The osteogenic capacity and lncRNA/mRNA expression profile of periosteal-derived mesenchymal stem cells of congenital pseudarthrosis of the tibia

Purpose: Congenital pseudarthrosis of the tibia (CPT) is an uncommon disease. The biological properties, molecular patterns, and regulatory mechanisms of periosteal-derived mesenchymal stromal cells (PDMSCs) remain unclear. Methods: PDMSCs were separated from patients with either CPT or control donors. The purity of stem cells was determined by flow cytometry. Using osteogenic induction culture and Co-culture THP1-derived macrophage and PDMSCs, the osteogenic and osteoclastic activities were determined by histological examination and western blot analysis. The differential expression of lncRNA/mRNA was analyzed through RNA-Seq. Results: The proportion of CD31 and CD34 positive cells were less than 1.5%, while CD44 and CD90 positive cells were higher than 95% in PDMSCs. After osteogenic differentiation induction, the calcium nodule formation and osteogenic protein indicators levels were lower in CPT-PDMSCs. Co-culture THP1-derived macrophage and PDMSCs, after osteoclast differentiation induction, dissolved hydroxyapatite plate activity, osteoclastic protein indicators levels of co-culture with CPT PDMSCs were shown to be higher than control. RNA-seq results revealed 822 differentially expressed mRNAs and 194 differentially expressed lncRNAs, respectively. Functional analysis showed that differentially expressed lncRNAs involved in tryptophan metabolism, steroid biosynthesis, and so on. Based on differential mRNAs and lncRNAs, we conducted correlation analysis of their cis regulation, and the results showed that 27 mRNAs were cis regulated with 24 lncRNAs, a total of 30 cis-regulated combinations. Conclusion: CPT-PDMSCs has weak osteogenesis ability, but stronger ability to promotes osteoclast differentiation, we found that certain lncRNAs and mRNAs were differentially expressed in CPT-PDMSCs which have important roles in regulated osteogenesis and osteoclast differentiation. Overall design: Comparison of lncRNA/mRNA expression profiles in control and Congenital pseudothrosis of the tibia patient-derived peristal-derived mesenchymal stromal cells by RNA-seq

研究背景：胫骨先天性假关节（Congenital pseudarthrosis of the tibia, CPT）是一种罕见疾病，目前骨膜来源间充质基质细胞（periosteal-derived mesenchymal stromal cells, PDMSCs）的生物学特性、分子特征及调控机制仍未阐明。
研究方法：本研究从胫骨先天性假关节患者及健康对照供者体内分离PDMSCs。采用流式细胞术检测干细胞纯度；通过成骨诱导培养、THP1来源巨噬细胞与PDMSCs共培养体系，分别利用组织学检测与蛋白质印迹（Western Blot）分析成骨与破骨活性；通过RNA测序（RNA-Seq）分析长链非编码RNA（long non-coding RNA, lncRNA）与信使RNA（messenger RNA, mRNA）的差异表达情况。
研究结果：PDMSCs中CD31、CD34阳性细胞占比均低于1.5%，CD44、CD90阳性细胞占比均高于95%。经成骨分化诱导后，CPT组PDMSCs的钙结节形成能力及成骨蛋白标志物水平均低于对照组。在THP1来源巨噬细胞与PDMSCs共培养体系中，经破骨分化诱导后，CPT组PDMSCs共培养体系的羟基磷灰石平板溶解活性及破骨蛋白标志物水平均高于对照组。RNA测序结果显示，共筛选得到822个差异表达mRNA与194个差异表达lncRNA。功能分析显示，差异表达lncRNA涉及色氨酸代谢、类固醇生物合成等通路。基于差异表达mRNA与lncRNA进行顺式调控关联分析，结果显示27个mRNA与24个lncRNA存在顺式调控关系，共计30组顺式调控组合。
研究结论：CPT患者来源的PDMSCs成骨能力较弱，但促进破骨细胞分化的能力更强；本研究发现CPT-PDMSCs中存在一批差异表达的lncRNA与mRNA，这些分子在成骨与破骨分化调控中发挥重要作用。
整体实验设计：通过RNA-Seq分析健康对照与胫骨先天性假关节患者来源的PDMSCs中lncRNA与mRNA的表达谱差异。