mRNA exhibited much higher transfection efficiency and less cytotoxicity than DNA. Fluorometholone SOX2, cMYC, KLF4 and SV40 large T (LT) and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES) cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5-aza-2-deoxycytidine) and cultured in human embryonic stem cell (ES) medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. == Conclusion/Significance == Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells. == Introduction == An approach to reprogram cell fate without genetic modification would be very useful for regenerative medicine. Currently, most methodologies go through DNA-based routes, with foreign genetic materials either permanently left in the genome of resulting cells, or later removed or lost after multiple rounds of cell division. In all the cases, stringent genome wide tests are needed to confirm the absence of potentially Fluorometholone harmful insertional mutagenesis[1],[2]. Protein transduction of recombinant transcription factors has been used for reprogramming[3], but proteins produced in bacteria may be mis-folded and lack essential modifications that only occur in mammalian cells, so that their in vivo functionality may be compromised. Kim and colleagues reported the generation of human iPS cells by incubating fibroblasts with lysates from HEK 293T cells expressing recombinant OCT4, SOX2, KLF4 and cMYC[4]. However, cell lysates contain many poorly defined factors that could also be taken up by the reprogrammed cells and give unpredictable consequences. Thus this method will be difficult to implement in clinical settings. While chemical compounds have been used to regulate cell fate or alter DNA and chromatin modifications, to date, no reprogramming or trans-differentiation has been achieved by small molecules alone[5]. An mRNA-based approach could offer several advantages: first, it does not lead to any genetic modification of the host genome. mRNAs are directly translated into functional proteins in the cytoplasm with proper mammalian post-translational modifications which would result in much higher functionality than recombinant proteins produced in the bacteria. Second, mRNAs are much smaller than DNAs, and as single strand nucleic acids without any flanking plasmid sequences they Rabbit Polyclonal to OR5I1 can be introduced into cells with higher efficiency and much lower cytotoxicity. It is also easier to combine several different mRNAs and to control their dosage than using multiple or multi-cistronic DNA constructs. An obvious disadvantage of mRNAs is that they are degraded by the cell in 2-3 days so that the expression window is very short. Nevertheless, an mRNA based approach could be a useful means to regulate cellular function, and to mediate trans-differentiation such as from fibroblast to neurons or cardiomyocytes[6],[7]that require shorter time. mRNA transfection has been used in hematopoietic progenitor cells, mesenchymal stroma cells, antigen presenting dendritic cells and lymphocytes[8],[9],[10],[11]. Activated B cells transfected with mRNAs encoding co-stimulatory molecules, cytokines and antigen showed enhanced proliferation and were able to induce antigen-specific cytotoxic Fluorometholone T lymphocyte responses[10]. dendritic cells transfected with mRNAs of viral antigen stimulated robust and specific T cell response[12]. Moreover, in a phase I/II clinical trial of dendritic cells vaccine, autologous dendritic cells loaded with autologous melanoma mRNA as tumor antigen produced vaccine specific response in the majority of patients[13]. This trial also showed that cells transfected with mRNA are safe for use in patients. In the present study, we set out to test the feasibility of using mRNA to Fluorometholone induce pluripotency. We found that microporation is highly effective for mRNA transfection. Moreover, Fluorometholone transient expression of OCT4, SOX2, KLF4, cMYC and LT together with small molecule treatment significantly increased the expression of embryonic stem cell specific genes in fibroblast cells. == Methods == == Ethics Statement == HuF1 (XX) was derived from an abortus obtained from a patient undergoing 1st trimester fetal termination using Mefipristone. The project was approved by the South Sheffield Research Ethics Committee (SSREC) and a fully informed patient consent (written) was obtained according to local and national guidelines. == Cell culture == Three human fibroblast lines were used in this study. HuF1 (XX, passage 4) is a human fetal skin fibroblast cell line (source described above). The derivation procedure was as follows: fetal skin was recovered.