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

描述(申请人提供)：剖析iPS细胞在特定合成底物上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)，在特定的培养条件下支持诱导多能干细胞(IPS)和人类胚胎干细胞(HES)在无异种污染的特定培养条件下处于未分化状态(自我更新)。因此，我们克服了通常依赖于小鼠胚胎饲养层细胞(MEF)或未定义基质(如MatrigelTM)支持的未知生长条件，到目前为止，这些条件严重限制了我们进行畅通无阻的机制研究的能力，并阻碍了我们使用这些干细胞治疗衰弱的人类疾病的能力。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确定的培养系统中获得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、无异种和完全定义的培养系统，该系统由PMEDSAH作为底物和无血清、定义的培养液组成，以阐明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前提。这个相互竞争的更新提案中概述的项目旨在直接解决这些目标，我们的成功应该会对使用多能干细胞再生人类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，因此在特定的目标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，我们将确定引导iPS细胞在合成聚合物基质上黏附并保持未分化状态的细胞受体机制，测试多能iPS细胞使用不止一个细胞黏附系统在特定聚合物基质上黏附和支持自我更新的假设。在具体目标3中，我们将证明患者特有的iPS细胞可以在没有异种污染的特定底物上获得，并能够分化为能够再生颅面部骨骼缺陷的间充质干细胞，以证明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。我们的方法是独特的，从根本上不同于最先进的状态，因为它使用合成成分作为细胞-底物相互作用的结构基元。通过完成我们的目标，我们将对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；即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。这一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。

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.