3D Bioengineering Strategies to Mimic Human Skeletal Muscle Progenitor Cell Niche

描述(由申请人提供)： 肌肉萎缩发生在衰老、艾滋病毒感染、癌症和许多其他病理情况下，导致生活质量显著下降，2000年经济负担达185亿美元。虽然皮肤和肺组织的3D体外模型已被证明在阐明体内平衡和疾病进展的机制方面是必不可少的，但骨骼肌的类似模型还不存在。我们提出了一个利用工程化细胞外基质(EECM)、趋化线索的梯度和细胞模式的原始人类骨骼肌的3D模型。这一合作方案结合了细胞微环境工程和人类肌肉祖细胞(HMuPC)和成肌细胞生物学方面的互补专业知识。目的1是开发和优化3D eECM以促进hMuPC的增殖。先前的研究结果表明，hMuPC对微环境的生物化学和生物力学有重要的反应，并在2D培养后减少了增殖和再生。定制的eECM将由一种蛋白质工程生物材料制成，这种材料可以独立调节生物力学(弹性模数=1-100 kpa)和细胞-配体密度(0-100,000个配体/微米3)。在使用相同的eECM的2D和3D培养中，hMuPC的活性、增殖和肌源性分化将被直接比较。目的2建立hMuPC体外迁移的三维模型。在体内，调节hMuPC迁移到再生部位的可溶信号知之甚少。将在微流控装置中进行hMuPC迁移速度、定向持久性和丝状孔道延伸的时移成像，从而能够形成稳定的浓度分布。迁移将在2D和3D eECM上进行比较，以响应假定的趋化线索的梯度和均匀浓度。对年轻人(18-25岁)、老年人(60-80岁)和营养不良的人骨骼肌活检组织的细胞裂解产物的迁移进行量化，以确定潜在的趋化新调节因子。目标3是开发一种模拟人体骨骼肌组织的3D图案。人成肌细胞将被培养在有图案的eECM上，以诱导肌管融合和排列。纤维融合率、成熟度、核指数和排列将在不同图案几何形状、生物力学和生化的eECM上进行比较。多片排列的肌管将与hMuPC分层在一起，以创建再生肌肉组织的动态模型。这些目标将导致组织培养的新3D技术，骨骼肌生物学的基本新见解，以及激活hMuPC并刺激在损耗和衰老过程中受损的肌肉再生的潜在新临床疗法。 (摘要结束)

DESCRIPTION (provided by applicant): Muscle wasting occurs during aging, HIV infection, cancer, and numerous other pathological conditions, resulting in a significant decrease in quality of life and a financial burden of $18.5 billion in 2000. While 3D in vitro models of skin and lung tissue have proven essential in elucidating mechanisms of homeostasis and disease progression, analogous models of skeletal muscle do not exist. We propose a 3D model of primary human skeletal muscle that utilizes an engineered extracellular matrix (eECM), gradients of chemotactic cues, and cellular patterning. This collaborative proposal combines complementary expertise in cell microenvironment engineering and human muscle progenitor cell (hMuPC) and myoblast biology. Aim 1 is to develop and optimize a 3D eECM to enhance the proliferation of hMuPCs. Previous results show that hMuPCs are critically responsive to the biochemistry and biomechanics of the microenvironment and have diminished proliferation and regeneration following 2D culture. Customized eECM will be made from a protein-engineered biomaterial that enables independent tuning of biomechanics (elastic moduli = 1-100 kPa) and cell-ligand density (0-100,000 ligands/micron3). Viability, proliferation, and myogenic differentiation of hMuPCs will be directly compared between 2D and 3D cultures utilizing identical eECM. Aim 2 is to develop a 3D in vitro model of hMuPC migration. Little is known about the soluble cues that regulate hMuPC migration to sites of regeneration in vivo. Time-lapse imaging of hMuPC migration speed, directional persistence, and filopodia extension will be performed in a microfluidic device that enables the formation of stable concentration profiles. Migration will be compared on 2D and in 3D eECM in response to gradients and uniform concentrations of putative chemotactic cues. Migration in response to cell lysates from young (18-25 years old), old (60-80 years old), and dystrophic human skeletal muscle biopsies will be quantified to identify potential novel regulators of chemotaxis. Aim 3 is to develop a 3D patterned mimic of human skeletal muscle tissue. Human myoblasts will be cultured on patterned eECM to induce myotube fusion and alignment. Fiber fusion rate, maturity, nuclear index, and alignment will be compared on eECM of varying pattern geometry, biomechanics, and biochemistry. Multiple sheets of aligned myotubes will be layered together with hMuPCs to create a dynamic model of regenerating muscle tissue. These aims will lead to new 3D technologies for tissue culture, fundamental new insights in skeletal muscle biology, and potential new clinical therapies to activate hMuPCs and stimulate regeneration of muscle damaged during wasting and aging. (End of Abstract)