97
Ann. N.Y. Acad. Sci. 1049: 97–106 (2005). © 2005 New York Academy of Sciences.
doi: 10.1196/annals.1334.010
How Wnt Signaling Affects Bone Repair by
Mesenchymal Stem Cells from the
Bone Marrow
CARL A. GREGORY, WILLIAM G. GUNN, EMIGDIO REYES,
ANGELA J. SMOLARZ, JAMES MUNOZ, JEFFREY L. SPEES,
AND DARWIN J. PROCKOP
Center for Gene Therapy, Tulane University Health Sciences Center,
New Orleans. Louisiana 70112, USA
ABSTRACT: Human mesenchymal stem cells (hMSCs) from bone marrow are a
source of osteoblast progenitors in vivo, and under appropriate conditions they
differentiate into osteoblasts ex vivo. The cells provide a convenient cell culture
model for the study of osteogenic tissue repair in an experimentally accessible
system. Recent advances in the field of skeletal development and osteogenesis
have demonstrated that signaling through the canonical wingless (Wnt) path-
way is critical for the differentiation of progenitor cell lines into osteoblasts. In-
hibition of such signals can predispose hMSCs to cell cycle entry and prevent
osteogenesis. Our investigation of the role of Wnt signaling in osteogenesis by
hMSCs ex vivo has demonstrated that osteogenesis proceeds in response to
bone morphogenic protein 2 stimulation and is sustained by Wnt signaling. In
the presence of Dkk-1, an inhibitor of Wnt signaling, the cascade is disrupted,
resulting in inhibition of osteogenesis. Peptide mapping studies have provided
peptide Dkk-1 agonists and the opportunity for the production of blocking an-
tibodies. Anti-Dkk-1 strategies are clinically relevant since high serum levels of
Dkk-1 are thought to contribute to osteolytic lesion formation in multiple my-
eloma and possibly some forms of osteosarcoma. Specific inhibitors of glycogen
synthetase kinase 3
(GSK3
), which mimic Wnt signaling, may also have a
therapeutic benefit by enhancing in vitro osteogenesis despite the presence of
Dkk-1. Antibodies that block Dkk-1 and GSK3
inhibitors may provide novel
opportunities for the enhancement of bone repair in a variety of human diseas-
es such as multiple myeloma and osteosarcoma.
KEYWORDS: mesenchymal stem cells; Wnt; Dkk-1; bone; osteogenesis; cancer
MESENCHYMAL STEM CELLS FROM BONE MARROW
The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by
Friedenstein,1 who described clonal, plastic adherent cells from bone marrow capa-
ble of differentiating into osteoblasts, adipocytes, and chondrocytes.2–5 These cells
Address for correspondence: Carl A. Gregory, Center for Gene Therapy, Tulane University
Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112. Voice: 504-988-7716;
fax: 504-988-7710.
ca_gregory@hotmail.com

98 ANNALS NEW YORK ACADEMY OF SCIENCES
were later found to differentiate into “stromal” cells, structural components of the
bone marrow that supported ex vivo culture of hematopoiesis by providing extracel-
lular matrix components, cytokines, and growth factors.6–10 Numerous laboratories
around the world have now demonstrated that multipotent MSCs can be recovered
from a variety of adult tissues and can differentiate into various tissue lineages. In
particular, Verfaille and colleagues report that a specific type of murine MSC isolat-
ed from bone marrow, muscle or brain termed multipotential adult progenitor cells
(MAPCs) differentiate into a variety of tissue lineages including myoblasts, hepato-
cytes, and even neural tissue.11–14
Although it is clear that single cell–derived colonies of MSCs can transdifferen-
tiate into multiple tissue lineages in culture, there have been indications that they can
also undergo cell fusion. Evidence supporting the case for cell fusion by MSCs
comes from a set of experiments conducted by Spees and colleagues15 in which
MSCs, labeled with green fluorescent protein, were co-cultured with pulmonary
small airway epithelial cells (SAECs) after a brief heat shock to mimic tissue dam-
age. After heat shock, a good proportion of the confluent SAEC monolayer died by
apoptosis. When introduced to the damaged SAEC cultures, the green MSCs ad-
hered to the tissue culture plastic, resulting in a confluent mixture of cells. Over a
few days, some of the MSCs differentiated into epithelial-like cells but others fused
with SAECs, producing chimeras. These investigators suggested that cell fusion may
be an acute response to tissue damage and may rescue dying cells by supplying ad-
ditional cytosolic metabolites and organellar components.
Because multipotent MSCs are easily expanded in culture, there has been much
interest in their clinical potential for tissue repair and gene therapy.16 As a result, nu-
merous studies have been carried out demonstrating the migration and multi-organ en-
graftment potential of MSCs in animal models and in human clinical trials.13,17–25
One trial in particular utilized hMSCs from compatible healthy donors to treat indi-
viduals with the brittle bone disease osteogenesis imperfecta, yielding encouraging
results.18–20
In addition to their ability to home, engraft, and differentiate into damaged tis-
sues, MSCs secrete cytokines and trophic factors that provide beneficial effects to
surrounding tissue. This characteristic has been exploited in the field of regenerative
neuroscience, where MSCs, when injected into the brain or damaged spinal cord, en-
graft and provide a permissive stromal microenvironment for the growth and repair
of existing neural tissue.22,26–29 In our laboratory, MSCs have been shown to ex-
press and secrete a variety of neurotrophic and neuroprotective proteins in culture
and when implanted into the brains of test animals (Munoz et al., unpublished ma-
terial). Expression of these beneficial factors persists from stably engrafted MSCs
for a number of weeks post implantation, and it is hoped that this discovery will lead
to a promising treatment for neural lesions caused by ischemia and spinal cord inju-
ry. Although the MSCs secrete their own extensive repertoire of cytokines, MSCs
can be genetically modified in a stable manner by a variety of retroviral and lentiviral
vectors and therefore could be induced to secrete agents at sites of tissue injury or
tumors. It is noteworthy that MSCs have been shown to preferentially migrate to tu-
mors and, when genetically modified to secrete interferon-β, they reduce the size of
the lesions.36
Graft and host compatibility is always an issue when transplanting heterologous
tissue into an immunocompetent host, and immunotolerance of implanted MSCs has

99GREGORY et al.: Wnt SIGNALING IN OSTEOGENESIS
been the subject of much debate in recent years. One remarkable facet of MSC phys-
iology is that the cells may actually inhibit inflammation and immunologic respons-
es in the host. In vitro, MSCs fail to induce allogeneic responses in mixed
lymphocyte reaction assays and they escape lysis by cytotoxic T cells and natural
killer cells. The immunomodulatory properties of MSCs are probably explained by
their lack of an HLA type II receptor and the secretion of cytokines.30–33 Indeed, al-
logenic MSC implants do not appear immunogenic in the non-human primate34 and
human recipients receiving MSCs from sibling donors.18,19 However, the require-
ment for fetal bovine serum (FBS) in the expansion medium of MSCs is a source for
concern when administering the cells to human recipients. Using an assay based on
fluorescently labeled FBS it was found that a dose of 10 million MSCs could be con-
taminated with milligram quantities of FBS. Furthermore, most of the FBS was in-
ternalized and could not be removed by washing the cells.35 This observation
explains the findings of Horwitz et al., who reported that after an infusion of allo-
genic MSCs, 5 of 6 children with ostoegenesis imperfecta showed improved growth
and skeletal durability, whereas one recipient showed no improvement (patient 6 in
the study) and reacted immunologically to the infusion of cells.20 Testing revealed
that the patient had developed a profound humoral response against bovine serum
albumin, the major bovine contaminant in MSC preparations. The solution to this
problem lies in the ability of MSCs to expand in adult human serum and retain all of
their beneficial properties. In the clinic, MSCs could be expanded in FBS to the de-
sired number, then grown for an additional 2 days in medium containing the patients’
own serum. Support for this strategy is based on findings from a rat model, where
repeated administrations of rat MSCs evoked a profound humoral response against
FBS. However, when expanded for a short time in medium containing rat serum, the
response did not occur.35 Furthermore, in human cultures of MSCs, FBS contamina-
tion could be reduced to nearly zero by a 2-day culture in medium containing human
serum35 and should therefore prevent the occurrence of immune reactions in subse-
quent trials.
MODULATION OF WNT SIGNALING AFFECTS GROWTH AND
OSTEOGENIC DIFFERENTIATION OF MSCS
Since human MSCs (hMSCs) are recovered from a donor by a straightforward il-
iac crest aspirate and simply enriched by virtue of their adherence to tissue culture
plastic, the cells provide a convenient ex vivo model for the study of mesenchymal
stem cell expansion and differentiation. Cultures of hMSCs classically display a lag
phase of about 4 days before they enter a phase of exponential growth.37–39 On fur-
ther investigation, it was found that conditioned medium from cultures of hMSCs re-
duced the lag period when added to freshly plated MSCs39 and ablated a shorter 12-
hour lag period when the medium of established hMSC cultures was replaced10 (see
FIG. 1a). The agent responsible for the lag-phase ablation was identified as Dickkopf-
1 (Dkk-1), an inhibitor of the canonical Wnt pathway.10,40,41 Dkk-1 was highly ex-
pressed in rapidly dividing hMSCs, but was absent or expressed at very low levels at
the stationary phase of growth and when proliferation was blocked by withdrawal of
serum (FIGS. 1b, c, and d). Recombinant Dkk-1 reduced the length of a lag phase in-
duced by a change of medium (FIG. 1e) and addition of antiserum against Dkk-1

100 ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 1. (a) Growth of hMSCs after medium replacement containing various pro-
portions of conditioned medium. (b) Western blot assay for Dkk-1 in hMSCs from early log
(E) and stationary (S) cultures of MSCs. (c) Hybridization ELISA analysis of Dkk-1 encod-
ing RT-PCR products generated from early, late, and stationary phase cultures. Dkk-1 ex-
pression is high during proliferation. Signals were normalized against GAPDH expression.
(d, upper 2 panels) During growth arrest by serum starvation, transcription of Dkk-1 is in-
hibited. Cell cycle analysis of hMSCs after 5 days in culture followed by addition of medium
containing no FCS or 20 % (v/v) FCS. The relative proportions of cells in G1, S phase, and
G2 phase are indicated on the histograms. Phase-contrast micrographs are presented with
each histogram illustrating cell density in each case.(d, lower panel) Hybridization ELISA

101GREGORY et al.: Wnt SIGNALING IN OSTEOGENESIS
extended the lag phase (FIG. 1f). Interestingly, these observations seem consistent
with the model for Wnt signaling during limb bud development proposed by Hart-
mann and Tabin,42 where positive signaling through the canonical Wnt pathway in-
duces differentiation and a postmitotic state by increasing the intracellular level of
stable β-catenin.
In the canonical pathway (FIG. 2), Wnt ligands bind to the transmembrane recep-
tor frizzled (Frz) and the co-receptor lipoprotein-related protein 5 and 6 (LRP-5/6).
Activation of frizzled recruits the cytoplasmic bridging molecule, disheveled (Dsh),
so as to inhibit glycogen synthetase kinase 3β (GSK3β). Inhibition of GSK3β de-
creases phosphorylation of β-catenin, preventing its degradation by the ubiquitin-
mediated pathway. The stabilized β-catenin acts on the nucleus by activating TCF/
LEF-mediated transcription of target genes.43,44 In the light of recent work demon-
strating that canonical Wnt signaling drives the differentiation of progenitor cell
lines into osteoblasts,45.46 it seems reasonable that Dkk-1 is acting to prevent termi-
nal differentiation of the MSCs in culture while they proliferate. Two observations
support this hypothesis: (i) when Wnt signaling is mimicked by culturing the MSCs
with the glycogen synthetase kinase-β inhibitor, LiCl, osteogenesis can be acceler-
ated (FIGS. 3a and b), and (ii) addition of exogenous Dkk-1 can inhibit osteogenesis
(FIG. 3c).
analysis of Dkk-1 encoding PCR products demonstrating Dkk-1 is only expressed during
proliferation. (e) Effect of Dkk-1 on the lag phase of hMSCs. Conditioned medium of lag-
phase cells was replaced with fresh medium containing vehicle or 0.01 µg mL−1 recombi-
nant Dkk-1. (f) Effect of anti-Dkk-1 polyclonal serum on proliferation of hMSCs after a
change of medium to induce a second lag period. (Data reproduced with permission from
Gregory et al.10)
FIGURE 2. Simplified scheme of the canonical Wnt pathway. Explanation of the ab-
breviations are given in the text.

102 ANNALS NEW YORK ACADEMY OF SCIENCES
MSCS, DKK-1 AND CANCER
There are surprising parallels in Dkk-1 expression and activity between MSCs
and some osteosarcoma cell lines. When assayed by RT-PCR, the osteosarcoma cell
lines MG-63 and SAOS both expressed high levels of Dkk-1 in culture.10 Further-
more, the MG-63 cell line exhibited a characteristic lag phase induced by fresh me-
dium that could be extended by addition of the blocking Dkk-1 antiserum.10 The role
of Dkk-1 in osteosarcoma proliferation is probably similar to its role in MSCs, but
this remains to be tested. Nevertheless, high Dkk-1 expression seems to be a hall-
mark of most osteosarcomas since screening of serum from newly diagnosed juve-
niles by ELISA revealed consistently high systemic levels of Dkk-1 in affected
individuals (Gregory et al. in preparation). One interesting possibility is that Dkk-1
may be inhibiting terminal differentiation of the hMSCs, which could result in the
FIGURE 3. Long-term treatment of hMSCs with 10 mM LiCl improves the differenti-
ation of hMSCs into osteoblasts. (a) RT-PCR assays for alkaline phosphatase (ALP), a mark-
er of osteogenesis, demonstrate that addition of 10 mM lithium causes earlier and higher
levels of ALP expression in mineralizing cultures of hMSCs. Data are presented in from two
seperate donors. (b) Extraction and colorimetric quantification of the calcium-binding dye,
Alizarin Red S (a marker of mineralization), confirms that lithium increases the rate of os-
teogenic differentiation by hMSCs. P values versus appropriate “supplemented medi-
um”control: P < 0.05 (*), P < 0.01 (**) for n = 6. Panel c: Addition of 500 ng mL−1
recombinant Dkk-1 to mineralizing hMSCs in the presence of osteogeic supplements, β-
glycerophosphate, ascorbate, and bone morphogenic protein 2 disrupts the Wnt-ediated dif-
ferentiation process. Histograms refer to alkaline phosphatase activity per cell. (Data repro-
duced with permission from Gregory et al.30)

103GREGORY et al.: Wnt SIGNALING IN OSTEOGENESIS
lack of mineralization and weakening of the bone seen in some cases. The signifi-
cance of high Dkk-1 levels in cases of osteosarcoma remains to be elucidated, but
anti-Dkk-1 strategies may provide a novel and effective means for control of the-
disease.
Multiple myeloma (MM) is a fatal malignancy of antibody-secreting plasma cells
(PCs) and it accounts for 10% of all hematologic malignancies.47 MM is the only
hematological malignancy consistently associated with widespread osteolytic
lesions (OLs) that cause a debilitating degenerative bone disease. Patients with overt
and symptomatic MM suffer from intractable bone pain at the site of the OLs, par-
ticularly in the spine and the long bones. In advanced cases, patients suffer spinal
cord compression and loss of mobility. There is no satisfactory treatment for OLs,
but in extreme cases, lesions are filled with a hardening resin that strengthens the
surrounding bone. OLs appear adjacent to clusters of malignant PCs, suggesting that
the MM cells secrete local factors that affect the osteoclasts and osteoblasts and their
regulation of normal coupled bone turnover. Initial studies have shown that the OLs
occur in response to hyperactivated osteoclasts resulting from the secretion of nu-
merous ligands secreted by the malignancy.48 However, recent studies have utilized
global gene expression profiling to identify molecular determinants of OLs in newly
diagnosed MM patients. Of the genes found to be reproducibly overexpressed in cas-
es of MM exhibiting osteolytic lesions, Dkk-1 was found to be the only secreted
product.49 Further investigation strongly correlated Dkk-1 serum levels with MRI-
diagnosed osteolytic lesions. In addition, in vitro experiments demonstrated that
Dkk-1 could reduce BMP2-induced alkaline phosphatase activity in osteoblast pro-
genitor cells,49 an observation confirmed by our laboratory with MSCs. It was there-
fore hypothesized that the abnormally high level of Dkk-1 probably prevents Wnt-
mediated terminal differentiation of progenitors into osteoblasts, further disrupting
the balance of normal coupled bone turnover, resulting in the persistence of OLs.
Clearly, Dkk-1 activity in individuals with multiple myeloma is a candidate target
for the reduction of the frequency and size of OLs. This could be accomplished by
at least two means: bypassing the action of Dkk-1 at the membrane by GSK3β in-
hibitors is an extremely attractive method since small molecule inhibitors are avail-
able. However, GSK3β is a multi-role enzyme in mammalian cells and its inhibition
could lead to undesirable side effects, especially in vivo. A more specific approach
involves the production of recombinant blocking antibodies to the Dkk-1 molecule
or its receptor. Some progress has been made in this field with the production of pep-
tide agonists of Dkk-1 that mimic the LRP6 binding site in Dkk-130 and these could
be used as antigens for specific antibody production.
SUMMARY
It is becoming clear that adult stem cells, and particularly hMSCs, will be pow-
erful tools for regenerative medicine and gene therapy in the near future. Elucidation
of the role of Dkk-1 and Wnt signaling in bone repair by MSCs has yielded valuable
insights into the mechanism of skeletal regeneration and how this can go wrong in
malignant disease. Agents that modulate Wnt signaling and Dkk-1 activity could
provide a new and valuable class of pharmaceutical agents for the enhancement of
tissue repair in humans.

104 ANNALS NEW YORK ACADEMY OF SCIENCES
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