https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/49782/1/Yamanaka_Cell_131_5.pdf

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

If it were possible to reprogram differentiated human somatic cells into a pluripotent state, patient-specific and disease-specific stem cells could be developed. Previous work generated induced pluripotent stem (iPS) cells capable of germline transmission from murine somatic cells by transd

/pdf/induction-of-pluripotent-stem-cells-from-adult-human-3jc4cxk5x5.pdf

Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors

Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.

http://web.mit.edu/7.31/restricted/pdfs/F05_and_earlier/Nichols.pdf

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells

Chimaeric combinations of normal and mutant embryonic tissues were used to investigate the lethal effect of the yellow gene. The homozygous mutant embryos could not be identified before implantation. Therefore, embryos from both intercross matings and control backcross matings were used to provide inner cell masses (ICMs) for injection into genetically marked blastocysts of the CFLP random bred stock. All conceptuses obtained from reimplanted blastocysts were analysed at mid-gestation for the presence of donor isozyme of glucose phosphate isomerase. A similar proportion of chimaeras were found in the experimental and control series, indicating rescue of the lethal Ay/Ay ICM tissue. The reciprocal experiment also produced a similar proportion of chimaeras but there was a 25% postimplantational loss of injected embryos evidenced by empty decidual swellings. The results suggest that the yellow mutation primarily affects the trophectoderm which cannot be rescued by a normal ICM, whereas Ay/Ay ICM is capable of survival in a chimaera at least until mid-gestation.

Investigation of the lethal yellow Ay/Ay embryo using mouse chimaeras.

BACKGROUND: Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent relationship to the zygote’s bilateral plane. METHODS: Spaced oil drops were injected into the zona pellucida to delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the zygotes within the zona pellucida. RESULTS: Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship to the zygote’s bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane. CONCLUSIONS: Bilateral symmetry may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.

An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse

Several cell and tumour lines were isolated from a single-embryo-derived teratocarcinoma and their karyotypes and differentiation in adult hosts recorded. The majority of cells contained normal karyotypes by banding. The cells were injected into blastocysts and although they sometimes colonized the yolk sac, they never colonized the embryo. Thus the possession of a normal karyotype is not a sufficient condition for embryo colonization. The loss of growth capacity was investigated by studying differentiation and tumourigenicity in a variety of circumstances. The change in appearance from an EC cell morphology to a big flat cell in culture leads to retardation of growth in adult hosts. When EC cells are injected into a blastocyst, the ability to grow progressively both in culture and in adult hosts is lost.

Growth and differentiation of an embryonal carcinoma cell line (C145b).

Disclosed are embryonic stem cell-derived dendritic cells, genetically modified immature dendritic cells capable of maturation, as well as methods for the production of such cells. In one embodiment, the cells made be produced by a method comprising the steps of providing a population of embryonic stem cells; culturing the embryonic stem cells in the presence of a cytokine or combination of cytokines which brings about differentiation of the embryonic stem cells into dendritic cells; and recovering the dendritic cells from the culture. In a further embodiment, the cells may be genetically modified.

Method for producing dendritic cells

The results of experiments in which horseradish peroxidase (HRP) was used to mark single trophectoderm or inner cell mass (ICM) cells in situ in mouse blastocysts have led to the proposal that growth of the trophectoderm depends on stem cells located in the inner cell mass. Thus, the finding that the visual centre of clones formed following labelling of the central polar trophectoderm cell in early or expanding blastocysts was consistently shifted towards or into the mural trophectoderm was attributed to their displacement by ICM-derived cells. However, the frequency with which central polar cells were displaced is likely to have been overestimated by using the visual centre of descendant clones as the index of their location. Also, the possibility that displacement of central polar cells was an artefact of the marked temporary interruption of their cycling that resulted from labelling was not discounted. Furthermore, no attempt was made to ascertain whether cells located elsewhere in the polar trophectoderm also moved murally, as expected if there is a general displacement of such cells. In the present study, labelling of either the central or a peripheral polar trophectoderm cell with HRP was achieved without obviously perturbing their subsequent proliferation. Moreover, displacement was assessed by recording the location of the proximal boundary rather than the visual centre of the resulting clones. Even by this conservative criterion, the majority of labelled central cells moved towards or into the mural trophectoderm. In marked contrast, however, labelled peripheral polar cells moved murally in only a minority of cases. The remainder either retained their original position or moved towards rather than away from the central polar region. Such an anisotropic pattern of growth of the polar trophectoderm is not readily explicable in terms of recruitment of cells from the ICM. Rather, it accords with the view that the polar trophectoderm is a proliferative centre, and suggests that movement murally of its surplus cells may be restricted circumferentially, possibly through anchorage of the junctional trophectoderm cells that extend processes over the free surface of the ICM.

/pdf/clonal-analysis-of-growth-of-the-polar-trophectoderm-in-the-3qi13awpud.pdf

Richard L. Gardner

Papers

Investigation of the lethal yellow Ay/Ay embryo using mouse chimaeras.

An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse

Growth and differentiation of an embryonal carcinoma cell line (C145b).

Method for producing dendritic cells

Clonal analysis of growth of the polar trophectoderm in the mouse.