Tissue-Specific Extracellular Matrix for Culturing Stem and Progenitor Cells

描述(申请人提供)：不同组织的细胞外基质不同。由于正常的健康细胞创造了它们自己的最佳环境，因此当这些细胞产生时，可以从组织中创造出适合细胞培养的最佳环境。令人惊讶的是，目前对研究特定组织再生细胞类型(所谓的成体干细胞)的生长和分化感兴趣的研究人员还没有这样的“培养器皿”。虽然有通用的涂层和3-D支架，但这些产品并不是许多研究人员正在研究的各种成年哺乳动物干细胞和祖细胞所特有的，因此它们不是针对给定系统进行优化的。几位研究人员最近发现，细胞外基质(ECM)成分的细微变化可以对培养中的生长、分化和组织形成产生巨大影响。为此，我们建议研究提供细胞-基质相互作用的组织特异性ECM涂层，以及这些相互作用可能对成体干细胞和祖细胞的生长和分化产生的影响。这样，我们将为哺乳动物细胞的体外生长和分化提供必要的工具，特别是干细胞和祖细胞，从而加快再生医学许多领域的发展步伐。在这项研究中，我们将首先致力于开发细胞培养涂层，以证明我们方法的可行性。我们将在3种相关的ECM涂层上测试3种细胞类型：1)皮肤来源的干细胞分化为角质形成细胞(脱细胞真皮ECM)，2)肌肉卫星细胞分化为骨骼肌成肌细胞(骨骼肌ECM)，3)羊水来源的干细胞分化为肝细胞(肝脏ECM)。这将通过使用我们的组织去细胞的标准方案，通过开发无细胞组织消化的新方案，最后通过将这些消化液涂覆到组织培养皿上来实现。细胞将在各自的底物以及其他底物上进行生长和分化潜力测试，以证明细胞外基质-细胞相互作用的特异性。鉴定将包括定量(如流式细胞术)、定性(如免疫细胞化学)和功能(如代谢)分析。我们推测，由组织特异性ECM介导的细胞-基质相互作用将促进更有效、更完整和更快速的分化，从而导致更多的功能性组织再生。这些新的生物材料最终可能导致细胞和组织疗法的更快发展，以及更有效的工业放大和临床翻译。

DESCRIPTION (provided by applicant): Extracellular matrix from different tissues is different. Since normal healthy cells create their own optimal environment, it follows that an optimal environment for cell culture can be created from the tissue when these cells were derived. Surprisingly, no such "cultureware" is currently available to researchers interested in investigating the growth and differentiation of tissue-specific regenerative cell types (so called adult stem cells). While there are generic coatings and 3-D scaffolds, these products are not specific to the various adult mammalian stem and progenitor cells being studied by many investigators, hence they are not optimized for a given system. Several researchers have recently shown that subtle changes to extracellular matrix (ECM) components can have a dramatic effect on growth, differentiation, and tissue formation in culture. To that end, we propose to study tissue-specific ECM coatings that provide cell-substrate interactions, and the effect these interactions may have on growth and differentiation of adult stem and progenitor cells. In so doing, we will provide essential tools for the in vitro growth and differentiation of mammalian cells, specifically stem and progenitor cells, thereby accelerating the pace of development in many areas of regenerative medicine. In this study, we will first aim to develop cell culture coatings to demonstrate the feasibility of our approach. We will test 3 cell types on 3 related ECM coatings as the substrate of interest: 1) skin-derived stem cells differentiated into keratinocytes (acellular dermis ECM), 2) muscle satellite cells differentiated into skeletal myoblasts (skeletal muscle ECM), and 3) amniotic fluid-derived stem cells differentiated into hepatocytes (liver ECM). This will be accomplished by using our standard protocols for tissue decellularization, by developing new protocols for acellular tissue digestion, and finally by coating these digestates onto tissue culture dishes. Cells will be tested for growth and differentiation potential on their respective substrates, as well as the other substrates, to demonstrate the specificity of the ECM-cell interactions. Characterization will include both quantitative (e.g. flow cytometry) qualitative (e.g. immunocytochemistry), and functional (i.e. metabolic) assays. We hypothesize that the cell-substrate interactions mediated by tissue-specific ECM will facilitate more efficient, complete, and rapid differentiation, thereby resulting in more functional tissue regeneration. These new biomaterials could eventually lead to more rapid development of cell and tissue-based therapies, as well as more efficient industrial scale-up and clinical translation.