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Robert C Robbins - One of the best experts on this subject based on the ideXlab platform.

  • safe Genetic Modification of cardiac stem cells using a site specific integration technique
    Circulation, 2012
    Co-Authors: Feng Lan, Junwei Liu, Kazim H Narsinh, Leng Han, Andrew S Lee, Marisa Karow, Patricia K Nguyen, Divya Nag, Michele P Calos, Robert C Robbins
    Abstract:

    Background—Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable Genetic Modification of the cells ex vivo. Safe Genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results—We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared...

  • cell transplantation and tissue regeneration safe Genetic Modification of cardiac stem cells using a site specific integration technique
    2012
    Co-Authors: Kazim H Narsinh, Marisa Karow, Patricia K Nguyen, Michele P Calos, Robert C Robbins, Shijun Hu, Joseph C Wu
    Abstract:

    Feng Lan, Junwei Liu, Kazim H. Narsinh, Shijun Hu, Leng Han, Andrew S. Lee, Marisa Karow, Technique Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2012 American Heart Association, Inc. All rights reserved. is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation doi: 10.1161/CIRCULATIONAHA.111.084913 2012;126:S20-S28 Circulation. http://circ.ahajournals.org/content/126/11_suppl_1/S20 World Wide Web at: The online version of this article, along with updated information and services, is located on the

Chisa Hidaka - One of the best experts on this subject based on the ideXlab platform.

  • osteoblastic differentiation of human and equine adult bone marrow derived mesenchymal stem cells when bmp 2 or bmp 7 homodimer Genetic Modification is compared to bmp 2 7 heterodimer Genetic Modification in the presence and absence of dexamethasone
    Journal of Orthopaedic Research, 2010
    Co-Authors: Ryan S Carpenter, Laurie R Goodrich, David D Frisbie, John D Kisiday, Beth Carbone, Wayne C Mcilwraith, Christopher J Centeno, Chisa Hidaka
    Abstract:

    Bone marrow-derived mesenchymal stem cells (BMDMSCs) have been targeted for use in enhancement of bone healing; and their osteogenic potential may be further augmented by genes encoding bone morphoGenetic proteins (BMP's). The purpose of this study was to compare the effect of Genetic Modification of human and equine BMDMSCs with BMP-2 or -7 or BMP-2 and -7 on their osteoblastogenic differentiation in the presence or absence of dexamethasone. The BMDMSCs were harvested from the iliac crest of three human donors and tuber coxae of three equine donors. Monolayer cells were Genetically modified using adenovirus vectors encoding BMP-2, -7 or both and cultured in the presence or absence of dexamethasone. Expression of BMPs was confirmed by enzyme linked immunosorbent assay (ELISA). To evaluate osteoblastic differentiation, cellular morphology was assessed every other day and expression and secretion of alkaline phosphatase (ALP), as well as expression levels of osteonectin (OSTN), osteocalcin (OCN), and runt-related transcription factor-2 (Runx2) were measured for up to 14 days. Human and equine BMDMSCs showed a capacity for osteogenic differentiation regardless of Genetic Modification or dexamethasone supplementation. Dexamethasone supplementation was more important for osteoblastogenic differentiation of equine BMDMSCs than human BMDMSCs. Genetic Modification of BMDMSCs increased ALP secretion with AdBMP-2 homodimer having the greatest effect in both human and equine cells compared to AdBMP 7 or AdBMP 2/7. BMP protein elution rates reached their maximal concentration between day 4 and 8 and remained relatively stable thereafter, suggesting that Genetically modified BMDMSCs could be useful for cell-based delivery of BMPs to a site of bone formation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1330–1337, 2010

  • Genetic Modification of chondrocytes with insulin like growth factor 1 enhances cartilage healing in an equine model
    Journal of Bone and Joint Surgery-british Volume, 2007
    Co-Authors: Laurie R Goodrich, Chisa Hidaka, Paul D Robbins, Christopher H Evans, Alan J Nixon
    Abstract:

    Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro . The purpose of this study was to determine whether arthroscopically-grafted chondrocytes Genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 107 AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 107 naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic Modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with Genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic Modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through Genetic Modification of chondrocytes prior to transplantation.

Thaddeus S Stappenbeck - One of the best experts on this subject based on the ideXlab platform.

  • in vitro expansion and Genetic Modification of gastrointestinal stem cells in spheroid culture
    Nature Protocols, 2013
    Co-Authors: Hiroyuki Miyoshi, Thaddeus S Stappenbeck
    Abstract:

    It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to Genetic Modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.

Michal J Besser - One of the best experts on this subject based on the ideXlab platform.

  • Genetic Modification of tumor infiltrating lymphocytes via retroviral transduction
    Frontiers in Immunology, 2021
    Co-Authors: Hadas Weinsteinmarom, Gideon Gross, Michal Levi, Hadar Brayer, Jacob Schachter, Orit Itzhaki, Michal J Besser
    Abstract:

    Adoptive T cell therapy (ACT) holds great promise for cancer treatment. One approach, which has regained wide interest in recent years, employs antitumor T cells isolated from tumor lesions ("tumor-infiltrating lymphocytes" or TIL). It is now appreciated that a considerable proportion of anti-melanoma TIL recognize new HLA-binding peptides resulting from somatic mutations, which occurred during tumor progression. The clinical efficacy of TIL can potentially be improved via their Genetic Modification, designed to enhance their survival, homing capacity, resistance to suppression, tumor killing ability and additional properties of clinical relevance. Successful implementation of such gene-based strategies critically depends on efficient and reproducible protocols for gene delivery into clinical TIL preparations. Here we describe an optimized protocol for the retroviral transduction of TIL. As the experimental system we employed anti-melanoma TIL cultures prepared from four patients, recombinant retrovirus encoding an anti-CD19 chimeric antigen receptor (CAR) as a model gene of interest and CD19+ and CD19- human cell lines serving as target cells. Transduction on day 7 of the rapid expansion protocol (REP) resulted in 69 ± 8% CAR positive TIL. Transduced, but not untransduced TIL, from the four patients responded robustly to CD19+, but not CD19- cell lines, as judged by substantial secretion of IFN-γ following co-culture. In light of the rekindled interest in antitumor TIL, this protocol can be incorporated into a broad range of gene-based approaches for improving the in-vivo survival and functionality of TIL in the clinical setting.

Takeshi Onitsuka - One of the best experts on this subject based on the ideXlab platform.