Rapid and efficient reprogramming of human amnion-derived cells into pluripotency by three factors OCT4/SOX2/NANOG.

Reprogramming human somatic cells to pluripotency represents a valuable resource for research aiming at the development of in vitro models for human diseases and regenerative medicines to produce patient-specific induced pluripotent stem (iPS) cells. Seeking appropriate cell resources for higher efficiency and reducing the risk of viral transgene activation, especially oncogene activation, are of significance for iPS cell research. In this study, we tested whether human amnion-derived cells (hADCs) could be rapidly and efficiently reprogrammed into iPS cells by the defined factors: OCT4/SOX2/NANOG. hADCs from normal placenta were isolated and cultured. The 3rd passage cells were infected with the lentiviral vectors for the delivery of OCT4, SOX2, and NANOG. Afterwards, the generated iPSCs were identified by morphology, pluripotency markers, global gene expression profiles, and epigenetic status both in vitro and in vivo. The results showed that we were able to reprogram hADCs by the defined factors (OCT4/SOX2/NANOG). The efficiency was significantly high (about 0.1%), and the typical colonies appeared on the 9th day after infection. They were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and the epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into various cell types of all three germ layers both in vitro and in vivo. These results demonstrate that hADCs were an ideal somatic cell resource for the rapid and efficient generation of iPS cells by OCT4/SOX2/NANOG.

Zhao HX ，Li Y ，Jin HF ，Xie L ，Liu C ，Jiang F ，Luo YN ，Yin GW ，Li Y ，Wang J ，Li LS ，Yao YQ ，Wang XH ... -  《-》

Generation of human-induced pluripotent stem cells from gut mesentery-derived cells by ectopic expression of OCT4/SOX2/NANOG.

Li Y ，Zhao H ，Lan F ，Lee A ，Chen L ，Lin C ，Yao Y ，Li L ... -  《-》

Analysis of co-expression of OCT4, NANOG and SOX2 in pluripotent cells of the porcine embryo, in vivo and in vitro.

To derive porcine embryonic stem (ES) cell lines, the time window during which porcine embryos contain pluripotent cells that are predisposed to undifferentiated self-renewal in vitro must be identified. Therefore we first studied the spatial and temporal expression pattern of key factors in pluripotency and lineage segregation of blastocyst-stage porcine embryos between embryonic days (E) 6.5 and E10.5 using whole mount in situ hybridization, quantitative reverse transcription (RT)-PCR and whole mount immunofluorescence. Expression of NANOG and SOX2 was detected in both the ICM and epiblast, while OCT4 expression became restricted to the epiblast at E9.5. Surprisingly ICM and epiblast cells also expressed CK18. Consequently, growth factors which sustain the undifferentiated growth of human ES cells and mouse epiblast stem cells (EpiSCs) were tested for their ability to sustain undifferentiated self-renewal of porcine ICM and epiblast cells in vitro. Cultures of ICM cells resulted in a higher percentage of primary colonies with an ES-like morphology compared to primary cultures derived from epiblast cells. These undifferentiated colonies sustained expression of OCT4, NANOG, SOX2 and CK18. The expression of CK18 suggests that these cells are more similar to human ES cells and mouse EpiSCs than to mouse ES cells. Although undifferentiated cultures were maintained for limited passages, ICM and epiblast cultures rapidly differentiated into cell types of mesodermal, ectodermal, and endodermal origin, as characterized by RT-PCR. These results demonstrate that porcine ICM and epiblast cells can not be cultured in vitro with currently used human ES cell culture conditions. Importantly however, the trio of OCT4, NANOG and SOX2, which are known to form an autoregulatory network for pluripotency in other systems, are co-expressed also by porcine epiblasts, and by undifferentiated primary colonies in culture.

du Puy L ，Lopes SM ，Haagsman HP ，Roelen BA ... -  《-》

The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines.

Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc. Given that iPS cell technology may be useful for medical applications, the quality of iPS cells needs to be maintained during prolonged cultivation. However, it is unclear whether there are any differences in stability among different iPS clones. We infected mouse embryonic and adult fibroblasts with retroviruses encoding Oct4, Sox2, Klf4, c-Myc, and green fluorescent protein (GFP). We obtained embryonic stem (ES) cell-like colonies with silenced retroviral transgenes and divided these colonies into two groups: ES cell-like colonies that underwent retroviral silencing (i) on around day 14 (called early iPS) or (ii) on around day 30 (called late iPS), after infection. We compared morphology, proliferation efficiency, pluripotency marker expression, and karyotype between early iPS and late iPS cells. Early iPS cells were more stable than late iPS cells. At passage 20, most of the early iPS clones maintained ES cell-like morphology, expressed pluripotency markers, and showed proliferation efficiency similar to ES cells. Furthermore, early iPS clones derived from both embryonic and adult fibroblasts gave rise to chimeras and could show germ line competency. In contrast, late iPS clones tended to lose their ES cell-like morphology and normal karyotype in prolonged culture. Our results provide useful information on the efficient derivation of stable iPS cells that may be useful for germline transmission in mouse. This study suggests that early completion of full reprogramming allows for superior iPS cell generation.

Okada M ，Yoneda Y 《-》

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Verma R ，Holland MK ，Temple-Smith P ，Verma PJ ... -  《-》