Immunoblot analyses were performed with whole cell lysates (100g protein each) from the indicated mPanstra stem cell strains grown in their maintenance medium. properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing. == 1. Introduction == Direct reprogramming of somatic cells into pluripotent stem cells (termed induced pluripotent stem cells (iPSCs)) has been achieved by inducing the expression of transcription factors associated with pluripotent cell states and cell transformation [1]. The first successful attempt used Rabbit Polyclonal to Serpin B5 over-expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, to convert somatic fibroblasts to iPSCs [24]. In subsequent studies, iPSCs were generated by overexpressing only the first three of these factors, and combinations using Cephalexin monohydrate other transcription factors (e.g., Nanog, Lin28, Nr5a2, Esrrb) were also successful [58]. Progress in iPSC technology development has faced two main problems: (1) risk of cell transformation and mutation by transduced gene expression constructs and (2) poor efficiency. Transduction of somatic cells with synthetic mRNAs or proteins has addressed the gene transfer problem [911]. Other strategies have been the use of one or two of the transcription factors in combination with small molecules to achieve reprogramming [1214]. On the other hand, the low efficiency of reprogramming continues to present a major challenge, especially with Cephalexin monohydrate gene-free strategies. With the stochastic model for reprogramming now generally accepted [15], Cephalexin monohydrate one approach to increasing reprogramming efficiency might be the use of cell populations whose active genetic networks inherently share similarities with those of pluripotent stem cells. Unipotent and multipotent tissue-specific stem cells may have this character. Previous iPSC studies have focused mainly on transcription factor combinations necessary for the induction and maintenance of a pluripotent state. To the authors’ knowledge, the possibility of tissue stem cells being involved as a predisposed population, though considered, previously has not been investigated specifically. In the closest example on the topic, cultures enriched for neural stem cells were shown to be more efficiently reprogrammed than routinely used fibroblast populations [1618]. However, this property was attributed to the expression of complementing factors in these cells, instead of their tissue stem cell phenotypeper se. Other specific investigations of the role of tissue stem cells may have been discouraged by the well-known difficulty of obtaining Cephalexin monohydrate quantities of these cells sufficient for informative experimental analyses [19]. Here, we report the investigation of readily available adult mouse pancreatic stem cell strains for predisposition for pluripotency reprogramming. These clonal tissue stem cells were expanded by the method of suppression of asymmetric cell kinetics (SACK). The SACK method is based on the concept that tissue stem cells are difficult to expandex vivobecause of their inherent asymmetric self-renewal kinetics [19,20].In vivo, asymmetric self-renewal allows tissue stem cells to maintain their Cephalexin monohydrate own number while continuously replenishing differentiated tissue cells. However,in vitro, the same process leads to the dilution and loss of tissue stem cells among their accumulating lineage-committed progeny cells. The SACK method uses specific guanine ribonucleotide precursors to shift tissue stem cells reversibly from asymmetric self-renewal kinetics to symmetric self-renewal kinetics, which promotes their exponential expansion in culture [21]. We found that, when simply transferred to culture conditions used for pluripotent stem cells, SACK-expanded pancreatic stem cells acquired properties of pluripotent stem cells. Moreover, inclusion of xanthine (Xn), the SACK agent used for their derivation, significantly increased their acquisition of properties characteristic of pluripotency. These findings suggest that targeting symmetrically self-renewing tissue stem cells could significantly increase the efficiency of pluripotency reprogramming when combined with other reprogramming strategies. == 2. Materials and Methods == == 2.1. Cell Culture == Mouse pancreatic cell strain (mPanstra) cells; (seeSection 3for detailed description) were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% dialyzed fetal bovine serum (DFBS; GIBCO, Invitrogen, Carlsbad, Calif, USA), 100 U/mL penicillin, 100g/mL streptomycin, and 1 mM xanthine (Xn, Sigma Chemical Co., St. Louis, Mo, USA), at 37C in a humidified incubator with 5% CO2. STO fibroblasts (American Type Culture Collection, Manassas,.