After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgenefree iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.regenerative medicine | nonintegrating RNA vector

SUMMARYMolecular mechanisms that generate distinct tissue layers in plant shoots are not well understood. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer, beginning at an early stage of development. The promoters of many epidermis-specific genes, including ATML1, contain an ATML1-binding site called an L1 box, suggesting that ATML1 regulates epidermal cell fate. Here, we show that overexpression of ATML1 was sufficient to activate the expression of epidermal genes and to induce epidermis-related traits such as the formation of stomatal guard cells and trichome-like cells in non-epidermal seedling tissues. Detailed observation of the division planes of these ectopic stomatal cells suggested that a near-surface position, as well as epidermal cell identity, were required for regular anticlinal cell division, as seen in wild-type epidermis. Moreover, analyses of a loss-of-function mutant and overexpressors implied that differentiation of epidermal cells was associated with repression of mesophyll cell fate. Collectively, our studies contribute new information about the molecular basis of cell fate determination in different layers of plant aerial organs.

No abstract

Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61–77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.