Dkk-1-derived synthetic peptides and lithium chloride for the control and recovery of adult stem cells from bone marrow
Dkk-1-derived synthetic peptides and lithium chloride for the control and recovery of adult stem cells from bone marrow
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DOI：
10.1074/jbc.m406275200
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发表时间：
2005-01-21
影响因子：
4.8
通讯作者：
Prockop, DJ
中科院分区：
文献类型：
--
作者：
Gregory, CA;Perry, AS;Prockop, DJ
已证实来自骨髓的人间充质干细胞（hMSCs）是体内成骨祖细胞的来源，并且在适当的条件下离体分化成成骨细胞。由于hMSC通过髂嵴抽吸物回收并凭借其粘附于组织培养塑料而富集，因此细胞为在实验可及系统中研究成骨组织修复提供了方便的离体模型。骨骼发育和成骨领域的最新进展已经证明，通过经典无翅（Wnt）途径的信号传导对于祖细胞系向成骨细胞的分化至关重要。这些信号的抑制可使MSC易于进入细胞周期并抑制骨生成。在这里，我们报告了来自经典Wnt抑制剂Dickkopf-1（Dkk-1）的第二个富含半胱氨酸的结构域的合成肽在控制骨髓基质中hMSCs的生长和恢复中具有实用性。Dkk-1中对应于残基217 - 269的三种肽各自被发现在培养中增强hMSCs的增殖超过2天。最具活性的肽表现出激动剂特性，因为它消除了当hMSC培养物接受新鲜培养基时观察到的增殖滞后。它还降低了内源性Dkk-1的表达（Gregory，C.一、辛格，H.，和Prockop，D. J.（2003）J. Biol. Chem. 278，28067 - 28078）。当通过向hMSC培养物中加入LiCl来提高β-连环蛋白的胞质水平时，该肽也加速了锂撤出时β-连环蛋白的降解。对应于残基184 - 204的第二肽在对数增殖期对hMSC具有优先和高亲和力。对hMSC裂解物的肽覆盖测定证实，所述肽结合至对应于LRP 6的分子量的184-kDa蛋白。与对照组相比，通过该肽回收的细胞具有增强的成骨潜力，但具有较低的成软骨潜力。因为Wnt拮抗剂增加培养物中非定向hMSC的数量，所以它们可以通过增加全身迁移hMSC的水平而用于增加体内骨伤口愈合的速率。因此，这样的分子可能有助于开发一种新的药物家族，用于改善人类的愈合过程。
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.