Document Detail


Long-Term Engraftment and Angiogenic Properties of Lentivirally Transduced Adipose Tissue-Derived Stromal Cells.
MedLine Citation:
PMID:  22492300     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Human adipose tissue-derived stromal cells (ADSCs) are being evaluated for cardiovascular repair. We developed an ex vivo method for producing angiogenic ADSCs transduced with a self-inactivating lentiviral vector (LV) expressing the enhanced green fluorescence protein (EGFP) from an internal cytomegalovirus (CMV) promoter to track these cells after in vivo engraftment. ADSCs from visceral adipose tissue were transduced using a LV incorporating the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) sequences and the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance EGFP gene expression. We compared infection protocols with non-concentrated lentiviral supernatant or pellet fractions after ultracentrifugation, testing transduction efficiency, and reporter gene expression by quantitative flow cytometry at 5 and 28 days. Transduction of ADSCs with pellet after ultracentrifugation provided the highest transduction rate [flow cytometry titers: 6.5 ± 0.3 × 10(5) transduction units (TU)/mL and 20 ± 1.2 × 10(6) TU/mL at day 5 with non-concentrated lentiviral supernatant and pellet, respectively, with titer in the supernatant after ultracentrifugation remaining undetectable]. Reporter gene expression did not affect cell viability, morphology, proliferation, differentiation, self-renewal, or angiogenic activity. Furthermore, reporter gene expression did not significantly affect Fas/CD95-induced apoptosis. The in vivo implantation of transduced ADSCs into a mouse ischemic leg model resulted in efficient engraftment and angiogenesis. ADSC gene labeling using LVs is feasible and efficient, without impairment of stem cell characteristics, cell engraftment, and angiogenic activity. Such transduced ADSCs can be efficiently tracked in vitro and in vivo and may serve as vehicle for therapeutic genes.
Authors:
Rosalinda Madonna; Roberto Bolli; Gregg Rokosh; Raffaele De Caterina
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-4-11
Journal Detail:
Title:  Molecular biotechnology     Volume:  -     ISSN:  1559-0305     ISO Abbreviation:  -     Publication Date:  2012 Apr 
Date Detail:
Created Date:  2012-4-11     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9423533     Medline TA:  Mol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Institute of Cardiology and Center of Excellence on Aging, "G. d'Annunzio" University Chieti, C/o Ospedale SS. Annunziata, Via dei Vestini, 66013, Chieti, Italy.
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