Association between in vivo bone formation and ex vivo migration capacity of human bone marrow stromal cells

Introduction: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues suggesting that only a subpopulation of hBMSC possess “homing” capacity. Thus, we tested the hypothesis that a subpopulation of hBMSC defined by ability to form heterotopic bone in vivo, is capable of homing to injured bone. Methods: We tested ex vivo and in vivo homing capacity of a number of clonal cell populations derived from telomerized hBMSC (hBMSC-TERT) with variable ability to form heterotopic bone when implanted subcutaneously in immune deficient mice. In vitro transwell migration assay was used and in vivo homing ability to bone fractures in mice was visualized by bioluminescence imaging (BLI). In order to identify the molecular phenotype associated with enhanced migration, we carried out comparative DNA microarray analysis of gene expression of hBMSC-derived high bone forming (HBF) clones versus low bone forming (LBF) clones. Results: In this study, clonal cell populations forming in vivo bone were shown to exhibit higher ex vivo transwell migration and following intravenous infusion, enhanced in vivo homing ability to bone fractures. Comparative microarray analysis of LBF versus HBF clones identified a significant enrichment of gene categories of cell chemo-attraction, adhesion and migration. Among these genes, platelet-derived growth factor receptor (PDGF-R) α and β were highly expressed in HBF clones. Further studies showed that the chemoattractive effects of PDGF in vitro was more enhanced in HBF clones compared to LBF clones and this effect was abolished in presence of a PDGFR-β-specific inhibitor: SU-16f. Sorted PDGFR-β+ cells from LBF clones showed that the PDGFR-β+ enriched cell population exhibited strong chemoattractance towards PDGF. Conclusion: Our data demonstrate phenotypic and molecular association between in vivo bone formation and migratory capacity of hBMSC. PDGFR-β can be used as a potential marker for the prospective selection of hBMSC populations with high migration and bone formation capacities suitable for clinical trials for enhancing bone regeneration. Total RNA obtained from telomerized human bone marrow derived mesenchymal stromal cells (T4P38a) with high in vivo bone forming capacity and (T4P74a) with low bone forming capacity and three single cell clones with high bone forming capacities (DD8, AD10 and BB10) and three clones with low bone forming capacities (CF1, CB4 and CD4). All cells were cultured under standard conditions and RNAs were isolated at baseline with no induction. each sample was represented in doublicate as a and b.

引言：在组织再生治疗中，开发应用人类骨骼干细胞（human skeletal stem cells，亦称骨髓基质干细胞，hBMSC）的系统性移植方案存在临床需求。在系统性移植研究中，仅有少量hBMSC能够归巢至受损组织，这表明仅部分hBMSC亚群具备“归巢”能力。因此，本研究提出并验证假说：以体内异位成骨能力为特征的hBMSC亚群，具备向受损骨组织归巢的能力。

方法：本研究对端粒酶转化hBMSC（hBMSC-TERT）来源的多个克隆细胞群的离体及体内归巢能力进行了检测，这些克隆细胞群在免疫缺陷小鼠皮下植入后，异位成骨能力存在差异。本研究采用体外Transwell迁移实验，并借助生物发光成像（bioluminescence imaging，BLI）可视化观测小鼠骨折模型中的体内归巢能力。为鉴定与迁移能力增强相关的分子表型，本研究对hBMSC来源的高成骨克隆（high bone forming clones，HBF）与低成骨克隆（low bone forming clones，LBF）的基因表达开展了DNA微阵列比较分析。

结果：本研究证实，能够在体内形成骨组织的克隆细胞群，其体外Transwell迁移能力更强；经静脉输注后，此类细胞向小鼠骨折部位的体内归巢能力也更为显著。对LBF与HBF克隆的微阵列比较分析显示，细胞趋化、黏附及迁移相关的基因类别存在显著富集。在这些基因中，血小板衍生生长因子受体（platelet-derived growth factor receptor，PDGF-R）α与β在HBF克隆中呈高表达。进一步研究表明，体外实验中血小板衍生生长因子（platelet-derived growth factor，PDGF）的趋化作用在HBF克隆中较LBF克隆更为显著，且该效应可被PDGFR-β特异性抑制剂SU-16f完全阻断。从LBF克隆中分选得到的PDGFR-β阳性细胞显示，PDGFR-β富集的细胞群对PDGF表现出较强的趋化反应。

结论：本研究数据证实，hBMSC的体内成骨能力与迁移能力存在表型及分子层面的关联。PDGFR-β可作为潜在标志物，用于前瞻性筛选具备高迁移能力与成骨能力的hBMSC群，以应用于增强骨再生的临床试验。

本研究提取的总RNA来自端粒酶转化的人骨髓来源间充质基质细胞：其中T4P38a具备高体内成骨能力，T4P74a具备低体内成骨能力；同时包含3株高成骨能力单细胞克隆（DD8、AD10与BB10）及3株低成骨能力克隆（CF1、CB4与CD4）。所有细胞均在标准培养条件下培养，未施加任何诱导因素，于基线状态下提取RNA。每个样本设置2个复孔，分别标记为a与b。