After serial passaging of ESC-like colonies, extensive spontaneous differentiation was observed. applications. receptor (Omerbasic et?al., 2016) and extreme resistance to hypoxia through fructose metabolism to avoid tissue damage (Park et?al., 2017). In addition, these animals do not maintain stable body temperature, can live at low oxygen and high carbon dioxide concentrations in the atmosphere, and show other features that are useful for biomedical research (Edrey et?al., 2011, Kim et?al., 2011). Open in a separate window Physique?1 Generation of Naked Mole Rat iPSCs (A) Naked mole rat (expression and mutation in the oncogene. They used CP-409092 a conventional human culture condition to derive NMR iPSCs and found that they could be generated at high oxygen and with a low efficiency of reprogramming in the case of adult fibroblasts. Moreover, chimeric contribution of NMR iPSCs has not been examined that would further support their pluripotency. Here, we report the development of NMR iPSCs from embryonic and adult fibroblasts using drug-inducible expression of OSKM with high efficiency. HVH-5 The iPSCs displayed the pluripotency and some non-canonical features such as a propensity for a tetraploid karyotype and resistance to forming teratomas. Interestingly, these?iPSCs contributed to interspecific chimera despite differences in physiological temperature and phylogenetic distance. Moreover, the transcriptomes of NMR iPSCs were more similar to those of human than mouse?iPSCs. These cells and the associated protocols should pave the way for generation of gene-targeted NMR models for biomedical research and provide much-needed cell culture systems to facilitate aging and cancer-related research at the cellular and molecular levels. Results Conventional Protocols that Support Preparation of Mouse iPSCs Do Not Favor NMR Cell Reprogramming To reprogram NMR cells, we employed a doxycycline-inducible lentiviral system, in which mouse or human OSKM were inserted downstream of a tetracycline operator (Carey et?al., 2009, Hockemeyer et?al., 2008). We first used?NMR embryonic fibroblasts (45?days postcoitum) (Physique?1A, top) and maintained them in a conventional mouse ESC medium following transduction (Physique?1A, bottom, blue letters). Reprogramming of somatic cells toward iPSCs is usually thought to proceed through three phases: initiation, maturation, and stabilization (Plath and Lowry, 2011). The initiation phase is marked by the mesenchymal-to-epithelial transition (MET) and bone morphogenic protein signaling (Li et?al., 2010, Samavarchi-Tehrani et?al., 2010), and E-cadherin impeding reprogramming (Chen et?al., 2010). and represent early markers that predict an eventual reprogramming event, whereas endogenous is usually a late-phase reprogramming factor (Buganim et?al., 2012). To gain insights into MET and NMR cell reprogramming, we quantified NMR-specific E-cadherin (transcripts on day 6 following transduction (Physique?1A; bottom). Mouse OSKM-transduced NMR cells showed higher expression of CP-409092 these transcripts compared with cells transduced with human OSKM, although both approaches induced expression of the marker genes. In particular, the levels were 30-fold higher than in control fibroblasts (Physique?S1A). We further found that cytokine treatment increased the expression of reprogramming-related genes, with LIF (leukemia inhibitory factor) being?a stronger inducer than basic fibroblast growth factor (Physique?S1B). Hence, mOSKM and LIF were chosen for further experiments. We screened for changes in marker gene expression until day 24 and also analyzed the initial reprogramming genes and (Physique?1B). expression increased starting from day 3 and was maximal at day 24. increased at day 6 and gradually decreased until day 24. were dramatically increased from days 3C6 and gradually decreased to day 24. was gradually increased to day 24. Thus, transcription factors and cytokines could alter reprogramming-associated gene expression in NMRs. With the same method, we generated mouse iPSCs from embryonic and adult fibroblasts (Figures S1C and S1D). However, NMR cells showed no visible morphological changes until day 24 (Physique?S1E), when we detected OCT4-expressed cells (Physique?S1F). Nevertheless, this approach did not result in viable ESC-like NMR colonies, suggesting that the conventional protocols, which readily support preparation of mouse iPSCs, are unsatisfactory for deriving NMR iPSCs. Development of Optimal Protocols to Support Generation of NMR iPSCs SV40 large T antigen has been reported to improve the efficiency of iPSC generation (Park et?al., 2008). Reducing p53 expression can also improve this process (Mali et?al., 2008, Utikal et?al., 2009, Hong et?al., 2009, Hanna et?al., 2009a). In fact, SV40 large T antigen may support iPSC generation by inhibiting p53 expression (Bao et?al., 2011). Also, unlike mouse cells, rat ESCs and iPSCs require specific culture conditions, such as serum-free defined culture CP-409092 medium (N2B27) with inhibition of the MEK (mitogen-activated protein kinase)/ERK (extracellular signal regulated kinases?1 and 2) pathway and glycogen synthase kinase 3 (GSK3) by small synthetic drugs PD0325901 (PD).
After serial passaging of ESC-like colonies, extensive spontaneous differentiation was observed