Craniofacial Tissue Regeneration Using Membrane Potential to Control Stem Cell Di

利用膜电位控制干细胞 Di 进行颅面组织再生

• 批准号: 8454966
• 负责人: Amy Thurber Moody
• 金额: $ 3.4万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454966
• 关键词: Adipocytes; Affect; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Clinical; Color; Defect; Dental Pulp; Development; Differentiation Antigens; Disease; Electrophysiology (science); Ensure; Future; Genes; Goals; Health; Human; Injury; Investigation; Ions; Knowledge; MAP Kinase Gene; Measures; Membrane; Membrane Potentials; Methods; Molecular; Monitor; Natural regeneration; Oral; Osteoblasts; Outcome; Outcome Study; Pathway interactions; Patients; Pattern; Phosphorylation; Procedures; Proteins; Regulation; Reporter; Research; Resolution; Role; Sampling; Signal Transduction; Staging; Stem cells; Subfamily lentivirinae; System; Testing; Time; Tissues; Work; cell type; clinical application; craniofacial; extracellular; functional restoration; high throughput analysis; high throughput screening; improved; in vivo; maxillofacial; novel strategies; osteogenic; protein expression; reconstruction; soft tissue; stem cell differentiation; stem cell fate; tissue regeneration; tool; transdifferentiation; voltage

DESCRIPTION (provided by applicant): Regeneration of craniofacial tissue provides one means to restore function to a disease- and injury-free state. Biochemical factors have successfully been used to differentiate stem cells to many cell types. However, little progress has been made in being able to spatially pattern tissue. Recent evidence indicates the ability to use bioelectric signaling as an alternative method to direct stem cell differentiation. Due to advances in specialized tools, it is now possible to spatially and temporally control bioelectric stimulation. The hypothesis of this project is that membrane potential (Vmem) is an instructive differentiation signal capable of inducing specific molecular pathways depending on the precise Vmem. Inducing tissue regeneration with Vmem stimulation could profoundly impact oral and maxillofacial defect treatment by for the first time enabling spatial patterning of hard and soft tissues. The goal of this project is to develop a system to screen modulation of Vmem for the ability to induce stem cell differentiation to a specific cell type and to study the mechanism behind Vmem regulation. The focus will be on the differentiation of human dental pulp stem cells (hDPSCs) to osteoblasts and adipocytes, two important craniofacial cell types. The first aim of this project is to develop a screen to identify beneficial Vmem for differentiation to particular cll types. The screen will require reproducible induction of Vmem and analysis of differentiation. Electrophysiological recordings from hDPSCs will be used to correlate media ion concentrations with induced membrane potentials. Currently available differentiation marker fluorescent protein (FP) reporter constructs will be validated as a means to monitor hDPSC differentiation. As a proof of principle, FP expression in hDPSCs cultured in decreased biochemical induction factors known to affect differentiation will be observed. It is expected that changes in FP expression will correlate with the effects on differentiation and provided similar resolution of monitoring differentiation as other forms of analysis, such as qPCR, but with the added benefit of being nondestructive. In aim 2, the screen will be used to identify membrane potentials beneficial and instructive to osteogenic and adipogenic differentiation. Hyperpolarizing and depolarizing membrane potentials will be induced and changes in FP expression will be used to identify effects on differentiation in media with or without biochemical induction factors. Changes in differentiation molecular pathways will be investigated to identify Vmem signal transduction mechanisms. The outcome of this aim will provide evidence of the utility of using bioelectric signaling to induce differentiation of specific cell types. This work will enable future developmen of patterned tissue regeneration for the purpose of craniofacial reconstruction and other clinical applications. PUBLIC HEALTH RELEVANCE: Despite much progress in stem cell and tissue regeneration research, little progress has been made in developing complicated multi-cell type tissues as would be required for many oral and craniofacial applications. This project will develop a screen to allow high throughput analysis of Vmem induced effects as well as expand fundamental knowledge of the role of membrane potential in directing stem cell fate. The outcome of the study will provide a new tool for regeneration with improved spatial control of tissue patterning, which would have a broad impact on craniofacial reconstruction.

描述（由申请人提供）：颅面组织的再生提供了一种将功能恢复到无疾病和无损伤状态的方法。生化因子已成功地将干细胞与许多细胞类型区分开。但是，能够在空间上进行图形组织方面几乎没有取得进展。最近的证据表明，将生物电信号传导用作引导干细胞分化的替代方法的能力。由于专门工具的进步，现在可以在空间和时间上控制生物电刺激。该项目的假设是膜电位（VMEM）是一个具有指导性的分化信号，能够根据精确的VMEM诱导特定的分子途径。通过VMEM刺激诱导组织再生可以首次通过实现硬组织和软组织的空间模式来深刻影响口服和上颌面缺损治疗。该项目的目的是开发一个系统，以筛选VMEM的调制，以诱导干细胞分化为特定细胞类型并研究VMEM调控背后的机制。重点将放在人类牙髓干细胞（HDPSC）与成骨细胞和脂肪细胞的分化，两种重要的颅面细胞类型。该项目的第一个目的是开发一个屏幕，以确定有益的VMEM，以区分特定的CLL类型。屏幕将需要可重现的VMEM诱导和分化分析。 HDPSC的电生理记录将用于将培养基浓度与诱导的膜电位相关联。当前可用的分化标记荧光蛋白（FP）报告基因构建体将被验证为监测HDPSC分化的手段。作为原理的证明，将观察到在已知影响分化的生化诱导因子中培养的HDPSC中的FP表达。预计FP表达的变化将会 与对分化的影响相关，并提供了与其他形式的分析（例如qPCR）相似的分辨率分辨率，但其额外的好处是无损。在AIM 2中，屏幕将用于确定对成骨和脂肪生成分化有益的膜电位。将诱导超极化和去极化膜电位，FP表达的变化将用于确定对有或没有生化诱导因子的培养基分化的影响。将研究分化分子途径的变化，以鉴定VMEM信号转导机制。该目标的结果将提供使用生物电信号传导诱导特定细胞类型的分化的证据。这项工作将使未来的图案组织再生发展中心，以实现颅面重建和其他临床应用的目的。 公共卫生相关性：尽管干细胞和组织再生研究取得了很大进展，但在开发复杂的多细胞类型组织中几乎没有取得进展，就像许多口腔和颅面应用所必需的那样。该项目将开发一个屏幕，以允许对VMEM诱导效应的高吞吐量分析，并扩大对膜潜力指导干细胞命运作用的基本知识。该研究的结果将提供一种新的工具，以改善组织模式的空间控制，这将对颅面重建产生广泛的影响。