https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/159777/1/j.cell.2006.07.024.pdf

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

https://science.sciencemag.org/content/sci/318/5858/1917.full.pdf

Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells

Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

/pdf/epigenetic-regulation-of-gene-expression-how-the-genome-55fnpf16ec.pdf

Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals

We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.

/pdf/pluripotency-of-mesenchymal-stem-cells-derived-from-adult-200s4qlizf.pdf

Pluripotency of mesenchymal stem cells derived from adult marrow

Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

Viable offspring derived from fetal and adult mammalian cells

Cloning is the asexual reproduction of an individual, such that the offspring have an essentially identical nuclear genome. Nuclear transfer and cloning have been achieved in a number of species, namely sheep, cows, goats, rabbits, cats and mice, but have been largely unsuccessful, so far, in dogs, primates and rats. Clearly, contributory factors which affect the outcome of successful cloning experiments are not universally applicable to all species. One theme common to all cloning experiments, however, is the overall inefficiency of the process, typically 0-4%. A number of factors contribute to nuclear transfer inefficiency, and we will review mouse cloning experiments, which address these problems, highlighting the importance of donor nucleus choice (somatic or ES cell, fetal or adult, quiescent or actively dividing). Finally, we will summarize the emerging principles which appear to govern nuclear reprogramming and production of clones, and will consider the application of nuclear transfer to the rat.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2003.049742

Nuclear transfer in rodents.

Mammalian eggs await fertilisation while arrested at the second metaphase stage of meiotic division. A network of signalling pathways enables the establishment and maintenance of this metaphase-II arrest. In the absence of fertilisation, mammalian eggs can spontaneously exit metaphase II when parthenogenetically stimulated, or sometimes without any obvious stimulation. Ovulated rat eggs abortively release from metaphase-II arrest once removed from egg donors. Spontaneously activated rat eggs extrude the second polar body and proceed to the so-called metaphase III-'like' stage, with clumps of condensed chromatin scattered in the egg cytoplasm. It is still unclear what makes rat eggs susceptible to spontaneous activation; however, a vague picture of the signalling pathways involved in the process of spontaneous activation is beginning to emerge. Such cell cycle instability is one of the major reasons why it is more difficult to establish nuclear transfer in the rat. This review examines the known predisposing factors and biochemical mechanisms involved in spontaneous activation. The strategies used to prevent spontaneous metaphase-II release in rat eggs will also be discussed.

Rat eggs cannot wait: Spontaneous exit from meiotic metaphase-II arrest.

Revolutionary new opportunities for the modification of milk composition have been created by the development of methods for gene transfer and targeted mutation of genes may extend the range of opportunities still further. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue-specific hormonally induced expression in the mammary gland. Fragments of the ovine beta-lactoglobulin gene fused to cDNA for the human therapeutic proteins clotting factor IX and alpha-1 antitrypsin have directed production of these proteins in the milk of transgenic mice and sheep. Factor IX was biologically active and co-migrated with authentic proteins, but was present at too low a concentration for commercial exploitation. Recent observations suggest that fusion genes containing genomic clones direct production of higher concentrations of protein. Mouse whey acidic protein genomic sequences also directed production of low concentrations of human tissue plasminogen activator in the milk of transgenic mice. Targeted expression of this kind may be used for the production of therapeutic and industrial proteins, to increase the concentration or modify the nature of milk proteins, reduce the concentration of lactose, change the composition of fat or direct production of bacteriocidal proteins in milk in order to combat mastitis.

Modification of milk composition.

Although it is only a decade since the use of transgenic livestock for the production of pharmaceutical proteins in milk was first suggested, great progress has been made. There is every reason to expect that, as methods for the transfer of genomic clones are applied with efficient regulatory elements, many proteins will be produced at high concentrations in the milk of livestock species. In most cases, these proteins will be biologically active, however, it remains to be shown whether they will be identical in structure and function to the natural product.

Strategies for production of pharmaceutical proteins in milk

Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce 'induced pluripotent stem cells' is providing revolutionary new opportunities in research and cell therapy.

https://royalsocietypublishing.org/doi/pdf/10.1098/rstb.2014.0366

Ian Wilmut

Papers

Nuclear transfer in rodents.

Rat eggs cannot wait: Spontaneous exit from meiotic metaphase-II arrest.

Modification of milk composition.

Strategies for production of pharmaceutical proteins in milk

Somatic cell nuclear transfer: origins, the present position and future opportunities.