Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic.
Second Faculty of Medicine, Charles University, Prague, Czech Republic.
Stem Cells Transl Med. 2019 Jun;8(6):535-547. doi: 10.1002/sctm.18-0223. Epub 2019 Feb 25.
An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1 rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase-like protein, cl-casp8), apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1 model of ALS. Stem Cells Translational Medicine 2019;8:535-547.
越来越多的研究表明，人骨髓间充质干细胞（hMSC）在治疗肌萎缩侧索硬化症（ALS）方面具有有益作用。我们比较了通过腰椎穿刺或肌肉（股四头肌）注射，或两者联合应用，重复鞘内给予 hMSC 或其条件培养基（CondM）对症状性 SOD1 大鼠的影响。我们进一步评估了该治疗对脊髓组织中三种主要细胞死亡途径（坏死性凋亡、细胞凋亡和自噬）的影响。所有动物均进行行为学测试（握力测试、Basso Beattie Bresnahan（BBB）测试和转棒测试），并通过免疫组织化学、qPCR 和 Western blot 分析组织。所有接受 hMSC 治疗的症状性 SOD1 大鼠的寿命均显著延长，运动能力提高，末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）阳性细胞数量减少。此外，联合 hMSC 给药可增加运动神经元存活，维持股四头肌中的神经肌肉接头，并显著降低参与坏死性凋亡（Rip1、混合谱系激酶样蛋白、cl-casp8）、细胞凋亡（cl-casp 9）和自噬（beclin 1）的蛋白水平。此外，联合 hMSC 治疗后星形胶质细胞增生和 Connexin 43 水平升高减少。重复给予 CondM 或单独肌肉内注射可改善运动活动；然而，这一改善在分子水平上并没有得到支持。我们的结果提供了新的证据，表明重复鞘内和肌肉内给予 hMSC 的联合应用可保护运动神经元和神经肌肉接头，不仅通过减少细胞凋亡和自噬，而且还通过坏死性凋亡途径，这在啮齿动物 SOD1 模型的 ALS 中与细胞死亡明显相关。干细胞转化医学 2019；8:535-547.
