Abstract
It is established that human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo and under appropriate conditions, differentiate into osteoblasts ex vivo. Because hMSCs are recovered by iliac crest aspirate and enriched by virtue of their adherence to tissue culture plastic, the cells provide a convenient ex vivo model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose MSCs to cell cycle entry and inhibit osteogenesis. Here, we report that synthetic peptides derived from the second cysteine-rich domain of the canonical Wnt inhibitor Dickkopf-1 (Dkk-1) have utility in controlling the growth and recovery of hMSCs from bone marrow stroma. Three peptides corresponding to residues 217-269 in Dkk-1 were each found to enhance the proliferation of hMSCs in culture over 2 days. The most active peptide exhibited agonistic characteristics in that it ablated the proliferation lag observed when cultures of hMSCs receive fresh medium. It also reduced the expression of endogenous Dkk-1 (Gregory, C. A., Singh, H., and Prockop, D. J. (2003) J. Biol. Chem. 278, 28067-28078). When the cytosolic level of beta-catenin was elevated by addition of LiCl to cultures of hMSCs, the peptide also accelerated degradation of beta-catenin on withdrawal of lithium. A second peptide, corresponding to residues 184-204 had preferential and high affinity for hMSCs in the log phase of proliferation. Peptide overlay assays on hMSC lysates confirmed that the peptide bound to a 184-kDa protein corresponding to the molecular mass of LRP6. Cells recovered by this peptide had enhanced osteogenic potential but less chondrogenic potential compared with controls. Because Wnt antagonists increase the number of non-committed hMSCs in culture, they may be of use in increasing the rate of osseous wound healing in vivo by increasing the level of systemically migrating hMSCs. Therefore, such molecules could contribute to the development of a novel family of pharmaceutical agents for the improvement of the healing process in humans.
Highlights
Mesenchymal stem cells or marrow stromal cells1 can differentiate into numerous mesenchymal tissue lineages including osteoblasts, chondrocytes, adipocytes, and neural precursors [1,2,3,4,5,6,7,8] making them attractive candidates for cytotherapy, bioengineering, and gene therapy [9]
Wnt signaling apparently plays an essential role in the differentiation of pluripotent cell lines into osteoblasts [12, 13] and natural inhibitors of Wnt signaling can inhibit osteogenesis [13, 14]
The rapid expansion of human mesenchymal stem cells (hMSCs) under simple culture conditions is an attractive characteristic of the cells and it has been the subject of much investigation [1, 8, 10]
Summary
Mesenchymal stem cells or marrow stromal cells (human MSCs) can differentiate into numerous mesenchymal tissue lineages including osteoblasts, chondrocytes, adipocytes, and neural precursors [1,2,3,4,5,6,7,8] making them attractive candidates for cytotherapy, bioengineering, and gene therapy [9]. In previous studies on secreted factors that regulate the growth of hMSCs, we found that synthesis of the canonical Wnt inhibitor Dickkopf-1 (Dkk-1) was required before the cells entered the cell cycle [10] Based on this observation, we investigated the role of canonical Wnt signaling in hMSC growth and differentiation into osteoblasts. The most active peptide exhibited agonistic characteristics in that it ablated the proliferation lag observed when cultures of hMSCs receive fresh medium It reduced the expression of endogenous Dkk-1 [10]. The results suggest the use of agents that regulate the canonical Wnt pathway for control and recovery of hMSCs. Peptides corresponding to Dkk-1, other Wnt inhibitors, or the Wnt ligands themselves could contribute to the development of a novel class of pharmaceutically active compounds for the control of tissue repair in humans. Previously described agents that affect the canonical Wnt pathway may be useful for controlling hMSCs in the treatment of bone fractures and skeletal lesions such as those observed in multiple myeloma [14]
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