CA2045630A1

CA2045630A1 - Proliferative action of leukaemia inhibitory factor on satellite cells

Info

Publication number: CA2045630A1
Application number: CA002045630A
Authority: CA
Inventors: Lawrence Austin
Original assignee: Individual
Current assignee: Monash University
Priority date: 1989-11-24
Filing date: 1990-11-20
Publication date: 1991-05-25
Also published as: NO922008L; DE69027123D1; US5435999A; AU6896791A; JPH05502224A; EP0502081A1; JP2000128791A; WO1991007992A1; EP0502081A4; AU624284B2; JP3040815B2; ATE138267T1; DE69027123T2; EP0502081B1; NO922008D0; HK1003475A1

Abstract

(57) Abstract The present invention relates to the use of leukaemia inhibitory factor (LiF), alone or in combination witj other cytokines such as interleukin-6 (IL-6) and/or transforming growth factor .alpha.(FGF.alpha.) and/or fibroblast growth factor (FGF), to stimulate the ?liferation and/or differentiation of mammalian satellite cells. THe present invention also contemplates a method comprising ?yoblast transfer therapy whereby LIF, alone or in combination with other cytokines, is/are employed to proliferate and/or dif-ferentiate mammalian satellite cells into myoblasts. The present invention is also directed to a cell activating composition and a pharmaceutical composition comprising LIF alone or in combination with other cytokines to promote proliferation and/or differ-entiation of mammalian satellite cells in vitro and in vivo, respectively.

Description

2 PCT/AU~/O~ ~
~563~

PROLIFER~TIVE ACT~ON OF L~URAEMIa IN~IBITO~Y
FACTOR ON SA~ELLITE CEL~S

The present invention rPlates to the use of leukaemia inhlbltory factor (LIF), alone or in combinatlon with other cytokines such as interleukin 6 (IL-6) and/or transforming growth factor a (TGFa) and/or fibroblast growth factor (FGF), to stimulate the proliferation and/or differentiation of ma~malian 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 ~o proliferate and/or diferentiate 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 wlth other cytokines to promote proliferation and/or d~fferentiation of mammalian satellite cells in vi~ro and in vivo, respec~ively.

Skeletal muscle consists of parallel arrays of multinucleated cel's which are innervated and attached to bone through tendons. Although th~se highly differentiated cells are not capable of replication, muccle has a high capaclty ~or reg~neration after in;ury or disease and this is achieved by the activation of stem c~lls, called satellite cells, which lie in close association with muscle fibres. It has been estimated that up to ~0~ o~ muscle cell nuclei ~re found in satellite c~

On actlvatlon, satellita cells dl~rentiate lnto elong~t~d mononuelaar myobla3~s. Th~e, when ln su~Plci~nt numbera, ~uS~ ~o ~orm mul~inucl~ated myo~ubes,~
~he progenitor of the muscle ~lbr~.

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WO91/07992 PC~/AU90/00556 2 ~ 63~
Prlmary cultures of muscle cells all originate from the satelllte cells. The muscle is minced and treated with trypsin to break up fibres and extracellular matrix.
Satellite eells, 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 prollferate and undergo dlfferentiation into rnyoblasts.
These also proliferate and when the culture reaches confluence, the cells be~in to fuse to form multinucleated myotubes. The cPlls 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 bindlng growth factor, fibroblast growth factor (FGF), stimula~es growth of satellite oells (Di Mario and 5tohman, Differentiation 39:42-49, lg88).

The prssen~ invention arose in part from a study into the effect of a variety of cytokines on the early stages of muscle cell ~rowth 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, stimulate the proliferatlon of sat~lllte cells and the subsequent development of myoblasts.

Accordingly, one aspect o~ the present invention relates to a method of stimulating the prolieration and/or diferentiatlon of mammalian satellite cells into myoblasts wh.tch method comprises cont~cting sald cells with a stlmula~lon-e~ctlve amount oX LIF ~or a tlme and un~er condl~ion~ su~iGient ~or said sa~elllte cells to proliferat~ ~nd/or diP~erentiate into m~oblasts.

WO91/07992 PCT/AU90/005~6 ;~0~i63~
Another aspect of the present invention r~latec ~o a method of stimulating the proliferation and/or d~fferentiation of mammalian satellite cells into myoblasts which method compri~es contacting said cells wlth a stimulation-effec~ive amount of LIF in simultaneous or se~uential combination with one or more other cytokines, for a tlme and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts.
Still another aspect of the present ~nvention contemplates a method of myoblast transfer therapy comprising con~acting mammallan satellite cells with a proliferation- and/or differentiation- effective amount of LIF for a time and under conditions sufficient for sald satellite cells to proliferate and/or dif~erentiate lnto myoblasts and then administering said myoblasts at multiple sites into muscles. In an alternative to this embodiment, LIF 1~ used in simul~aneous or sequential combination w~th one or more ot~er cytokines.

Yet ano~her aspect of ~he present invention relates to a cell activating composition comprising LIF in combination with one or more other cytokines, and one or more physlologically acceptable carrieræ and/or diluents.

Still yet another aspect of the present inventlon relates to the use o~ LIF, alone or ln combination with one or more other cytokines, in the manufactur~ of a cell activatlng composition for stlmulating the proliferation and/or dif~erentlation o~ mammal~an satellite cells into myoblasts.

Xn ~tlll yat ano~her a~p~ct o~ ~he pre8ent inventlon ~S ~here is provided a pha~maceutical composition for ~lmulating the proll~ration and/or dl~f~r~ntiatlon of ~at~llite calls comprising LIF and one or more other . . i., ..
; ;. ,, . : ~ ~ ` ' - - . .. . .
:. .. ..

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WO9l/079g2 , PCT/AU90/0056 63i[~

cytokines and one or more pharmaceutically acceptable carriers and/or diluents.

In one preferred embodiment, the cytokines in optional combination with LIF include IL-6 and/or TGFa and/or F5F.

The satellite cells and cytokines may originate from homologous or heterologous mammals. If the same mammal is used, satelllte cells and cytoklnes may originate from the same or different species of the same ma~nal.
Mammals contemplated herein include but are not llmited to humans, mice and livestock animals.

The presen~ lnvention extends to naturally occurring (native), recombinant and/or synthetic oytokines and/or to their derivatives and/or analogues and/or to any combinations thereof. For example, recombinant murine and human LIF are disclosPd in International Patent Appllcat~on No. PCT/AU88/00093. Use of the term ~LIF"
herein encompasses all such forms of LIF and its derivatives and analogues and includes single or multiple amino acid substltutions, d~letions and/or addltlons to the polypeptlde portion of the LIF molecule and sin~le or mult~ple substltutions, deletions and~or additions to the carbohydrate portlon of the molecule (when present).
Derivatives and analogues of LIF includ~ portions of native, recombinant and/or synthetic LIF which have the desired activity.

By "~lmul~aneou~ or ~equen~lal combinatlon" as u~ed h~reln m~an~ the additlon oP LIF and one or more other cy~okine~ at ~hs same time, 1.~. in a single composltion or the administratlon of each active molecule or groups th~reo~ one a~er the othe~. ~y way e~ non-limi~ing 3S H~ample, LIF may be used flr~t followed by a ~econd cytokin~ ~ollowed b~ a thlrd cytoklne ~nd ~o on.
Al~ernatlvely, LIF may be u~ed irst ~ollow~d by a ,:

W091/079~2 ~CT/~U~/~05~6 ;~ 563q~
combination of other cyto~ines. In another em5Odiment, other cytokines are usad first (slmultaneously or sequentially), followed by ~IF.

The present invention is of medical significance especially in relation to primary, genetically determined, muscle myopathies. There are considerable numbers of these, the most severe and the most common o which, being Duchenne muscular dystrophy (DMD). The affected gene is known and ~ts protein product has been deduced. The protein product, dystrophin, is probably a a component o the cytos~eleton, membrane linkage. It is large, 425,000 daltons, and the gene is the largest of the human genes. B~cause of its complexity, it is unlikely that genetic manlpulation will be possible in the near future. However, another approach has been shown to be effecttve in mouse models o~ muscular dystrophies, including the mdx mouse.

~0 ~his approach involves th~ growin~ of myoblasts in culture derived from normal mice and injecting them, at multiple sites, into muscles of mutant mice. The results have ~hown that not only has rejection been minimal, but the muscles contaln dystrophin whereas previously there was none.

In a mouse strain showing very severe muscle wastage (not dystrophin deficient, but a mutant wlth an unknown de~ect) muscle stren~th returned to near normal.
Thus, a proc~dur~ is contemplated termed myoblast transfer ther~py, whereby human myobla~ts, grown in culture, are inJected at multlple site~ into muscles oP
DMD, Thi~ approach i8 appllcable to all primary 3~ myopathi~s and not only DMD.

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W091/07992 PCT/AU9~/00556 The process involves lnJection of many myob ~asts a~ ~
multlple sites in a large number of muscles. It ls time-consuming and the cost of myoblast cul~ure is high. At present, techniques of culturing myoblasts utilize medium to long term culture wlth varying concentrations of the expensive reagent feo~al calf serum (FCS). Thus, any factor which may accelerate the myoblast differentiation and growth should be significant in reducing the cos-t o ~.
myoblast production. In accordance with the present invention, therefore, LIF alone, or in combinatlon with other cytokines such as IL-6 and/or TGFa and/or FGF, will fulfill this ne2d.

Accordingly, one aspect of the present invention is directed to a method of stimulating the prollferation and/or dlfferentiation of mammalian satellite cells into myoblasts comprising contacting said satelllt~ oells wi~h a stimulation-effective amount of LIF, alone or in combination with other cytokines such as IL-6 and/or ~GFa and/or FGF, for a time and under conditions sufficient to stimulate the satelllte cells.

The present invention is also dtrected to cell act~vating compositions comprising LIF with or without other cytokines such as IL-6 and/or TGFa and/or FGF and one or more physlologically acceptable carriers and/or diluents.
Preferably, the compositions comprise LIF in combination wlth one or more other cytokines.

~he present inventlon is also directed to a pha~maceutical composition ~or stimulating the prollferation and/or dif~erentiation of mammallan ~atell~te cell~ whiCh compositlon compri~e~ LI~ with one or mor~ oth~r cytokines and one or more pharmaceutlcally 3S accaptable carriers and/or diluents. In one ambodiment, tho cy~okines o~her than L~F contemplat~d herein include IL-6 and/or TGFa and/or FGF. Methods :Eor preparing a . ,.

WO9l/07992 PCT/AU90/005~6 7 2(1 45~
pharmaceutical composition are known in the art such as described in Remin~ton's Phanmaceutical Sciences 16th ed.~ 1980, Mach Publishing Co., edited by Osol et al. and hereby incorporated by reference. The route of administration and the effect~ve amount of active component will be determined by the sltuation but one preferred route is intramuscular although other routes of administration may be employed. For tAe purposes of exemplification of this embodiment of the present invention, the effective amount of ~IF used is ~rom about 0.1 to about 1000 U/ml, preferably about 1 to about 100 U/ml and most preferably from about 10 to about 50 U/ml.
A unlt of ~IF is defined in PCT/AU88~00093. In general, other cytokines will be used at from about 1 to about: 100 ng/ml. More specifically, IL-6 may be preferably used at a concentration of about 60 to about 100 ng/ml; TGFa preferably at about 1 to about 20 ng/ml; and FGF
pre~erably at about 20 to about 50 ng/ml. These concentrations may vary depending on the circumstances and it is not the intention to necessarily limit the present invention to these effective amounts.

The present invention is further described by the following non-lim~ting Figures and Examples.
In the Figu~es:

Figure 1 is a graphical representation showing the response o~ pa~aged cells to LIF at several concentrations of FCS. Passaged mouse myoblasts (P2) were plated into 96 well pla~es at 2500 c~lls per well, ln Ham Fl2 medium contalning 5, 7.5 or 10~ v/v FCS. LIF
was addad at ~he concen~rations ~hown and -the cell grown w.ithou~ further change o~ medium~ Cell numbers were counted a~ the time~ shown and are axpre~sed as cells/mm2 o~ w~ll sur~aca.

8 ~o~s6~01 F~gure 2 i~ a graphlcal representation 3howing the effect of ~IF on human myoblasts. Human myoblasts were derived from a sample of human skeletal muscle, grown to about 80~ confluence and passaged. These passaged cells were grown in the pr~sence of LIF, as described ~or Figure 1.

F~gure 3 ls a graphlcal representatlon showing th~ effect of TGF~a replaced LIF at the concentrations shown.

Figure 4 is a graphlcal representation show~ng the effect of FCS concentration of LIF activity. LIF and FCS human myoblasts were grown as described for Figure 2. FCS, ranging from O to 20~ v/v was added to the ~am F12 medium before addition of LIF. (a) 0~ - S% v/v FCS, (b) 7.5~ -20~ v/v FCS.

F$gure 5 ls a photographic representation showing fu;ionof LIF-supported myoblast~ in culture. Human myoblasts were grown either in the absence or presence of 30 units/ml LIF. These were harvested, suspended in PBS and injected into one extensor digltorum longus of mdx mice.
The mice wera killed 5 weeks la~er and the muscle was embedded for cryostat sectioning. Sections were treated with a~tl-dystrophin antibodies and the presence of dystrophin was visualised using fluorescein labelled anti-sheep antibodies. tA) Musc}e of C57-BL-10 normal, dystrophin positive mouse. (B) Muscle o~ non~injectecl mdx, dystrophin negative mouse. (C) Muscle from mdx mouse ln~ect~d with myoblasts grown in the presence of L~F. No~e patches of dystrophin - positive areas underlying the sarcolemma.

Figu~e 6 i~ a photographic reprasen~atlon .showing L~F
recep~or~ on myoblaYts. Autoradiography o~ mou~o m~oblas~ sub~ected to ~2sI-~IF. (a) ~2sI-~IF ~lona. (b) l~sI~LIF in the pre~nce o~ a lOO0-~old exceY3 o non-l~b~ d L~F.

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WO91/07992 F'CT/AU~0/00556 2 014~i63 a3 ~XA~PLE 1 MATERIaLS AND METHODS

Mouse musc~e cells:
The muscle used was from the hind legs of mouse strain CS7/BL/10. A mutant from the same strain was also used.
This is a mdx mutant ln whlch the muscle protein dystrophin is missing. This is an excellent model to study muscular dystrophy since the same gene is affected in the human condition.

Primary cultures of these cells were grown as described by Gurusingha et al. Muscle and Nerve 11: 1231-1239, 1988, except that 5 - 10~ v/v FCS was used. When at about 80~ confluence, the cells were washed briefly with P8S and treated wlth 0.025% w/v trypsin in dissociation buffer to detach them. Foe~al calf serum (FCS) was added to 5~ v/v concentration to lnhiblt the t~ypsln, the cells centrifuged at l~O0 rpm for lO minutes and washed twice with PBS. They were then plated at 10% confluence in Ham F12, 20% v/v FCS to provide passaged cells.

Human muscle cells:
Under these conditions, many cells survive and differentlate, but at a rate lower than those under optimum condltlons in which 20% (v/v) FCS ls used.
Vsually there is a lag period of 3 - 4 days during which time cell numbers t~nd to decrease followed by khe appearance of myoblasts ~t day 5 - 7. The cells are initially ~lated lnto 96 well clu~ter plates at a density of 3-5000 cells/well. Growth factors are added 3 days a~er initlatlon of ~he culture and ~he ePPect qu~t;ltated by countlng cells as ~atQllitQ cells or myobl~ts over a perlod.

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WO91/07992 PcT/AU90/00556 ~563~

Samples (0.5 - 1.5g) o~ h~man skeletal muscle were removsd by collaborating surgeons from consenting patients during operative procedures. ~luman Ethics Commlttee Approvals are held from Monash University, the Monash Medical Centre, Clayton and the Royal'Childre~'s Hospital, Parkvill2. These samples were transported back to the laboratory and cultures commenced, essentially as described for mouse cells.

In vivo myoblast transfer:
Passaged mouse or human myoblasts were grown to ~0 - 90%
confluence in the absence or presence of 30 uni~s/ml LIF
and harvested as descrihed above. They were suspended in PBS at 3 x 108 cells/ml. Mutant mdx mice, 25 - 32 days of age were anaesthetised by I.P. injection of a mixture of hypnorm (O.3 ml/~g~ and diazapam (5mg/kg). One extensor digitorum longus (EDL~ muscle was exposed and myoblasts were injacted. These cells were delivPred in 1~1 lots from a SGE syringe fi~ted with a needle that had been electropolish~d at the tip to 27 gauge. The syringe was mouted on a mlcromanipulator to control position and depth of in~ection. F`our or five in;ections were made into each muscle, at intervals of 1.5 - 2mm. Control injections were carried out using P~S ~lone. The wound was sutured a~d the mouse allowed to recover.

After 4 to 6 weeks, ~he animals were killed by breakin~3 the neck, the EDL muscle re-exposed, cooled with ice cold PBS, removed and embed~ed in Tissue - Tek OCT and imme~ia~ly qu~nahed ~n isopentane at liquid N~
temp~rature. ~he blocks wer~ trimm~d at -25 and transv~r~e 8~tions cut in a cryos~at at -20 to 3 - 4 ~m.
When ~ir-dried, the s~ct.lons were treated wi~h ei~her ~0 Kd or 30 Kd anti-dy~rophin antibodies (Ho~man et al.
~Ql~ 51: 919-923, 1987) diluted 1/200 with P~S. Pre-i~une sQrum wa.s ~imilarly diluted. APter incubation at R.T. for 30 minute~ at 100% humidity, the sections were WO91/07992 PCT/AU90/OOS~6 11 2~563~
washed three times and then subjec~ed to FITC donkey -antisheep antibodies ~Silenius, Melbourne) at 1/40 dilution. ~hey were again washed and mounted under coverslip~.

E~AMPLE 2 Mouse Myoblasts:
Primary cul~ures of mouse myoblasts were passaged and 10 grown in medium containing LIF at various concentrations.
This procedure was carried out three times in medium contalning 5~, 7.5% and lO~ v/v FCS. Thus, lt was possible to test whether pa~saged cells respond to LIF in the same manner as primary cultures and also to examine the response to ~IF under va~ious yrow~h conditions.

Figure l shows that passaged mouse oells respond t- LXF
at several cell concentrat~on~ of FCS. The optim:~
concentration of LI~ is 30 units/ml (14pg/ml) as ~ was for primary cultures. There was a greater effect of LIF
at 10% v/v FCS concentration than at lower concentrations, this being a 13 fold lncrease over controls.

25 Human mYoblasts:
These cells were grown ~rom donor human muscle and passaged cells were seeded at 2-3000 cells per well in a 96 well cluster plate in Ham Fl2 medium containing 7 . 5%
v/v FCS as was carried out for initlal mouse call cul~ur~s. ~hQ modlum con~ained v~rylng concontrations o LI~ C~ll number~ were countod a~ tim~s up ~o 12 days ~nd thQ re~ults ~r0 shown in Flyuro 2. As with mous~
cells, ~h~ra wa9 a marked stimulation oP proll~ratlon of m~obl~s~s by LIF. A~ain ~he optlmum conaant~ation ound ~5 ~or LIF wa~ 30 unl~ml. This show~ that hum~n myoblasts re~pond to LIF ln a mannor similar to mouse myoblas~.
~.

WO91/07992 PCT/AU~/0~56 2~563~

It has previously been found that TGF-a also stlmulates mouse myoblasts. Human cells were grown also in the presence of this cytokine at concentrations rang~ng up to 10 ng/ml. Figure 3 shows that there was early response to TGF-a, with an optimum concentration of 1 ng/ml, the same as that found for mouse cells. As was the case for LIF, higher concentrations were less efflective than that at the optimum. Unlike mouse cells, the response occurred early. This may be due to species differences or passaging of cells or some other unknown factors.

Effects of_FCS concentrations of LIF activi~y:
Passaged human myoblasts were grown in Ham F12 medium containing FCS concentrations ranging from 0-20% v/v YCS.
At each of the FCS concentrations, LIF was added at 0, 30 or 100 units/ml. Figure 4 shows that at 0 and 1% v/v FCS
there is no cell growth in the absence or presence of LIF. When the FCS concentration is 2% v/v, again there is no growth in the absence of LIF, but some growth occurs in its presence. At increasing concentrations of FCS, LIF provides increased growth and as shown previously, 30 units/ml of LIF is more effective that L00 units/ml, at all FCS levels. Optimum stimulation of growth by LIF was found to occur at 15% v/v FCS.
~5 In iv~ v~bl-st transfer:
Mlce were lnJected wlth either mouse or hu~an myoblasts cultured ln the presence o LIF as de~3cribed in Example 1 at the r~te o 1 - 1.5 x 106 c:clls per EDL mus::le. ~hese 3~ w~ killed and the muscles prepar~d for immuno cytochemistry 4 - 6 w~eks la~er. Figure 5A shows the pre~enc:~ o~ dy~trophin in an E~L muscle of ~he C57-BL-10 dy~trophln positive control mousa s~raln. As descrlbed by other3 (Partridge ~ al. Nature 337: 176-179, 1989) 35 the dystrophin is located under the ~ur~ace of the sarcolemma membrane. The mdx dystroph~ n negatlve muscle is ~hown ln Flgure SB. No immunoreaction is evident.

W091/07992 PCT/AU~/~56 13 2~5~ 3~
Flgure 5C shows EDL muscle sections from an mdx mouse lnjected 6 weeks earlier with human myoblasts. That fusion has occurred Can be seen from dystrophin pcsitive patches lorated at the sarcolemma of the fibres.
Posltive fusion was also found when mouse myoblasts were injected into the EDL muscles.

LIF reCeDtOrS on myoblasts:
Mouse myoblasts were grown on glass slides, pretreated with fibronectin to ensure good adhesion. After eight days in culture, they w~re subjected to lZsI-labelled LIF
ln the absence and present of a 1000 fold excess of unlabelled LIF. Figure 6A shows that LIF receptors are present on myoblasts, whereas Figure 6B shows low non-specific binding.

Those skilled in the :?.r~ will appreciate that theinvention described herein is susceptible to variations and modifica~lons other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The inventlon also includes all of the steps, features, compositions and compounds re~erred to or indicated in thls specification, lndividually or collectlvely, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. 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 leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for said satellite cells to proliferate and/or differentiate into myoblasts.

2. The method according to claim 1 which further comprises the addition of one or more other cytokines in simultaneous or sequential combination with LIF.

3. The method according to claim 1 or 2 wherein the LIF, cytokine and/or satellite cells are from the same mammal.

4. The method according to claim 3 wherein the LIF
cytokine and/or satellite cells are from the same species of mammal.

5. The method according to claim 1 or 2 wherein the LIF, cytokine and/or satellite cells are from different mammals.

6. The method according to any one of claims 1 to 5 wherein the mammal is human, mouse or a livestock animial.

7. The method according to claim 1 or 2 wherein the LIF
and/or cytokine is prepared by recombinant or synthetic means.

8. The method according to any one of the preceding claims wherein the cytokine is one or more of IL-6, TGF.alpha.
and/or FGF.

9. The method according to any one of the preceding claims wherein LIF is provided at a concentration of from about 0.1 to about 1000 U/ml and the cytokine is provided at a concentration of from about 1 to about 100 ng/ml.

10. The method according to claim 9 wherein LIF is provided at a concentration of from about 1 to 100 U/ml.

11. The method according to claim 10 wherein LIF is providsd at a concentration of from about 10 to about 50 U/ml.

12. 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.

13. The method according to claim 12 which further comprises the addition of one or more other cytokines in simultaneous or seguential combination with LIF.

14. The method according to claim 12 or 13 wherein the LIF, cytokine and/or satellite cells are from the same mammal.

15. The method according to claim 14 wherein the LIF, cytokine and/or satellite cells are from the same species of mammal.

16. the method according to claim 12 or 13 wherein the LIF, cytokine and/or satellite cells are from different mammals.

17. The method according to any one of claims 12 to 16 wherein the mammal is human, mouse or a livestock animal

18. The method according to claim 12 or 13 wherein the LIF and/or cytokine is prepared by recombinant or synthetic means.

19. The method according to any one of the preceding claims wherein the cytokine is one or more of IL-6, TGF.alpha.
and/or FGF.

20. The method according to any one of claims 12 to 19 wherein LIF is provided in a concentration of from about 0.1 to about 1000 U/ml and the cytokine is provided at a concentration of from about 1 to about 100 ng/ml.

21. The method according to claim 20 wherein LIF is provided at a concentration of from about 1 to about 100 U/ml.

22. The method according to claim 21 wherein LIF is provided at a concentration of from about 10 to about 50 U/ml.

23. The method according to any one of claims 12 to 20 wherein the myoblasts so obtained are administered by intramuscular injection.

24. A cell activating composition comprising LIF in combination with one or more other cytokines and one or more physiologically acceptable carriers and/or diluents.

25. The composition according to claim 24 wherein the LIF, cytokine and/or satellite cells are from the same mammal.

26. The composition according to claim 25 wherein the LIF, cytokine and/or satellite cells are from the same species of mammal.

27. The composition according to claim 24 wherein the LIF, cytokine and/or satellite cells are from different mammals.

28. The composition according to any one of claims 24 to 27 wherein the mammal is human, mouse or a livestock animal.

29. The composition according to claim 24 wherein the LIF and/or cytokine is prepared by recombinant or synthetic means.

30. The composition according to any one of the preceding claims wherein the cytokine in combination with LIF is one or more of IL-6, TGF.alpha. and/or FGF.

31. The use of LIF in the manufacture of a cell activating composition for stimulating the proliferation and/or differentiation of mammalian satellite cells into myoblasts.

32. The use according to claim 31 further comprising the use of one or more other cytokines in simultaneous or sequential combination with LIF.

33. The use according to claim 31 or 32 wherein the mammal is human, mouse or livestock animal.