title: Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cells.
abstract: Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into various tissue types, including bone, cartilage, tendon, adipocytes, and marrow stroma, making them potentially useful for human cell and gene therapies. Our objective was to demonstrate the utility of glass needle-mediated microinjection as 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 fetal bovine serum, prescreened for its ability to promote cell adherence, proliferation, and osteogenic differentiation. Successful delivery of Oregon Green-dextran via intranuclear microinjection was achieved, yielding a postinjection viability of 76 +/- 13%. Excellent short-term gene expression (63 +/- 11%) was achieved following microinjection of GFP-containing vectors into hMSCs. Higher efficiencies of short-term gene expression ( approximately 5-fold) were observed when injecting supercoiled DNA, pYA721, as compared with the same DNA construct in a linearized form, YA721. Approximately 0.05% of hMSCs injected with pYA721 containing both the GFP and neomycin resistance genes formed GFP-positive, drug-resistant colonies that survived >120 days. Injection of linearized YA721 resulted in 3.6% of injected hMSC forming drug-resistant colonies, none of which expressed GFP that survived 60-120 days. These studies demonstrate that glass needle-mediated microinjection can be used as a method of delivering macromolecules to hMSCs and may prove to be a useful technique for molecular and cell biological mechanistic studies and future genetic modification of hMSCs.
authors.affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, USA. ttsulaia@hotmail.com
authors.forename: Tamara V
authors.initials: TV
authors.lastname: Tsulaia
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Prokopishyn
authors.forename: Aqing
authors.initials: A
authors.lastname: Yao
authors.forename: N D Victor
authors.initials: ND
authors.lastname: Carsrud
authors.forename: M Clara
authors.initials: MC
authors.lastname: Carou
authors.forename: David B
authors.initials: DB
authors.lastname: Brown
authors.forename: Brian R
authors.initials: BR
authors.lastname: Davis
authors.forename: Judith
authors.initials: J
authors.lastname: Yannariello-Brown
mesh_terms: D002478:Cells, Cultured; D003911:Dextrans / Q000493:pharmacokinetics*; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors / Q000493:pharmacokinetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008845:Microinjections / Q000379:methods*; D008856:Microscopy, Fluorescence
chemical_list: D003911:Dextrans; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins