in vitro and the distinguishing features of human osteosarcomas in vivo. These included atypical nuclei, aberrant mitoses, production of alkaline phosphatase, secretion of osteoid extracellular matrix, and striking neovascularization. Although with a lower tumorigenicity, this phenotype was superimposable to that observed after transfer of the MET gene activated by mutation. Both transformation and tumorigenesis were fully abrogated when MET expression was quenched by short-hairpin RNA or when signaling was impaired by a dominant-negative MET receptor. These data show that MET overexpression is oncogenic and that it is essential for the maintenance of the cancer phenotype. (Cancer Res 2006; 66(9): 4750-7)

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“…26 We are aware that other constitutive promoters like elongation factor 1 would allow an even better expression, but no direct comparisons have been reported in literature yet. 27,28 Analysis and comparison of the BLI and FLI performances in vivo were possible, since fusion of the reporters ensured equimolar expression in cells. Our results showed that the limit of detection using BLI is approximately 1 Â 10 3 hMSCs in the brain cortex using a dose of 25 mM/kg of D-Luc.…”

Section: Discussionmentioning

confidence: 99%

Optimized longitudinal monitoring of stem cell grafts in mouse brain using a novel bioluminescent/near infrared fluorescent fusion reporter

et al. 2017

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Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood-brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 mM/kg or 943 mM/kg) or CycLuc1 (25 mM/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 Â 10 5 cells using both fluorescence and BLI. The highest bioluminescent signals (*1 Â 10 7 photons per second) were achieved 15 min after the injection of D-Luc (943 mM/kg). This allowed us to monitor as low as 1 Â 10 5 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.

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Morphological Analysis and Lentiviral Transduction of Fetal Monkey Bone Marrow-Derived Mesenchymal Stem Cells

Cited by 47 publications

References 47 publications

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

MET Overexpression Turns Human Primary Osteoblasts into Osteosarcomas

Optimized longitudinal monitoring of stem cell grafts in mouse brain using a novel bioluminescent/near infrared fluorescent fusion reporter

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