MATERIALS AND METHODS FOR ENHANCING BONE CELL DIFFERENTIATION

Abstract

This document provides materials and methods for enhancing bone cell differentiation. For example, compositions containing an epigenetic drug, an actin modulator, and/or one or more osteogenic differentiation factors that can be used to enhance bone cell differentiation are provided.

Claims

1. A composition comprising an epigenetic drug, an actin modulator, and one or more osteogenic differentiation factors.

2. The composition of claim 1, wherein the epigenetic drug inhibits enhancer of zeste homolog 2 (EZH2) polypeptide activity.

3. The composition of claim 2, wherein the epigenetic drug is GSK126.

4. The composition of claim 1, wherein the actin modulator inhibits actin polymerization.

5. The composition of claim 4, wherein the actin modulator is cytochalasin D (CytoD).

6. The composition of claim 1, wherein the one or more osteogenic differentiation factors are pro-osteogenic differentiation factors.

7. The composition of claim 6, wherein the one or more osteogenic differentiation factors are selected from the group consisting of Wnt10b, a sclerostin antibody, BMP2, BMPS, teriparatide, and abaloparatide.

8. The composition of claim 1, wherein the epigenetic drug is GSK126, wherein the actin modulator is CytoD, and wherein the one or more osteogenic differentiation factors are selected from the group consisting of Wnt10b, a sclerostin antibody, BMP2, BMPS, teriparatide, and abaloparatide.

9. A composition comprising GSK126, cytochalasin D (CytoD), and one or more of Wnt10b, a sclerostin antibody, BMP2, BMPS, teriparatide, and abaloparatide.

10. A method for enhancing bone cell differentiation, the method comprising administering to a stem cell a composition comprising an epigenetic drug, an actin modulator, and one or more osteogenic differentiation factors, wherein the stem cell is differentiated into a bone cell.

11. The method of claim 10, wherein the stem cell is an adult stem cell.

12. The method of claim 11, wherein the adult stem cell is selected from the group consisting of a mesenchymal stem cell, an adipose-derived stem cell, and a bone marrow-derived stem cell.

13. The method of claim 12, wherein the adult stem cell is an adipose-derived stem cell.

14. The method of claim 10, wherein the bone cell is an osteocyte.

15. The method of claim 10, wherein the stem cell is from a mammal.

16. The method of claim 15, wherein the mammal is a human.

17. The method of claim 10, wherein the epigenetic drug is GSK126.

18. The method of claim 10, wherein the actin modulator is cytochalasin D (CytoD).

19. The method of claim 10, wherein the one or more osteogenic differentiation factors are selected from the group consisting of Wnt10b, a sclerostin antibody, BMP2, BMPS, teriparatide, and abaloparatide.

20. The method of claim 10, wherein the stem cell is in vivo.

21. The method of claim 10, wherein the stem cell is in vitro.

22. The method of claim 21, wherein the stem cell is on a substrate.

23. The method of claim 22, wherein the substrate comprises a bone-related extracellular matrix protein.

24. The method of claim 23, wherein the bone-related extracellular matrix protein is a collagen.

25. A method for increasing polypeptide expression in a stem cell, the method comprising contacting the stem cell with a composition comprising an epigenetic drug, an actin modulator, or one or more osteogenic differentiation factors, wherein expression of one or more osteogenic polypeptides is increased.

26. The method of claim 25, wherein the stem cell is an adult stem cell.

27. The method of claim 26, wherein the adult stem cell is selected from the group consisting of a mesenchymal stem cell, an adipose-derived stem cell, and a bone marrow-derived stem cell.

28. The method of claim 27, wherein the adult stem cell is an adipose-derived stem cell.

29. The method of claim 25, wherein the bone cell is an osteocyte.

30. The method of claim 25, wherein the stem cell is from a mammal.

31. The method of claim 30, wherein the mammal is a human.

32. The method of claim 25, wherein the composition comprises GSK126.

33. The method of claim 25, wherein the composition comprises cytochalasin D (CytoD).

34. The method of claim 25, wherein the composition comprises Wnt10b, a sclerostin antibody, BMP2, BMPS, teriparatide, or abaloparatide.

35. The method of claim 25, wherein the stem cell is in vivo.

36. The method of claim 25, wherein the stem cell is in vitro.

Description

EXAMPLES

Example 1

Modulation of the Histone H3K27 Methyltransferase EZH2 Stimulates WNT, PTH and BMP2-Related Paracrine Signaling to Promote Osteogenesis

[0027] Bone stimulatory therapeutics that promote bone formation include bone morphogenetic proteins (e.g., BMP2) and intermittent treatment with parathyroid hormone (PTH) or PTH related protein, as well as antibody-suppression of WNT inhibitors (e.g., SOST). Furthermore, inactivation of EZH2, an epigenetic regulator with histone 3 lysine 27 (H3K27) methyltransferase activity, using a pharmacological inhibitor (GSK126) is bone anabolic in skeletally mature mice and osteo-protective in estrogen-depleted (ovariectomized) mice. These biological effects are directly related to the ability of EZH2 inhibition to promote osteogenic differentiation and inhibit adipogenic differentiation of mesenchymal stem cells.

[0028] The molecular mechanisms by which EZH2 inhibition promotes osteogenic differentiation were assessed. Results from mRNAseq and ChIP-seq analyses suggested that EZH2 inhibition is anti-proliferative and generates a quiescent cellular state by upregulating the CDK inhibitory protein CDKN2A/p16 and downregulating expression of genes required for mitosis. This quiescent state is conducive for expression of bone-related extracellular matrix proteins (e.g., collagens) that support matrix mineralization. It was found that EZH2 inhibition modulates WNT, PTH and BMP signaling. Several Wnt ligands (e.g., Wnt10b, Wnt10a, and Wnt6) are robustly expressed in differentiating MC3T3 cells. Interestingly, the pro-osteogenic Wnt10b was greatly up-regulated by EZH2 inhibition. Similarly, the PTH receptor (Pthr1h) as also enhanced by GSK126 in preosteoblasts.

[0029] Western blotting analysis demonstrated that EZH2 inhibition enhanced Smad1/5 phosphorylation, a well-established biomarker for the activation of BMP2 signaling, in MC3T3 cells. Furthermore, EZH2 inhibition stimulated the expression of BMP2-responsive genes, including several genes involved in osteoblast differentiation (e.g., PTH1R, DLX5, SP7, and IBSP).

[0030] These results demonstrated that EZH2 controlled paracrine signaling in osteoblasts involving the WNT, PTH and BMP2 pathways to stimulate osteogenic differentiation, and suggested that inhibitors of EZH2, which include well-tolerated and orally available drugs, may be effective in stimulating bone acquisition by supporting the endogenous local activation of natural bone stimulatory ligands at physiological doses in bone.

Example 2

Cytochalasin D Improves the Osteogenic Potential of Human Adipose-Derived Mesenchymal Stem Cells Concomitant with Repression of EZH2 and Heterochromatin-Related H3K27me3 Marks

[0031] The effect of CytoD on adipose tissue-derived MSCs (AMSCs) was investigated. AMSCs offer several advantages over other sources of MSCs, particularly in the ease of tissue harvest, isolation and expansion to generate sufficient cell numbers for therapy. Because AMSCs have limited osteogenic potential, it is necessary to design molecular strategies to improve their ability to attain a mature osteoblastic phenotype. The depolymerization of the actin cytoskeleton with CytoD had marked effects to enhance osteogenic differentiation of AMSCs throughout the cell culture time-course, as reflected by significant increases in alkaline phosphatase activity and mineralization, as well as the expression of osteogenic polypeptides RUNX2, ALP, OPG and TGFβ3. RNA-seq analyses of both AMSCs and BMSCs in response to CytoD (24 hour) revealed significant upregulation of a program of other osteogenic markers, including those linked to the BMP2-RUNX2 axis (e.g., SP7). Furthermore, CytoD decreased protein levels of Enhancer of Zeste Homolog 2 (EZH2), an epigenetic suppressor of osteogenic differentiation that mediates heterochromatinization of bone-related genes by trimethylation of histone 3 lysine 27 (H3K27me3). This loss of EZH2 protein is reflected by decreased levels of H3K27me3 marks indicating a global reduction in heterochromatin.

[0032] These results demonstrated that actin polymerization is linked to epigenetic mechanisms that control the acquisition of the osteogenic phenotype in AMSCs, and suggested that CytoD advanced the osteogenic potential of AMSCs facilitating their use in skeletal regenerative strategies.

OTHER EMBODIMENTS

[0033] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.