Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Patent
1991-08-12
1995-07-25
Elliott, George C.
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
4352402, 514 2, A61K 3534, A61K 3819, C12N 508
Patent
active
054359996
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the use of leukaemia inhibitory factor (LIF), alone or in combination with other cytokines such as interleukin-6 (IL-6) and/or transforming growth factor .alpha. (TGF.alpha.) and/or fibroblast growth factor (FGF), to stimulate the proliferation and/or differentiation of mammalian satellite cells. The present invention also contemplates a method comprising myoblast transfer therapy whereby LIF, alone or in combination with other cytokines, is/are employed to proliferate and/or differentiate mammalian satellite cells into myoblasts. The present invention is also directed to a cell activating compositon and a pharmaceutical composition comprising LIF alone or in combination with other cytokines to promote proliferation and/or differentiation of mammalian satellite cells in vitro and in vivo, respectively.
Skeletal muscle consists of parallel arrays of multinucleated cells which are innervated and attached to bone through tendons. Although these highly differentiated cells are not capable of replication, muscle has a high capacity for regeneration after injury or disease and this is achieved by the activation of stem cells, called satellite cells, which lie in close association with muscle fibres. It has been estimated that up to 20% of muscle cell nuclei are found in satellite cells.
On activation, satellite cells differentiate into elongated mononuclear myoblasts. These, when in sufficient numbers, fuse to form multinucleated myotubes, the progenitor of the muscle fibre.
Primary cultures of muscle cells all originate from the satellite cells. The muscle is minced and treated with trypsin to break up fibres and extracellular matrix. Satellite cells, released as a result of this process, are harvested and placed under cell culture conditions.
After a lag period of about three days, the cells proliferate and undergo differentiation into myoblasts. These also proliferate and when the culture reaches confluence, the cells begin to fuse to form multinucleated myotubes. The cells may be passaged many times, but this must be done at the myoblast stage, before fusion.
The nature of the control of proliferation of satellite cells and subsequent differentiation into myoblasts is not well known although it has been discovered that the heparin binding growth factor, fibroblast growth factor (FGF), stimulates growth of satellite cells (Di Mario and Stohman, Differentiation 39:42-49, 1988).
The present invention arose in part from a study into the effect of a variety of cytokines on the early stages of muscle cell growth in culture. In accordance with the preset invention, it has been discovered that LIF and to a lesser extent other cytokines such as IL-6 and TGF.alpha., stimulate the proliferation of satellite cells and the subsequent development of myoblasts.
Accordingly, one aspect of the present invention relates to a method of stimulating the proliferation and/or differentiation of mammalian satellite cells into myoblasts which method comprises contacting said cells with a stimulation-effective amount of LIF for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts.
Another aspect of the present invention relates to a method of stimulating the proliferation and/or differentiation of mammalian satellite cells into myoblasts which method comprises contacting said cells with a stimulation-effective amount of LIF in simultaneous or sequential combination with one or more other cytokines, for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts.
Still another aspect of the present invention contemplates a method of myoblast transfer therapy comprising contacting mammalian satellite cells with a proliferation- and/or differentiation- effective amount of LIF for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts and then administering said myoblasts at multiple sites into muscles. In an alternative to this em
REFERENCES:
patent: 5166065 (1992-11-01), Williams et al.
Baumann, et al.; Journal of Immunology, vol. 143, pp. 1163-1167; Aug. 15, 1989.
Baumann, et al. (1989) "Hepatocyte-Stimulating Factor III Shares Structural and Functional Identity with Leukemia-Inhibitory Factor", J. Immunol. 143, 1163-1167.
Yamamori et al. (1989) "The Cholinergic Neuronal Differentiation Factor From Heart Cells Is Identical to Leukemia Inhibitory Factor" Science 246, 1412-1416.
Elliott George C.
Monash University
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