Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.
Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.
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DOI：
10.22203/ecm.v041a07
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发表时间：
2021-01-19
影响因子：
3.1
通讯作者：
Purmessur D
中科院分区：
文献类型：
--
作者：
Tang S;Salazar-Puerta A;Richards J;Khan S;Hoyland JA;Gallego-Perez D;Walter B;Higuita-Castro N;Purmessur D
椎间盘（IVD）退变的特征是细胞结构和蛋白多糖合成减少，炎症、软骨病和神经/血管向内生长增加。用于IVD变性的再生方法主要是基于细胞治疗或涉及病毒载体，其与诱变和不期望的免疫应答相关。本研究使用大量电穿孔和工程化细胞外囊泡（EV）将叉头盒F1（FOXF 1）mRNA递送到退化的人髓核（NP）细胞中，作为IVD再生的微创治疗策略。大量电穿孔用于研究在3D琼脂糖构建体中培养4周期间FOXF 1对人NP细胞的影响。确定了FOXF 1到单层人IVD细胞中的工程化EV递送，随后在试验小鼠IVD穿刺模型中进行体内验证。FOXF 1转染通过上调健康NP标志物[FOXF 1，角蛋白19（KRT 19）]，减少炎性细胞因子[白细胞介素（IL）-1β，-6]，分解代谢酶[金属蛋白酶13（MMP 13）]和神经生长因子（NGF）显著改变基因表达，并显著增加人NP细胞中的糖胺聚糖积累。装载有FOXF 1的工程EV证明成功包封FOXF 1货物并被单层培养的人NP细胞有效摄取。与注射后第7天的对照相比，体内将负载FOXF 1的EV注射到小鼠IVD中导致FOXF 1和Brachyury的显著上调，没有细胞毒性的证据。这是第一项证明FOXF 1的非病毒递送和体外人NP细胞和体内小鼠IVD细胞重编程的研究。该策略代表了治疗IVD变性和相关背痛的非成瘾性方法。负责这篇论文的科学编辑是Sibylle格拉德。
Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, −6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain. The Scientific Editor responsible for this paper was Sibylle Grad.