- 作者: Tamara V. Tsulaia Nicole L. Prokopishyn Aqing Yao N.D. Victor Carsrud M. Clara Carou David B. Brown Brian R. Davis Judith Yannariello-Brown
- 作者服務機構: Department of Human Biological Chemistry and Genetics, and Microbiology and Immunology, and Sealy Center for Oncology and Hematology, University of Texas Medical Branch, Galveston, Tex., Gene-Cell, Inc., Houston, Tex., and Tissue Transformation Technologies, Inc., Edison N.J., USA
- 中文摘要: --
- 英文摘要: Human mesenchymal stem cells (hMSCs) are multipo-tent cells that can differentiate into various tissue types,including bone, cartilage, tendon, adipocytes, and mar-row stroma, making them potentially useful for humancell and gene therapies. Our objective was to demon-strate the utility of glass needle-mediated microinjectionas a method to deliver macromolecules (e.g. dextrans,DNA) to hMSCs for cell and molecular biological studies.hMSCs were isolated and cultured using a specific fetalbovine serum, prescreened for its ability to promote celladherence, proliferation, and osteogenic differentiation.Successful delivery of Oregon Green-dextran via intra-nuclear microinjection was achieved, yielding a postin-jection viability of 76 ± 13%.Excellent short-term geneexpression (63 ± 11%) was achieved following microin-jection of GFP-containing vectors into hMSCs. Higherefficiencies of short-term gene expression(~5-fold)were observed when injecting supercoiled DNA,pYA721, as compared with the same DNA construct in alinearized form, YA721.Approximately 0.05% of hMSCsinjected with pYA721 containing both the GFP and neo-mycin resistance genes formed GFP-positive, drug-resis-tant colonies that survived>120 days. Injection of linear-ized YA721 resulted in 3.6% of injected hMSC formingdrug-resistant colonies, none of which expressed GFPthat survived 60-120 days. These studies demonstratethat glass needle-mediated microinjection can be usedas a method of delivering macromolecules to hMSCsand may prove to be a useful technique for molecularand cell biological mechanistic studies and future genet-is modification of hMSCs.
- 中文關鍵字: --
- 英文關鍵字: Gene dilivery methods. Gene Therapy. Mesenchymal Stem cells. Microinjection