创伤和运动神经元疾病引起的肌肉神经支配会导致致残性疾病。功能恢复的一个限制步骤是神经肌肉接头(NMJ)的再生以用于神经支配。干细胞有可能促进这些再生过程,但是目前的方法成功有限,干细胞的最佳类型仍有待确定。神经c干细胞(NCSC),作为周围神经系统的发育前兆,具有独特的优势,但NCSC在神经肌肉再生中的作用尚不清楚。此外,能够在移植后维持NCSC存活的细胞递送方法至关重要.
我们建立了一个简化的协议来推导,隔离,并表征来自人iPSC的功能性p75+NCSC,而没有重编程因子的基因组整合。为了提高体内递送时的存活率,将NCSC在微孔板中离心以通过控制悬浮细胞密度形成期望尺寸的球状体。还研究了人骨髓间充质干细胞(MSC)以进行比较。将NCSC或MSC球体注射到神经支配损伤的腓肠肌中,并研究了对NMJ形成和功能恢复的影响。还将球状体与工程化神经肌肉组织共培养以评估对体外NMJ形成的影响。
在球状体中培养的NCSC显示参与神经肌肉再生的可溶性因子的分泌增强。球体的肌内移植使NCSC能够长期存活和保留,与单细胞悬液的移植相反。此外,如步态分析所示,NCSC球体在四周后显着改善了功能恢复。电生理学,和NMJ神经支配的速率。MSC球体,另一方面,有微不足道的影响。NCSC或MSC球体与工程化肌管和运动神经元的体外共培养进一步证明了NCSC球体的神经支配NMJ形成的改善。
我们证明干细胞类型对于神经肌肉再生至关重要,并且NCSC具有独特的优势和治疗潜力,可促进周围神经损伤后的神经支配。球形培养的生物物理效应,特别是,使NCSC在体内递送后能够长期存活。此外,合成神经肌肉组织,或者“芯片上的组织,“可能提供一个平台来评估干细胞的神经肌肉再生。
Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical.
We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro.
NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids.
We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or \"tissues-on-a-chip,\" may offer a platform to evaluate stem cells for neuromuscular regeneration.