Yoon Hyung Ho, Lee Hyang Ju, Min Joongkee, Kim Jeong Hoon, Park Jin Hoon, Kim Ji Hyun, Kim Seong Who, Lee Heuiran, Jeon Sang Ryong
Department of Neurological Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea.
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea.
J Korean Neurosurg Soc. 2021 Sep;64(5):705-715. doi: 10.3340/jkns.2021.0003. Epub 2021 May 28.
Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI.
Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining.
Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups.
Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.
通过我们之前的临床试验发现，间充质干细胞（MSC）对慢性脊髓损伤（SCI）的治疗效果并不充分。因此，开发具有更高疗效的干细胞制剂的需求日益增加。我们在脊髓损伤6周后将增强型分泌Wnt3a的人间充质干细胞（hMSC）移植到损伤的脊髓中，以改善慢性脊髓损伤大鼠模型中的轴突再生。我们假设增强的Wnt3a蛋白表达可以促进脊髓损伤后的神经再生。
36只Sprague-Dawley大鼠在T9-10椎体水平使用Infinite Horizon（IH）撞击器造成损伤，并分为五组：1）磷酸盐缓冲盐水注射组（仅损伤组，n = 7）；2）hMSC移植组（MSC组，n = 7）；3）用pLenti载体（无Wnt3a基因）转染的hMSC移植组（pLenti-MSC组，n = 7）；4）用Wnt3a基因转染的hMSC移植组（Wnt3a-MSC组，n = 7）；5）用增强型Wnt3a基因（Wnt3a mRNA表达增加1.7倍）转染的hMSC移植组（1.7 Wnt3a-MSC组，n = 8）。脊髓损伤6周后，使用立体定位和微量注射泵在两个点注射每5×105个细胞/15 μL。为了评估脊髓损伤后的功能恢复情况，大鼠在损伤后的第1、2和3天接受Basso-Beattie-Bresnahan（BBB）运动测试，然后每周进行一次，共14周。使用生长相关蛋白43（GAP43）、微管相关蛋白2（MAP2）和神经丝（NF）免疫染色评估轴突再生情况。
损伤14周后（移植8周后），1.7 Wnt3a-MSC组的BBB评分（15.0±0.28）显著高于仅损伤组（10.0±0.48）、MSC组（12.57±0.48）、pLenti-MSC组（12.42±0.48）和Wnt3a-MSC组（13.71±0.61）（p<0.05）。免疫染色显示Wnt3a-MSC组和1.7 Wnt3a-MSC组中轴突再生标志物GAP43、MAP2和NF的表达增加。
我们的结果表明，hMSC中Wnt3a基因表达的增强可促进慢性脊髓损伤大鼠模型中的轴突再生并改善功能恢复。
