Defining pluripotency

Retroviral marking of single human embryonic stem cells shows that cultures of these cells contain subpopulations with distinct functional properties.     

Capturing pluripotency

In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.     

Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.     

Primed for pluripotency

To date, pluripotent epiblast stem cells (EpiSCs) had only been derived from postimplantation mouse embryos. In this issue of Cell Stem Cell, Najm et al. (2011) demonstrate that EpiSCs can be routinely derived from preimplantation embryos, showing that both human and mouse blastocysts can produce the same class of primed pluripotent cells.     

Roads to pluripotency

One of the hottest technologies used for nuclear repro-gramming in these days is the induction of pluripotent stem (iPS) cells by defined molecules. This is exemplified by the recent generation of viable, live-born, and fertile mice through tetraploid complementation by Zhou's lab (Zhao et al., 2009), thus having demonstrated for the first time the true pluripotency of iPS cells as ES cells.     

Splicing up pluripotency

In this issue of Cell, Gabut and colleagues (2011) identify a new splice variant of FOXP1 that directly regulates the expression of pluripotency genes. It endows human embryonic stem cells with their pluripotent nature and is required for the reprogramming of somatic cells to induced pluripotent stem cells.     

Reprogramming of sheep fibroblasts into pluripotency under a drug-inducible expression of mouse-derived defined factors

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.