WO1998048830A1

WO1998048830A1 - Method for promoting myogenesis using osteogenic proteins

Info

Publication number: WO1998048830A1
Application number: PCT/US1998/008466
Authority: WO
Inventors: Charles P. Emerson; Anne-Gaelle Borycki; Brian Brunk
Original assignee: Trustees Of The University Of Pennsylvania
Priority date: 1997-04-28
Filing date: 1998-04-27
Publication date: 1998-11-05
Also published as: AU7260598A

Abstract

Methods and compositions are provided for stimulating lineage commitment of adult or embryonic myogenic stem cells. The methods comprise treating the cells with the osteogenic protein, OP-1, at a concentration effective to induce myogenesis of stem cells. The methods are useful for therapeutic purposes in embryonic development and in repair/regeneration of mature muscle.

Description

METHOD FOR PROMOTING MYOGENESIS USING OSTEOGENIC PROTEINS

FIELD OF THE INVENTION

The present invention relates to the field of tissue morphogenesis, regeneration and repair, and more specifically to methods and compositions for stimulating lineage commitment of embryonic or adult myogenic stem cells.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.

Vertebrate skeletal muscle fibers are formed by the cellular fusion of progenitor myoblasts, which are embryonic cells that proliferate and populate the muscle-forming regions of embryos. Myogenic lineages become determined during somite morphogenesis, leading to the formation of myoblasts. Myoblasts are a stably determined cell type, capable of extensive cell division, the progeny of which faithfully inherent their myoblast identity and can express their potential to differentiate into muscle fibers.

Thus, in embryological development, myogenesis involves "determination" of myogenic stem cells to become the myoblast lineages in the somite and "differentiation" of myoblasts to become myocytes and myofibers in the muscle-forming regions of the embryo. A similar process occurs in repair or regeneration of adult muscle. Muscle tissue which has been compromised either by disease or trauma undergoes a repair process that involves, in part, activation of satellite cells (a distinct population of post-mitotic stem cells closely associated with skeletal muscles) and their migration to the site of injury or damage, determination of these stem cells into myoblasts, and differentiation of myoblasts into mature muscle cells. The process involves the fusion of the mononucleated myogenic cells (myoblasts) to form a multinucleated syncytium (myofiber or myotube) .

Several mammalian genes that regulate the determination of the skeletal lineage have been identified. These genes (myoD , myogenin , myf-5 and MRF-4 ) encode transcription factors comprising a subgroup within the basic helix-loop-helix (bHLH) superfamily of Myc-related DNA binding proteins. MyoD is a muscle regulatory gene transcription factor activated in early somitic mesoderm, which is composed of multipotential cells that give rise to cartilage, dermis, as well as skeletal muscle progenitor cells in response to signals from adjacent tissues (Emerson, 1993) . In combination with myf5 , a closely related gene, myoD is essential for the formulation of skeletal myogenic cell lineages in the mouse embryo (Rudnicki et al., 1993) . Thus, it would be advantageous for research and therapeutic purposes to identify agents capable of enhancing expression of myf5 and myoD in early muscle development.

Acting alone, myoD is an essential gene for the regeneration of skeletal muscle from satellite cells in adults (Megeney et al., 1996). The myoD gene is one of the earliest genes activated in satellite cells in response to muscle injury. Therefore, identification of signal molecules that activate myoD is important for therapeutic purposes, to increase the population of activated satellite cells to promote muscle regeneration and repair.

The activation of myoD and myf5 genes in somite cells that form the myoto al body wall muscle lineage is controlled by developmental signal molecules (Pownall et al., 1996; Cossu et al., 1996; Munsterberg et al., 1995). These include growth factors, such as basic fibroblast growth factor (bFGF) and transforming growth factor-/? (TGF-β) , which comprises a superfamily of related growth factors. Also included is sonic hedgehog (Shh) protein, which has been found to enhance myoD expression and somite yogenesis in zebrafish embryos (Currie & Ingham, 1996) and avian somite culture (Munsterberg et al., 1995) .

One class of the TGF-/3 superfamily of growth and differentiation factors includes the bone orphogenetic proteins (BMPs) , also referred to interchangeably as osteogenic proteins (OPs) . Bone morphogenetic proteins are well know and extensively described (see, e.g., U.S. Pat. No. 5,610,021 and references cited therein; see also Vukicevic et al., 1996) . Originally isolated from bone, these proteins are capable of inducing the full developmental cascade of bone formation when implanted into a mammalian body under appropriate conditions. The bone morphogenetic protein BMP7 (osteogenic protein OP-1) has also been implicated in metanephric differentiation during early embryonic kidney development (Vukicevic et al., 1996), as well as in the alleviation of tissue destructive effects associated with the inflammatory response to tissue injury (WO 93/04692) . However, osteogenic proteins have not been implicated in myoblast determination or muscle cell differentiation. SUMMARY OF THE INVENTION

It has been discovered in accordance with the present invention that certain osteogenic proteins, in a concentration-dependent manner, stimulate myogenesis of stem cells. Accordingly, the present invention is comprised of promoting myogenesis of stem cells by treating the cells with a composition that includes the osteogenic protein. For purposes of this invention, the term "myogenesis" is intended to include any step in the process of myogenic determination of stem cells into committed lineages (sue as myoblasts) and differentiation of those committed cells into muscle cells and fibers.

According to one aspect of the invention, methods are provided that comprise the step of administering to a subject an osteogenic protein as defined herein, for a time and at a concentration effective to stimulate myogenesis of ste cells, including embryonic stem cells and adult myogenic stem cells (such as satellite cells) .

In one embodiment, the invention features compositions and therapeutic methods for stimulating muscle regeneration and/or repair, which comprises administering to a subject, for delivery to the site of muscle disease or injury, a therapeutically effective amount of the osteogenic protein defined herein.

In another embodiment, the invention features compositions and methods for stimulating determination of myogenic cells in a developing embryo, which comprises treating the embryo an effective amount of the osteogenic protein defined herein.

According to another aspect of the invention, methods are provided that comprise treating cultured stem cells with an osteogenic protein as defined herein, for a time and at a concentration effective to stimulate myogenesis of the cells, including embryonic stem cells (such as pre-somitic cells) and adult myogenic stem cells (such as satellite cells) . In one embodiment, the invention provides ex vivo treatment methods for expanding populations of determined myogenic cells, which comprises removing myogenic stem cells from a donor ( which, in a preferred embodiment is the patient in need of such treatment) , treating the cells with an effective amount of osteogenic protein as defined herein, and replacing the cells in the patient.

In the present invention, the term "patient" or "subject" is intended to refer to a human or an animal.

According to another aspect of the invention, methods are provided to induce expression of the myoD gene in vitro or in vivo , which comprise exposing the gene in a appropriate biological environment to an osteogenic protein of the invention in an amount and for a time effective to induce expression of the gene.

Other features and advantages of the invention will be apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Histograms showing the effects of sonic hedgehog (Shh) and BMP proteins on presomitic mesoderm (ps ) explant cultures. Fig. 1A shows the results of presomitic mesoderm with the overlying ectoderm cultured in the presence of the indicated purified proteins. Fig. IB shows the results of culturing psm with the adjacent notochord and neural tube (NCNT) in the presence of the overlying ectoderm. Results are presented with the bars representing the average number of cells/explant (10-70 explants per dataset) . The P values of each condition relative to base media are located just above each bar.

Figure 2. Histogram showing that BMP7 at low concentration, but not BMP2 and GDF5, acts in synergy with Shh to induce MyoD expression in presomitic mesoderm (psm) explants. Results are presented with the bars representing the average number of cells/explant (10-70 explants/dataset) . The P values of each condition relative to base media are located just above each bar.

Figure 3. Histogram showing that BMP7 at high concentration inhibits MyoD induction by the neural tube / notochord complex on presomitic mesoderm (psm) explants. Results are presented with the bars representing the average number of cells/explant (10- 70 explants/dataset) . The P values of each condition relative to base media are located just above each bar .

Figure 4. Histogram showing that BMP7 at low concentration acts synergistically with Shh and Wntl but not with Wnt4 to induce MyoD expression in presomitic mesoderm (psm) explants. Results are presented with the bars representing the average number of cells/explant (10-70 explants/dataset) . The P values of each condition relative to base media are located just above each bar.

Figure 5. Histogram showing that the surface ectoderm is required to mediate BMP7 and Shh signals in the psm culture system. Results are presented with the bars representing the average number of cells/explant (10-70 explants/dataset) . The P values of each condition relative to base media are located just above each bar. DETAILED DESCRIPTION OF THE INVENTION

It has been discovered in accordance with the present invention that the osteogenic protein OP-1 (BMP7) is a new signal molecule that has myoD-inducing activities, either independent of or in combination with other signal molecules, such as sonic hedgehog (Shh) protein or Wntl proteins. OP-1 induces myoD expression and myogenic lineage determination in a concentration-dependent manner: low concentrations (e.g., between about 0.1 and 1.0 ng/ l) induce myoD expression, while higher concentrations (e.g., greater than about 10 ng/ml) , which, notably are concentrations required to stimulate bone formation) , inhibit myoD expression. Without limiting the invention to any particular mechanism of OP-1 activity, it is believed that, in embryos, myoD activation occurs through one or a combination of alternative pathways: (l) a dependent pathway through which Shh controls OP-1 expression, which in turn activates myf5 , which in turn activates myoD; (2) a co-dependent pathway, wherein Shh is required for inducing mesoderm to express myf5 , and then OP-1 functions together with myf5 to activate myoD; and (3) a parallel pathway, wherein two lineages of presomitic mesoderm cells exist, one responding to Shh and the other responding to OP-1, both lineages thereafter inducing myoD expression through myf5. In regeneration and repair of adult muscle tissue, OP-1 at low concentration is expected to induce myoD expression in satellite cells, thereby promoting muscle fiber formation.

Thus, OP-1 as a myogenic inducer will find broad utility, not only in further research to understand the mechanisms controlling normal muscle development and regeneration, but also in therapies for the treatment of muscle disease and injury. Osteogenic (bone morphogenetic) proteins are fully described in the art (See U.S. Patent No. 5,610,021 and references cited therein). In addition, a soluble form of osteogenic proteins has been disclosed (U.S. Patent No. 5, 610,021).

An exemplary osteogenic protein of the present invention is OP-1 (BMP7) . OP-1 is a member of a family of proteins (osteogenic proteins) , which are a subclass of the TGF-/3 super-family of proteins. The members of this family share characteristic structural figures (described in the art, e.g. U.S. Patent No. 5,610,021), as well as substantial amino acid sequence homology in their C-terminal domains, including a conserved seven cysteine structure. The proteins are translated as a precursor polypeptide sequence, having an N-terminal signal peptide sequence (typically less than about 30 residues, referred to as the "pre-pro" region) , followed by a "pro" region, which is cleaved to yield the mature protein sequence. The mature sequence comprises both the conserved C- terminal seven cysteine domain and N-terminal sequence which varies significantly between the various osteogenic proteins. The polypeptide chains dimerize and the dimers typically are stabilized by at least one interchain disulfide bond linking the two polypeptide chain subunits. The mature subunits produced from mammalian cells typically have molecular weights in the range of about 15-23 kDa, depending on the degree of glycosylation and N-terminal truncation. The dimeric species typically have a molecular weight in the range of about 30-40 kDa.

OP-1 refers generically to a sub-family of osteogenically active proteins produced by expression of part or all of the hOPl gene. It is referred to interchangeably herein as BMP7. OP-1 and its allelic variants, species and other naturally occurring and biosynthetic sequence variants are contemplated to be useful in the present invention.

OP-1 may be isolated from bone and other tissue as described in the art, or they may be produced by expression of recombinant genes encoding the proteins, which are also described in the art. The invention is intended to include osteogenic proteins encoded by, and produced by expression of, nucleic acids which hybridize to DNA or RNA sequences encoding the mature OP-1 protein under stringent hybridization conditions. As used herein, stringent hybridization conditions are defined as the following conditions, or their equivalent as would be understood by one skilled in the art: hybridization in 40% formamide, 5X SSPE, 5X Denhardt's Solution, 0.1% SDS at 37 °C overnight, and washing in 0.1X SSPE, 0.1% SDS at 50°C (See, e.g., Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory, 1989) .

The osteogenic proteins of the invention can be used for a wide variety of therapeutic purposes, as well as for research purposes. The protein can be administered alone or in combination with other agents, such as inducers of proliferation (growth factors) , antibiotics or anti-inflammatory agents. The proteins can be used to treat injury or trauma of mature muscle, including surgical trauma. They also can be used to treat degenerative diseases of muscle, including muscular dystrophy, myasthenia gravis, multiple sclerosis, nerve block injury, muscle atrophy, embryonic failure of myotomes to migrate, and rhabdomyosarcoma .

The osteogenic proteins can be administered in vivo or ex vivo . For in vivo administration, they can be administered directly to the location of myogenic stem cells in the body, or to the site of injury or trauma. In a preferred embodiment, the proteins are implanted in a controlled release device at a selected site containing the stem cells.

For ex vivo treatment, appropriate stem cells are obtained, either from normal donors or from an unaffected site in the subject to be treated, induced to proliferate, myogenesis induced by the proteins of the invention, then returned to the diseased or injured individual, e.g. via myoblast transfer. Myoblast transfer involves injecting myoblast cells into the muscle of a patient requiring treatment. Myoblasts transferred into mature muscle tissue will proliferate to a limited extent, and will undergo differentiation into mature muscle fibers (Dhawan et al., 1991). The osteogenic proteins of the invention are administered to patients or to cultured cells in combination with an acceptable pharmaceutical carrier. An effective dose in cultured cells will be in the range of between about 0.1 to 5.0 ng/ml , and preferably less than 1.0 ng/ml. An effective dose in vivo can be calculated from the cultured cell dosage according to standard methods, and will be of a several-fold (e.g., about ten-fold) lesser dosage amount than that needed to stimulate bone formation in vivo . If an implantable or injectable controlled delivery vehicle is used to deliver the protein, dosages can be calculated according to methods in the art for use of OP-1 in bone formation. For instance, U.S. Patent Nos. 4,960,590 and 5,354,557 disclose that 25 mg of an osteogenic device (an implantable matrix) implanted into rats will contain at least 5 ng of purified osteogenic protein for an observable bone- forming effect, 21-25 ng for a half maximal effect, and 25-50 ng for a full effect. Accordingly, a myogenic delivery vehicle may be formulated to contain about 10-fold less dosage per 25 mg vehicle (e.g., 0.5 - li ng minimum, 2.1-2.5 ng for half maximal effect, 2.5- 5.0 ng for a full effect).

The osteogenic proteins of the invention may be administered in a liquid medium to a patient or to cultured cells. Preferably though, for in vivo administration, the protein is formulated in a delivery vehicle that provides sustained or controlled release of the protein and other active ingredients. Such delivery vehicles preferably comprise a bioerodible, surface eroding polymer matrix that degrades in situ , releasing the protein and other active ingredients to the surrounding biological environment. A delivery vehicle that comprises a solid or semi-solid matrix (e.g. gel or beads) can be implanted at the site where muscle repair or regeneration is desired or where myogenic stem cells are located.

Particularly preferred delivery vehicles comprise reversible gelling compositions, such as Pluronic™ (BASF, New Jersey) . These vehicles are liquid at low temperature and form semi-solid gels at higher temperature (e.g. body temperature) . Thus they can be injected, rather than implanted, into the desired site in the body. The foregoing discussion has focused on therapeutic uses of the osteogenic proteins of the invention for stimulating myogenesis in developing embryos or to effect repair/regeneration of mature muscle. The proteins of the invention will also find broad utility as myogenic agents in other areas. For instance, they can be used for research purposes to study regulatory features of embryological muscle development, or fundamental mechanisms of satellite cell activation in muscle regeneration and repair, or the coordination of muscle and bone regeneration in response to injury. This latter research utility may also have a therapeutic aspect, i.e. for limb or digit regeneration. As another example, the proteins will find utility in the agricultural area, for increasing muscle mass in meat animals. Such a utility can be realized by various of the ex vivo techniques described above, especially as combined with ex vivo genetic engineering of myogenic stem cells (e.g. to over-express myoD or other recombinant genes, as described, for example, in WO 93/21347) , followed by stimulation of myogenesis and re-introduction into animals.

The following examples are provided to describe the invention in greater detail. The examples are intended to illustrate, rather than to limit, the invention. In the examples, OP-1 is referred to as BMP7.

EXAMPLE 1 Mouse presomitic mesoderm (psm) Ex lant Model

A mouse embryo tissue explant model has been developed for investigation of myoD regulation and the determination of skeletal muscle cell lineages in the mammalian embryo. This culture system was developed to investigate and define signal molecules that direct the activation of myoD and myf5 and determine presomitic mesoderm (psm) cells to the skeletal muscle lineage.

Explants of presomitic mesoderm were dissected from 9.5 day mouse embryos with sharpened tungsten knives and plated into 48 well gelatin coated plates. During the initial culture period, tissues attach, and approximately 2000 cells spread to form a monolayer on the gelatin-coated surface. After three days in culture, explants were fixed with 4% paraformaldehyde and stained with antibodies or LacZ, as described for individual experiments below. We have investigated three different culture explants: 1) psm dissociated from other associated tissues by dispase digestion (psm) ; 2) psm isolated in association with ectoderm (psm+ect) ; and 3) psm isolated in association with neural tube/notochord (psm+ncnt) , with and without ectoderm.

Psm explants were derived from two transgenic strains of mice that carry lacZ transgenes that express β-galactosidase fused to a nuclear localization domain, under the control of the human and the quail myoD transcription enhancers (Goldhamer et al., 1995; Pinney et al., 1995). The human and quail myoD enhancers drive lacZ expression in somitic lineages in the developing mouse embryos, as visualized by lacZ staining of embryo whole mounts. We have also examined lacZ activation in cultures derived from transgenic mice that carry the entire 24 kb region 5' of the human myoD gene, including the core enhancer at -22kB (Goldhamer et al., 1992; Goldhamer et al., 1995; Faerman et al., 1995), and the distal regulatory region (DRR) at -5kB (Tapscott et al., 1992; Asakura et al., 1995). Cultures derived from lacZ transgenic embryos were stained for β- galactosidase expression to assay myoD enhancer activity, as well as with myosin heavy chain antibodies, to assay muscle differentiation.

Psm cultures were also derived from non- transgenic embryos. Cultures derived from non- transgenic mice were stained with MyoD antibodies using immunoperoxidase assays to detect MyoD expression in activated nuclei.

When the procedures described above were performed, it was found that both the human and quail myod enhancer transgenes are expressed in presomitic mesoderm cultures in a subset of myosin-positive cells in response to co-culture with the neural tube / notochord. EXAMPLE 2 Immunohistological Assays of myoD Activation in psm Cultures Cultures of psm, psm+ect and psm+ncnt were treated with a variety of signaling molecules and assayed for MyoD expression using immunohistological assays. After three days in culture, explants were fixed in 4% paraformaldehyde and stained with the MyoD 5.8a antibody (Dias et al., 1992) using a Vectastain Elite kit (Vector Labs, immunoperoxidase stain) .

Figure 1 shows results of the above- described experiments. As can be seen from Fig. 1A, both 200 ng/ml sonic hedgehog (Shh) and 1 ng/ml BMP7 give approximately a 2 fold increase over base and the P value shows high significance while low concentrations of two other BMP family members (BMP2 and GDF5) decrease the number of positive cells. The two proteins together show nearly a 3.5 fold increase over base. Both 200 ng/ml Shh and 1 ng/ml BMP7 give greater than three fold increase over base in the presence of media conditioned on RatBla-Wnt4 cells while the conditioned media itself shows no effect. As can be seen in Fig. IB, high concentrations of BMP proteins (100 ng/ml) decrease the number of myoD positive cells in psm/NCNT cultures to levels that, in the case of BMP7 , are comparable to the background psm alone. Interestingly, culturing in the presence of 1 ug/ml of BMP7 blocking antibody increases the number of myoD positive cells nearly two fold.

The results of the experiments shown in Figure 1 and in other experiments (not shown) are summarized below.

1) Psm shows no MyoD expression, either when cultured alone or in the presence of the variety of signaling molecules that induce or inhibit MyoD in psm+ect and psm+ncnt cultures. These studies suggest that the ectoderm and/or neural tube/notochord produce as yet unidentified signal molecules that are required along with BMP7 and sonic hedgehog protein to induce expression of myoD and myf5 (it is believed that these as-yet unidentified molecules may function in maintenance of upstream genes, such as pax3 ) . 2) Psm+ect in base medium has a low basal level of MyoD expressing cells (30 cells per explant) , whereas psm+ncnt in base medium has a higher basal level of MyoD expressing cells (400 cells per explant) . These findings and those described above are consistent with the findings of Cossu et al. (1996) that both the ectoderm and neural tube/notochord have myoD inducing activities on psm.

3) Sonic hedgehog (Shh) , a signal molecule produced by the ncnt, induces MyoD expression in psm+ect cultures by 2-3 fold above basal expression. Shh recombinant protein used in these assays includes the N-terminal 198 amino acids of the protein (Marti et al., 1995a; Marti et al., 1995b). Shh in combination with conditioned medium derived from Wnt4-expressing Rat Bla cells induces MyoD expression by 4-5 fold above basal expression.

As shown in Fig. 1A, Wnt4 conditioned medium along has no MyoD induction activity, and base medium in combination with Shh and Wnt4 conditioned medium does not induce MyoD expression in cultures of psm alone, as predicted from the work on avian psm (Munsterberg et al., 1995). In these avian psm studies, MyoD was assayed using RT-PCR, which monitors overall MyoD mRNA levels, whereas in our experiments MyoD protein was assayed using immunostaining, which monitors numbers of cells induced to produce MyoD. It should be noted that conditioned medium prepared from Rat Bla cells alone (uninfected with Wnt retroviral expression vectors) can enhance the activity of Shh as well as BMP7 (see below) in MyoD induction (data not shown) . 4) At low concentrations, BMP7 induces MyoD 2-3 fold above basal levels in psm+ect cultures, and BMP7 in combination with either Shh or Wnt4 conditioned medium further enhances the inductive effect of low concentrations of BMP7. Those results suggest that BMP7 and Shh, in the presence of ectoderm, have independent inductive activities (though they both may regulate myoD expression by a common downstream signal, myf5 , as discussed in Example 4) . This conclusion is further supported by the observation that BMP7 blocking antibodies do not inhibit MyoD induction in psm+ncnt cultures, but actually enhance expression by as much as two fold, consistent with the findings, discussed below, that high concentrations of BMP7 as well as high and low concentrations of BMP2 and GDF5, strongly inhibit MyoD expression in psm+ncnt cultures.

5) High and low concentrations of BMP2 and GDF5 and high concentrations of BMP7 inhibit basal expression of MyoD, both in psm+ect and psm+ncnt cultures, consistent with idea that BMPs are general inhibitors of MyoD and myogenesis, and that the inductive effects of low concentrations of BMP7 are specific. The concentration-dependent effect of BMP7 on myoD expression is described in greater detail in Example 3 below.

Based on the above observations and considerations, we believe that, in the mouse embryo, BMP7 along with other as yet to be defined ectoderm factors, including Wnts, induce MyoD and initiate myogenic lineage determination. MyoD also can be activated indirectly by Myf5 (see Example 4) .

In the avian embryo, myoD and myf5 are co- activated in medial somite cells adjacent to the ncnt complex (Pownall et al, 1992) . Therefore, Shh could be a direct regulator of both myoD and myf5 (though it appears that myf5 is required for Shh induction of myoD expression, as described in Example 4) , although the data do not exclude a role for BMP7 in myoD regulation in avian embryos, which seems likely. However, we have discovered that BMP7 is expressed in the dorsal neural tube (but not in the overlying ectoderm) near the site of myoD activation, so it appears that BMP7 also has a role in myoD activation in the avian as well as the mammalian embryo.

EXAMPLE 3

Further Characterization of the Role of BMP7 in myoD Regulation

The results reported in this example, using the above-described psm assay, provide further characterization of the role of BMP7 in myoD regulation. Culture methods and other methods are as described in Examples 1 and 2 , with the addition of methods relating to Wnt treatments, as set forth below.

In assays to test Wntl and Wnt4, Rat Bla cells infected with control retrovirus (MV7) or with retroviruses expressing Wntl or Wnt4 cDNAs (obtained from Anthony Brown, Cornell Medical School) were seeded at low density on microtiter dishes in base medium minus bFGF. Psm explants were plated on top of the low density Rat Bla cells in base medium plus bFGF and co-cultured for 3 days, at which time the Rat Bla infected cells had formed a monolayer. Experimental results are shown in Figures 2-

5. The data shown in Figure 2 (in part presented in Figure 1A described in Example 2) show that low concentrations of BMP7 and sonic hedgehog (Shh) enhance myoD expression in psm (with associated ectoderm) cultures, and that BMP7 and Shh act synergistically. Moreover, the BMP7 inductive effect is specific in that two other BMPs (BMP2 and GDF5) do not induce myoD alone or in combination with Shh. The data shown in Figure 3 show that high concentrations of BMP7 , as well as BMP2 and GDF5, inhibit induction of myoD in psm co-cultured with neural tube/notochord and ectoderm, which induces high levels of myoD presumably because the neural tube and notochord produce high levels of Shh. (When ectoderm is removed, myoD activation occurs, although at lower levels, consistent with the idea that the signals are coming from the neural tube/notochord) . It is believed that the effect of high concentrations of BMP7 is to activate other regulatory genes in psm, such as paxl , that push the psm cells along the bone differentiation pathway.

The significant conclusion from the data in Figures 2 and 3 is that BMP7 shows specific, concentration-dependent activities for the induction and inhibition of myogenic determination, as assayed by the activation of myoD .

The data set forth in Figure 4 show that Wntl can greatly potentiate the myoD inducing activity of low concentrations of BMP7 on psm/ectoderm cultures and that the effects of Wntl are specific in that Wnt4 does not work cooperatively with BMP7. These data are significant in that they show that BMP7 in combination with Wntl is a very strong inducer of myoD and that, in combination with Shh, these signals synergize to induce myoD to the high levels achieved in control culture in which psm is co-cultured with both neural tube/notochord and ectoderm (see Figure 3) . Our interpretation of these data (as discussed in Example 2) is that myoD activation occurs through two pathways, which may or may not be independent of one another (see Example 4) : a Shh pathway from the neural tube/notochord, and a BMP7 pathway from the ectoderm. Wntl appears to play a general role as its activity enhances both Shh and BMP7. The data set forth in Figure 5 show that ectoderm is required for both Shh and BMP7 induction of myoD in pre-somitic mesoderm explants. Note that the basal expression of myoD in psm ectoderm co- cultures in 30 cells per well (see Figure 4) . In the absence of ectoderm, activity is reduced to less than 4 cells per psm explant, and combinations of BMP7, Shh and Wntl do not stimulate myoD expression. In these studies ectoderm is separated by dispase, which does not damage tissue as shown by reconstitution of ectoderm in cultures after dispase treatment (not shown) . These results imply the existence of additional general maintenance factors provided by the ectoderm, which are necessary for Shh and BMP7 induction of myoD in pre-somitic cells. Since ectoderm is not a part of adult tissue, satellite cells involved in repair and regeneration of adult muscle either do not require these general maintenance factors, or the factors are provided by a variety of tissues in the satellite cells' surrounding environment .

EXAMPLE 4 Role of myf5 in Shh and BMP7 induction of myoD

In the previous Examples, we reported the discovery that low concentrations of BMP7 (OPl) induce the myogenic determination gene, myoD , in tissue culture explants of day 9.5 presomitic mesoderm. Sonic hedgehog (Shh) , another embryonic signaling molecule, also induces myoD , and together, Shh and OPl have cooperative inductive activities. High concentrations of OPl block the ability of Shh to induce myoD , as do high and low concentrations of BMP2 and GDF5. The independent and cooperative activities of Shh and OPl in myoD induction led us to propose that these signal molecules are working through two distinct pathways to activate myoD and initiate skeletal myogenesis in presomitic mesoderm.

The data reported in this Example indicates that Shh and OPl actually function together in a common jny.f5-dependent pathway to activate myoD in presomitic mesoderm from early stage embryos. In addition, we have preliminary evidence that Shh activates the synthesis of OPl protein in mesoderm cells. However, because myoD also is activated in later stage embryos in myf5 mutant mice, a myf5- independent inductive mechanism also must exist to activate myoD .

Specific Findings: myf5 is essential for Shh and BMP7 inductive activity. Explant cultures of presomitic mesoderm were prepared from day 9.5 mouse embryos that were heterozygous and homozygous for mutant myf5 , as assayed by single embryo PCR. Cultures from individual embryos were tested for myoD induction by Shh and BMP7. (myf5 mutant mice, provided by Dr. M Rudnicki, McMaster University, were generated by gene targeting) . These experiments showed that both the independent and cooperative activities of Shh and BMP7, as well as neural tube/notochord signals, are completely blocked in mesoderm prepared from embryos that are mutant for myf5 and reduced in mesoderm of embryos that are heterozygous for myf5 , compared to wild type mesoderm. Thus, the myoD-inducing activities of Shh and BMP7 require myf5 and, therefore, function upstream in a my s-dependent pathway. As myoD can be activated in later developmental stages in ovo in my-f5-mutant embryos, a second myf 5-independent pathway is likely to exist.

Shh induces BMP7. Immunostaining of presomitic mesoderm cultures with anti-BMP7 monoclonal antibody reveals that Shh increases expression of BMP7 in mesodermal cells by 10 fold. These finding will be confirmed by using additional BMP7 antibodies and use double immunostaining to identify whether BMP7- expressing cells are muscle progenitors express myoD, myfδ or other muscle genes or distinct cell population.

pax3 is essential for Shh and BMP7 inductive activity. Presomitic mesoderm explants from splotch embryos (pax3 null, provided by J. Epstein at Penn) do not respond to Shh, BMP7 or neural tube/notochord signals to activate myoD , consistent with the recent findings by others that pax3 is essential for a mesoderm functions required for myoD activation. We think that pax3 has a general function in mesoderm.

The findings set forth in this example and the previous examples suggest three alternative models for Shh/BMP7 control of myoD activation in presomitic mesoderm explants of the early mouse embryo, as outlined below.

1. A dependent pathway assumes that Shh controls BMP7 expression, which in turn activates myf5 , which in turns activates myoD. This model accounts for the BMP7 inductive activity of Shh on BMP7 expression and the essential requirement of myf5 , but does not explain the cooperative induction by Shh and BMP7. 2. A co-dependent pathway assumes that Shh is required for inducing mesoderm to express myf5 , and then BMP7 functions together with myf5 to activate myoD . This model assumes that some explanted mesoderm cells already express a myf5 because of earlier Shh signaling from neural tube/notochord, and Shh added to tissue culture further increases the number of myf5 positive cells. 3. A parallel pathway model assumes that there are two linages of presomitic mesoderm cells, a Shh-responsive lineage and a BMP7 responsive lineage, which respond independently to these signals to induce myoD through myfδ , which may be expressed in these cultures or be induced by these signal molecules.

REFERENCES

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While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. Therefore, the invention is not limited to the particular embodiments described or exemplified, but is capable of variation and modification without departure from the scope of the appended claims.

Claims

What is claimed is:

1. A method of promoting myogenesis of stem cells in culture or in a patient in need of such treatment, comprising administering to the cells an osteogenically active OP-1 protein, for a time and at a concentration effective to stimulate said myogenesis.

2. The method of claim 1, wherein said stem cells are of embryonic origin.

3. The method of claim 1, wherein said stem cells are satellite cells.

4. The method of claim 1, wherein said protein is administered to an embryo.

5. The method of claim 1, wherein the protein is administered in combination with a therapeutically effective amount of a compound selected from the group consisting of growth factors, proliferative agents, antibiotics and anti-inflammatory agents.

6. The method of claim 1, wherein said administering step is performed by implanting or injecting in the patient the protein incorporated into a delivery vehicle that provides controlled release of the protein at a site where the myogenesis of stem cells is desired.

7. The method of claim 1, wherein said administering step is performed by obtaining the stem cells by removal from a donor, administering the protein to the removed cells, and implanting the removed cells into the patient at a site where muscle regeneration is desired.

8. A method of inducing expression of a myoD gene, which comprises exposing the gene to an osteogenically active OP-1 protein in an amount and for a time effective to induce expression of the gene.

9. The method of claim 8 , wherein said myoD gene is exposed to a concentration of said protein of between about 0.1 ng/ml and about 1.0 ng/ml.

10. The method of claim 8, wherein said myoD gene is exposed to said protein in combination with a compound selected from the group consisting of growth factors and proliferative agents.

11. The method of claim 8, wherein said myoD gene is exposed to said protein in combination with at least one compound selected from the group consisting of sonic hedgehog protein, Wntl protein and myf5 protein.

12. The method of claim 8, wherein said myoD gene is disposed within a cultured stem cell.

13. The method of claim 8, wherein said myoD gene is disposed within a stem cell of an intact, living embryo.

14. The method of claim 8 , wherein said myoD gene is disposed within a cultured satellite cell.

15. The method of claim 8, wherein said myoD gene is disposed within a satellite cell of a patient.

16. A pharmaceutical composition for promoting myogenesis of stem cells in a patient in need of such treatment, comprising a myogenic amount of an osteogenically active OP-1 protein in a medium biologically compatible with the stem cells.

17. The pharmaceutical composition of claim 16, which further comprises a therapeutically effective amount of one or more compounds selected from the group consisting of growth factors, proliferative agents, antibiotics and anti-inflammatory agents.

18. The pharmaceutical composition of claim 16, wherein the biologically compatible medium comprises an injectable liquid.

19. The pharmaceutical composition of claim 16, wherein the biologically compatible medium comprises an implantable solid or semi-solid matrix.