| Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells
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Author:
Date:
2020-09-20
[Abstract] Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha ...
[摘要] [摘要] 角膜缘干细胞移植已成功用于治疗角膜缘干细胞缺乏症的患者。但是，由于移植物质量低或移植细胞特性不足，长期临床结果常常不能令人满意。为了通过保留干细胞表型来增强人角膜缘上皮干细胞或祖细胞（LEPC）的体外扩增，并提高后续移植效率，离体细胞-基质相互作用应模拟体内条件。在角膜缘利基中优先表达的层粘连蛋白同工型可以用作上皮组织工程的培养基质。我们最近发表了LEPC在各种层粘连蛋白同工型上的扩增，并观察到与组织培养板和其他层粘连蛋白同工型相比，层粘连蛋白α5衍生的基质通过保留干/祖细胞表型，支持LEPC的有效扩增。在这里，我们描述了通过重组人层粘连蛋白-511 E8片段（LN-511E8）作为培养底物，通过胶原酶消化和LEPC的有效扩增从尸体角膜缘组织分离LEPC的优化方案。
[背景] 角膜上皮干/祖细胞（LEPC）负责角膜上皮的持续更新，位于高度专业化和复杂的环境中。它包括特定的细胞外基质组分和在支撑角膜缘细胞小生corneo -scleral缘（Schermer 等人，1986; Cotsarelis 。等人，1989; 奥多涅斯。等人，2012 ;梅。等人，2012）。这种干/祖细胞储库的损坏或损伤可导致角膜新生血管形成，慢性炎症和与角膜混浊和视力丧失相关的基质瘢痕形成（Kenyon和Tseng，1989； Sangwan 和Tseng ...
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| SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
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Author:
Date:
2020-08-05
[Abstract] DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single ...
[摘要] [摘要] DNA双链断裂（dsb）是影响基因组完整性的最具毒性的损伤之一。dsb主要通过非同源末端连接（NHEJ）和同源重组（HR）进行修复。HR过程的一个关键步骤是通过DNA末端切除，产生一个长的3′单链DNA链，这是在DNA链入侵后，以同源区域为模板进行DNA合成所必需的。DNA末端切除抑制NHEJ并触发同源定向的DSB修复，最终保证DNA的可靠修复。已建立的评价DNA末端切除过程的方法是免疫荧光法分析磷酸化S4/8rpa32蛋白病灶（DNA末端切除的标志物）或磷酸化S4/8rpa32蛋白水平。近年来，切除轨迹单分子分析（SMART）被认为是一种可靠的方法，可以通过免疫荧光法观察S期特异性DNA损伤剂（如喜树碱）处理细胞后产生的长3′单链DNA尾。然后，通过图像分析软件（如Photoshop）测量DNA束长度，评价DNA末端切除机的处理能力。DNA纤维的制备是在非变性条件下进行的，因此免疫荧光只检测DSB处理产生的特定的3′单链DNA尾。
[背景] ...
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| Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells
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Author:
Date:
2020-07-20
[Abstract] The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and ...
[摘要] [摘要] 骨骼肌是身体活动和运动表现的关键，但是衰老和疾病通常会由于肌肉细胞的浪费或变性而导致肌肉质量的逐步丧失。肌卫星细胞（MuSCs）代表的组织STE群体米细胞小号居住在骨骼肌和负责骨骼肌的体内平衡维持和再生。生长，损伤和变性信号激活MuSC，然后增殖（增殖的MuSC被称为成肌细胞），分化并与现有的多核肌肉细胞（肌纤维）融合，以介导肌肉的生长和修复。在这里，我们描述了从小鼠骨骼肌中分离MuSC的体外实验方案分析。此外，我们提供了有关如何将原代成肌细胞培养和分化成肌管的详细协议，以及用于表征细胞的免疫荧光染色程序。这些方法对于在体外模拟再生肌生成以了解MuSC 的动力学，功能和分子调控至关重要。
[背景] 通过多种细胞功能维持肌肉的动态平衡，对于保持肌肉的完整性至关重要。组织特异性成体干细胞能够在整个生命中连续不断地再生局部组织。在成年骨骼肌中，称为肌肉卫星细胞（MuSC）的干细胞群具有强大的再生能力，这是肌肉动态平衡的关键（Yin 等人，2013； Dumont 等人，2016）。静态MuSC位于与肌肉纤维并列的基底层下方的壁iche中，负责肌肉的生长和再生（Yin 等人，2013； Dumont 等人，2016）。
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