Abstract
The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.
Highlights
The feasibility of reprogramming somatic cells to induced pluripotent stem cells [1,2,3,4] has led to the possibility of developing disease-specific iPS cells for improved disease modeling in vitro [5,6,7] and potential use in clinical applications [8, 9]
Human dermal Huntington fibroblasts were transfected with different factor combinations including oct4 + sox2 + klf4 (OSK), oct4 + nanog + sox2 (ONS), and oct4 + nanog + hTERT (ONT), oct4 + nanog + cmyc (ONM), oct4 + nanog + klf4 (ONK), oct4 + nanog + sox2 + hTERT + klf4 + c-myc (ONSTKM)
All factors in different combinations were transfected in a ratio of 1 : 1. The foreskin fibroblasts were transfected with OSK exclusively
Summary
The feasibility of reprogramming somatic cells to induced pluripotent stem cells (iPS) [1,2,3,4] has led to the possibility of developing disease-specific iPS cells for improved disease modeling in vitro [5,6,7] and potential use in clinical applications [8, 9]. The potential therapeutic application of initial iPS cell lines was hampered by the fact that applied methods of iPS cell derivation modified the host genome through the integration of DNA sequences [3, 10,11,12,13,14,15]. It has been shown that the modified mRNA-mediated delivery of reprogramming factors based on nucleofection is an efficient and nontoxic alternative approach to cell modification [18] which has recently facilitated the derivation of iPS cell lines [19,20,21]. We investigate in vitro transcribed mRNA transfection as a method for producing iPS cells that does not bear any risk with respect to genetic modification. We show the reprogramming of fibroblasts from Huntington disease donors, the use of alternative reprogramming cocktails, alternative factor combinations, and the application of different mRNA-transfection techniques
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