Dissecting Self-renewal Mechanisms of iPS Cells on Defined Synthetic Substrates

剖析 iPS 细胞在特定合成基质上的自我更新机制

• 批准号: 8240773
• 负责人: Paul Hugo Krebsbach
• 金额: $ 38.35万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240773
• 关键词: Address; Adhesions; Alkanesulfonates; Animals; Binding; Biological; Biological Assay; Biological Process; Biomimetics; Biotechnology; Cell Adhesion; Cell Differentiation process; Cell Line; Cell surface; Cells; Charge; Chemical Engineering; Chemistry; Clinical; Communities; Culture Media; Defect; Derivation procedure; Development; Embryo; Environment; Extracellular Matrix; Goals; Growth; Growth Factor; Heparin; Heparitin Sulfate; Human; In Vitro; Integrins; Knowledge; Label; Lead; Learning; Maintenance; Mediating; Medical; Mesenchymal Stem Cells; Methods; Mission; Modification; Molecular; Molecular Probes; Mus; Natural regeneration; Nature; Osteoblasts; Patients; Phase; Phenotype; Pluripotent Stem Cells; Polymers; Positioning Attribute; Property; Proteomics; Protocols documentation; Public Health; RGD (sequence); Receptor Cell; Recombinant Proteins; Research; Research Design; Resistance; Serum; Side; Solid; Somatic Cell; Stem cells; Sterilization; Structure; Subcellular Fractions; Substrate Interaction; Support System; Supporting Cell; Surface; System; Testing; Thick; Undifferentiated; United States National Institutes of Health; Vertebral column; Xeno; abstracting; base; bone; chemical property; clinical application; clinically relevant; craniofacial; density; design; extracellular; heparin proteoglycan; human disease; human embryonic stem cell; human tissue; induced pluripotent stem cell; innovation; matrigel; novel; phosphonate; physical property; pluripotency; regenerative therapy; research study; self-renewal; skeletal; stem; stem cell biology; stem cell differentiation; stem cell fate; success; sugar; synthetic biology; therapeutic development; tool

DESCRIPTION (provided by applicant): Dissecting Self-renewal Mechanisms of iPS Cells on Defined Synthetic Substrates Abstract ) We developed a synthetic polymer matrix substrate (PMEDSAH) that supports induced pluripotent stem (iPS) cell and human embryonic stem (hES) cell expansion in an undifferentiated state (self-renewal) in defined culture conditions that are free from xenogeneic contamination. We have thus overcome the undefined growth conditions that typically depend on the support of mouse embryonic feeder cells (MEFs) or an undefined matrix such as MatrigelTM and that until now have severely limited our ability to perform unimpeded mechanistic studies and hindered our ability to use these stem cells to treat debilitating human diseases. Our goals for this proposal are to define the molecular mechanisms that maintain iPS cells in an undifferentiated state, the derivation of iPS cell lines in our xeno-free and fully defined culture system, and the controlled differentiation of these cells towards a mesenchymal stem cell (MSC) phenotype. We will take full advantage of our unique, xeno-free and fully defined culture system, which consists of PMEDSAH as the substrate and serum-free, defined culture medium to elucidate the mechanisms responsible for self-renewal on this substrate. Accomplishing these goals is an important prerequisite to the development of therapeutic protocols using pluripotent stem cells to regenerate human tissues. The projects outlined in this competing renewal proposal are designed to directly address these goals and our success should have a significant impact in methods to regenerate human tissues using pluripotent stem cells. It is well recognize that the microenvironment influences the fate of stem cells, thus in Specific Aim 1 we will define the structural and/or physico-chemical properties of PMEDSAH that lead to iPS cell self-renewal and maintenance of the undifferentiated state. In Specific Aim 2 we will determine the cell receptor mechanisms that direct adhesion and maintain iPS cells in an undifferentiated state on synthetic polymer substrates, testing the hypothesis that pluripotent iPS cells use more than one cell adhesion system to adhere to and support self-renewal on a defined polymer substrate. In Specific Aim 3 we will demonstrate that patient-specific iPS cells can be derived on defined substrates free of xenogeneic contamination and are able to differentiate into mesenchymal stem cells capable of regenerating craniofacial skeletal defects, as a proof of concept that our system has the potential of getting the medical and scientific community closer to "clinical-grade" pluripotent stem cells. Our approach is unique and fundamentally different than the state of the art because it uses synthetic components as the structural motifs in cell-substrate interactions. By accomplishing our goal, we will make significant contributions to the understanding one of the major unresolved issues in pluripotent stem cell biology; that is, learning how pluripotent stem cells interact with their extracellular environment to: 1) remain in a unique undifferentiated state and 2) make fate changing lineage decisions. This knowledge is important for both understanding basic stem cell biology and developing consistent and safe regenerative therapies. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because we will determine how to generate of patient- specific puripotent stem cells (iPS) in fully defined and xenogeneic-free conditions that can be differentiated to osteoblasts capable of regenerating clinically relevant skeletal defects. Our innovation is derived from our so- called synthetic biology approach where synthetic PMEDSAH takes on biological functions; for example, supporting cell adhesion, enabling self-renewal and heparin-like binding of growth factors. Because we will derive patient-matched stem cells in completely defined and xengeneic free conditions we will positively impact both the understanding of basic stem cell biology and contribute to the development of consistent and safe regenerative therapies. As such, successful completion of the projects should enable new horizons important to the mission of the NIH.

描述（由申请人提供）：剖析在确定的合成基质上iPS细胞的自我更新机制摘要）我们开发了一种合成聚合物基质基质（PMEDSAH），其支持诱导多能干（iPS）细胞和人胚胎干（hES）细胞在无异种污染的确定培养条件下以未分化状态（自我更新）扩增。因此，我们已经克服了通常依赖于小鼠胚胎饲养细胞（MEF）或不确定的基质（如MatrigelTM）的支持的不确定的生长条件，并且到目前为止，这些条件严重限制了我们进行不受阻碍的机制研究的能力，并阻碍了我们使用这些干细胞治疗使人衰弱的疾病的能力。 我们的目标是确定维持iPS细胞处于未分化状态的分子机制，在我们的无异种和完全定义的培养系统中衍生iPS细胞系，以及这些细胞向间充质干细胞（MSC）表型的受控分化。我们将充分利用我们独特的、无异种和完全确定的培养系统，该系统由PMEDSAH作为底物和无血清、确定的培养基组成，以阐明负责在该底物上自我更新的机制。实现这些目标是开发使用多能干细胞再生人体组织的治疗方案的重要先决条件。在这个竞争性的更新提案中概述的项目旨在直接解决这些目标，我们的成功应该对使用多能干细胞再生人体组织的方法产生重大影响。众所周知，微环境影响干细胞的命运，因此在具体目标1中，我们将定义导致iPS细胞自我更新和维持未分化状态的PMEDSAH的结构和/或物理化学性质。在具体目标2中，我们将确定细胞受体机制，指导粘附和维持iPS细胞在合成聚合物基质上处于未分化状态，测试多能iPS细胞使用一种以上的细胞粘附系统粘附并支持在定义的聚合物基质上自我更新的假设。在具体目标3中，我们将证明患者特异性iPS细胞可以在无异种污染的限定基质上衍生，并且能够分化成能够再生颅面骨骼缺陷的间充质干细胞，作为我们的系统具有使医学和科学界更接近“临床级”多能干细胞的潜力的概念证明。 我们的方法是独特的，从根本上不同于最先进的，因为它使用合成成分作为细胞基质相互作用的结构基序。通过实现我们的目标，我们将为理解多能干细胞生物学中尚未解决的主要问题之一做出重大贡献;也就是说，了解多能干细胞如何与其细胞外环境相互作用：1）保持独特的未分化状态，2）做出改变命运的谱系决定。这些知识对于理解基本的干细胞生物学和开发一致和安全的再生疗法都很重要。 公共卫生相关性：拟议的研究与公共卫生有关，因为我们将确定如何在完全定义和无异种的条件下产生患者特异性的纯能干细胞（iPS），这些干细胞可以分化为能够再生临床相关骨骼缺陷的成骨细胞。我们的创新源于我们所谓的合成生物学方法，其中合成PMEDSAH具有生物学功能;例如，支持细胞粘附，实现自我更新和生长因子的肝素样结合。因为我们将在完全定义和无异种的条件下获得患者匹配的干细胞，我们将积极影响对基础干细胞生物学的理解，并有助于开发一致和安全的再生疗法。因此，项目的成功完成将为NIH的使命开辟新的视野。