JP2002506623A - Composition for regulating cell differentiation, comprising lipid and morphogen - Google Patents

Abstract

  (57) [Summary] Disclosed are compositions and methods for modulating the differentiation state of a cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention is generally in the field of cell differentiation. In particular, the present invention provides compositions that modulate the process of cell differentiation and methods of using the same.

BACKGROUND OF THE INVENTION The process of cell differentiation is a continuous process that makes cells more specialized as evidenced by different functions, biochemical capabilities and morphology. Such a collection of differentiated cells gives rise to characteristic, functional tissues. In the absence of disease or trauma, once a cell has acquired a particular differentiated state, its fate is typically irreversible, and the characteristic phenotype of the differentiated cell is a particular When a differentiated cell maintains its ability to divide, it is passed on to its progeny.

[0003] A well-known example of cell differentiation is hematopoiesis. Here, progenitor stem cells are directed down one of many possible differentiation pathways into specific types of blood cells (eg, red blood cells,
Basophils, eosinophils, neutrophils, monocytes, platelets). Various hematopoietic growth factors have been identified. These can affect cells at certain steps in the process,
As a result, the cells are destined to follow an appropriate pathway leading to a particular differentiated blood cell type.

[0004] Morphogens are known to induce the proliferation and differentiation of precursor cells in a number of soft and hard tissues. As morphogens, bone morphogenic proteins (BM) identified by their ability to induce ectopic endochondral bone morphogenesis
P) family members. Morphogens are also referred to as osteogenic proteins, but are generally classified as a subgroup of growth factors of the TGF-β superfamily (Hogan (1996) Genes & D
evelment 10: 1580-1594). Members of the morphogen family of proteins include mammalian bone morphogenetic protein 1 (OP-1, BM
P-7 and Drosophila homolog 60A), bone morphogenetic protein 2 (OP-2, also known as BMP-8), bone morphogenetic protein 3
(OP-3), BMP-2 (BMP-2A or CBMP-2A, and Dro
sophila homolog DPP), BMP3, BMP4 (BM
P-2B or CBMP-2B), BMP5, BMP6, and their mouse homologs Vgr-1, BMP-9, BPM-10, BMP-
11, BMP-12, GDF3 (also known as Vgr2), GDF8, GD
F9, GDF10, GDF11, GDF12, BMP-13, BMP-14, B
MP-15, GDF-5 (also known as CDMP-1 or MP52), G
DF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), Xenopus homolog Vgl and NODAL, UNIVIN
, SCREW, ADMP, or NEURAL.

[0005] Mature morphogens result from processing through a "pro-form" to obtain a mature polypeptide chain capable of dimerizing and contain a carboxy-terminal active domain of about 97-106 amino acids. All members share a conserved pattern of cysteines in this domain, and the active form of these proteins can be either a disulfide-linked homodimer of a single family member or a heterodimer of two different members. (For example, Massag
ue (1990), Annu. Rev .. Cell. Biol. 6: 597; Sa
mpath et al. (1990) J. Am. Biol. Chem. 265: 13198. ). US Pat. No. 5,011,691, US Pat. No. 5,266,683
Ozkaynak et al. (1990), EMBO J .; 9: 2085-2093,
Wharton et al., (1991) PNAS 88: 9214-9218, Ozk.
aynak, (1992) J. Am. Biol. Chem. 267: 25220-25
227 and US Pat. No. 5,266,683, Celeste et al. (1991).
, PNAS 87: 9843-9847, Lyons et al. (1989), PNAS.
86: 4554-4558. These disclosures describe the amino acid and DNA sequences of osteogenic proteins, as well as chemical and physical properties. Wozney et al. (1988) Science 242: 1528-153.
4; BMP9 (WO93 / 00432, published January 7, 1993); DPP (P
adgett et al. (1987) Nature, 325: 81-84; and Vg-.
1 (Weeks (1987) Cell 51: 861-867).

[0006] Proper tissue repair, prevention, and regeneration depend on the ability of cells to enter and follow down certain pathways of cell differentiation. Therefore, repair of healthy tissue,
It is desirable to control the fate of cells that promote regeneration or maintenance.

SUMMARY OF THE INVENTION It has now been discovered that morphogenic proteins can be combined with other factors to direct cell fate. The present invention provides compositions and methods that affect cell fate. In particular, the compositions provided herein modulate the process of cell differentiation to promote the regeneration, repair or maintenance of healthy tissue.

[0008] The compositions of the present invention comprise a morphogen in combination with a lipid. Such compositions influence the down-regulation of a cell's particular pathway of differentiation. Lipid molecules useful in the present invention are organic compounds that are extractable from biological material by non-polar solvents such as ether, chloroform, benzene, and are not readily extractable by aqueous solvents. Such lipids include neutral lipids (eg, steroids (including cholesterol and glucocorticoids such as dexamethasone)); charged and polar lipids (eg, phospholipids, fatty acids, and sphingolipids); and eicosanoids (eg, , Prostaglandins, thromboxanes, leukotrienes and lipoxins). In a preferred embodiment, the lipid in the composition of the invention is a glucocorticoid (eg, dexamethasone).

[0009] The compositions of the present invention may further comprise a biocompatible matrix (eg, hydroxyapatite), collagen (particularly bovine bone collagen), or other biocompatible synthetic matrix. The compositions of the present invention may also include a carrier such as carboxymethylcellulose or fibrin glue. The morphogen / lipid compositions of the present invention can be administered in a physiologically acceptable buffer, such as an acetate buffer or a saline buffer.

[0010] The invention also provides a method of regulating cell differentiation, the method comprising regenerating tissue,
Treating the cells with an effective amount of a composition comprising a morphogen and a lipid to direct the population of cells down a differentiation pathway for repair or conservation. According to the methods of the present invention, the compositions can be applied to cells in vitro, ex vivo, or in vivo. The cells can be autologous, allogeneic, or xenogeneic. The methods of the invention are useful for influencing the differentiation of any cell type at any stage of development. However, such methods are particularly useful for altering the differentiation of pluripotent cells, or pluripotent stem cells. Such methods include, for example, mesenchymal cells, dedifferentiated cells, cancer cells, epithelial cells, hematopoietic cells, red blood cells,
And for effecting the differentiation or redifferentiation of a cell type selected from other stem cells.

DETAILED DESCRIPTION Morphogens have previously been described for tissue morphogenesis, including osteogenesis and adipogenesis.
(Eg, Sampath et al., J. Biol. C.
hem. 267: 20352-20362 (1992); Asahina et al., E.
xp. Cell Res. 222: 38-47 (1996); Reddi, Na
see Biochemistry, 1998). It has now been discovered that compositions comprising morphogens and lipids can regulate cell differentiation. Cell differentiation is the process by which multiple or pluripotent mesenchymal cells or other progenitor (stem) cells change into differentiated cells identified by characteristic proteins, biochemical activities and morphology .

[0012] The compositions described herein are particularly useful for affecting the cell fate of multiple or pluripotent mesenchymal cells. Such mesenchymal cells have the ability to differentiate into a particular cell type under the influence of differentiation factors present in a particular local environment. The compositions of the present invention can affect the direction and extent of development of such pluripotent cells. Such terminal effects can be, for example, fully differentiated cells found in the liver, heart or bone. Alternatively, the composition-induced differentiation can result in precursor cells for a given cell line. Precursor cells of such lineage
Includes muscle precursor cells, adipocytes and bone precursor cells. Precursor cells of such lineage retain the ability to further differentiate, but have characteristic morphological markers and / or
Alternatively, it can be identified by biochemical markers (eg, Asahina et al., E.
xp. Cell Res. 222: 38-47 (1996)).

[0013] The composition of the invention comprises a morphogen and a lipid. In its mature native form, the naturally occurring morphogen is a glycosylated dimer, typically about 30-36 as determined by SDS-PAGE.
It has an apparent molecular weight of kDa. When reduced, the 30 kDa protein yields two glycosylated peptide subunits, which are approximately 16 kD
a and an apparent molecular weight of about 18 kDa. In the reduced state, the protein has no detectable osteogenic activity. Non-glycosylated proteins, which also have osteogenic activity, have an apparent molecular weight of about 27 kDa. When reduced, the 27 kDa protein gives rise to two non-glycosylated peptide chains, which have a molecular weight of about 14 kDa to about 16 kDa. Typically, naturally-occurring bone morphogenetic proteins are translated as precursors, which comprise an N-terminal signal peptide sequence (typically less than about 30 residues), followed by cleavage of mature C It has a "pro" domain giving rise to a terminal domain. The signal peptide is rapidly cleaved at the cleavage site during translation. This cleavage site is called Von He
ijne (1986) Nucleic Acids Research 14:
4683-4691 and can be predicted in a given sequence. This prodomain is typically about 3 times larger than the fully processed C-terminal domain.
Twice as large.

[0014] Morphogens useful herein include any known naturally occurring native proteins (allelic variants, phylogenetic counterparts and other variants thereof, whether naturally occurring, Biosynthetically generated (for example, "
Muteins "or" mutant proteins ")), as well as morphogenic members of the new, common osteogenic family of proteins.

Particularly useful sequences are DPP (from Drosophila), Vgl (Xeno
pus), Vgr-1 (from mouse), OP1 and OP2 proteins, proteins (U.S. Pat. No. 5,011,691 and Upperman
and BMP2, BMP3, BMP4 (WO 88/002).
05, U.S. Pat. No. 5,013,649 and WO 91/18098), BMP5 and BMP6 (WO90 / 11366, PCT / US90 / 0).
1630), which include the C-terminal 96 or 102 amino acid sequence of BMP8 and BMP9. Other proteins useful in the practice of the present invention are OP1, OP2, OP3, BMP2, BMP3, BMP4, BMP5, BM
P6, BMP9, GDF-5, GDF-6, GDF-7, DPP, Vgl, Vg
r, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6, G
DF-7, BMP10, BMP11, BMP13, BMP15, UNIVIN,
Includes active forms of NODAL, SCREW, ADMP, or NURAL, and amino acid variants thereof. In one presently preferred embodiment, the osteogenic protein is OP1, OP2, OP3, BMP2, BMP4, BMP5, BM
P6 and BMP9, and their amino acid sequence variants and homologs (
This includes species homologs). OP-1 and O
Publications disclosing the P-2 sequences, and their chemical and physical properties, are described in U.S. Patent Nos. 5,011,691 and 5,266,683, which are incorporated herein by reference. Incorporated).

In a preferred embodiment, the morphogen for use in the method of the invention has at least 70% homology with the amino acid sequence of the C-terminal 7 cysteine domain of human OP-1 (SEQ ID NO: 2), And Re described in this specification
Includes proteins that have the ability to induce endochondral bone formation in ddi and Sampath assays. Compounds meeting these requirements are considered to be functionally equivalent to known response morphogens. The candidate sequence and the reference sequence are aligned to determine whether the candidate amino acid sequence is functionally equivalent to the reference morphogen. The first step in performing the alignment is to use an alignment tool (eg, N
eedleman et al., 48J. Mol. Biol. 443 (1970), and DNAstar, Inc. ,
Align Prog, a commercial software package manufactured by
ram). These teachings are incorporated herein by reference. After performing an initial alignment, it is then refined by comparison with a multiple sequence alignment of a family of related proteins (eg, those shown in FIGS. 1A-1L). This alignment is a multiple sequence alignment of a family of known morphogens (including hOP-1). Once the alignment between the candidate sequence and the reference sequence is performed,
Then, upon refinement, a percent homology score is calculated. The individual amino acids of each sequence are sequentially compared according to their similarity to each other. Similarity factors include similar size, shape and charge. Determining amino acid similarity 1
One particularly preferred method is described by Dayhoff et al., 5 Atlas of Prot.
ein Sequence and Structure 345-52 (19
78 and an addendum, which is a PAM250 matrix described in U.S. Pat. The similarity score is first calculated as the sum of the aligned pair amino acid similarity scores. Insertions and deletions are ignored for purposes of percent homology and identity. Therefore, the gap penalty is not used in this calculation. The raw score is then compared to the candidate compound and hOP-
Normalize by dividing by the geometric mean of the scores of one of the seven cysteine domains. The geometric mean is the square root of the product of these scores. The normalized raw score is the percent homology.

In a preferred embodiment, the functionally equivalent morphogen sequence shares at least 60% amino acid homology with the reference sequence. That is, any 60% of the aligned amino acids are either identical to the corresponding amino acid in the reference sequence or are conservative substitutions. Any of one or more of the naturally occurring or biosynthesized morphogens disclosed herein can be used as a reference sequence to determine whether a candidate sequence belongs to the morphogen family. In a preferred embodiment, the reference sequence is the C-terminal 7 of human OP-1 shown in SEQ ID NO: 2.
A cysteine backbone sequence. Examples of conservative substitutions for use in the above calculations include substitution of one amino acid for another with similar characteristics,
For example, substitutions within the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; Serine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine are well known. The terms "conservative variant" or "conservative variation" also mean that an antibody having binding specificity for the resulting substituted polypeptide chain also has a binding specificity with the unsubstituted or original polypeptide ( That is, "cross-reaction" or "
Immune response ") involves the use of a substituted amino acid in place of the original unsubstituted amino acid in a given polypeptide chain.

In a preferred embodiment, morphogens useful in the present invention are defined by a generic amino acid sequence that represents a modification in a known morphogen. For example, SEQ ID NOs: 4 and 5 encompass the modifications observed between preferred morphogens. This includes OP-1, OP-2, OP-3, CBMP-2A, CBMP-
2B, BMP-3, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr
-1 and GDF-1. SEQ ID NO: 5 includes all of SEQ ID NO: 4,
It also contains the five amino acid sequences of SEQ ID NO: 8 at its N-terminal. The global sequence is C
Terminal domain (defined by 6 cysteine skeleton and 7 cysteine skeleton)
Includes both the amino acid identity shared by these sequences in (SEQ ID NOS: 4 and 5, respectively), and alternative amino acids for the variable positions within the sequence. Positions where alternative amino acids are possible are denoted by “Xaa”. FIGS. 2A and B show alternative amino acids for each “Xaa” position in SEQ ID NOs: 4, 5 and 8. For example, referring to SEQ ID NO: 5 and FIGS. 2A and B, the "X" at position 2
“aa” can be tyrosine or lysine. The global sequence provides the appropriate cysteine backbone for intermolecular or intramolecular disulfide bonds, and includes certain key amino acids that are likely to affect the tertiary structure of the protein. Additionally, position 36 in SEQ ID NO: 4, or 41 in SEQ ID NO: 5
The position "Xaa" may be an additional cysteine, thereby encompassing the morphogenic active sequences of OP-2 and OP-3.

In another embodiment, a useful morphogen is SEQ ID NO: 6 or SEQ ID NO: 7
And those defined by These are complex amino acid sequences of the following morphogens: human OP-1, human OP-2, human OP-3, human BMP-2
, Human BMP-3, human BMP-4, human BMP-5, human BMP-6, human B
MP-8, human BMP-9, human BMP-10, human BMP-11, Droso
phila 60A, Xenopus Vg-1, sea urchin UNIVIN, human CD
MP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6, human B
MP-13), human CDMP-3 (human GDF-7, human BMP-12), mouse GDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN
, Drosophila dpp, Drosophila SCREW, mouse N
ODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GD
F-10, human GDF-11, mouse GDF-11, human BMP15, and rat BMP-3b. SEQ ID NO: 7 includes all of SEQ ID NO: 6 and also, at its N-terminus, the five amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 6 corresponds to a C-terminal 6 cysteine skeleton and SEQ ID NO: 7 corresponds to a 7 cysteine skeleton. Positions that allow for alternative amino acids are represented by “Xaa”. FIG. 3A, B
And C indicate alternative amino acids for each "Xaa" position in SEQ ID NOs: 6, 7 or 9.

As noted above, certain preferred morphogen sequences useful in the present invention have greater than 60% identity, preferably greater than 65%, with the amino acid sequence defining the preferred reference sequence for hOP-1. Have identity. These particularly preferred sequences include allelic and morphological variants of the OP-1 and OP-2 proteins (Drosophila 60A protein, and the closely related proteins BMP-5, BMP-6, and Vgr-1). Including). Therefore,
In certain particularly preferred embodiments, useful morphogens include proteins comprising the generic amino acid sequence SEQ ID NO: 3 (referred to herein as "OPX"). This amino acid sequence defines a seven cysteine backbone and OP-1 and O
Accommodates homology between several identified variants of P-2. Positions that allow for alternative amino acids are represented by “Xaa”. FIG. 4 shows alternative amino acids for each “Xaa” position in SEQ ID NO: 3.

In yet another preferred embodiment, useful morphogens include those having an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with DNA or RNA encoding a reference morphogen. Standard stringency conditions are characterized in standard molecular biology textbooks. Generally, Molecular Cloning A Laboratory Manual (Sambrook et al., Eds., 1989);
DNA Cloning, Volumes I and II (ed by DN Glover, 1
Oligonucleotide Synthesis (MJ.
Gait, eds., 1984); Nucleic Acid Hybridizat.
ion (BD Hames and SJ Higgins eds., 1984); B
. Perbal, A Practical Guide To Molecular
See ar Cloning (1984).

[0022] Lipids useful in the compositions and methods of the present invention can be extracted from biological materials by non-polar solvents (eg, ether, chloroform, benzene), but not readily by aqueous solvents. There are no organic compounds. Such lipids can be isolated from natural sources or synthesized and can be neutral, polar or charged molecules. Lipids useful in the compositions and methods of the present invention include steroids (eg, cholesterol and glucocorticoids); fatty acids; phospholipids; and sphingolipids; Including, but not limited to. In one embodiment of the invention, the lipid component of the composition is a lipid characteristic of a differentiated cell that is desired to be maintained.
In another embodiment, the lipid component is present in an amount sufficient to provide a desired differentiated state.

The compositions provided herein can also include various other components. For example, in addition to morphogens and lipids, the compositions of the invention can include a biocompatible, biodegradable matrix, such as collagen or hydroxyapatite. Such a matrix allows cells to invade and proliferate into the matrix material during differentiation. As an alternative in applications where the structural integrity of the resulting tissue is less important, the compositions of the invention can be prepared in non-matrix formulations. This includes carboxymethylcellulose, fibrin glue,
Alternatively, a physiologically acceptable buffer (eg, acetate buffer or physiological buffer) is included.

The cells may be exposed to the compositions of the invention either in vitro or in vivo, or ex vivo applications. The resulting degree of cell differentiation can be monitored by assaying for characteristic phenotypic markers, such as morphological or biochemical activity. The cells to which the compositions of the invention are exposed can be autologous, allogeneic, or xenogeneic.

The compositions of the present invention are useful in regenerating, repairing, and maintaining tissue that is affected by disease, trauma, or aging. Further, the compositions and methods described herein are useful in tissue reconstitution procedures and in maintaining the integrity or viability of transplanted tissue. For example, the compositions of the present invention can be applied topically to regulate the differentiation of cells in specific areas of the tissue in need of maintenance, recovery or repair. Alternatively, the mesenchymal cells can be cultured in vitro or ex vivo in the presence of the composition of the present invention until the desired differentiation state is achieved,
It can then be transplanted into an individual in need of such differentiated cells.

The compositions of the present invention are useful for maintaining the integrity of a transplanted tissue or cell. For example, a composition of the present invention is administered to a patient before, after, or simultaneously with a transplant procedure to maintain the differentiated state of the transplanted organ, tissue or cell so as to match the recipient environment.

The compositions of the present invention can be administered directly to cells in culture or in vivo (by any of a variety of known modes of administration, including intraperitoneal, intravenous, intramuscular, subcutaneous, and sublingual).
Can be administered.

Example: Composition comprising Morphogen and Lipids for Regulating Pluripotent Mesenchymal Cell Differentiation The following experiment demonstrates that the composition of OP-1 and dexamethasone was obtained from the murine mesenchymal cell line D1 Was tested for its ability to regulate bone and adipogenesis.

Materials and Methods Pluripotent mesenchymal cells, D1, were cloned from mouse bone marrow stromal cells.
D1 cells were plated in 35 mm culture wells and maintained in DMEM with 10% fetal calf serum. After the cells reached confluence, they were treated either with increasing concentrations (10, 50, 100 ng / ml) of OP-1 or dexamethasone (10 ^-7 M) simultaneously. Cells were stained with either von Kossa staining to demonstrate matrix mineralization or with Sudan IV to identify lipid vesicles. Northern blots of total RNA were analyzed for osteocalcin cDNA, collagen type I cDNA and 4
Hybridization was carried out using 22 (aF2) cDNA and a fat-specific gene. In addition, cells were treated with OP-1 (50 μg / ml), antibody IB-12 against OP-1, or 10 ⁻⁷ M dexamethasone and IB-12, and steroid-induced adipogenesis was reduced by OP- 1 was mediated.

(Results) OP-1 induced both bone formation and adipogenesis changes in D1 cells. Accumulation of lipid vesicles in the cells started 4 days after treatment with OP-1. The number of adipocytes increased with increasing concentrations of OP-1 and time of treatment. 42
2 (aP2) expression is dose dependent and increases with time of treatment,
It reached the highest in 10 days. In control cultures not treated with OP-1, no adipocytes were observed, and no fat-specific gene expression was detected. OP-1 enhanced the osteoblast properties of D1 cells by increasing the number of mineralized nodules and osteocalcin gene expression, but the effect was 10 ng /
There was no dose dependency between ml and 100 ng / ml. However, OP-1
Inhibited the expression of collagen type I mRNA. Dexamethasone caused adipogenesis by stimulating 422 (aF2) gene expression in D1 cells, which reduced the expression of the osteoblast genes osteocalcin and type I collagen.

Stimulation treatment with OP-1 and dexamethasone has shown a synergistic effect in stimulating adipogenesis and on inhibiting the expression of the type I collagen gene. Dexamethasone also offset the stimulatory effect of OP-1 on osteocalcin gene expression. These results indicate that compositions comprising OP-1 and its lipid dexamethasone have a synergistic effect in stimulating cell differentiation.

[Sequence list]

[Brief description of the drawings]

FIG. 1A shows a multiple sequence alignment of a family of related proteins.

FIG. 1B is a continuation of FIG. 1A.

FIG. 1C is a continuation of FIG. 1B.

FIG. 1D is a continuation of FIG. 1C.

FIG. 1E is a continuation of FIG. 1D.

FIG. 1F is a continuation of FIG. 1E.

FIG. 1G is a continuation of FIG. 1F.

FIG. 1H is a continuation of FIG. 1G.

FIG. 1I is a continuation of FIG. 1H.

FIG. 1J is a continuation of FIG. 1I.

FIG. 1K is a continuation of FIG. 1J.

FIG. 1L is a continuation of FIG. 1K.

FIG. 1M is a continuation of FIG. 1L.

FIG. 2A shows alternative amino acids for each “Xaa” position in SEQ ID NOs: 4, 5 and 8.

FIG. 2B is a continuation of FIG. 2A.

FIG. 3A shows alternative amino acids for each “Xaa” position in SEQ ID NOs: 6, 7 or 9.

FIG. 3B is a continuation of FIG. 3A.

FIG. 3C is a continuation of FIG. 3B.

FIG. 4 shows alternative amino acids for each “Xaa” position in SEQ ID NO: 3.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C12N 5/06 C12N 5/00 EF term (reference) 4B024 AA20 BA21 CA04 DA02 EA04 GA11 HA01 4B065 AA90X AA93X AB01 BA02 CA24 CA44 CA46 4C081 AB11 CD022 CD111 CD122 CD28 CD29 CF032 DA15 4C084 AA01 BA01 BA02 BA22 BA44 MA01 NA13 ZB212 ZC022

Claims (16)

[Claims] 1. A composition comprising a lipid and a morphogen, wherein the morphogen comprises: (a) at least 70% amino acid homology to the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). And b) a protein having at least 60% amino acid identity with the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). 2. The composition according to claim 1, wherein said morphogen is OP-1. 3. The method according to claim 1, wherein the morphogen is BMP-2, BMP-7, or BMP-5.
The composition of claim 1, wherein the composition is selected from the group consisting of: and BMP-6. 4. The composition according to claim 1, wherein said morphogen is BMP-4. 5. The composition of claim 1, further comprising a biocompatible matrix. 6. The composition of claim 5, wherein said matrix comprises hydroxyapatite. 7. The composition of claim 5, wherein said matrix comprises collagen. 8. The composition according to claim 1, further comprising carboxymethylcellulose. 9. The composition of claim 1, further comprising fibrin glue. 10. The composition of claim 1, wherein the morphogen is non-covalently bound to one or more morphogen pro regions. 11. The composition according to claim 1, 5 or 8, further comprising a physiologically compatible buffer. 12. The composition according to claim 11, wherein said buffer is an acetate buffer. 13. A method for regulating cell differentiation, comprising: (a) a protein having at least 70% amino acid homology to the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). And b) exposing the cell to a composition comprising: (b) a protein having at least 60% amino acid identity with the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). 14. The method of claim 13, wherein said cells are selected from the group consisting of pluripotent mesenchymal cells, pluripotent stem cells, dedifferentiated cells, and precursor cells. 15. A method for generating precursor cells, comprising: (a) having at least 70% amino acid homology to the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). Exposing the undifferentiated cells to a composition comprising: a protein; and (b) a protein having at least 60% amino acid identity with the C-terminal 7 cysteine domain of OP-1 (SEQ ID NO: 1). Causing the undifferentiated cells to differentiate into precursor cells. 16. The method of claim 15, wherein said precursor cells are selected from the group consisting of muscle precursor cells, adipocytes, and preosteoblasts.