Transcriptome mapping of bone fracture identifies Notch signaling as an important regulator of skeletal repair

We present a transcriptomic analysis that provides a better understanding of regulatory mechanisms within the healthy and injured periosteum leading to novel translational approaches for bone healing. The focus of this work is on the early regulatory control of bone healing by completing a transcriptomic analysis of forming periosteal callus cells on day 3 post fracture. Based on our previous work indicating that induced Notch1 signaling in osteoprogenitors leads to better healing, we contrasted samples in which Notch 1 intracellular domain (NICD1) is overexpressed by periosteal stem/progenitor cells with control unperturbed periosteum. We determined molecular mechanisms and changes in skeletal stem/progenitor cells (SSPC) and other cell populations within callus including hematopoietic lineages. Notch ligands were differentially expressed in endothelial and mesenchymal populations, with Dll4 restricted to endothelial cells while Jag1 expressed by various mesenchymal populations. When targeting deletion of Dll4 in SSPCs using a-smooth muscle actin (aSMACreER) in mesenchymal cells there was no phenotype, while deletion in EC using Cdh5CreER exhibited negative effects on the early fracture healing. Translation of these observations into clinically relevant model of bone healing revealed the positive effects of combination of Notch ligands delivery with currently used osteogenic inducer BMP2. Single cell RNA sequencing (sc RNA-seq) was performed from intact and fractured periosteal cells (3 days after the fracture) in aSMACre+/NICD1 and Cre-/NICD1 mice. To induce NICD1 overexpression Cre- and Cre+ mice were injected with tamoxifen (75 mg/kg) on a day of fracture and 2 dpf (or if intact, 3 and 1 day before sacrificing animals). Three animals of each group were sacrificed and periosteum (intact) or periosteal callus (fractured) were pooled and digested as previously described (13, 42) using Collagenase P (Roche, IN, USA), hyaluronidase (Sigma Aldrich, St Louis, MO, USA) digestion for 1 h on 37°C with shaking. Live, mesenchymal (CD45-) and hematopoietic (CD45+) cells were sorted and single cell RNA-seq performed (10x Genomics). Periosteal cells were digested, stained for CD45-eFluor 450, Ter119-APC and propidium iodide to exclude dead cells. CD45-Ter119- and CD45+Ter119+ were sorted (BD FACSSymphonyTM S6 Cell Sorter) and twelve thousand live cells were loaded into 10X Chromium X controller with ~10,000 cells cell barcoded for RNA-seq using a Chromium Single Cell 3' Reagent Kits v3.1. Libraries were sequenced on a NovaSeq 6000 S4 flow cell lane (Illumina) with an average sequencing depth of 100,000 reads per cell for CD45- and 50,000 reads per cell for CD45+. For FASTQ generation and alignments, Illumina basecall files were converted to FASTQs using bcl2fastq v2.20.0.422 (Illumina) and FASTQ files associated with the gene expression libraries aligned to the mm10 genome using the version 3.1.0 Cell Ranger count pipeline (10x Genomics).

本研究提供了一项转录组学分析（transcriptomic analysis），旨在加深对健康及损伤状态下骨膜（periosteum）内调控机制的理解，进而为骨修复开发全新的转化研究路径。本研究的核心聚焦于骨修复的早期调控机制：我们对骨折后第3天形成的骨膜骨痂（periosteal callus）细胞开展转录组学分析。基于本团队前期研究发现：在骨祖细胞（osteoprogenitors）中诱导激活Notch1信号通路可改善骨修复效果，我们将骨膜干/祖细胞（periosteal stem/progenitor cells）中过表达Notch1胞内域（Notch1 Intracellular Domain, NICD1）的样本，与未经过干预的正常骨膜对照组样本进行了对比分析。我们解析了骨痂内骨骼干/祖细胞（Skeletal Stem/Progenitor Cells, SSPC）及包括造血谱系（hematopoietic lineages）在内的其他细胞群的分子调控机制与表达变化。Notch配体在内皮细胞（endothelial cells）与间充质细胞群（mesenchymal populations）中存在差异化表达：Dll4仅局限于内皮细胞，而Jag1则由多种间充质细胞群表达。我们利用间充质细胞中的α-平滑肌肌动蛋白-CreER（α-smooth muscle actin-CreER, αSMACreER）靶向敲除骨骼干/祖细胞中的Dll4，未观察到明显表型；而利用Cdh5-CreER在内皮细胞（Endothelial Cells, EC）中敲除Dll4，则会对骨折早期修复产生负面影响。将上述研究结果转化至临床相关骨修复模型后发现：联合递送Notch配体与当前临床常用的成骨诱导剂（osteogenic inducer）骨形态发生蛋白2（Bone Morphogenetic Protein 2, BMP2），可产生积极的修复效果。我们对αSMACre+/NICD1与Cre-/NICD1小鼠的完整骨膜细胞及骨折后第3天的骨膜骨痂细胞开展了单细胞RNA测序（Single Cell RNA Sequencing, scRNA-seq）。为诱导NICD1过表达，我们在骨折当天及骨折后天数（days post fracture, dpf）第2天（若为完整骨膜样本，则在处死动物前3天与前1天）向Cre-与Cre+小鼠注射他莫昔芬（tamoxifen），剂量为75 mg/kg。每组处死3只小鼠，收集完整骨膜或骨折后的骨膜骨痂，按照此前报道的方法（13, 42）进行混合消化：使用胶原酶P（Collagenase P）（罗氏，美国印第安纳州罗切斯特）与透明质酸酶（hyaluronidase）（Sigma Aldrich，美国密苏里州圣路易斯）在37℃摇床中消化1小时。分选得到活细胞、间充质细胞（CD45-）与造血细胞（CD45+），并利用10x Genomics平台开展单细胞RNA测序。对骨膜细胞进行消化后，使用CD45-eFluor 450、Ter119-APC与碘化丙啶（propidium iodide）进行染色，以排除死细胞。分选得到CD45-Ter119-与CD45+Ter119+细胞（使用BD FACSSymphony™ S6细胞分选仪），随后将12000个活细胞加载至10X Chromium X控制器中，利用Chromium单细胞3'端试剂试剂盒v3.1（Chromium Single Cell 3' Reagent Kits v3.1）对约10000个细胞进行RNA测序条形码标记。构建的文库在NovaSeq 6000 S4测序流动槽（Illumina）上进行测序：CD45-细胞的平均测序深度为每细胞100000条reads，CD45+细胞则为每细胞50000条reads。在FASTQ文件生成与比对环节：我们使用bcl2fastq v2.20.0.422（Illumina）将Illumina碱基识别文件转换为FASTQ文件，并利用Cell Ranger count v3.1.0分析流程（10x Genomics）将基因表达文库对应的FASTQ文件比对至mm10小鼠参考基因组。