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 《-》

Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells.

Kunisato A ，Wakatsuki M ，Kodama Y ，Shinba H ，Ishida I ，Nagao K ... -  《-》

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 ... -  《-》

Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.

Zhou W ，Freed CR 《-》

Successful Derivation of an Induced Pluripotent Stem Cell Line from a Genetically Nonpermissive Enhanced Green Fluorescent Protein-Transgenic FVB/N Mouse Strain.

Abbey D ，Singh G ，Verma I ，Derebail S ，Kolkundkar U ，Chandrashekar DS ，Acharya KK ，Vemuri MC ，Seshagiri PB ... -  《-》