WO2010115053A2 - Methods of using canonical wnts as enhancers for bone formation by human bone-marrow derived mesenchymal stem cells - Google Patents

Abstract

The invention is directed to methods of using canonical Wnt ligands as enhancers for bone formation by bone-marrow derived mesenchymal stem cells (MSCs). More specifically, disclosed herein is the use of the canonical Wnt ligands, such as Wnt1 and Wnt3a, for the enhancement of ectopic bone formation.

Description

METHODS OF USING CANONICAL WNTS AS ENHANCERS FOR BONE FORMATION BY HUMAN BONE-MARROW DERIVED MESENCHYMAL STEM

CELLS

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part in the course of research sponsored by the NCI (CA71672), the Breast Cancer Research Foundation from NY State Department of Health. Accordingly, the U.S. government may have certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to methods of using canonical Wnt ligands as enhancers for bone formation by bone-marrow derived mesenchymal stem cells (MSCs). More specifically, disclosed herein is the use of the canonical Wnt ligands, such as Wntl and Wnt3a, for the enhancement of ectopic bone formation.

BACKGROUND OF THE INVENTION

Growing evidence indicates that Wnt signaling plays a critical role in stem/progenitor self-renewal in adult tissues (Reya and Clevers, 2005), in which these cells serve as reservoirs for tissue renewal in response to trauma, disease and aging. In the canonical pathway, Wnts signal through frizzled and LRP5/6 coreceptors, leading to inactivation of Axin/GSK3 β complex, which otherwise phosphorylates and directs degradation of β-catenin. Stabilized β-catenin translocates into the nucleus, forms complex with TCF/LEF transcription factors to activate Wnt target genes (Reya and Clevers, 2005). Some Wnts lack this ability and stimulate noncanonical pathways through effectors including c-Jun N-terminal kinase (JNK), Rho GTPase, or Ca²⁺ (Veeman et al., 2003).

Adult mesenchymal stem cells (MSCs) isolated from bone marrow are multipotent and give rise to tissues including bone, cartilage, muscle and adipose (Pittenger et al., 1999). Recent studies have revealed critical transcription factors involved in the commitments of different MSC-derived lineages. For example, osteoblastic differentiation is controlled by Runx2, Osterix, and Dlx5, whereas PPARγ is involved in adipocyte commitment (Harada and Rodan, 2003). Genetic studies have also established that Wnt/β-catenin activity is essential for normal osteogenesis (Day et al., 2005; Hill et al., 2005). Enhancement of Wnt signaling either by Wnt overexpression (Bennett et al., 2007) or deficiency of Wnt antagonists (Morvan et al., 2006; ten Dijke et al., 2008) is associated with increased bone formation in mice and humans. Loss or gain of function mutations in LRP5 also cause osteoporosis or high bone mass phenotypes, respectively (Boyden et al., 2002; Gong et al., 2001; Little et al., 2002).

The in vitro effects of Wnt signaling on osteogenic differentiation of MSCs are controversial. Wnt/ β-catenin signaling has been reported to stimulate differentiation of mouse MSCs towards the osteoblastic lineage (Gaur et al., 2005; Gong et al., 2001). However, both stimulatory (Gregory et al., 2005) and inhibitory (Boland et al., 2004; de Boer et al., 2004) effects of canonical Wnt signaling on osteogenic differentiation by human MSCs have been observed. Because of their osteogenic potential, human adult bone marrow MSCs are one of the most promising stem cell populations for bone regeneration as well as repairing critical-size bone defects that fail to undergo spontaneous healing (Meijer et al., 2007). Thus, there is a great need in the art to develop ways for influence MSC commitment along osteoblastic and other lineages.

SUMMARY OF THE INVENTION

The present invention addresses these and other needs by providing a method of enhancing bone formation in a subject in need thereof comprising administering to the subject a canonical Wnt ligand in an amount effective to enhance bone formation. Non-limiting examples of canonical Wnt ligands useful in the method of the present invention include Wntl, Wnt2, Wnt3, Wnt3a, Wnt7b, Wnt8, and WntlOb. In a preferred embodiment, such canonical Wnt ligand is Wntl or Wnt3a.

In the method of the invention, the canonical Wnt ligand can be administered in the form of a polypeptide or a nucleic acid encoding such polypeptide. Alternatively, the canonical Wnt ligand can be administered in the form of a bone-marrow derived mesenchymal stem cell (MSC) expressing such canonical Wnt ligand. In a preferred embodiment, such MSC is human MSC (hMSC).

In one embodiment of the invention, the canonical Wnt ligand is administered in the form of a matrix forming a bone transplant. Such matrix forming a bone transplant can be, for example, coated or mixed with (i) the canonical Wnt ligand or (ii) with MSCs expressing such canonical Wnt ligand or (iii) with a mixture of canonical Wnt ligand-expressing and non-expressing MSCs. Non-limiting examples of matrices useful in the method of the present invention include resorbable and non-resorbable matrices (e.g., tricalcium phosphate, tetracalcium phosphate, hydroxyapatite, and mixtures thereof) and biodegradable polymers (e.g., PLG, polylactic acid, polyglycolic acid, and mixtures thereof), and mixtures thereof.

In one embodiment, the amount of the canonical Wnt ligand effective to enhance bone formation is in the range from 5 to 50 ng/ml. In a preferred embodiment, the amount of the canonical Wnt ligand effective to enhance bone formation is approximately 25 ng/ml.

In a preferred embodiment, the subject to which canonical Wnt ligand is administered is human. In one embodiment, the subject has a condition selected from the group consisting of bone fracture, bone injury, bone implant, disorder characterized by low bone mineral density (BMD) or bone fragility (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), and avascular necrosis (osteonecrosis)), and bone loss due to other disorders (e.g., HIV infection, cancers, and arthritis).

In one embodiment, the canonical Wnt ligand is administered locally to or near the site of bone fracture or injury. In another embodiment, the canonical Wnt ligand is administered systemically.

In a specific embodiment, the invention provides a method for enhancing bone formation in a subject in need thereof comprising administering to the subject a bone transplant which has been ex vivo pre -treated with a canonical Wnt ligand in an amount effective to enhance bone formation.

In another specific embodiment, the invention provides a method for enhancing bone formation in a subject comprising administering to the subject a mixture of (i) mesenchymal stem cells (MSCs) with unmodified Wnt ligand expression and (ii) MSCs which heterologously (exogenously) express a canonical Wnt ligand. In a preferred embodiment, the heterologously (exogenously) expressed canonical Wnt ligand is Wntl . In one embodiment, the ratio of (i) MSCs with unmodified Wnt ligand expression and (ii) MSCs which heterologously (exogenously) express a canonical Wnt ligand is in the range 1: 1-20:1. In a preferred embodiment, such ratio is approximately 3:1.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Differential sensitivity of hMSCs to Wnt inhibition of osteogenic TO adipogenic differentiation. A) Scanned images of alkaline phosphatase (ALP) (upper) or Alizarin Red staining (lower) in hMSCs after 3 weeks in osteogenic medium (OS) in the presence of different concentrations (ng/ml) of recombinant Wnt3a. B) Relative ALP activity and calcium deposition under conditions as in A. C) Quantification of adipocyte differentiation in response to Wnt3a. Bars represent relative extracted oil Red-0 staining after 3 weeks in adipogenic medium. D-G) ALP and oil Red-0 staining in hMSCs after 3 weeks in a mixed medium containing equal volumes of OS and adipogenic media. Wnt3a was added at 0 (D), 5 (E), 25 (F), or 100 ng/ml (G). Bar, 20 micromolar. H) Relative ALP activity in hMSCs cultures as in D-G. I) Relative extracted oil Red-0 staining and calcium deposition in hMSCs cultures as in D-G. Results represent mean values ± s.d. from two independent experiments in triplicate. *P<0.05.

Figure 2. Mechanisms involved in Wnt inhibition of osteogenic differentiation. A) Real-time PCR analysis of Runx2, Osx, Dlx5 expression in vector or Wntl expressing hMSCs grown in basal (Undiff), or OS medium for 4 and 14 days. B-C) Representative immunoblots for MAPK activation (B) and Ro r2 expression (C), in vector or Wntl expressing hMSCs grown in basal, or OS medium for 2 and 6 days. D) Immunoblots of MAPK activation in control or Ror2 shRNA expressing hMSCs grown as in B. E) Scanned images of ALP and Alizarin red staining in hMSCs after osteogenic induction in the presence or absence of 20 μM SP600125.

Figure 3. Functions of endogenous Wnt signaling in hMSCs osteogenic differentiation. A) TCF reporter analysis in hMSCs after infection with Top or Fop luciferase virus, and renilla luciferase virus, together with either vector or dnTCF4 virus. Luciferase activity was measured at 48 h and normalized to renilla activity. B) Parental or Wntl expressing hMSCs were transduced with Top- or Fop-GFP virus, and parental cells were treated at 24 h with or without Wnt3a CM. GFP was visualized by fluorescence microscopy at 72 h. Bars, 10 micromolar. C) Top-luciferase reporter activity (left), and Axin2 and Dkkl expression by real-time PCR (right) in hMSCs at 14 d in osteogenic medium. Fold changes were all relative to levels in undifferentiated cells. D) Scanned images of ALP or Alizarin red staining (left) in hMSCs expressing vector or dnTCF4 after 3 weeks in osteogenic medium. Quantification of relative ALP activity and calcium content are shown (right). Results represent mean values ± s.d. from two independent experiments in triplicate.

Figure 4. Effects of canonical Wnt signaling on osteogenesis by hMSC in an ectopic bone formation model. A) hMSC-Vec cells alone; B) hMSC-Wntl cells alone; C-F) hMSC-Vec cells mixed with hMSC-Wntl cells at ratio of 1: 1 (C), 3:1 (D), 10:1 (E), or 20:1 (F). b, bone; HA, HA/TCP. Arrows designate osteocytes within bone structures in eosin stained sections. G) Quantitative analysis of bone formation by hMSCs in the recovered implants of A-F from triplicate experiments. Bars, 100 micromolar.

Figure 5. Canonical Wntl stimulates bone formation by hMSCs in a paracrine mode in vivo. A) hMSC-Wntl alone; B-C) hMSC-LacZ mixed with hMSC-Wntl at ratio of 1 : 1 (B) or 3: 1 (C). Sections in A-C were stained for WNTl protein (brown) and LacZ (blue), with higher magnification images in D-F as indicated. Dashed lines outline bone. Red arrowheads indicate LacZ positive osteocytes. G) β-catenin staining (brown) in section with 1 :1 mixture as in B. Black arrows indicate Wntl cells, and red arrows indicate LacZ cells with positive β-catenin staining. H) A section with bone structures surrounded by mixed hMSCs as in B, was stained for Wntl (brown), LacZ (blue) and PCNA (red), with higher magnification image in I. Red and black arrows indicate positive PCNA staining in Wntl and LacZ expressing hMSCs, respectively. All sections were counterstained with hematoxylin (dark blue). Bars, 100 micromolar. J) Quantification of PCNA staining in implants with hMSC-LacZ alone, or 1: 1 mixed hMSCs as in B. K) Quantification of cell numbers in implants of LacZ and Wntl expressing hMSCs. Numbers shown are mean values ± s.d. of triplicate experiments in J and K. L) Model for the effects of a canonical Wnt gradient on bone formation by hMSCs in vivo.

Figure 6. Effects of canonical Wnts on hMSCs differentiation and proliferation. A) Canonical Wnts inhibit osteogenic differentiation by hMSCs. hMSCs in 12-well plates were stimulated with osteogenic medium (OS) in the presence of either control conditioned medium (CM) or Wnt3a CM, or after stable lentiviral transduction of vector or Wntl. Staining for alkaline phosphatase (ALP) activity (upper panel), or mineralization by Alizarin red (lower panel), was performed after 2 and 3 weeks of differentiation, respectively. B) Real-time PCR analysis of expression of genes associated with osteoblastic differentiation in vector or Wntl expressing hMSCs after induction with OS for 0 (undiff), 4 or 14 days. Genes include alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN) and collagen IAl (CoIlAl). C) Comparison of effects of Wnt3a and PDGF on hMSC proliferation and differentiation. Upper panel shows phase contrast images of hMSCs grown in Con CM (50%), Wnt3a CM (50%) or PDGF (50 ng/ml) containing media. Middle and bottom panels show ALP and alizarin red staining of hMSCs induced with osteogenic medium containing of Con CM (50%), Wnt3a CM (50%) and PDGF (50 ng/ml) for 3 weeks, respectively. D) Inhibition of β-catenin signaling antagonizes Wntl inhibition of osteogenic and adipogenic differentiation. hMSCs were transduced with lentivirus expressing vector, Wntl, or dnTCF4 in addition to Wntl, and then subjected to osteogenic or adipogenic differentiation for 3 weeks. Cultures were stained at 3 weeks with alizarin red (Top) or oil-Red-0 (bottom). Bar, 20 micromolar.

Figure 7. Inhibition of endogenous Wnt/ β-catenin signaling on hMSCs differentiation in vitro. A) dnTCF4 enhances gene expression associated with osteogenic differentiation as determined by real-time PCR in vector or dnTCF4 expressing hMSCs grown in basal, or OS medium for 4 and 14 days. B) Spontaneous hMSC adipogenic differentiation in basal medium stimulated by dnTCF4 expression (upper panel). DnTCF4 expression enhanced adipocyte differentiation after 3 weeks in adipogenic medium (lower panel, stained with oil-Red-O). C) Real-time PCR analysis of PPARγ expression in vector or dnTCF4 expressing hMSCs grown in basal (0 day), or adipogenic medium for 6 and 14 days. D) dnTCF4 expressing hMSCs were subjected to osteogenic differentiation as in Fig.3D, and stained for ALP and oil-Red-O. Arrows indicate adipocytes. Bar, 20 micromolar. Results in A and C represent mean values ± s.d. from two independent experiments in triplicate.

Figure 8. Paracrine Wnt signaling stimulates ectopic bone formation by hMSCs in vivo. A) hMSC-LacZ cells alone; B-C) hMSC-LacZ mixed with hMSC- Wntl cells at ratio of 1:1 (B), or 3: 1 (C). Sections in A-C were stained for LacZ (blue) and eosin (red). Arrows indicate osteocytes with positive LacZ staining. D) PCNA staining of a section as in A. Black arrows indicate hMSC-LacZ cells, and red arrows indicate mouse cells stained positive for PCNA. E-F) β-catenin staining in implants with 1: 1 mixture of hMSCs expressing stabilized β-catenin (S33Y) and LacZ (E), or with hMSC-LacZ cells alone (F). Black arrows indicate LacZ staining (blue). Red arrows indicate β-catenin staining (brown). Sections in D-F were counterstained with hematoxylin. Bars, 100 micro molar. G) Quantification of cell numbers in implants with hMSCs expressing β-catenin (S33Y) alone, or mixed with hMSC-LacZ cells at 1 :1 ratio. H) Quantitative analysis of bone formation in recovered implants with hMSCs expressing stabilized β-catenin (S33Y) alone, or mixed with vector hMSCs at 1 :1 ratio. Results in G and H reflect mean values ± s.d. of triplicate experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on unexpected observation of differential sensitivities of adult multipotent human mesenchymal stem cells (hMSCs) to canonical Wnt inhibition of osteogenesis vs adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous Wntl but not stabilized β-catenin expression markedly stimulates bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo.

As used herein, the term "Wnt ligand" refers to a family of secreted signaling glycopoproteins that play essential roles in embryonic development and postnatal tissue homeostasis and comprises 19 members. Within this family, some Wnt ligands signal to increase the stability of β-catenin, which leads to activation of β-catenin dependent gene expression. These Wnt ligands, which are referred to as "canonical Wnt ligands", include Wntl, Wnt2, Wnt3, Wnt3a, Wnt7b, Wnt8, and Wntl Ob.

As used herein, the term "enhance bone formation" refers to any acceleration in bone fracture or injury repair or healing process, or any increase in bone surface, mass or density.

The term "amount effective to enhance bone formation" is used herein to refer to that quantity of a compound (e.g., a canonical Wnt ligand) or a pharmaceutical composition containing such compound or a cell (e.g., MSC) expressing such compound that is sufficient to enhance bone formation. As disclosed in the Examples section, below, for canonical Wnt ligands such as Wnt3a, amounts effective to enhance bone formation are preferably in the range from 5 to 50 ng/ml, most preferably, around 25 ng/ml.

In the context of the present invention insofar as it relates to any of the disease conditions recited herein, the terms "treat", "treatment", and the like mean to positively affect in any way a healing of a bone injury or fracture (e.g., by speeding up the healing process or by improving the bone condition and/or mass at the injury or fracture site) or to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. The term "protect" is used herein to mean prevent, delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject. Within the meaning of the present invention, disease conditions include without limitation treatment/healing of bone fractures, other bone injuries and implants (e.g., dental implants and hip implants), disorders characterized by low bone mineral density (BMD) and/or bone fragility (such as, e.g., primary and secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (01), avascular necrosis (osteonecrosis)), and bone loss due to other disorders (e.g., associated with HIV infection, cancers, or arthritis).

As used herein the term "therapeutically effective" applied to dose or amount refers to that quantity of a compound (e.g., a canonical Wnt ligand) or a pharmaceutical composition containing such compound or a cell (e.g., MSC) expressing such compound that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present invention, the term "therapeutically effective dose or amount" refers to that quantity of a canonical Wnt ligand or a pharmaceutical composition containing such canonical Wnt ligand or MSC expressing such canonical Wnt ligand that is sufficient to reduce or eliminate at least one symptom of a disease specified above. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. Therapeutically effective dosages according to the present invention can be determined stepwise by combinations of approaches such as, e.g., (i) characterization of effective doses in in vitro assays followed by (ii) characterization in animal studies using target tissue effects as a readout, followed by (iii) characterization in human trials using target tissue effects as a readout.

The phrase "pharmaceutically acceptable", as used in connection with the compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

The terms "administering" or "administration" as used herein are intended to encompass all means for directly and indirectly delivering a compound to its intended site of action. The canonical Wnt ligands of the invention or MSCs of the invention can be administered locally to the affected site. In methods for enhancing bone formation, a canonical Wnt ligand or MSC can be administered locally to or near the site of bone fracture or injury. Preferably, such local administration should occur shortly following the fracture or injury. In some embodiments a canonical Wnt ligand or MSC is administered within about two days, usually within about one day of fracture or injury, and is provided for not more than about two weeks, not more than about one week, not more than about 5 days, not more than about 3 days, etc. The canonical Wnt ligands of the invention or MSCs of the invention can be administered in the form of resorbable or non-resorbable matrices (e.g., tricalcium phosphate, tetracalcium phosphate, hydroxyapatite) or biodegradable polymers (e.g., PLG, polylactic acid/polyglycolic acid) which are coated or mixed with canonical Wnt ligands or with MSCs expressing such ligands or with mixtures of canonical Wnt ligand expressing and non-expressing MSCs. Alternatively, such matices forming bone transplants can be treated with canonical Wnt ligands ex vivo and can be later administered to the site of fracture or injury. The canonical Wnt ligands of the present invention can be also administered systemically in the form of polypeptides or nucleic acids encoding such polypeptides or various delivery vehicles comprising such polypeptides or nucleic acids (e.g., viral vectors such as, e.g., adenoviral vectors or lentiviral vectors, or non-viral gene delivery systems such as, e.g., liposomes or a high pressure gene delivery system). The term "systemic" as used herein includes, for example, parenteral, oral, transdermal, transmucosal, intranasal, and buccal administration. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intra-arteriole, intradermal, intraperitoneal, intraventricular, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial administration. Preferred routes of administration according to the present invention are local administration to the site of fracture or injury and ex-vivo transplant treatment.

The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" is implicit and in this context means within an acceptable error range for the particular value.

The term "subject" means any animal, including mammals and, in particular, humans.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (herein "Sambrook et ah, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. 1994; among others. EXAMPLES

The in vitro biological effects of canonical Wnts on differentiation of adult hMSCs were first analyzed. Consistent with previous studies (Boland et al., 2004; de Boer et al., 2004), treatment of hMSCs with Wnt3a conditioned medium (CM), or lentiviral-mediated transduction of Wntl, strongly inhibited osteogenic differentiation compared to that of respective control cultures as reflected by reduced staining for the early osteoblastic marker, alkaline phosphatase (ALP) and reduced mineralization as detected by Alizarin red staining (Fig. 6A). Moreover, exogenous Wntl resulted in dramatic downregulation of expression of several markers associated with osteoblastic differentiation (Harada and Rodan, 2003), including ALP, bone sialoprotein (BSP), and osteocalcin (OCN) (Fig. 6B).

In addition to its inhibition of osteogenic differentiation, Wnt stimulation was associated with increased cell proliferation (Fig. 6C). Since terminal differentiation is commonly associated with exit from cell cycle, it was assessed whether Wnt inhibition of differentiation might be due to enhanced proliferation blocking exit from cell cycle. However, PDGF, which also increased hMSC proliferation and resulted in higher saturation density (Fig. 6C), did not inhibit osteogenic differentiation, arguing that the Wnt effects could not be explained by stimulation of cell proliferation alone.

The osteogenic inhibitory function of canonical Wnts seemed inconsistent with the fact that this pathway plays a positive role in bone homeostasis in vivo. It has been observed that aging leads to a decrease of bone-forming osteoblasts and an increase of marrow adipocytes (Verma et al., 2002). These findings are consistent with the possibility that alterations in hMSC lineage differentiation may have profound effects on bone formation. Since Wnt signaling can also inhibit adipogenic differentiation (Ross et al, 2000), it was assessed whether canonical Wnts might modify hMSC lineage commitment to favor osteoblastic differentiation. As shown in Fig. IA and B, osteogenic differentiation was partially inhibited at 100ng/ml of Wnt3a, while concentrations of 25 ng/ml or lower failed to detectably inhibit either ALP activity or mineralization. In contrast, Wnt3a was much more potent in inhibiting adipocyte differentiation, with approximately 40% inhibition observed at 5 ng/ml, and complete inhibition at 100 ng/ml (Fig.1C). As might be expected for transcriptional signaling through Wnt/β-catenin, a dominant negative TCF4 (dnTCF4) antagonized exogenous Wntl mediated inhibition of both osteogenic and adipogenic differentiation (Fig. 6D).

To directly compare hMSC sensitivity to Wnt inhibition of commitment to the osteogenic vs adipogenic lineage under the same conditions, these cells were cultured in a binary differentiation medium that efficiently induced differentiation along both lineages. Wnt3a significantly blocked adipogenic differentiation at 5 ng/ml, and completely abolished adipogenesis at 100 ng/ml (Fig. 1, D-G & I). In marked contrast, treatment with Wnt3a at both 5 and 25 ng/ml resulted in a significant relative increase in osteogenic differentiation as manifested by increased ALP activity and mineralization (Fig. 1 H&I). At 100 ng/ml, Wnt3a partially inhibited osteogenic differentiation as well (Fig. 1 H&I). These results indicate that under conditions permissive for binary lineage differentiation, differences in sensitivity to Wnt inhibition may alter the equilibrium and shift the commitment from adipocytes towards osteoblasts.

One mechanism underlying osteogenic inhibition by Wnt/β-catenin signaling has been attributed to direct interaction and inhibition of Runx2 transcriptional activity by β-catenin/Lefl complex (Kahler and Westendorf, 2003). However, it was also reported that Wnt/β-catenin signaling increases Runx2 level in mouse mesenchymal cells (Gaur et al., 2005). Thus, the expression of osteoblastic transcription factors including Runx2, Osterix and Dlx5 was examined in hMSCs. As shown in Fig. 2A, the expression levels of these genes were reduced in Wntl expressing hMSCs in basal medium as well as under osteogenic conditions compared to that in vector cells, consistent with high levels of Wnt signaling acting to downregulate these osteoblastic transcription factors.

ERK and p38 MAP kinase pathways have been shown to play critical roles in osteogenesis (Kratchmarova et al., 2005). As shown in Fig. 2B, p38 and ERK, as well as JNK, were activated as evidenced by their increased phosphorylation in hMSCs undergoing osteogenic differentiation. Activation of p38 was detectable as early as 2 days while increased p-JNK was obvious by 6 days. Wntl expression largely inhibited activation of both kinases under osteogenic conditions, whereas there was no significant change in p-ERK levels in response to Wntl expression under the same conditions (Fig. 2B). The Ror2 receptor tyrosine kinase plays an important role in skeletal development as well as mediates Wnt5a signaling to the JNK pathway (Oishi et al., 2003). Other studies have shown that Wnt5a stimulates osteogenic differentiation (Baksh et al, 2007) and Ror2 shRNA inhibits hMSC osteogenic differentiation (Liu et al, 2007). When Ror2 expression in response to osteogenic stimulation was examined, an increase was observed, which was blocked by exogenous Wntl (Fig. 2C). Moreover, when Ror2 expression was inhibited with shRNA under osteogenic conditions, neither p38 nor ERK activation was affected, while JNK activation was effectively blocked (Fig. 2D) and osteogenic differentiation partially inhibited as well. Similarly, treatment of hMSCs under differentiation conditions with SP600125, a small molecule inhibitor of JNK, partially inhibited osteogenic differentiation (Fig. 2E). These findings support that canonical Wnt inhibition of Ror2 upregulation and JNK activation contributes to its osteogenic inhibitory functions.

Canonical Wnt signaling has been implicated in stem/progenitor cell maintenance in a number of adult tissues (Reya and Clevers, 2005). Thus, it was assessed whether hMSCs demonstrated endogenous Wnt signaling activity. Using a lentiviral based TCF luciferase reporter system, hMSCs showed a approximately 7-fold TCF reporter activity over that of the control reporter, and this activity could be completely inhibited by dnTCF4 expression (Fig. 3A). It wasobserved further that endogenous TCF activity in hMSCs was heterogeneous, as only approximately 5% of cells exhibited high activity employing a GFP-based reporter system (Fig. 3B). Moreover, this endogenous Wnt signaling was downregulated during osteogenic differentiation as monitored by decreased levels of TCF reporter activity and expression of a known β-catenin target gene Axin2 (Fig. 3C). In contrast, expression of the Wnt antagonist Dkkl was markedly increased during osteogenic differentiation (Fig. 3C), suggesting that Dkkl may participate in downregulating endogenous Wnt signaling during osteogenic differentiation.

To assess the biological effects of endogenous Wnt signaling, the differentiation responses of dnTCF4 and vector expressing hMSCs were compared. In contrast to the osteogenic inhibition by exogenous Wntl, dnTCF4 enhanced osteogenic differentiation (Fig. 3D), as well as increased expression of genes associated with osteogenic differentiation (Fig. 8A). DnTCF4 also enhanced adipogenic differentiation (Fig. 7B) and increased PPARγ expression (Fig. 7C), with evidence of spontaneous adipocyte commitment both in basal medium (Fig. 7B), and under osteogenic conditions (Fig. 7D), neither of which responses were observed in vector hMSCs. All of these results argue that endogenous Wnt signaling plays an important role in maintaining hMSCs in a relatively undifferentiated state.

To investigate how canonical Wnt inhibitory effects on osteogenic differentiation in vitro might translate into a positive function in bone formation in vivo, a widely used ectopic bone formation model in immune deficient mice was employed (Krebsbach et al., 1997). When mixed with ceramic powder of HA/TCP (hydroxyapatite and tricalcium phosphate), vector expressing hMSCs formed detectable, small fragments of ectopic bone constituting approximately 1% of the total scaffold area (Fig. 4A and G). Consistent with the inhibitory effects of high Wnt signaling levels on hMSC osteogenic differentiation in vitro, exogenous Wntl expressing hMSCs exhibited little if any detectable bone forming capacity under the same conditions (Fig. 4B and G). Based on the observations of heterogeneous endogenous Wnt activity in hMSCs, higher levels of heterogeneous Wnt activity were modeled by mixing vector-expressing hMSCs and Wntl -expressing hMSCs at different ratios in the implants. These conditions led to a striking enhancement of bone formation with as much as 25% bone formation observed (Fig. 4C-G).

To directly identify which cell population was responsible, hMSCs were labeled with LacZ by viral transduction. LacZ expressing hMSCs alone formed only small amounts of bone (Fig. 8A), similar to that observed with vector hMSCs (Fig. 4A). When bone formation was significantly enhanced in implants with mixed LacZ and Wntl expressing hMSCs (Fig. 8B and C), the bone structures were predominately comprised of LacZ staining cells. In contrast, WNTl immunostaining was observed in dense cell clusters surrounding bone structures (Fig. 5A-F). These results strongly support the conclusion that Wntl expressing hMSCs act primarily to stimulate osteogenesis by Wntl negative hMSCs.

As a next step, the Wnt signaling activity was examined in mixed implants by immunonostaining for β-catenin, since the presence of non-membrane bound β-catenin is a hallmark of canonical Wnt signaling activation. As shown in Fig. 5G, cytoplasmic β-catenin was apparent within cells containing both LacZ positive (hMSC-LacZ) and negative (hMSC-Wntl) cells surrounding bone-like structures but was generally undetectable in osteocytes embedded within the ectopic bone. These findings argue for both autocrine and paracrine Wnt signaling but that only naϊve hMSCs with low paracrine Wnt signaling activity capable of differentiating into bone-forming osteocytes.

Immuno staining for PCNA (proliferating cell nuclear antigen) revealed significant increases in both LacZ and Wntl expressing hMSCs in mixed implants (Fig. 5H, I and J) compared to hMSC-LacZ cells alone (Fig. 5J and Fig. 8D). These results correlated with the increased numbers of both Wntl and LacZ staining cells observed in mixed implants (Fig. 5K). These results establish that canonical Wntl specifically stimulated in vivo proliferation of hMSCs in both autocrine and paracrine modes, with the latter increasing the population of naϊve hMSCs capable of undergoing osteogenesis under low or undetectable Wnt signaling.

To address whether or not the enhanced bone formation by Wntl was caused by other factors induced by activated β-catenin signaling, stabilized mutant β-catenin was expressed in hMSCs and effects on bone formation in vivo were examined, β-catenin was specifically detected in hMSCs expressing stabilized β-catenin but not in mixed LacZ expressing hMSCs in the same implants (Fig. 8E), or implants with hMSC-LacZ cells alone (Fig. 8F). Of note, expression of stabilized β-catenin significantly increased cell numbers in implants but had relatively little effect on the number of naive hMSC-LacZ cells under mixed conditions (Fig. 8G). Consistent with the in vitro osteogenic inhibitory function of β-catenin as reported previously (Jian et al., 2006), little if any bone formation was observed by hMSCs expressing mutant β-catenin alone, and no increased bone formation was observed when mixed with hMSC-LacZ cells (Fig. 8H). These results establish that canonical Wnt ligands acting in a cell non-autonomous manner are required for enhanced bone formation in this context.

Without wishing to be bound by any theory, based on the present findings and previous studies, a model for canonical Wnt modulation of osteogenesis by hMSCs in vivo can be proposed (Fig. 5L), in which a Wnt activity gradient results from the asymmetrical localization of Wnts and antagonists between hMSC stem/progenitor and osteoblasts/osteocyte compartments. For example, Dkkl upregulation was observed during osteogenesis, and previous studies have indicated increased expression of other Wnt antagonists sFRPs (Boland et al., 2004) and Sost by osteoblasts/osteocytes (Winkler et al., 2003). Such a Wnt gradient likely plays an important role in stimulating self-renewal and expansion of hMSCs/progenitors at high Wnt activity, while permitting osteoblastic differentiation where Wnt activity decreases. Differential sensitivity to Wnt inhibition of osteogenesis vs adipogenesis in vitro also provide a possible mechanism to favor osteoblastic commitment within certain range of Wnt activity by restricting hMSCs from adopting adipocytic cell fate. The present findings point to a direct role of canonical Wnt in bone formation, but do not exclude a role for LRP5 on bone formation through regulation of the hormonal acting serotonin as recently reported (Yadav et al., 2008).

Despite their potential clinical use in regenerative medicine and tissue engineering, bone formation by hMSCs is poor, and repair of critical size bone defects has not so far been achieved (Meijer et al., 2007). A recent report showed that pre-exposure of hMSCs to db-cAMP, an activator of the PKA pathway, can enhance ectopic bone formation by these cells from 1.5% in the control group up to 6% (Siddappa et al., 2008). The present findings indicate that canonical Wnts can have a major positive impact on hMSC mediated bone formation in HA/TCP transplants, increasing bone formation up to 25% under the present experimental conditions.

Thus, appropriate manipulation of canonical Wnt ligand expression can lead to significant improvement in the efficiency of tissue engineering and enhance the therapeutic value of MSCs for restoration of bone defects. Appropriate manipulation of a canonical Wnt ligand expression resulting in the formation of a Wnt concentration gradient, can be achieved by, e.g.,

(1) transient canonical Wnt expression in MSCs, or by other supporting cells such as fibroblasts;

(2) direct administration of canonical Wnt ligand proteins into bone tissue or engineered bone transplants; or (3) asymmetrical distribution of stably Wnt expressing cells into naive MSC-containing bone tissue. This approach allows both expansion of MSCs within regions with high Wnt concentration, and proper differentiation of these cells into bone-forming cells as such cells encounter decreasing Wnt concentration. This approach supports sustained and enhanced bone growth by these cells to improve the repair of bone defects.

MATERIALS AND METHODS

Cell culture, conditioned medium, growth factors and inhibitors

Primary hMSCs cultures were obtained from three independent donors (Lonza) and expanded in basal medium, or induced with osteogenic or adipogenic medium (Lonza) according to manufacturer's instructions. CM of control or Wnt3a, was collected from L-cell transfectant as described (Boland et al., 2004). Recombinant Wnt3a (R&D Systems), PDGF-BB (PeproTech), and SP600125 (Calbiochem) were commercially obtained.

Assays for in vitro osteogenic and adipogenic differentiation

Calcium deposition was analyzed by staining with 2% Alizarin red (Sigma); or extracted with 0.5N HCl and quantified with Liquicolor Kit (StanBio Laboratory). ALP activity was detected histochemically with Leukocyte Alkaline Phosphatase Kit (Sigma); or quantified with a chemiluminescent substrate (Roche) and normalized to total proteins. Adipocytes were stained with oil Red-0 (Sigma) as previously described (Ross et al., 2000). For quantification, the incorporated dye was extracted with isopropanol and measured at 500 nm for optical density.

Constructs, virus production and transduction of hMSCs

Mouse Wntl, mutant β-catenin (S33Y), dnTCF4, and LacZ cDNA were all cloned into lentiviral vectors. Short hairpin RNA targeting Ror2 (Liu et al., 2007), or a control shRNA, was also expressed by lentiviral vector. Viruses were produced in HEK293T cells by transfection with lentiviral and packaging plasmids. hMSCs were selected for stable expression after viral transduction.

Western Blot and Luciferase Reporter Assay

Western Blot was performed as previously described (Liu et al., 2005). Antibodies for p-JNK, JNK, p-P38, p38, p-ERK and ERK were all from Cell Signaling, α-Ror2 was from R&D systems. A lentiviral-based TCF reporter system containing 14 repeats of TCF binding sites (TOP) was used to assay Wnt signaling, with FOP reporter consisted of mutated TCF binding sites as control. Cells were co-infected with Renilla luciferase virus, and assayed using Dual Luciferase Assay System (Promega).

Quantitative real-time PCR

cDNA was synthesized from total RNA extracted with TRIzol (Invitrogen). Real-time PCR was conducted with approximately 50 ng of cDNA using FastStart SYBR green master mix (Roche). Two independent experiments were performed for each reaction in triplicate. All data were normalized to TBP. Primer sequences are available upon request.

In vivo transplantation and histological analysis of bone formation

Detailed procedures followed protocols described previously (Krebsbach et al., 1997). For each transplant, IxIO⁶ cells in total were loaded onto 40 mg of HA/TCP powders (Zimmer), and transplanted subcutaneously into immune deficient 8- to 15-week-old female mice. Transplants were recovered at 12 weeks, fixed, decalcified and embedded into paraffin blocks. Consecutive sections were prepared from three different levels, and subjected to hematoxylin and eosin (H&E) staining.

LacZ detection and immunostaining of implant sections

For LacZ staining, implants were fixed in 4% paraformaldehyde, 0.8% glutar aldehyde, 0.02% NP40, 1 mM MgCl₂ in PBS for Ih, stained overnight at 37°C with 400 micrograms/ml X-gal, 0.1 mM MgCl₂, 20 mM potassium ferrocyanide, 20 mM potassium ferricyanide in PBS, and subjected to decalcification and histological processing. WNTl or PCNA was immunostained with α-Wntl (Zymed), or α-PCNA (Santa Cruz), followed by secondary antibodies (Invitrogen) according to manufacturer's protocol.

Microscopy imaging

Images of hMSCs (Fig. 1 D-G, Fig. 6C-D, and Fig. 7B and D) were acquired with the PixCell II laser capture system (Arcturus Engineering, Inc.) with a 10x objective for Fig. ID-G, and a 2Ox objective for other images (except Fig. 6C middle panel at 4Ox), using Arc200 software. Top- and Fop-GFP experiments (Fig. 3B) were analyzed with a fluorescence microscope (Eclipse TE200; Nikon) using a 2Ox objective. Images were acquired with a camera (Diagnostic Instruments, Inc.) using Spot advanced software. Sections (Fig. 4 and 5, Fig. 8) were analyzed with a microscope (Eclipse E200; Nikon) with a 2Ox objective (Fig. 4A-F, Fig. 5A-C, Fig. 8A-C), a 4Ox objective (Fig. 5D-H, Fig. 8D-F), and a 10Ox objective (Fig. 51). Images were acquired with a camera (Diagnostic Instruments, Inc.) using Spot advanced software. The present invention is also described and demonstrated by way of the above examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

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Claims

WHAT IS CLAIMED IS:

1. A method of enhancing bone formation in a subject in need thereof comprising administering to the subject a canonical Wnt ligand in an amount effective to enhance bone formation.

2. The method of claim 1, wherein the canonical Wnt ligand is selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt7b, Wnt8, and WntlOb.

3. The method of claim 1 , wherein the canonical Wnt ligand is Wntl or Wnt3a.

4. The method of claim 1 , wherein the canonical Wnt ligand is administered in the form of a polypeptide or a nucleic acid encoding such polypeptide.

5. The method of claim 1 , wherein the canonical Wnt ligand is administered in the form of a bone-marrow derived mesenchymal stem cell (MSC) expressing such canonical Wnt ligand.

6. The method of claim 5, wherein the MSC is human MSC (hMSC).

7. The method of claim 1 , wherein the canonical Wnt ligand is administered in the form of a matrix forming a bone transplant.

8. The method of claim 7, wherein the matrix forming a bone transplant is coated or mixed with (i) the canonical Wnt ligand or (ii) with MSCs expressing such canonical Wnt ligand or (iii) with a mixture of canonical Wnt ligand-expressing and non-expressing MSCs.

9. The method of claim 7, wherein the matrix forming a bone transplant is selected from the group consisting of resorbable matrices, non-resorbable matrices, biodegradable polymers, and mixtures thereof.

10. The method of claim 7, wherein the matrix forming a bone transplant is selected from the group consisting of tricalcium phosphate, tetracalcium phosphate, hydroxyapatite, PLG, polylactic acid, polyglycolic acid, and mixtures thereof.

11. The method of claim 1, wherein the amount of the canonical Wnt ligand effective to enhance bone formation is in the range from 5 to 50 ng/ml.

12. The method of claim 1 , wherein the subject is human.

13. The method of claim 1, wherein the subject has a condition selected from the group consisting of bone fracture, bone injury, bone implant, disorder characterized by low bone mineral density (BMD) or bone fragility, and bone loss due to other disorders.

14. The method of claim 13, wherein the disorder characterized by low bone mineral density (BMD) or bone fragility is selected from the group consisting of primary osteoporosis, secondary osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI), and avascular necrosis (osteonecrosis).

15. The method of claim 13, wherein the other disorder is selected from the group consisting of HIV infection, cancers, and arthritis.

16. The method of claim 13, wherein the canonical Wnt ligand is administered locally to or near the site of bone fracture or injury.

17. A method for enhancing bone formation in a subject in need thereof comprising administering to the subject a bone transplant which has been ex vivo pre -treated with a canonical Wnt ligand in an amount effective to enhance bone formation.

18. The method of claim 17, wherein the canonical Wnt ligand is selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt7b, Wnt8, and WntlOb.

19. The method of claim 17, wherein the canonical Wnt ligand is Wntl or Wnt3a.

20. A method for enhancing bone formation in a subject comprising administering to the subject a mixture of (i) mesenchymal stem cells (MSCs) with unmodified Wnt ligand expression and (ii) MSCs which heterologously express a canonical Wnt ligand.

21. The method of claim 20, wherein the canonical Wnt ligand is selected from the group consisting of Wntl, Wnt2, Wnt3, Wnt3a, Wnt7b, Wnt8, and WntlOb.

22. The method of claim 20, wherein the canonical Wnt ligand is Wntl .

23. The method of claim 22, wherein the ratio of (i) MSCs with unmodified Wnt ligand expression and (ii) MSCs which heterologously express a canonical Wnt ligand is in the range 1 :1-20:1.

24. The method of claim 22, wherein the ratio of (i) MSCs with unmodified Wnt ligand expression and (ii) MSCs which heterologously express a canonical Wnt ligand is approximately 3:1.