Craniofacial tissue engineering with human & adult ESCs

描述(由申请人提供)：我们建议利用我们在生物材料开发方面的成熟和有充分记录的历史作为组织替换的支架，作为干细胞方法在颅面组织再生中的平台。这项工作的主要假设包括：i)胚胎来源的人类干细胞可分化为头面部组织产生细胞，如成骨细胞和软骨细胞；ii)高通量技术可用于筛选大量可能用于组织工程支架的介质和聚合物候选材料；iii)干细胞与三维聚合物支架的适当组合可用于生产复杂的头面部组织。为了验证这些假设，我们提出了以下假设：目的1：研究人类胚胎干细胞在体外分化为颅面组织前体细胞的情况。我们之前研究人类胚胎干细胞体外分化为各种细胞类型和组织的工作将继续进行，以评估分化为颅面组织形成所需细胞类型所需的条件。 目的2：利用高通量筛选技术研究人干细胞与聚合物表面的相互作用。最近，我们开发了使用各种单体和聚合物的纳升级微阵列来快速筛选细胞/聚合物相互作用的技术。成功与否将通过细胞增殖和头面部组织的组织特异性标志物的存在来评估。目的3：评价已分化和未分化的人胚胎干细胞种子聚合物支架材料的体外组织生成能力。支持人类干细胞向颅面组织分化的聚合物材料将被制成含有各种基因和生长因子的三维支架，并通过各种标记的组织学染色进行分析。目的4：将先前目标的最佳候选细胞接种于已分化和未分化的细胞，并将其植入大鼠的皮下或临界大小的缺损区模型中。无菌大鼠的皮下模型将用于评估干细胞种子支架在体内环境中的组织产生，包括细胞分化为多种表型的支架。无菌大鼠颅骨缺损模型是一种临床上相关的模型，通常被用作骨再生的衡量标准。

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.