Differentiation of Bone-Marrow Stem Cells in a Laser Patterned Myocyte Coculture

描述（由申请人提供）：本提案描述了为具有物理和工程背景的PI提供的心脏细胞生物学培训。培训内容包括：1)细胞生物学的研究生课程；2)分子和细胞生物学实验技能培训；3)学术发展，包括建立研究网络和撰写手稿/提案。目前的目标是提高Pi获得NIH资助的能力，特别是R01资助。长期目标是帮助PI在生物医学和工程技能之间取得更好的平衡，发展他在生物医学工程方面的学术生涯。为了实现这些目标，PI建议在工程心肌细胞共培养模型中研究骨髓干细胞的功能分化。在这个模型中，将制造相同的细胞培养微孔，通过应用激光束在每个孔中创建一个明确的干细胞-肌细胞界面。该模型将为不同类型共培养肌细胞对干细胞分化的调控提供具有统计学意义的数据。为了验证微环境将调节干细胞向心肌细胞功能分化的假设，将解决以下具体目标：1)通过改变细胞外基质、切换共培养细胞类型和基因修饰细胞-细胞结合蛋白表达来确定微环境在调节干细胞向心肌细胞分化中的作用；2)利用原子力显微镜测定干细胞的力学性能，通过测量细胞刚度，利用牵引力显微镜评价细胞的力学耦合；3)利用膜片钳技术研究动作电位，利用微电极阵列评估电耦合，确定干细胞的电学特性。心脏病是美国男性和女性死亡的主要原因，占每年死亡人数的近40%。通过增加我们对骨髓干细胞向心肌细胞的功能分化的了解，Pi的研究将有助于当前再生医学的努力，即使用患者自己的细胞恢复受损心脏的功能。Pi的机构将提供超过2500平方英尺的实验室空间，并无限制地使用所有核心设施，这些设施配备了最先进的设备，此外还有75%的释放时间。

DESCRIPTION (provided by applicant): This proposal describes training in cardiac-cell biology for a PI whose background is in physics and engineering. The training includes 1) graduate courses in cell biology; 2) experimental-skill training in molecular and cellular biology; and 3) academic development, including developing a research network and writing manuscripts/proposals. The immediate goal is to improve the Pi's ability to obtain NIH funding, particularly an R01 grant. The long-term goal is to assist the PI in developing his academic career in biomedical engineering with a better balance between his biomedical and engineering proficiencies. To achieve these goals, the PI proposes to investigate the functional differentiation of bone-marrow stem cells in an engineered cardiac myocyte-coculture model. In this model, identical cell-culture microwells will be fabricated to allow creation of a defined stem cell-myocyte interface in each well through application of a laser beam. This model will provide statistically significant data on the regulation of stem-cell differentiation by various types of cocultured myocytes. To test the hypothesis that the microenvironments will regulate the functional differentiation of stem cells into myocytes, the following specific aims will be addressed: 1) Determine the microenvironment's role in regulating stem-cell differentiation into cardiac cells by changing the extracellular matrix, switching the coculture cell types, and genetically modifying the cell-cell juctional protein expressions; 2) Use atomic force microscopy to determine the stem cells' mechanical properties by measuring cell stiffness and traction force microscopy to evaluate the cells' mechanical coupling; 3) Determine the stem cells' electrical properties by studying action potentials using the patch clamp technique and evaluating electrical coupling using microelectrode arrays. Heart disease is America's leading cause of death for both men and women, accounting for nearly 40% of all annual deaths. By adding to our knowledge of the functional differentiation of bone-marrow stem cells into heart muscle cells, the Pi's research will contibute to the current effort in regenerative medicine to restore the the function of a damaged heart using the patient's own cells. The Pi's institution will provide more than 2,500 sq feet of lab space and unlimited access to all core facilities, which are staffed and provided with state-of-the-art equipment, in addition to 75% release time.