Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1990-05-31
1992-11-24
Doll, John J.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4352402, 4352403, C12N 500, C12N 506
Patent
active
051660652
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the use of a previously discovered and characterised molecule, leukaemia inhibitory factor (LIF), in the isolation and propagation of embryonic stem cells in vitro.
Embryonic stem (ES) cells, the pluripotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute normally to all tissues including the germline (for review see Robertson, E. J. (1986) Trends in Genetics 2:9-13). Not only can ES cells propagated in vitro contribute efficiently to the formation of chimaeras, including germline chimaeras, but in addition, these cells can be manipulated in vitro without losing their capacity to generate germ-line chimaeras (Robertson, E. J. et. al. (1986) Nature 323:445-447).
ES cells thus provide a route for the generation of transgenic animals such as transgenic mice, a route which has a number of important advantages compared with more conventional techniques, such as zygote injection and viral infection (Wagner and Stewart (1986) in Experimental Approaches to Embryonic Development. J. Rossant and A. Pedersen eds. Cambridge: Cambridge University Press), for introducing new genetic material into such animals. First, the gene of interest can be introduced and its integration and expression characterised in vitro. Secondly, the effect of the introduced gene on the ES cell growth can be studied in vitro. Thirdly, the characterised ES cells having a novel introduced gene can be efficiently introduced into embryos by blastocyst injection or embryo aggregation and the consequences of the introduced gene on the development of the resulting transgenic chimaeras monitored during pre- or post-natal life. Fourthly, the site in the ES cell genome at which the introduced gene integrates can be manipulated, leaving the way open for gene targeting and gene replacement (Thomas, K. R. and Capecci, M. R. (1987) Cell 51:503-512).
However, it is known that ES cells and certain EC (embryonal carcinoma) cell lines will only retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts (such as murine STO cells, e.g. Martin, G. R. and Evans, M. J. (1975) Proc. Natl. Acad. Sci. USA 72:1441-1445) or when cultured in medium conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H. (1984) Exp. Cell Res. 154:233-242; Smith, A. G. and Hooper, M. L. (1987) Devel.Biol. 121:1-91). In the absence of feeder cells or conditioned medium, the ES cells spontaneously differentiate into a wide variety of cell types, resembling those found during embryogenesis and in the adult animal. The factors responsible for maintaining the pluripotency of ES cells have, however, remained poorly characterised.
In work leading to the present invention, it has been found that LIF has the capacity to substitute for, or be added to, feeder layers (or conditioned medium) in supporting the maintenance of pluripotential ES cells in vitro.
LIF is a protein that has previously been purified, cloned and produced in large quantities in purified recombinant form from both Escherichia coli and yeast cells. (International Patent Application No. PCT/AU88/00093, filed Mar. 31, 1988.) LIF has been defined as a factor, the properties of which include:
1. it has the ability to suppress the proliferation of myeloid leukaemic cells such as M1 cells, with associated differentiation of the leukaemic cells; and
2. it will compete with a molecule having the defined sequence of murine LIF or human LIF (defined in International Patent Application No. PCT/AU88/00093) for binding to specific cellular receptors on M1 cells or murine or human macrophages. In addition to the biological properties previously disclosed for murine and human LIF, LIF has now been found to have the following properties: of the pluripotential phenotype in vitro of ES cells. presence of LIF, to contribute to somatic and germline cell tissues of chimaeric animals such as mice when re-introduced into the embryonic environment; ES (EKcs-1 (previously known as CS1) and D3) and EC (PCC3-3A an
REFERENCES:
Doetschman et al. (1988); Establishment of transfer blastocyst-derived embryonic stem cells (ES) Devel. Bio. 127:224-277.
Evans et al. (1990); Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts; Theriogenology 33:125-128.
Handyside et al. (1987); Toward the isolation of embryonal stem cell lines from the sheep Roux's Archives Development Biology 196:185-190.
Piedrahita et al. (1990); Influence of feeder layer type on the efficiency of isolation of porcine embryo derived cell lines; Theriogenology 34:865-877.
Piedrahita et al. (1990); On the isolation of embtryonic stem cells: Comparative behaviour or murine, porcine and ovine embryos; Theriogenology 34:879-901.
Strojek et al. (1990); A method for cultivating morphologenically undifferentiated embryonic stem cells from porcine blastocysts; Theriogenology 33:901-913.
Smith et al. Buffalo Rat Liver Cells Produce a Diffusible Activity which Inhibits the Differentiation of Murine Embryonal . . . Developmental Biology vol. 121, No. 1, pp. 1-9, 1987.
Martin Isolation of a Pluripotent Cell Line from Early Mouse Embryos Cultured in Medium Conditioned by Teratocarcindma Prod. Natl. Acad. Sci. (USA) vol. 78, No. 12, pp. 7634-7638 1981.
Gough Nicholas M.
Hilton Douglas J.
Williams Robert L.
Amrad Corporation Limited
Cooper Iver P.
Doll John J.
Elliott George C.
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