Efficient Transfection of Embryonic and Adult Stem Cells

Abstract

The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid-based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10-fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard-to-transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue-specific differentiation of stem cells. Stem

A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells

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Electrophysiological evaluation of engrafted stem cell-derived neurons

“…(ii) the specifics of construct and driving promoters [36][37][38][39][40][41] , as well as (iii) the transfection method employed and the resulting integration sites [42][43][44][45] . Single-cell RT-PCR analysis as well as post hoc immunohistochemistry could be used to exclude subpopulation analysis and to demonstrate additional features of recorded cells.…”

Advancements in reprogramming strategies for the generation of induced pluripotent stem cells

“…Gonzales et al [61] simplified the protocol by the delivery of "all-inone" single plasmid containing all the four reprogramming factors, using nucleofection transfection technology. Nucleofection is a proprietary technology provided by Amaxa [Cologne, Germany] based on electroporation technique that combines specific cell-type solutions with specific electrical parameters generated by the Nucleofector™ device to deliver DNA directly to the nucleus, which may consequently lead to enhanced gene expression [62]. Unlike the serial transfection procedure by Okita et al [60], the iPS cells generated using nucleofection did not show evidence of transgene insertion in their genome.…”