WO2019241462A1 - Stem cells for the treatment of conditions and diseases - Google Patents

Abstract

In one particular embodiment, the present invention uses dental pulp-derived stem cells for the treatment of osteoporosis. In one particular embodiment, the present invention uses dental pulp-derived stem cells for the treatment of ischemic stroke. In one particular embodiment, the present invention uses dental pulp-derived stem cells for the reduction of inflammations associated with inflammatory bowel disease.

Description

STEM CELLS FOR THE TREATMENT OF CONDITIONS AND DISEASES

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates in general to the field of stem cell therapies and applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62/684,617, filed June 13,

2018, the entire contents of which are incorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under R01AR068279, 1R41EY024217, and 1R41AG057242 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] The present application includes a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on _ ,

2019, is named _ .txt and is _ , _ bytes in size.

BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with osteoporosis.

[0006] Osteoporosis is a silent systemic skeletal disease of progressive bone loss that each year causes 9 million fractures worldwide [1, 2] and about 1.5 million fractures in the United States [3] In the US alone, over 10 million people aged 50 or older suffer from osteoporosis and an additional 43 million have low bone mass that may progress into osteoporosis [4] The incidence of osteoporosis is roughly twice greater in women than in men [5] A number of environmental, endocrine, and genetic factors contribute to the development of osteoporosis [6] Other contributing factors include nutritional deficiencies, smoking, lack of exercise, and the presence of other diseases or medications [7] Several treatment options for osteoporosis exist currently, including calcium and vitamin D supplements as preventative measures [6], bisphosphonates as the first line of therapy [8], and other hormonal and biologic treatments.

[0007] Under normal physiologic conditions, bone undergoes a constant process of remodeling whereby it is simultaneously resorbed and deposited by osteoclasts and osteoblasts, respectively [17] Osteoporosis results from excessive resorption by osteoclasts coupled with inadequate osteoblast activity that is insufficient to properly restore bone [18], leading to structural alterations and reduction in BMD. Osteoblasts are derived from mesenchymal precursors and osteoclasts are derived from myeloid cells. Dental pulp stem cells (DPSCs) are mesenchymal in nature, retain self-renewal and mutipotential capacity, and are capable of mediating tissue regeneration [19, 20] These cells have been shown to maintain their“sternness” without changes in either morphology or expression of stem cell markers over time in culture [21] DPSCs have been used to regenerate lost dental pulp as well as dentin [22] The potential of DPSCs for bone tissue engineering has been demonstrated both in vitro and in vivo [23, 24] Osteogenic differentiation has been enhanced by the addition of bone morphogenetic protein (BMP)-2 [25] and by growth on various polymeric and biologic scaffolds [26] However, it is not well established how DPSC are regulated during osteogenic differentiation.

[0008] Wnt signaling plays essential roles in cell proliferation and differentiation during embryogenesis, post-natal development, and tissue homeostasis [27] Wnt signaling is also important for maintenance and expansion of stem cells [28, 29] Moreover, it is important in regulating the osteogenic process [30, 31], and its disruption is associated with several bone diseases, including osteoporosis [32]

[0009] Thus, a need remains for improved methods for creating differentiated cells useful for transplantation and treatment of diseases requiring new cells derived from stem cells.

SUMMARY OF THE INVENTION

[0010] In one embodiment, the present invention includes a method of making a differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, comprising: growing dental pulp-derived stem cells (DPSC) in a growth media; and treating the DPSC with: a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell; a chondrogenic medium to differentiate the DPSC into the chondrogenic cell, or an adipogenic induction medium to differentiate the DPSC into the adipogenic cell. In one aspect, the DSPCs are differentiated into an osteogenic cell with the phytoestrogen Ferutinin. In another aspect, the chondrogenic medium comprises modified Eagle’s media F12, 1-glutamine, fetal bovine serum, 1- proline, ascorbic acid, sodium pyruvate, insulin, transferrin, selenium, antibiotics, and dexamethasone. In another aspect, the adipogenic induction medium comprises Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum, insulin, and at least one of: dexamethasone, indomethacin, M3-isobutyl-l-methylxanthin, or pioglitazone. In another aspect, the method further comprises formulating the osteogenic, chondrogenic or adipogenic cells into a transplant. In another aspect, the method further comprises expanding the number of the osteogenic, chondrogenic or adipogenic cells for transplantation. In another aspect, the method further comprises delivering the osteogenic, chondrogenic or adipogenic cells to a subject. In another aspect, the DPSC are obtained by a method comprising: obtaining a molar; extracting from the molar the pulp; mincing the pulp; growing the cells in the pulp in a cell culture media; and isolating the cells that migrate from the pulp, wherein the cells are the pulp-derived stem cells. In another aspect, the DPSC are grown and a supernatant is obtained from the cells, and the supernatant is contacted with monocytes to induce M2 polarization of the monocytes. In another aspect, the DPSC are provided to a subject to treat an ischemic stroke. In another aspect, the DPSC are provided to a subject reduce an inflammation associated with an inflammatory bowel disease.

[0011] In another embodiment, the present invention includes a composition and method of transplantation of a differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, comprising: identifying a subject in need of transplantation with an osteogenic, chondrogenic, or adipogenic cell; growing dental pulp-derived stem cells (DPSC) in a growth media; and treating the DPSC with: a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell; a chondrogenic medium to differentiate the DPSC into the chondrogenic cell; or an adipogenic induction medium to differentiate the DPSC into the adipogenic cell; and transplanting the osteogenic, a chondrogenic or adipogenic cell into the subject. In one aspect, the DSPCs are differentiated into an osteogenic cell with the phytoestrogen Ferutinin. In another aspect, the chondrogenic medium comprises modified Eagle’s media F12, L-glutamine, fetal bovine serum, L-proline, ascorbic acid, sodium pyruvate, insulin, transferrin, selenium, antibiotics, and dexamethasone. In another aspect, the adipogenic induction medium comprises Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum, insulin, and at least one of: dexamethasone, indomethacin, M3-isobutyl- 1-methylxanthin, or pioglitazone. In another aspect, the method further comprises expanding the number of the osteogenic, chondrogenic or adipogenic cells prior to transplantation. In another aspect, the DPSC are obtained by a method comprising: obtaining a molar; extracting from the molar the pulp; mincing the pulp; growing the cells in the pulp in a cell culture media; and isolating the cells that migrate from the pulp, wherein the cells are the pulp-derived stem cells. In another aspect, the molar is syngeneic, allogeneic, or xenogeneic. In another aspect, the DPSC are grown and a supernatant is obtained from the cells, and the supernatant is contacted with monocytes to induce M2 polarization of the monocytes. In another aspect, the DPSC are provided to a subject to treat osteoporosis. In another aspect, the DPSC are provided to a subject to treat an ischemic stroke. In another aspect, the e DPSC are provided to a subject reduce an inflammation associated with an inflammatory bowel disease.

[0012] In another embodiment, the present invention includes a differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, made by a method comprising: growing dental pulp-derived stem cells (DPSC) in a growth media; and treating the DPSC with: a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell; a chondrogenic medium to differentiate the DPSC into the chondrogenic cell; or an adipogenic induction medium to differentiate the DPSC into the adipogenic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing and other objects, features, and advantages of the present invention will be apparent from the following description of embodiments as illustrated in the accompanying drawings, in which reference characters refer to the same parts throughout the various views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating principles of the present invention.

[0014] FIGS. 1A-1B show dental pulp-derived stem cells (DPSC) morphology for human cells during expansion. Dental pulp tissues were plated to expand stem cells. Left panel shows early growth of stem cells and right panel shows stem cells after confluence.

[0015] FIG. 2 shows the DPSC phenotype. Flowcytometric analysis reveals that expanded cells represent a homogeneous population characterized by expression of CD90, CD105, and CD73. They do not express CD133 and CD34 (hematopoietic stem cell markers), CD31 (endothelial progenitor cell marker), CD45R (B cell marker), CD 14 (monocyte marker), CD l ib (dendritic cell marker), CXCR4 (chemokine receptor), or MHC class II (antigen presentation protein).

[0016] FIGS. 3A-3C Expanded DPSCs maintain multipotential differentiation abilities. DPSCs were differentiated towards the osteogenic, chondrogenic, and adipogenic lineages. (FIG. 3 A) Micrographs at various magnifications of differentiated DPSCs following alizarin red staining. (FIG. 3B) Chondrogenic differentiation visualized by microscopy after Alcian blue staining. (FIG. 3C) Micrographs of adipogenic differentiation shown after oil red o staining.

[0017] FIG. 4 shows that Ferutinin modulates mRNA expression of osteoblast specific and key Wnt pathway genes. RT-PCR was carried out to evaluate gene expression of COL1A1, BGLAP, LRP6, DVL3, GSK3B, and CTNNB 1. Expression is shown as fold difference ± SEM derived from calculated AACT values. Statistical significance, p<0.05 was shown (*), compared to vehicle-treated controls. 4B. Ferutinin modulates Wnt/p-catcnm signaling pathway molecules in DPSCs. Various Wnt/p-catenin signaling pathway proteins were evaluated in DPSCs after stimulation with ferutinin (10 pg/mL) for 12, 24, and 48 h using western blot methods. GAPDH was used as an internal loading control. Band density is presented as protein expression relative to GAPDH ± SEM.

[0018] FIGS. 5A and 5B show DPSCs treated with ferutinin express osteogenic molecules. Immunostaining was performed to visualize expression levels of (5 A) collagen 1A1, and (5B) osteocalcin in ferutinin-treated DPSCs compared to vehicle-treated cells. DAPI was used for nuclear staining.

[0019] FIG. 6 shows that GSK3 inhibition modulates Wnt/p-catenin signaling pathway molecules in DPSCs. Various Wnt/p-catcnin signaling pathway molecules in DPSCs were evaluated after stimulation with a GSK3 inhibitor molecule CHIR-98014 for 12, 24 and 48 h. Western blot results are shown.

[0020] FIGS. 7A and 7B show that Ferutinin regulates Wnt3a and Dvl3 genes epigenetically. Wnt signaling pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP) quantitative PCR methods to assess epigenetic regulation in DPSCs after stimulation with ferutinin for 24 h. Promoter site active marks of Wnt3a and Dvl3 genes for both H3K9 (FIG. 7A) acetylation and H3K4 (FIG. 7B) trimethylation were evaluated and shown graphically. (* indicates p < 0.05, ** indicates p < 001).

[0021] FIGS. 8 A to 8D show the dose-dependent inhibition of osteoclast differentiation and related molecules by DPSCs. FIG. 8A. Images of induced differentiated RAW 264.7 cells determined by TRAP staining at day 6 in the absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation. FIG. 8B. The number of TRAP positive multinucleated osteoclasts present in each group shown graphically in absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation. FIG. 8C. Quantitative real-time PCR analysis of osteoclast differentiation-related marker genes such as Nfatcl, Ctsk, Rank, Trap, and Mmp9 expressions were shown graphically at day 6 in the absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation of RAW 264.7 cells. In all cases, Gapdh expressions were kept as internal controls. FIG. 8D. Western blotting of osteoclast differentiation-related molecules such as NFATcl, cathepsin K, MMP9, and p65 protein levels, keeping GAPDH as an internal control, were shown at day 6 in the absence or presence of DPSCs (contact-free co culture) during osteoclast differentiation.

[0022] FIG. 9 shows that DPSCs inhibited osteoclast differentiation-related molecules in myeloid cells. Immunocytochemical staining images of osteoclast differentiation determined for NFATcl, cathepsin K, TRAP, or MMP9 molecules at day 6 in the absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation of RAW 264.7 cells.

[0023] FIGS. 10A and 10B show that DPSCs inhibited expression of pro-inflammatory, and induced anti inflammatory genes along with M2 phenotype molecules. FIG. 10A. Quantitative real-time RT-PCR analysis of pro- and anti-inflammatory marker genes such as Tnf- a, and IL-4Ra respectively expressions were shown graphically at day 6 in the absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation of RAW 264.7 cells. FIG. 10B. Quantitative real-time RT-PCR analysis of M2 polarization marker genes such as Argl and Yml(Chil3) expressions were shown graphically at day 6 in the absence or presence of DPSCs (contact-free co-culture) during osteoclast differentiation of RAW 264.7 cells. In both cases, Gapdh expressions were kept as internal controls.

[0024] FIGS. 11A to 1 IE show that DPSC secreted OPG during osteoclast differentiation and induced OPG expressions in osteoclast precursor cells. RAW 264.7 cells were cocultured with two different concentrations of DPSCs, or without DPSCs (assigned as osteoclast, OC) in osteoclast induction medium (sRANKL, M- CSF, and 10% FBS containing DMEM) using a trans-well culture system, or cultured alone in 10% FBS DMEM assigned as monocyte. Total RNA and proteins were isolated after 6 days of culture from the RAW 264 7 cells, and supernatants were collected at different time points during the course of differentiation. FIG. 11A. The mRNA expression of OPG was determined by real-time PCR from cultured RAW 264.7 cells keeping b-actin as internal controls. FIG. 1 IB. Western blot analyses of OPG protein level shown at day 6 in the absence or presence of various concentrations of DPSC during osteoclast differentiation of RAW 264.7 cells. FIG. 11A. 11C-E. Secreted OPG in culture supernatants were measured by ELISA collected at different time points during the course of osteoclast differentiation. FIG. 11C. After 1 day of culture. FIG. 11D. After 2 days of culture. FIG. 11E. After 6 days of culture. *= P< 0.05 when compared with OC. All values are represented as mean ± SEM of triplicate samples from one of the three independent experiments.

[0025] FIGS. 12A to 12E show that DPSC constitutively express and secrete osteoprotegerin (OPG). DPSCs were cultured in different conditions (1% FBS in DMEM, 10% FBS in DMEM, osteoclast stimulating media, or 1 x PBS) for various time points (0, 3, 6, 24, 48 or 72 h), and supernatants were collected either for quantification of OPG, or cells were harvested for total RNA isolation. FIG. 12A. The mRNA expression of OPG was determined by real-time PCR from cultured DPSCs keeping b-actin as internal controls. FIGS. 12B-E. Quantification of secreted OPG in DPSC culture supernatants collected at various time points from different culture conditions. FIG. 12B. In 1% FBS containing DMEM. FIG. 12C. In 10% FBS containing DMEM. FIG. 12D. In 1 x PBS. FIG. 12E. In osteoclast differentiating medium. *= P< 0.05 when compared with OC. All values are represented as mean ± SEM of triplicate samples from one of the three independent experiments.

[0026] FIGS. 13A and 13B show recombinant OPG inhibited osteoclast differentiation related molecules in a dose-dependent manner. FIG. 13A. Quantitative real-time RT-PCR analysis of Nfatcl, Ctsk, Rank, Trap, and Mmp9 expressions were shown graphically at day 6 in the absence or presence of various concentrations of recombinant OPG during osteoclast differentiation of RAW 264.7 cells. In all cases, Gapdh expressions were kept as internal controls. Gapdh expressions were kept as internal controls in all quantitative RT-PCR analyses. (# indicates p<0.05 when compared between control and day 6 of osteoclast differentiated samples, * indicates p<0.05 when compared between day 6 of osteoclast differentiated samples in absence and presence of either of the concentrations of recombinant OPG.) FIG 13B Western blot analyses of NFATcl, cathepsin K and MMP9 molecules shown at day 6 in the absence or presence of various concentrations of OPG during osteoclast differentiation of RAW 264.7 cells.

[0027] FIGS. 14A to 14H shows that DPSC mimicked the OPG-mediated signaling pathway (PI3K) to inhibit osteoclast differentiation. FIG. 14A. TRAP staining images of RAW 264.7 cells at day 6 of osteoclast differentiation in the absence or presence of various concentrations of OPG during osteoclast differentiation of myeloid cells. FIG. 14B. The number of TRAP positive multinucleated osteoclasts present in each group shown graphically in absence or presence of various concentration of OPG during osteoclast differentiation. FIG. 14C. Western blot analyses of pAKT and total AKT molecules from PI3K pathway shown at day 6 in the absence or presence of two different concentrations of OPG during osteoclast differentiation of RAW 264.7 cells. FIG. 14D. Western blot analyses of pAKT and total AKT molecules shown at day 6 in the absence or presence of two different concentrations of DPSCs (contact-free co-culture) during osteoclast differentiation of RAW 264.7 cells. FIG. 14E. TRAP staining images of RAW 264.7 cells at day 6 of osteoclast differentiation in the absence or presence of various concentrations of PI3K inhibitor (LY294002) during osteoclast differentiation of myeloid cells. FIG. 14F. The number of TRAP positive multinucleated osteoclasts present in each group shown graphically in absence or presence of various concentration of PI3K inhibitor (LY294002) during osteoclast differentiation. FIG. 14G. TRAP staining images of RAW 264.7 cells at day 6 of osteoclast differentiation in the absence or presence of DPSC (10: 1) with or without anti-OPG antibody (1 pg/ml) during osteoclast differentiation of myeloid cells. FIG. 14H. The number of TRAP positive multinucleated osteoclasts present in each group shown graphically in absence or presence of DPSC and/or with or without anti-OPG antibody (1 pg/ml) during osteoclast differentiation. Representative TRAP staining images are shown from three independent experiments. DESCRIPTION OF THE INVENTION

[0028] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0029] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as“a”,“an” and“the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0030] In one embodiment, the present invention relates to a method of improving the efficacy of a cell transplant, and more specifically, to the development of differentiated cells of an osteogenic, a chondrogenic, an adipogenic, or myeloid lineage for transplantation, wherein the stem cells are from a dental origin, namely, dental stem cells.

[0031] As used herein, the term“stem cell” is an undifferentiated cell that is capable of essentially unlimited propagation either in vivo or ex vivo and capable of differentiation to other cell types. The stem cell can be then be differentiated, committed, immature, progenitor, or mature cell types present in the tissue from which it was isolated, or dramatically differentiated cell types, such as for example the erythrocytes and lymphocytes that derive from a common precursor cell, or even to cell types at any stage in a tissue completely different from the tissue from which the stem cell is obtained. For example, dental stem cells are differentiated into cells of osteogenic, a chondrogenic or adipogenic lineage. Further, it is taught herein that these differentiated cells can be further used to differentiate cells into cells of myeloid lineage.

[0032] As used herein, the term“improved efficacy of a transplant” refers to the capability of a transplant, in particular, a stem cell transplant, to repair damaged tissue, in particular, neuronal tissue.

[0033] As used herein, the term“culturing” refers to propagating or nurturing a cell or a collection of cells, by incubating for a period of time in a cell culture media under conditions that support cell viability or propagation. Culturing can include one or more steps selected from, e g., expanding and proliferating a cell, collection of cells according to the present invention.

[0034] As used herein, the term“isolating” a stem or a differentiated cell refers to the process of removing a stem cell from a tissue sample and separating away other cells that are not stem cells. An isolated stem cell will generally be free from contamination by other cell types and will generally have the capability of propagation and differentiation to produce mature cells of the tissue from which it was isolated. However, when dealing with a population of stem cells, e g., a culture of stem cells, it is understood that it is practically impossible to obtain a collection of stem cells that is 100% pure. Therefore, an isolated stem cell can exist in the presence of a small fraction of other cell types that do not interfere with the use of the stem cell for analysis, production of other, differentiated cell types, and/or transplantation. Isolated stem cells will generally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% pure. For example, the isolated stem cells according to the present invention will be at least 98% or at least 99% pure.

[0035] Typically, a stem cell is“expanded” by propagation in cell culture by cell division to other stem cells and/or progenitor cells. Expansion of stem cells may occur spontaneously as stem cells proliferate in a culture or it may require certain growth conditions, such as a minimum cell density, cell confluence on the culture vessel surface, or the addition of chemical factors such as growth factors, differentiation factors, or signaling factors.

[0036] As used herein, the term“stem cell transplant” refers to a composition comprising stem cells, wherein the composition is suitable for administration by transplantation into a subject

[0037] The stem cell transplant of the disclosure may advantageously be obtained from a tissue biopsy, such as peripheral blood, umbilical cord blood or bone marrow. Collection of bone marrow or peripheral blood for use in autologous or allogeneic stem cell transplantation therapies is common practice, and methods to collect bone marrow or peripheral blood biopsies are well known in the art.

[0038] As used herein, a“mammal” refers to any mammal including but not limited to human, mouse, rat, cat, dog, sheep, monkey, goat, rabbit, hamster, horse, cow, or pig.

[0039] As used herein,“allogeneic” refers to genetically different members of the same species.

[0040] As used herein,“isogeneic” or“syngeneic” refers to cells of an identical genetic constitution.

[0041] As used herein,“xenogeneic” refers to cells of a different species.

[0042] As used herein, a“recipient” refers to a mammal that receives an organ, tissue or cells taken from a donor, in this case, stem cells or differentiated cells can be transplanted into the recipient.

[0043] As used herein, a“donor” is a mammal from which organs, tissues or cells are taken for transplant into a recipient.

[0044] As used herein, the phrase "in need thereof means that the patient has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by a diagnosis that certain cells are in need of replacement. In any of the methods and treatments described herein, the animal or mammal can be in need thereof, e.g., transplantation.

[0045] Example 1. Ferutmin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling

[0046] Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. The inventors have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, the inventors show that ferutinin activates DPSCs by modulating the Wnt/p-catcnin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, the inventors inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP) -quantitative PCR approaches. The inventors confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.

[0047] ft was found herein that: isolation and homogeneous expansion of multipotent human dental pulp- derived stem cells (DPSCs); Ferutinin activates DPSCs via the Wnt/p-catenin signaling pathway, and induces osteocalcin and collagen 1A1 both mRNA and proteins; GSK3 inhibitor also activates DPSCs through the Wnt/p-catenin signaling pathway; and Ferutinin induces H3K9 acetylation and H3K4 trimethylation in the promoter sites of the WNT3A and DVL3 genes in DPSCs.

[0048] Ferutinin is a daucane phytoestrogen found in plants of the Ferula genus which binds to both isoforms of the estrogen receptor [33] Ferutinin has been shown to prevent bone loss in rats [34] Furthermore, its efficacy in promoting the recovery of bone density following ovariectomy-induced osteoporosis has also been demonstrated [35] The molecular pathways by which Ferutinin promotes osteogenesis have yet to be elucidated. Furthermore, its potential therapeutic efficacy is currently unknown.

[0049] This study was undertaken to develop an effective potential stem cell therapy using human DPSCs for the treatment of osteoporosis. The pharmacological compound ferutinin was tested to activate and promote osteogenesis in DPSCs. Furthermore, molecular pathways and epigenetic mechanisms by which ferutinin promotes DPSC differentiation were evaluated.

[0050] Materials and Methods. DPSC isolation and expansion. Human dental pulp derived stem cells (DPSC) were isolated from discarded third molar teeth, which were obtained after surgical extraction from a healthy adolescent donor with prior approval from the Institutional Review Board (IRB) and consent from donor. Teeth were thoroughly (at least 3 times) washed with phosphate buffered saline (PBS) containing 1% Penicillin-Streptomycin-Glutamine (PSG) (Gibco, Thermo Fisher, Waltham, MA). Teeth were cut open to harvest the pulp, which was then minced into approximately 1 mm cubes and plated onto 60 mm cell culture plates and cultured with alpha (a) Modified Eagle Medium (MEM) (Gibco) with 20% FBS (Hy clone, Thermo Fisher, USA) and 1% PSG. Fresh medium was added every third day of culture after removing old medium. Cells that migrated from the pulp tissues and became confluent were collected by dissociation by scraping and were re-cultured as passage 1 and maintained using the same medium. Cell viability was determined using the trypan blue exclusion method. Experiments were performed using cells between 3-7 passages.

[0051] Flow cytometry. Fluorescently labeled antibodies for cell surface markers included CD73, CD90, and CD 105 (eBioscience, San Diego, CA), CD 133 and CD34 (Miltenyi Biotec, San Diego, CA), IgG, CD31, CD45R, CD14, CDl lb, CXCR4, and MHC class II (BD Biosciences, San Jose, CA). Flow cytometry was carried out according to a previously described method [36] The DPSC aliquots were incubated at 4°C for more than 30 min in 2% FBS containing Hanks’ buffer, each in presence of one of the aforementioned antibodies. After antibody staining, cells were washed twice using 2% FBS containing Hanks’ buffer and fixed in 1% paraformaldehyde. Flowcytometric analysis was performed by using a FACS Calibur analyzer machine (BD Biosciences). Relevant isotype controls were also included for compensation setting. At least 20,000 events were acquired for each sample for analysis using CellQuest Pro software (BD Biosciences).

[0052] Induced differentiation. The multi-differentiation potential of DPSCs was evaluated in vitro using mesenchymal stem cell (MSC) differentiation kits (Millipore, Burlington, MA). Cells (passage 3) were plated in 10 cm cell culture dishes and grown to confluency in otMEM medium with 20% FBS and 1% PSG before induced differentiation.

[0053] Osteogenic induction and Alizarin red S staining. Alizarin red S staining was carried out according to a previously described procedure [37] DPSCs were cultured in osteogenic induction medium (Millipore) consisting of basic growth medium with 0.1 mM dexamethasone, 0.2 mM ascorbic 2-phosphate, and 10 mM glycerol 2-phosphate for 14 days on a 6-well collagen-coated plate. Medium was replaced every 3rd day. After 14 days of differentiation, cells were subjected to Alizarin red S staining, which stains calcified deposits produced by osteoblast cells and thus provides evidence of osteogenic differentiation. For staining, cells were fixed with ice-cold 70% ethanol for 1 h at room temperature. Ethanol was removed and cells were washed twice with water. Water was aspirated and Alizarin red solution was added; cells were then incubated for 30 minutes in the dye at room temperature. Cells were then washed 4 times with water and left in water to avoid drying. Images were captured under a light microscope at various magnifications.

[0054] Chondrogenic differentiation and Alcian blue staining. Chondrogenic differentiation of hMSCs was accomplished by a modification of the protocol outlined by Johnstone et al. [38] In brief, aliquots of 250,000 DPSCs suspended in 0.5 mL medium were distributed to 15 mL conical polypropylene centrifuge tubes (VWR, West Chester, PA). The cells were centrifuged for 5 min at 600 g and pelleted at the bottom of the tube, and cultured in serum-free chondrogenic medium (Millipore). Tubes were placed in an incubator with caps loosened to permit gas exchange. The sedimented cells formed a spherical mass at the bottom of the tube within 24 h. Medium was replaced three times per week. Cell pellets were harvested by rinsing in D- PBS followed by fixation for 1 h in 4% formaldehyde in D-PBS, made fresh. Samples were then transferred into 70% ethanol, dehydrated in ethanol and xylene series, and paraffin-embedded. Sections of 5 mih were cut through the center of each pellet. Sections were stained with Alcian blue stain and images were captured with a light microscope.

[0055] Adipogenic induction and Oil Red O staining. DPSCs were cultured in adipogenic induction medium (Millipore) for 14 days. Differentiated cells produced lipid droplets that were subsequently stained using Oil Red O dye. To stain cells, medium was removed and cells were washed 3 times for 5 minutes each with IX PBS. Cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. The fixative was aspirated and the cells were washed 3 times for 5 minutes each with IX PBS, then washed twice with water. The water was aspirated and Oil Red O solution was added; cells were incubated for 50 minutes at room temperature. The Oil Red O solution was removed and cells were washed 3 times with water. Nuclei were stained with hematoxylin solution for 3 minutes. Images were captured under a light microscope at various magnifications.

[0056] Quantitative RT-PCR. Total RNA was isolated from DPSCs treated with ferutinin for 12, 24, and 48 h and from vehicle-treated DPSCs using a TRIzol purification method (Thermo Fisher). Complementary (c) DNA synthesized from the mRNA was used for quantitative PCR using a Bio-Rad CFX96 Real-Time System. Primers for COL1A1, BGLAP (Osteocalcin), LRP6, DVL3, GSK3B, CTNNB1 (b-catenin), and GAPDH were purchased from Integrated DNA Technologies (Coralville, IA). Sequences are presented in Supplemental Table IF Cq measurements were obtained, and data are presented as fold difference of AACT values corrected with GAPDH expression.

[0057] Western blot. Whole cell lysates were obtained from DPSCs cultured under control conditions or stimulated with ferutinin (10 pg/mL in aMEM) or CHIR-98014 (50 nM) for 12, 24, and 48 h Protein was quantified by colorimetric assay using the Bradford method (Bio-Rad, Hercules, CA) and the proteins were separated in a polyacrylamide gel. Briefly, a polyacrylamide gel was cast and denatured proteins (20 pg) were loaded and separated through the gel by electrophoresis; a protein ladder was loaded as a marker (Sigma, St. Louis, MO). The proteins were transferred from the gel to a 0.45 pm nitrocellulose membrane (Bio-Rad) at 4 °C. The membrane was blocked for 1 h at room temperature (RT) with a blocking buffer composed of 5% nonfat milk in TBS-Tween-20 (TBST) (Boston BioProducts, Ashland, MA). The membrane was washed and incubated with primary antibody against LRP6, Dvl3, Axinl, Nakedl, b-catenin, GAPDH (Cell Signaling, Danvers, MA), and GSK3 (Santa Cruz Biotechnology, Dallas, TX) (1 : 1000 diluted in a solution of 5% BSA in TBST) for 2 h. The membrane was washed, then incubated in secondary antibody (1:3000 in a solution of 5% milk in TBST) (Cell Signaling). The membrane was then washed, placed in the cassette holder and incubated briefly in chemiluminescent substrate (Sigma). Films were then exposed and developed. Densitometric quantification of bands was performed using ImageJ software (NIH). [0058] Immunostaining. DPSCs were plated at medium density on glass cover slips in 6 well plates and with 1.5 mL complete medium (aMEM, 20% FBS, 1% PSG). Next day, medium was changed for complete medium containing 10 pg/mL ferutinin or 1 pL/mL DMSO in complete medium as a vehicle -treated control. 24 h later, medium was aspirated and cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton-X 100 at room temperature (RT). After washing with PBS, cells were blocked in 5% FBS in PBS overnight at 4 °C. Next day, they were washed with wash buffer (PBS with 0.5% FBS and 0.5% TBST) and incubated in primary antibodies (a-Osteocalcm and a-CollagenlAl; diluted 1: 100 in wash buffer) for 2 h at RT; two samples were incubated in secondary antibody alone to serve as negative controls. Samples were washed with wash buffer and were incubated with fluorophore conjugated secondary antibody (Alexa Fluor 488 a-Rabbit and TX Red a-Goat as necessitated by the primary antibodies used; diluted 1 :2000 in wash buffer) for 1 h at RT. Cover slips were mounted upon glass microscopy slides with ProLong Gold Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA). Upon drying, fluorescence microscopy was performed and images were obtained.

[0059] Chromatin immunoprecipitation (ChIP) and quantitative PCR. ChIP analysis was performed using Imprint® Chromatin Immunoprecipitation Kit (Sigma) according to previously described methods [39] Briefly, after chromatin cross-linking with 1% formaldehyde and DNA shearing, chromatin-protein complexes were immunoprecipitated from DPSCs with or without stimulation of ferutinin for 24 h, with antibodies against H3K9Ac (Millipore Sigma), H3K4me3 (Millipore Sigma). Antibody against goat IgG (Abeam, Cambridge, UK) was used as a negative control. Quantitative PCR analysis was performed with the primers described (in Supplementary Table-I and Figs. 1A-1B) using SYBR green PCR master mix (Thermo Fisher) and a real-time PCR machine (Bio-Rad CFX96 Real-Time System). Values obtained from the ChIP assay were normalized to the background obtained from the precipitation with a non-specific antibody. Percent (%) of input was analyzed by following standard formula. Each experiment was performed in triplicate at least three times.

[0060] DPSC isolation, expansion and characterization. DPSCs were isolated from the pulp of donor human third molar teeth and were expanded in plentiful numbers in vitro. These cells display a fibroblast-like morphology during expansion. Captured images were shown during early stage of expansion (Fig. 1A, left panel), and after confluence of the culture (Fig. IB, right panel). The phenotype of DPSCs is elucidated by flow cytometry. Flowcytometric analysis revealed that the expanded DPSCs are a population of homogenous cells (Fig. 2). These cells express all progenitor stromal stem cell biomarkers; 99.97% were positive for CD90, 99.94% were positive for CD105, and 99.96% were positive for CD73. They were negative for hematopoietic stem cell markers CD133 (2.03%) and CD34 (3.19%). They were negative for platelet endothelial cell adhesion molecule CD31 (2.01%). They were negative for CD45R (5.67%), a marker present on B cells and other antigen presenting cells. They were negative for CD14 (2.47%), a monocytic marker. They were negative for CD l ib (2.38%), a dendritic cell marker. They were negative for chemokine receptor CXCR4 (3.14%). They were also negative for the antigen presenting molecule MHC class II (1.48%).

[0061] Differentiation potential of DPSCs. To investigate whether expanded DPSCs retain their multipotential capabilities, the inventors induced them along the osteogenic, chondrogenic and adipogenic lineages in vitro for 14 days. Osteogenic differentiation was detected by using Alizarin red S staining, which stains calcium deposited by differentiated cells. Images at various magnifications were shown (Fig. 3A, upper panels). Red staining indicates that DPSCs have differentiated to osteoblastic cells, which deposit calcium extracellularly. Chondrogenic differentiation was detected by Alcian blue staining that stains acidic polysaccharides found in cartilage. Following induced differentiation, DPSCs were found to possess chondrogenic properties as demonstrated by positive Alcian blue staining (Fig. 3B, middle panels).

[0062] Adipogenic differentiation was detected by Oil Red O dye, which stains neutral lipids in cells. After induced differentiation, lipid droplets within cells were stained red, while cell nuclei were stained black by hematoxylin. Abundant oil droplets were observed, indicating that DPSCs are capable of differentiation along the adipogenic lineage (Fig. 3C, lower panels).

[0063] Effect of ferutinin on DPSC activation and differentiation. To further elucidate the role of ferutinin in DPSC activation and differentiation, expression of osteoblast specific genes (COL1A1 and BGLAP), and key Wnt pathway genes (LRP6, DVL3, GSK3B, CTNNB 1), and GAPDH as an internal control were evaluated by quantitative RT-PCR. Quantitative RT-PCR data revealed that all tested genes were elevated upon stimulation with Ferutinin at various degrees during the time course study (Fig. 4). The effects of ferutinin on expression of Wnt/p-catcnin pathway molecules were evaluated by western blot analysis (Fig. 4). Increased expression of LRP6 was observed at 12 and 24 h of stimulation. Dvl3 expression increased at 24 h of stimulation. Wnt3a expression was markedly increased at 12 and 24 h of stimulation and was completely attenuated by the 48 h time point. Wnt5a/b expression did not substantially change over the course of the experiment. Expression of Naked 1 increased at 12 and 24 h of stimulation, before returning to levels akin to the control condition by 48 h. Axin was very minimally expressed throughout, but was observed at slightly higher levels at the 24 h time point. GSK3 expression was attenuated at 12, 24, and 48 h of stimulation. Correspondingly, expression of b-catenin increased at 12 and 24 h of stimulation before returning to control levels by 48 h. GAPDH expression was assessed as a control to ensure equal loading (quantified data is presented in Supplemental Fig. 2). Data at the gene expression level corroborate protein-level observations. A notable exception is the observation of significantly increased mRNA expression of GSK3B at the 24 and 48 h time points following ferutinin stimulation, while at the same time points a marked decrease is observed at the protein level. This discrepancy may be due to post-translational modification of GSK3 or other regulatory mechanisms [32]; however, this stands to be evaluated in greater detail in the future.

[0064] Effect of ferutinin on osteogenic markers. Expression of key matrix proteins secreted by osteoblasts was evaluated by immunostaining techniques in DPSCs stimulated with ferutinin (10 pg/mL) for 24 h. Immunocytochemical evaluation revealed that the level of expression of collagen 1A1 was far greater in ferutinin-treated cells compared to cells treated with the vehicle (Fig. 5A). In addition, expression of osteocalcin was also increased in stimulated cells compared to vehicle treated cells (Fig. 5B). Samples were visualized and photographed using a Leica DMi8 microscope and Leica Application Suite X software.

[0065] Effect of GSK3 inhibitor on Wnt/p-Catenin signaling pathway molecules in DPSCs. The effect of CHIR-98014, a pharmacological compound that inhibits GSK3 was tested on Wnt/p-Catenin signaling pathway molecules of DPSCs at various time points (Fig. 6). In untreated DPSCs, LRP6 is expressed faintly; after 24 h of stimulation, however, it was substantially upregulated. Dvl3 was minimally expressed in untreated cells and was increased over time. Wnt3a was significantly upregulated over the course of differentiation. Wnt5a/b expression was upregulated following 12 and 24 h of stimulation before attenuating by 48 h. Nakedl was upregulated at all time points after stimulation. Axinl was most strongly expressed at 24 h following stimulation. Decreased expression of GSK3 was observed in stimulated cells. Expression of b-catenin was increased over all three time points. GAPDH served as a loading control and minimal changes were observed (quantified data is presented in Supplemental Fig. 3). These results indicate that inhibition of GSK3 pathway causes modulation of Wnt pathway molecules similar to that observed when DPSCs are stimulated with ferutinin.

[0066] Epigenetic evaluation of Wnt3a and DVL3 molecules upon ferutinin stimulation. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling in DPSCs, the pathway molecules Wnt3a and Dvl3 were analyzed by chromatin immunoprecipitation (ChIP) quantitative PCR approaches with and without stimulation by ferutinin. ChIP analysis revealed that the active marks of both histone 3 lysine 9 (H3K9) acetylation and histone 3 lysine 4 (H3K4) trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after stimulation with ferutinin (Figs. 7A-7B).

[0067] Osteoblasts are derived from mesenchymal precursor cells and are the key mediators of bone regeneration in health and disease states. Human dental pulp tissues from the wisdom teeth contain clonogenic, highly proliferative stem cells capable of tissue regeneration [19] These DPSCs show fibroblast- like morphology and express markers similar to those of human bone marrow stromal cells (BMSC). DPSCs have also been shown to self-renew in vivo after in vitro expansion [20] These cells have been shown to maintain their“sternness” without changes to morphology or expression of stem cell markers over time in culture [21] DPSCs have been used to regenerate lost dental pulp as well as dentin by differentiation into odontoblasts [22] DPSCs show promise for bone tissue engineering, as their capacity for osteogenic differentiation has been demonstrated both in vitro and in vivo [23, 24] However, the molecular mechanism by which DPSCs differentiate has yet to be defined. In this study, the inventors have demonstrated that DPSCs can be cultured successfully in vitro (Figs. 1A-1B) while maintaining their MSC-like multilineage differentiation capacity along the osteogenic, chondrogenic and adipogenic lineages (Figs. 3A-3C). They possess specifically mesenchymal properties, as they express the cell surface markers CD73, CD90, and CD105, and do not express markers of hematopoietic lineage cells (CD34, CD133, CDl lb, CD14, CD31, CD45R, CXCR4, MHC class II) (Fig. 2). Because they do not express MHC class II, they are considered to possess low immunogenicity and thus be suited for transplantation with low risk of rejection. Although they possess multilineage differentiation potential, the goal is to direct them towards the osteogenic lineage for the regeneration of bone. For that purpose, the inventors have investigated several compounds and found a phytoestrogen, ferutinin, which could be a potential molecule of interest.

[0068] Phytoestrogens are plant-derived polyphenols that share structural similarity to the endogenous estrogen 17 -estradiol. Many such compounds have been shown to share the osteoprotective effects of estrogens without possessing their carcinogenic side effects [40] Ferutinin is a daucane phytoestrogen found in plants of the Ferula genus and has affinity for both isoforms of the estrogen receptor; it is therefore speculated that the compound may be useful as a selective estrogen receptor modulator [41] The effects of ferutinin on bone metabolism have previously been evaluated in ovariectomized rats with known protective effects on bone density [34] Furthermore, it has been shown to aid in recovery of bone density in animal models of osteoporosis [35] However, the mechanisms by which ferutinin promotes bone density have not yet been evaluated. The inventors found that ferutinin promotes osteogenic differentiation by modulating the various molecules of the Wnt/fl-catcnm signaling pathway, which critically regulates osteogenesis (Fig. 4).

[0069] Wnt signaling plays essential roles in embryogenesis, post-natal development, and tissue homeostasis as a regulator of cell proliferation and differentiation [27] In the absence of the Wnt ligand, the transcription factor b-catenin is held within the destruction complex, composed of Axin, adenomatous polyposis coli protein (APC), and GSK3. GSK3 phosphorylates b-catenin, marking it for ubiquitination and proteasomal degradation [42] . When Wnt binds to the Frizzled/LRP receptor complex on the cell surface, the destruction complex is inhibited. Hypophosphorylated b-catenin is released from its interaction with destruction complex proteins and subsequently accumulates in the cytoplasm and enters the nucleus, where it acts to regulate transcription [43] Moreover, its importance as a pathway involved in regulating osteogenic differentiation of mesenchymal stem cells has been demonstrated [30, 31] Wnt signaling is central to bone modeling and especially to osteoblast function; its disruption is associated with several bone diseases including osteoporosis [32] The observations of the effect of ferutinin on molecules of the Wnt^-catenin signaling pathway are in accord with the canonical mechanism of this pathway’s activation.

[0070] Observing Wnt pathway dynamics alone is insufficient to state that cells are undergoing osteoblastic differentiation, as this pathway is involved in myriad cell growth, proliferation, and differentiation processes. Expression of key osteogenic markers was thus assessed to confirm DPSC differentiation toward the osteogenic lineage. Collagen 1A1 is expressed in osteoblasts from an early stage of differentiation and is a key component of the organic bone matrix [44, 45]; the inventors observed its upregulation in DPSCs as early as 24 h after stimulation with ferutinin (Fig. 5 A). Osteocalcin is an osteoblast-specific protein with important metabolic functions in bone [46]; the inventors observed that it was markedly upregulated in ferutinin-stimulated DPSCs (Fig. 5B). Expression of these key proteins indicates that ferutinin promotes osteoblastic differentiation of DPSCs.

[0071] Recent reports of DPSC activation and subsequent tooth repair by GSK3 antagonists [47] For this reason, the inventors employed the GSK3 inhibitor CFIIR-98014 to explore the role of Wnt signaling in osteoblastogenesis and validate the role of ferutinin as a Wnt pathway modulator (Fig. 6). When CFIIR- 98014 was administered, protein expression patterns similar to those seen with ferutinin were observed. This leads us to propose that ferutinin may act by a similar mechanism of GSK3 inhibition.

[0072] To confirm further, the inventors have performed molecular analysis at the epigenetic level to assess the regulation of the genes that the inventors found were most changed in DPSCs after stimulation with ferutinin. The inventors evaluated histone 3 lysine 9 acetylation (H3K9ac) and histone 3 lysine 4 trimethylation (F13K4me3). Both of these are hallmarks of active promotors and therefore indicate that transcription of the target gene is active [48, 49] The inventors found that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after stimulation with ferutinin (Figs. 7A-7B).

[0073] Though the Wnt pathway plays a significant role in osteoblastogenesis, it is one of the several key pathways in this process. The BMP2 pathway, for instance, is a critical regulator at various stages of osteoblastic differentiation and maturation [43] Signaling via estrogen receptors is also of great importance in osteoblastogenesis and bone health [44] Moreover, these pathways hardly act independently of one another; there is growing evidence of cross-talk among the many pathways involved [45]

[0074] These results demonstrate that ferutinin activates and promotes osteogenic differentiation of DPSCs canonical Wnt/fl-catcnin signaling pathway by activating marks of both H3K9 acetylation and H3K4 trimethylation in the promoter regions of WNT3 A and DVL3. It can be used as an inducer to modulate DPSCs towards osteogenic differentiation for a potentially effective stem cell therapy for osteoporosis.

[0075] Example 2. Dental pulp-derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and inducing M2 polarization of monocytes

[0076] Osteoclasts (OC) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodeling both in homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, the inventors show underlying molecular mechanism of activation of myeloid cells, and try to control using dental pulp-derived stem cells (DPSCs), which has potential for future therapeutic application for bone-related disorders. The inventors found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. In addition, DPSCs reduced expression of key OC markers, such as, NFATcl, cathepsin K, TRAP, RANK, and MMP-9 assessed by quantitative RT-PCR, western blotting, and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of myeloid cells. To define molecular mechanisms, the inventors found that osteoprotegerin (OPG), an OC inhibitory factor, was upregulated in myeloid cells in presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing expression of OC differentiation markers, NFATcl, cathepsin K, TRAP, RANK, and MMP9 in myeloid cells. The inventors further found that the downstream activation of PI3K signaling pathway (AKT) of OPG-mediated inhibition was similar to the DPSC co- cultured-mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.

[0077] Osteoclasts (OC) are multinucleated cells, differentiated from the monocyte/macrophage lineages under the influence of pathological stimulation that contribute to the bone resorption and remodeling. Bone remodeling is primarily regulated by interactions between bone-forming cells, osteoblasts (OB) and bone resorbing cells, OCs [1] A precise and orchestrated molecular communication among osteoblasts, osteoclasts, bone cells, and other bone marrow cells is necessary in regulating bone formation and resorption [2] Osteoblasts, stromal cells, and T cells express receptor activator for NF-icB-ligand (RANKL) and secrete M-CSF and TNF simultaneously. The binding of RANKL and M-CSF to RANK and CSF-1 receptor, respectively, induces osteoclastogenesis [3] The main switch for osteoclast-mediated bone resorption is the RANK-L, a cytokine that is released by activated osteoblasts. It’s action on the RANK receptor is regulated by osteoprotegerin, a decoy receptor that is also derived from osteoblasts [2] . RANK is highly expressed on the surface of osteoclast progenitors and mature osteoclasts, which translate osteoclastogenesis signals by binding to RANKL [4, 5] In pathological conditions, these essential signals for OC differentiation initiate from myeloid cells, specifically, inflammatory monocytes, macrophages, and dendritic cells [6, 7] In normal condition, OCs are essential for skeletal morphogenesis and restructuring. However, OC mediated excessive resorption of bone is evident during various pathological conditions, such as, arthritis, osteoporosis, and Paget's bone disease [2, 8] .

[0078] Inflammation is a cellular defense mechanism against foreign molecules that plays a critical role in maintaining cellular response. However, sustained inflammatory responses result in development of pathological condition such as rheumatoid arthritis (RA) that is often associated with bone and cartilage destruction. The presence of proinflammatory cells and cytokines in the synovium initiates the inflammatory process, and leads to the damage of cartilage and bone, resulting in deformity of the joints and compromised quality of life. The progressive nature of bone destruction is specifically involved activating osteoclasts (OCs) by interaction with synovial fibroblasts and immune cells [9, 10] Differentially activated macrophages play a key role in the pathophysiology of various types of cytoskeletal and other inflammation related diseases [11]. During initiation of inflammation, macrophages are activated and termed as proinflammatory Ml phenotype, which promote osteoclast differentiation and accelerate tissue damage by releasing high levels of pro-inflammatory cytokines, such as TNFa [9] In contrast, the anti-inflammatory macrophages are called as M2 phenotype, which counteract pro-inflammatory conditions by secreting anti-inflammatory cytokines, and scavenging cellular debris. The M2 macrophage can be induced by the cytokine interleukin-4 (IL-4) [12, 13] In general, Ml and M2 myeloid cells are involved in initiating and resolving inflammation respectively [14] Therefore, cell therapeutic strategy that modulates macrophage polarity may provide significant advantage by influencing uncontrolled osteoclast differentiation in diseases like osteoporosis and RA.

[0079] Osteoprotegerin (OPG) is a secretory glycoprotein of the TNF receptors superfamily, and is essential for osteoblast differentiation. OPG is produced in various tissues, such as, bone, skin, liver, lung, and stomach. OPG was initially found in human embryonic fibroblasts, and was termed as osteoclastogenesis inhibitory factor (OCIF) because of its role in inhibition of osteoclast differentiation [15] Treatment of OPG to the overectomized mice resulted in increased bone mineral density and bone volume with simultaneous reduction in number of active osteoclasts [16] OBs negatively regulate OC differentiation and function by producing OPG, a cytokine receptor of the tumor necrosis factor (TNF) receptor superfamily encoded by the TNFRSF11B gene. It was shown that OPG inhibits the OC differentiation and acts as a decoy receptor by sequestering RANKL [17] RANK mediated signaling pathways are also involved in TNF receptor- associated factors (TRAFs) and lead to activation of nuclear factor KB (NF-KB), c-Jun N-terminal kinase (JNK), P38, Src and AKT pathways [18] The activation and regulation of these signaling molecules are evident in osteoclast differentiation and resorptive activity.

[0080] Dental pulp-derived stem cells (DPSCs) are mesenchymal in nature, retain self-renewal and mutipotential capacity, and have potential for tissue regeneration [19, 20] These cells have been shown to maintain their“sternness” without changes in either morphology or expression of stem cell markers over long period of time in culture [21] This multipotency, especially the ability to differentiate towards the osteogenic lineages, is attractive for development of cell-based therapeutics for bone and cartilage in various pathological disorders, including RA, osteoarthritis, and osteoporosis [22, 23] In addition, DPSCs also have been shown to possess immunosuppressive effects [24], suggesting the potential use of human DPSCs for inflammatory and autoimmune diseases. Furthermore, DPSCs secrete a variety of growth factors and cytokines that employ paracrine activities on various cells for their functionality and differentiation [25] [0081] Current investigation focuses on elucidating underlying mechanisms by which DPSCs exert its inhibitory effects on myeloid cell differentiation into osteoclasts, which are responsible for bone resorption in various disorders of bones and cartilages.

[0082] Materials and Methods. Reagents and antibodies. Monocytic/macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (ATCC^® TIB-71). Soluble (s) RANKL (315-11-100 UG) and M-CSF (315-02-100 UG) were obtained from Pepro Tech Incorporation. The tartrate-resistant acid phosphatase (TRAP) assay kit (387A-1KT) was obtained from Sigma- Aldrich Corporation. DAPI (D1306) was purchased from Invitrogen Corporation. Antibodies for NFAT2/NFATcl (8032) and GAPDH (2118S) were purchased from Cell Signaling Technology. Antibody for osteoprotegerin (sc-390518) was purchased from Santa Cruz Biotechnology. The cathepsin K (abl88604), TRAP (abl85716) and MMP9 (ab38898) antibodies were purchased from Abeam.

[0083] Cell culture. Human dental pulp derived stem cells (DPSC) were isolated from discarded third molar teeth, which were obtained after surgical extraction from a healthy adolescent donor in clinic with prior approval from the Institutional Review Board (IRB), TTUHSC, Amarillo and consent from donors and parents as applicable. Teeth were thoroughly washed (at least 3 times) with phosphate buffered saline (PBS) containing 100 U/ml of penicillin, 100 pg/ml streptomycin and 2.5 pg/ml of amphotericin B (antibiotic- antimycotic solution, 15240062; Gibco, Thermo Fisher, Waltham, MA). Teeth were cut open to harvest the pulp, which was then minced into approximately 1 mm cubes and plated onto 60 mm cell culture plates and cultured with alpha (a) modified eagle medium (MEM, Gibco) supplemented with 20% FBS (Hyclone, Thermo Fisher Scientific, USA), 2 mM glutamate (Gibco) and 100 U/ml of penicillin, 100 pg/ml streptomycin and 2.5 pg/ml of amphotericin B (all from Gibco). Fresh medium was added every third day of culture after removing the old medium. Cells that migrated from the pulp tissues and became confluent were collected by dissociation by scraping or non-enzymatic dissociation buffer (Cellgro, 25056C1), and were re cultured as passage 1. Cell viability was determined using the trypan blue exclusion method. Experiments were performed using cells between 3 and 10 passages. RAW 264.7 cells were cultured and grown in DMEM containing 10% heat inactivated FBS and antibiotics (100 U/ml of penicillin and 100 pg/ml streptomycin) at 37°C in a 5% CO2 atmosphere according to the standard protocol and used for various experiments.

[0084] Osteoclast differentiation. RAW 264.7 cells were cultured at a density of 2^c 10⁵ cells/well in 6-well plates in DMEM containing 10% FBS and antibiotics (100 U/ml of penicillin and 100 pg/ml streptomycin) in the presence of 50 ng/ml recombinant mouse sRANKL and 20 ng/ml recombinant mouse macrophage colony stimulating factor (M-CSF/CSF1). M-CSF and sRANKL was replenished along with medium once every two days for 6 days. Cultures were maintained at 37°C in a humidified 5% C0₂ atmosphere. On day 6, culture medium was collected for the ELISA assay, and cells were either harvested for total RNA or protein extraction for qPCR and western blot (WB) assays respectively, or fixed for TRAP and immunofluorescence staining.

[0085] Osteoclast differentiation in presence of OPG. Recombinant mouse OPG (TNFRSFl lB-Fc) was purchased from BioLegend (552602, CA, USA). Various concentrations of recombinant OPG (5, 25, 50 and 100 ng/ml) were added to the M-CSF and sRANKL containing DMEM medium to evaluate the effects of OPG on osteoclast differentiation. Above-mentioned concentrations of OPG were added while changing the M-CSF and sRANKL containing DMEM medium to the respective wells. On day 6, cells were either harvested for protein extraction for WB assays or fixed for TRAP staining. [0086] Osteoclast differentiation in presence of PI3K inhibitor. PI3K inhibitor (LY294002) was purchased from Cayman Chemical (MI, USA). Various concentrations of LY294002 (1.5, 7.5 and 15 mM) and 0.01% DMSO (vehicle control) was added to the osteoclast differentiation medium containing M-CSF and sRANKL during the process of osteoclast differentiation. Above-mentioned concentrations of LY294002 and 0.01% DMSO were added while changing the osteoclast differentiation medium to the respective wells. On day 6, cells were fixed for TRAP staining.

[0087] Contact-free coculture assays. A coculture assay with RAW 264.7 and DPSCs was performed to evaluate the effects of DPSCs on osteoclast differentiation. RAW 264.7 cells (2.5 x lO⁵) in 10% DMEM were seeded in the lower wells and human DPSCs (2.5 x 10⁴) or (2.5 x 10³) in the upper chamber of 6-well Trans well plates (Coming) with 0.4 pm diameter of pore size. After 16-18 hours of seeding, upper chambers were placed on the respective co-culture wells and simultaneously adding 20 ng/ml M-CSF and 50 ng/ml sRANKL-containing medium to the respective wells. Anti-human OPG monoclonal antibody (1 pg) or isotype control IgGl (sc390518, from SantaCmz, and 5415 from Cell Signaling) was added to the respective wells along with osteoclast differentiating medium. On day 6, culture medium was collected for the ELISA assay, and cells were either harvested for RNA or protein extraction for qPCR and WB assays respectively or fixed for TRAP and immunofluorescence staining.

[0088] RNA extraction and real time PCR analysis. Total RNA was isolated from RAW 264.7 cell cultured alone, or in presence 20 ng/ml M-CSF and 50 ng/ml sRANKL containing DMEM medium, or from DPSC coculture for 6 days with RAW 264.7 cell in a trans-well system, or cocultured with DPSC in 10% DMEM medium at 4, 24 and 48 h of culture, or human DPSCs cultured in 10% and 1% FBS containing DMEM medium for 48 h and 72 h, using TRIzol reagent (Invitrogen Corporation, 15596026). One microgram of RNA was used for synthesis of cDNA using high capacity RNA to cDNA kit according to the manufacturers’ protocols (4387406; Applied Biosystems, Thermo Fisher Scientific). Real-time PCR amplification reactions were performed with the SYBR Green PCR Kit (Applied Biosystem, 4309155). The relative expression of each target gene was quantified by calculating Ct (threshold cycle) values and normalized by b-Actin levels. Each sample was analyzed in triplicate. Human primer denoted as (h). Primer sets purchased from Sigma Aldrich Corporation and Integrated DNA technologies were used in accordance to the manufacturer’s instructions: b-Actin 5’-GGCACCACACCTTCTACAATG-3’ (forward) (SED ID NO: l), 5’- GGGTGTTGAAGGTCTCAAAC-3’ (reverse) (SED ID NO:2); Ctsk 5’-

ATGTGAACCATGCAGTGTTGGTGG-3’ (forward) (SED ID NO:3); 5’- ATGCCGCAGGCGTTGTTCTTATTC-3’ (reverse) (SED ID NO:4); Nfatcl 5’- AGATGGTGCTGTCTGGCCATAACT-3’ (forward) (SED ID NO:5); 5’- TGCGGAAAGGTGGTATCTCAACAA-3’ (reverse) (SED ID NO:6); Trap 5’- GCCTTGTCAAGAACTTGCGACCAT-3’ (forward) (SED ID NO:7); 5’- TTCGTTGATGTCGCACAGAGGGAT-3’ (reverse) (SED ID NO: 8); Rank 5’- TAGGACGTCAGGCCAAAGGACAAA-3 (forward) (SED ID NO:9); 5’- AGGGCCTACTGCCTAAGTGTGTTT-3’ (reverse) (SED ID NO: 10); Mmp9 5’- TGAACAAGGTGGACCATGAGGTGA-3 (forward) (SED ID NO: 11); 5’- TAGAGACTTGCACTGCACGGTTGA-3’ (reverse) (SED ID NO: 12); Tnf-a 5’- ATTCACTGGAGCCTCGAA-3’ (SED ID NO: 13) (forward); 5’ -TGCACCTCAGGGAAGAATCTGGAA - 3’ (reverse) (SED ID NO: 14); IL-4Ra 5 -CAAGCTCTGACCTCTGGATTA-3’ (forward) (SED ID NO: 15); 5’- AATGATGGGAGCGGGTATAAG-3’ (reverse) (SED ID NO: 16); Argmase l 5’-

CCAGGGACTGACTACCTTAAAC-3’ (forward) (SED ID NO: 17); 5’ -GAAGGCGTTTGCTTAGTTCTG - 3’ (reverse) (SED ID NO: 18); Yml 5’ -GCTAAGGACAGGCCAATAGAA-3’ (forward) (SED ID NO: 19); 5’-GCATTCCAGCAAAGGCATAG-3’ (reverse) (SED ID NO:20); OPG 5’-

GCCGAGAGTGTAGAGAGGATAA-3’ (forward) (SED ID NO:21); 5’-

CTTCACCATTTCCTGGTCTCTG-3’ (reverse) (SED ID NO: 22); hOPG 5’-

CATTCTTCAGGTTTGCTGTTCC-3’ (forward); (SED ID NO:23) 5’ -CTCTCTACACTCTCTGCGTTTAC- 3’ (reverse) (SED ID N0 24); hGAPDH 5’-CCCTTCATTGACCTCAACTACA-3’ (forward) (SED ID NO:25); 5’ -ATGACAAGCTTCCCGTTCTC-3’ (reverse) (SED ID NO:26).

[0089] TRAP staining. Differentiated osteoclasts were determined by tartrate-resistant acid phosphatase (TRAP) Assay Kit (387A-1KT, Sigma- Aldrich) following the manufacturer's protocol. Briefly, monocytes were cultured on the cover slips in a 6-well plate for differentiation into osteoclasts in presence or absence of DPSC, or OPG or LY294002. On day 6 of the culture, coverslip was removed from plate and cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and then washed with PBS. Next, mixture of solution was prepared by using sodium nitrite, Fast Garnet GBC base solution, acetate solution, naphthol AS-BI phosphate solution, tartrate solution and deionized water (pre-warmed to 37°C) according to the manufacturer’s protocol. This solution was added to each of the coverslips and incubated for 1 h at 37°C in water bath protected from light. Finally, the cover slips were rinsed with deionized water thoroughly, mounted on a glass slide, and examined under a light microscope, (Olympus Corporation of the Americas, Waltham, MA, ix8 l). TRAP-positive cells (purple) containing at least three nuclei were counted as an osteoclast cell.

[0090] Western blot analysis. Western blot was performed to determine the levels of NFATcl, MMP9, cathepsin K, OPG, pAKT, AKT keeping GAPDH as an internal control in RAW 264.7 cells during the course of differentiation in presence or absence of various concentration of DPSCs or OPG. The cells were lysed in 100 mΐ pre-cooled RIPA lysis buffer (Millipore, Sigma Aldrich Corporation, 20-188) for 30 min on ice and centrifuged at 12,000 g for 10 min. The supernatant was collected, and protein concentrations were estimated with Bradford's reagent (Bio-Rad Incorporation, 5000006) using bovine serum albumin (BSA) (Sigma Aldrich Corporation, A7906-10G) as a standard. Equal amounts of protein (40 pg) were separated by SDS-PAGE gels electrophoretically and transferred to polyvinylidene difluoride membrane (Bio-Rad Incorporation, 1620115). After blocking with 5% BSA for 1 h at room temperature, the membranes were probed with primary antibodies for 12-16 h at 4°C. Then membranes were incubated with appropriate HRP (horseradish peroxidase)-labeled secondary antibodies (Cell Signaling Technology, 7074, and 7076) for 2 h at room temperature. Immunoreactive protein bands were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotechnology, RPN2232), and the band detections were kept within the linear range.

[0091] Immunofluorescence staining. To determine the protein expressions after osteoclast differentiation in presence or absence of DPSCs, immunofluorescence analysis was performed for NFATcl, MMP9, cathepsin K and TRAP proteins. In brief, RAW 264.7 cells were grown on sterile coverslips inserted into a 6- well plate. After 18 h of culture, cells were induced to osteoclast differentiation with MCSF and sRANKL in presence or absence of DPSC in a trans-well culture system. After 6 days of differentiation, cells were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology, sc-281692) for 30 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma Aldrich Corporation, T8787) and blocked with 1% BSA. Then, cells were incubated with 200 m\ of primary antibody (1:200) overnight at 4°C. After washing with PBS, cells were incubated with 200 m\ of secondary anti-rabbit or anti-mouse antibodies (Alexa Fluor 488, A11001 or Alexa Fluor 594, A21235; 1 :2000 dilution; Invitrogen Corporation) for 45 min in the dark. After incubation, cells were washed thrice with PBS (GIBCO, Thermo Fisher Scientific, 70013-032) and mounted with 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen Corporation, D1306) on glass slides and sealed with transparent nail varnish. Slides were viewed under fluorescence microscope and images were captured digitally using an Olympus ix81 microscope with Slidebook 5.0x64 software.

[0092] ELISA assays. The levels of secreted OPG were determined using human OPG enzyme-linked immunosorbent assay (ELISA) kit in accordance with the manufacturer’s recommended protocols (ELH- OPG-1, Ray Biotech, USA). To quantify OPG secretion from DPSCs, medium was collected from different experimental conditions, such as, DPSC cultured in 10% FBS in DMEM and 1% FBS in DMEM for 48 h or 72 h; DPSCs cultured in 1 x PBS for 3, 6, or 24 h; DPSC cultured in M-CSF and sRANKL containing DMEM medium; medium collected at various time point of osteoclast differentiation in presence or absence of DPSCs. Whereas 10% DMEM, 1% DMEM, 1 x PBS medium only without cells were used as a control in respective experiments as applicable.

[0093] Statistical analysis. All experiments were performed at least 3 times in triplicate, and the results were displayed as mean ± SEM. Statistical significance was ascertained by Student’s t-test, and p values less than 0.05 were considered significant.

[0094] Effect of DPSCs on osteoclast differentiation of myeloid cells. To investigate the effect of DPSCs on osteoclast differentiation, the osteoclast precursor cells (RAW 264.7) were cultured with or without DPSCs under contact-free co-culture system. Simultaneously, cell culture medium was supplemented with M-CSF and sRANKL to induce osteoclast differentiation. After 6 days of induction, TRAP staining was performed to evaluate the morphology of osteoclast cells. The morphology of OCs was confirmed by multinucleated TRAP positive cells observed under brightfield microscope. TRAP staining revealed that multinucleated osteoclast cells were observed in cell culture plates that were stimulated with M-CSF and sRANKL, labeled as OC (Fig. 8A). Whereas, RAW 264.7 cells cultured without M-CSF and sRANKL, labeled as control (monocytes) did not show any multinucleated TRAP positive cells (Fig. 8A). However, the abundance of multinucleated TRAP positive cells at the same time point were markedly decreased in presence of DPSCs, which were cultured under the same osteoclast stimulants, M-CSF and sRANKL (Fig. 8A). A dose-dependent reduction of the abundance of osteoclast cells was observed when cultured at 1: 10 ratio of DPSC:RAW 264.7 cells that inhibited more effectively than that of 1 : 100 ratio of DPSC:RAW 264.7 cells. The number of TRAP positive multinucleated cells were found significantly lower in RAW 264.7 cells that were differentiated in presence of DPSCs in contact-free co-culture condition compared to RAW 264.7 cells that were differentiated in the absence of DPSC (Fig. 8B). These observations indicate that the DPSCs are capable of inhibiting osteoclast differentiation.

[0095] Effect of DPSCs on the expressions of osteoclast related marker molecules. To further validate the effect of DPSCs on osteoclast differentiation, the mRNA expressions of various osteoclast-related marker genes in RAW 264.7 cells were evaluated after 6 days of induced differentiation in presence or absence of contact-free DPSCs. Quantitative PCR analysis revealed that key osteoclast related genes, such as, NFATcl (Nfatcl), cathepsin K (Ctsk), Rank, Trap, MMP9 (Mmp9) were significantly up-regulated in differentiated osteoclast cells compared to undifferentiated cells (Fig. 8C). These results confirm that the successful differentiation of RAW 264.7 cells into osteoclasts. However, mRNA expressions of osteoclast-related marker genes, such as Nfatcl, Ctsk, Rank, Trap, and Mmp9, were significantly decreased in differentiated osteoclast cells in presence of DPSCs in a dose dependent manner, compared to respective differentiated cells (without DPSC, Fig. 8C). To confirm the translation of the gene expressions to proteins, western blot analysis was also performed from isolated total proteins after 6 days of differentiation. Western blot analysis revealed that protein levels of NFATcl, cathepsin K, MMP9, and p65 were significantly increased in osteoclast differentiated cells compared to undifferentiated cells. However, protein levels were markedly decreased when induced differentiation was performed in the presence of DPSCs in a dose-dependent manner (Fig. 8D). Further, immunocytochemical staining confirmed that NFATcl, cathepsin K, MMP9, and TRAP positive multinucleated osteoclasts were markedly increased in differentiated cells compared to monocytes, whereas the same marker positive multinucleated cells were decreased in the presence of DPSCs (Fig. 9).

[0096] Effect of DPSCs on expressions of pro-inflammatory and anti-inflammatory genes in myeloid cells. DPSCs play immune modulatory role, and may further regulate the osteoclast differentiation process. Hence, the inventors tested whether DPSCs have any immune modulatory role in myeloid cells. To test that, the inventors performed contact-free co-culture of RAW 264.7 cells in presence or absence of DPSCs. Quantitative RT-PCR analysis of RNA harvested from RAW 264.7 cells at various time points showed that the expressions of TNF-a, one of the inflammatory genes was significantly downregulated in presence of DPSCs compared to control (Fig. 10A). Conversely, mRNA expressions of IL-4Ra, an anti-inflammatory gene was upregulated in RAW 264.7 cells in presence of DPSCs compared to control (Fig. 10A).

[0097] In addition, Arginasel (Argl), Yml ( Chil3 ), which are characteristics of M2 phenotype of myeloid cells, were significantly increased in RAW 264.7 cells in presence of DPSCs compared to controls (Fig. 10B). These data suggest that DPSCs may have the ability to polarize macrophages towards the anti inflammatory phenotype, which negatively regulate the osteoclast differentiation.

[0098] Effect of DPSCs on osteoprotegerin (OPG) expressions and secretions. To further understand the mechanism by which DPSCs are regulating myeloid cell differentiation, the gene and protein expressions of osteoclast inhibitory factor, osteoprotegerin (OPG) in RAW 264.7 were evaluated cells after 6 days of induced differentiation in the presence or absence of DPSC in contact-free co-culture condition. Quantitative PCR analysis showed that OPG m-RNA was significantly decreased in RAW 264.7 cells after 6 days of induced osteoclast differentiation compared to RAW 264.7 cells. However, when RAW 264.7 cells were induced to differentiate into osteoclasts in presence of DPSCs, mRNA expression of OPG was significantly increased in a dose -dependent manner (Fig. 11A). This observation was further confirmed by western blot studies using isolated proteins from myeloid cells from similar experimental designs. It was found that OPG protein expressions were increased in RAW 264.7 cells in presence of contact-free DPSC co-cultures compared to differentiated osteoclasts (Fig. 11B). Next, the inventors determined which cells secrete OPG during OC differentiation using a trans-well coculture system. Culture supernatants were measured by ELISA assays, and found that there was no detectable range of OPG in supernatants collected from monocytes or differentiated osteoclasts. However, the level of OPG was significantly higher in cultures where DPSC was added, and remained higher levels through out the differentiation process (Figs. 11C-E). These results indicate that myeloid cells might not be secreting OPGs.

[0099] DPSC’s ability to express and secrete OPG. To determine whether DPSC can express and secrete constitutively DPSCs were cultured under various culture conditions. In a serum-starvation condition (1% FBS containing DMEM) cells were harvested and supernatants were collected after 48 and 72 h of culture. The mRNA expression of OPG revealed that serum starvation significantly induced OPG expression in DPSCs in both time points compared to DPSCs cultured with 10% FBS containing medium, considered as control (Fig. 12A). The amount of secreted OPG in supernatants was measured and found that significantly higher levels of OPG after serum starvation (Fig. 12B). To investigate whether DPSC secrete OPG in 10% FBS containing medium, the inventors found that DPSC indeed constitutively secreted OPG in complete medium (Fig. 12C). The inventors further investigated whether DPSC can secrete OPG in compromised environment using PBS and osteoclast differentiation medium. Results indicate that in both conditions DPSCs secreted OPG (Figs. 12D & 12E), however, the levels of OPG were lower in PBS compared to other condition, possibly due to the decreased cellular survivability in PBS. These observations indicate that DPSCs can secrete OPG constitutively and under stressed conditions, and culture medium did not have significant effect on OPG secretion.

[00100] Effect of OPG on osteoclast differentiation. To investigate the effects of OPG on osteoclast differentiation-related molecules, the inventors induced osteoclast differentiation of RAW 264.7 cells in the absence or presence of various concentrations of recombinant OPG, and real-time RT-PCR and western blot analyses were performed. Quantitative RT-PCR analysis revealed that key osteoclast related genes, such as, Nfatcl, Ctsk, Rank, Trap, and Mmp9 were significantly reduced in presence of OPG compared to without treatment after 6 days of osteoclast differentiation (Fig. 13A). Further, western blot analysis confirmed that key osteoclast related proteins NFATcl, cathepsin K, and MMP9 levels were decreased in presence of OPG compared to differentiated osteoclasts (Fig. 13B). These observations support the ability of OPG in inhibiting osteoclast differentiation related marker molecules.

[0100] DPSCs in mimicking OPG-mediated signaling pathways of osteoclast differentiation. To determine the molecular mechanism of DPSC-mediated inhibition of osteoclast differentiation of myeloid cells, RAW 264.7 cells were induced to differentiate in absence or presence of various concentrations of recombinant OPG, or PI3K inhibitors, or DPSCs. First, TRAP staining was performed after 6 days of differentiation, and revealed that a concentration-dependent effects of OPG in inhibition of osteoclast differentiation. Higher concentrations (50 ng/ml and 100 ng/ml) of OPG significantly inhibited the differentiation of osteoclasts, where as, lower concentrations (5 ng/ml and 25 ng/ml) of OPG was not much effective in reducing osteoclast differentiation (Figs. 14A & 14B).

[0101] To identify the regulation of key signaling molecules involved in osteoclast differentiation, total proteins were isolated after six days from RAW 264.7 cells induced for OC differentiation in presence or absence DPSC or OPG as described earlier and subjected to western blot analysis. Western blot analysis showed that activated AKT(phosphorylated AKT) was upregulated in the osteoclasts, however, addition of OPG inhibited the activation of AKT in a concentration-dependent manner (Fig. 14C). Inhibition of AKT activation (phosphorylation) was also observed in presence of DPSCs in a contact-free co-culture (Fig. 14D). However, total AKT level was mostly unaltered. There was no significant difference in MAP kinase molecules (P38, ERK1/2) tested in these samples (data not shown). These data indicated that DPSC-mediated inhibition of osteoclast differentiation mimicked the signaling cascade of OPG-mediated inhibition of osteoclast differentiation. The inventors further confirmed the involvement of PI3K-AKT pathways during osteoclast differentiation by using PI3K specific inhibitor. The inventors found that PI3K inhibitor significantly reduced osteoclast differentiation in a dose-dependent manner (Figs. 14E & 14F).

[0102] To determine whether DPSC-mediated inhibition of osteoclast differentiation of myeloid cells was through OPG, the inventors induced osteoclast differentiation in the absence or presence of DPSC or DPSC plus anti-OPG Ab in a trans-well culture system. TRAP staining revealed that presence of DPSC reduced osteoclast differentiation and addition of anti-OPG Ab in the culture along with DPSC partially restored the osteoclast differentiation (Figs. 14G & 14H). These results confirmed that OPG-mediated inhibition of osteoclast differentiation by DPSC in part.

[0103] Indiscriminate osteoclast differentiation of myeloid cells play critical role in various bone related diseases such as arthritis, osteoporosis and Paget's bone disease [2, 8], hence it is important to limit uncontrolled osteoclast differentiation to prevent excessive bone resorption in bone-related diseases. Bone- related diseases are often associated with compromised life style, which causes significant health and socioeconomic burdens. Even though, various pharmacological agents temporarily suppress joint erosion, regeneration of the deformed bones is quite limited and persists as a lifelong disability. Regenerative cell therapeutic approach would be a novel and alternative strategy to treat arthritis and osteoporosis [26-29] .

[0104] To investigate potential use of adult stem cells, the inventors isolated and expanded DPSCs from the third molar teeth using standard procedures following our earlier established protocol [22] These cells are mesenchymal in nature and express the cell surface markers CD73, CD90, and CD105, but do not express markers (CD34, CD133) of hematopoietic lineage cells and retain their ability to differentiate into multiple lineages including osteoblastic lineage [22] . Given the inherent multipotency and immunomodulatory effects of DPSCs [24], the inventors considered that these cells could be a new source for treating bone-related diseases. As osteoclasts play an important role in bone resorption and joint destruction in bone-related diseases, the inventors sought to investigate the possible effect of DPSC treatment on osteoclastogenesis because it is still remained unclear how DPSCs affect on myeloid cells. DPSCs are shown to exert trophic effects on various cells, and is considered to be a potential therapeutic avenue for a number of musculoskeletal and autoimmune degenerative diseases [30]

[0105] The inventors focused on generating osteoclasts using established murine myeloid cells, RAW 264.7 cells with M-CSF and sRANKL for osteoclast differentiation using earlier documented methods [31, 32] After 6 days of differentiation, the inventors observed multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells confirming the ability to generate osteoclasts. TRAP is a widely used osteoclast marker, which is known to be localized in the transcytolytic vesicles of resorbing osteoclasts that destroy collagen by producing reactive oxygen species [33] Interestingly, when the inventors co-cultured DPSCs with RAW 264.7 cells, a significant reduction in osteoclast differentiation was observed, suggesting that DPSC has the ability to inhibit osteoclast differentiation process. Next, the inventors wanted to know whether DPSC has any effect on osteoclast-related molecules such as NFATcl, cathepsin K, RANK, TRAP and MMP9, which are critically involved in osteoclast maturation and resorption process. Specifically, NFATcl is a transcription factor and a master regulator of RANKL-induced osteoclast differentiation [34] Osteoclast specific genes, such as, TRAP [35] and cathepsin K [36] are directly regulated by NFATcl, indicating the significance of the NFATcl in osteoclastogenesis. The induction and activation of NFATcl integrate RANKL signaling in terminal differentiation of osteoclasts [35] In addition, high levels of both the cathepsin K and MMP-9 (gelatinase B) expressions in osteoclast play central role in the bone resorption process [37- 39] The inventors found that inhibition of osteoclast differentiation was associated with the reduction in expression of NFATcl, TRAP, cathepsin K and MMP9 after co-culture with DPSCs. This result confirmed that the DPSC s ability to suppress the terminal differentiation of osteoclast precursors to mature osteoclasts, which is in consistent with the previous related findings [40]

[0106] Next, the inventors sought to find the mechanisms by which DPSCs inhibit osteoclast differentiation. These data shows that DPSC-mediated inhibition of osteoclast differentiation through at least two different mechanisms. DPSCs are known to contribute tissue regeneration in a paracrine fashion by secreting various factors [25] The inventors found that DPSCs markedly suppressed osteoclast differentiation by the production of OPG, along with induction of OPG in myeloid cells. Osteoprotegerin (OPG) is known to be an inhibitory molecule for RANKL-dependent osteoclast differentiation and function [41] and RANKL neutralization improved bone resorption in osteoporosis and rheumatoid arthritis [27-29] In addition, it was shown that OPG-deficiency exhibited severe osteoporosis in mice due to excessive bone resorption by osteoclasts [42, 43] Our findings demonstrated that one of the possible mechanisms of DPSC-mediated inhibition of osteoclastogenesis was through secretion of OPG. The inventors further determined that the addition of recombinant OPG inhibited induced osteoclast differentiation of myeloid cells.

[0107] Sustained inflammatory responses result in developing chronic inflammatory diseases like rheumatoid arthritis that are often associated with cartilage and bone destruction [9, 10] Inflammatory cytokines that are abundant in the synovial fluid and synovium of arthritic patients induce RANKL expression on synovial fibroblasts resulting in increased RANKL signaling [9, 10] Osteoclastogenesis process could be controlled by limiting inflammatory responses in the arthritic synovium. Our data revealed that co-culture of DPSCs with osteoclast precursor cells lead to reducing inflammatory molecular expression in myeloid cells and polarization of M2 phenotype-associated molecules in the myeloid cells. These findings are in alignment with the others showing strong immunosuppressive functions of MSCs and DPSCs caused by soluble mediators such as anti-inflammatory cytokines [44], and based on this intriguing property, the mesenchymal nature of DPSCs might have potential for therapeutic application in bone-related diseases.

[0108] Signaling mechanism of osteoclast differentiation is well elucidated. M-CSF and RANKL stimulation leads to osteoclast differentiation of precursor cells. M-CSF is known to stimulate cell survival signaling mainly by activating extracellular signal regulated kinase (ERK) through growth factor receptor bound protein 2 (Grb-2) and thymoma viral proto-oncogene 1 (popularly called as AKT) through phosphatidylinositol 3-kinase (PI3K) pathway [45] Similarly, RANKL stimulation leads to the formation of the RANK-TRAF6 complex, that leads to the activation of the AKT, NF-kB, and MAPK pathways, including c-jun N-terminal kinase (JNK) and p38 [18, 45] However, the signaling cascade that DPSCs or mesenchymal stem cells influence in regulating osteoclast differentiation is not well established. Here, the inventors demonstrate that the secretory product of DPSC was able to suppress activation of AKT and further inhibit differentiation in presence M-CSF and RANKL during osteoclastogenesis possibly through secretion of OPG. To further confirm the role of OPG, the inventors show that AKT activation was inhibited by addition of recombinant OPG in presence of osteoclast stimulants M-CSF and RANKL.

[0109] It was found herein that human DPSCs inhibit osteoclastogenesis by reducing expression of key OC markers, such as, NFATcl, cathepsin-K, TRAP, RANK, and MMP-9 in myeloid cells through constitutive secretion of OPG. OPG potentially blocks the RANK-RANKL interaction, which is essential for differentiation of osteoclasts. Moreover, DPSCs reduced inflammatory signals of myeloid cells and polarized myeloid cells towards M2 phenotype resulting inhibition of osteoclast differentiation. The inventors show that OPG-mediated inhibition of the activation of PI3K signaling pathway (AKT) during OC differentiation was similar to the DPSC mediated inhibition of OC differentiation. The possibility of OPG-mediated inhibition of OC differentiation by DPSCs was confirmed by rescue experiment using anti-OPG antibody. This study shows the DPSC-mediated inhibition of osteoclastogenesis.

[0110] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated herein by reference in their entirety, to the extent that they provide exemplary, procedural, or other details supplementary to those set forth herein.

[0111] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0112] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0113] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0114] The use of the word“a” or“an” when used in con_junction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0115] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“includes” and“include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with“consisting essentially of’ or“consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term“consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.

[0116] The term“or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example,“A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0117] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as“about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0118] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. [0119] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words“means for” or“step for” are explicitly used in the particular claim.

[0120] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

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Claims

What is claimed is:

1. A method of making a differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, comprising:

growing dental pulp-derived stem cells (DPSC) in a growth media; and

treating the DPSC with:

a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell;

a chondrogenic medium to differentiate the DPSC into the chondrogenic cell; or an adipogenic induction medium to differentiate the DPSC into the adipogenic cell.

2. The method of claim 1, wherein the DSPCs are differentiated into an osteogenic cell with the phytoestrogen Ferutinin.

3. The method of claim 1, wherein the chondrogenic medium comprises modified Eagle’s media FI 2, 1- glutamine, fetal bovine serum, 1-proline, ascorbic acid, sodium pymvate, insulin, transferrin, selenium, antibiotics, and dexamethasone.

4. The method of claim 1, wherein the adipogenic induction medium comprises Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum, insulin, and at least one of: dexamethasone, indomethacin, M3- isobutyl-l-methylxanthin, or pioglitazone.

5. The method of claim 1, further comprising formulating the osteogenic, chondrogenic or adipogenic cells into a transplant.

6. The method of claim 1, further comprising expanding the number of the osteogenic, chondrogenic or adipogenic cells for transplantation.

7. The method of claim 1, further comprising delivering the osteogenic, chondrogenic or adipogenic cells to a subject.

8. The method of claim 1, wherein the DPSC are obtained by a method comprising:

obtaining a molar;

extracting from the molar the pulp;

mincing the pulp;

growing the cells in the pulp in a cell culture media; and

isolating the cells that migrate from the pulp, wherein the cells are the pulp-derived stem cells.

9. The method of claim 1, wherein the DPSC are grown and a supernatant is obtained from the cells, and the supernatant is contacted with monocytes to induce M2 polarization of the monocytes.

10. The method of claim 1, wherein the DPSC are provided to a subject to treat an ischemic stroke.

11. The method of claim 1, wherein the DPSC are provided to a subject reduce an inflammation associated with an inflammatory bowel disease.

12. A method of transplantation of a differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, comprising:

identifying a subject in need of transplantation with an osteogenic, chondrogenic, or adipogenic cell; growing dental pulp-derived stem cells (DPSC) in a growth media; and

treating the DPSC with:

a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell;

a chondrogenic medium to differentiate the DPSC into the chondrogenic cell; or an adipogenic induction medium to differentiate the DPSC into the adipogenic cell; and transplanting the osteogenic, a chondrogenic or adipogenic cell into the subject.

13. The method of claim 12, wherein the DSPCs are differentiated into an osteogenic cell with the phytoestrogen Ferutinin.

14. The method of claim 12, wherein the chondrogenic medium comprises modified Eagle’s media F12, L-glutamine, fetal bovine serum, L-proline, ascorbic acid, sodium pymvate, insulin, transferrin, selenium, antibiotics, and dexamethasone.

15. The method of claim 12, wherein the adipogenic induction medium comprises Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum, insulin, and at least one of: dexamethasone, indomethacin, M3- isobutyl-l-methylxanthin, or pioglitazone.

16. The method of claim 12, further comprising expanding the number of the osteogenic, chondrogenic or adipogenic cells prior to transplantation.

17. The method of claim 12, wherein the DPSC are obtained by a method comprising:

obtaining a molar;

extracting from the molar the pulp;

mincing the pulp;

growing the cells in the pulp in a cell culture media; and

isolating the cells that migrate from the pulp, wherein the cells are the pulp-derived stem cells.

18. The method of claim 17, wherein the molar is syngeneic, allogeneic, or xenogeneic.

19. The method of claim 12, wherein the DPSC are grown and a supernatant is obtained from the cells, and the supernatant is contacted with monocytes to induce M2 polarization of the monocytes.

20. The method of claim 12, wherein the DPSC are provided to a subject to treat osteoporosis.

21. The method of claim 12, wherein the DPSC are provided to a subject to treat an ischemic stroke.

22. The method of claim 12, wherein the DPSC are provided to a subject reduce an inflammation associated with an inflammatory bowel disease.

23. A differentiated cell selected from an osteogenic, a chondrogenic or adipogenic cell, made by a method comprising: growing dental pulp-derived stem cells (DPSC) in a growth media; and

treating the DPSC with:

a phytoestrogen provided in an amount sufficient to differentiate the DPSC into the osteogenic cell; a chondrogenic medium to differentiate the DPSC into the chondrogenic cell; or

an adipogenic induction medium to differentiate the DPSC into the adipogenic cell.