抗体介导的诱导多能干细胞成骨向分化机制及骨再生的研究

The discovery of induced pluripotent stem cells (iPSCs) is not only a breakthrough in biology theory, but also holds great promise as a new seeding cell source for customized cell therapy. Our previous study showed that differentiated cells derived from iPSCs via routine osteogenic protocols are lack of maturity. How to differentiate iPSCs into osteogenic lineage and promote bone regeneration in a safe, efficient and reliable way is an important issue that needs to be addressed before iPSCs could be used in clinics. Based on the new concept of "Antibody-mediated osseous regeneration" and our previous study of iPSCs, we hypothesize that immobilized anti-BMP-2 antibodies(Abs) could capture endogenous BMP-2 in local sites to mediate osteogenic differentiation of iPSCs and bone regeneration. We first screen Abs that demonstrate the ability not only to bind BMP-2 with high affinity, but also allow the BMP-2 immune complex to bind to iPSCs, and activate the BMP-2/Smad/Runx2 signaling pathway through immunofluorescent labeling and flow cytometry. Alginate microspheres pre-loaded with Abs and iPSCs are transplanted in nude rats subcutaneously and in critical-size calvarial defects to test the hypothesis and the mechanism. This study is a promising modality for optimizing osteogenic differentiation protocol of iPSCs. It is not only important for gaining insights into the differentiation mechanism of iPSCs, but also fundamental to the application of iPSCs in bone tissue engineering and customized cell therapy.

诱导多能干细胞（iPSCs）的发现，不仅在生物学理论上有重大突破，更为个体化细胞治疗带来了新希望。前期研究发现经常规成骨向诱导的iPSCs分化程度欠佳。如何将iPSCs诱导成为高质量成骨细胞是其在骨再生应用中亟待解决的问题。基于抗体介导骨再生的新理念及前期研究基础，我们设想利用BMP-2抗体募集内源性BMP-2到达移植区，诱导iPSCs成骨向分化，促进骨再生。本研究通过荧光标记及流式细胞筛选出既与BMP-2高亲和力结合，又不干扰BMP-2与iPSCs表面受体结合及下游信号通路激活的抗体，以裸大鼠皮下移植和原位骨缺损为动物模型，利用藻酸盐微球作为载体包埋iPSCs及BMP-2抗体，筛选最佳成骨诱导方案，考察其成骨向分化机制及参与骨再生的潜能。本研究对于优化iPSCs成骨诱导方案，提高分化效率，探索iPSCs成骨向分化机制具有重要意义，为iPSCs在骨组织工程中的应用及个体化治疗奠定研究基础。

诱导多能干细胞（iPSCs）的发现，不仅在生物学理论上有重大突破，更为个体化细胞治疗带来了新希望。前期研究发现经常规成骨向诱导的iPSCs分化程度欠佳。如何将iPSCs诱导成为高质量成骨细胞是其在骨再生应用中亟待解决的问题。基于抗体介导骨再生的新理念及前期研究基础，我们设想利用BMP-2抗体募集内源性BMP-2到达移植区，诱导iPSCs成骨向分化，促进骨再生。本研究首先通过荧光标记及流式细胞分析证明3G7 抗BMP2抗体既与BMP-2高亲和力结合，又不干扰BMP-2与iPSCs来源的MSC样细胞（iMSCs）表面受体结合。将包埋了iMSCs与BMP-2抗体的藻酸盐微球植入裸大鼠皮下，结果表明该组合能显著促进骨新生与血管形成，而直接包埋外源性BMP-2时局部血管数目显著低于前者，表现为营养不良性钙化。体外研究结果表明BMP-2抗体与BMP-2复合物可通过激活BMP2/Smad1/Runx2信号通路高效诱导iMSCs成骨向分化。本研究首次证明了抗体可通过募集内源性生物活性因子，介导iMSCs体内骨向分化。本研究对于优化iPSCs成骨诱导方案，提高分化效率，探索iPSCs成骨向分化机制具有重要意义，为iPSCs在骨组织工程中的应用及个体化治疗奠定研究基础。