Embryonic Stem Cell Models to Study the Axial Skeletal Lineage

描述（由申请人提供）：我们工作的中心目标是使用胚胎干细胞（ES）作为来源，从中获得治疗相关的骨骼祖细胞，以治疗与骨和软骨损伤相关的疾病。基于细胞的治疗领域在很大程度上处于起步阶段，面临着巨大的挑战，包括分离特定细胞类型的能力，在培养中适当维持细胞，在有限的谱系中分化细胞，以及移植后评估移植细胞的功能。这一领域绝对需要产生必要的研究工具来帮助研究人员解决这些挑战。胚胎干细胞分化为软骨和骨的过程，就像胚胎发育一样，将涉及几个中间分化步骤，目前尚不清楚在分化过程中的哪一点会存在理想的祖细胞群。因此，测试和比较在一个谱系内短暂形成的不同祖细胞群在移植后分化为软骨细胞和成骨细胞的能力将是至关重要的。本研究的目的是鉴定和测试轴向骨骼谱系的祖细胞类型。为了实现这一点，我们建议产生荧光蛋白报告ES细胞系，将标记祖细胞和成熟骨骼细胞类型。目的1重点培养了一种具有三种荧光蛋白报告基因的ES细胞系，以明确识别早期中胚层、体前旁轴胚层和硬核组的细胞类型，使我们能够研究轴向骨骼祖细胞的形成。Aim2将产生胚胎干细胞系，将不同的轴向骨骼祖细胞标记与软骨细胞/成骨细胞胚胎干细胞系结合，从而使我们能够测试和比较祖细胞类型在移植后分化为软骨和骨细胞的能力。公共卫生相关性：本应用的目的集中在胚胎干（ES）细胞衍生的轴向骨骼祖细胞的研究和测试。我们建议产生新的胚胎干细胞系，使用荧光蛋白报告来帮助鉴定软骨和骨谱系的祖细胞和成熟骨骼细胞类型。这些胚胎干细胞系将使我们能够研究轴向骨骼祖细胞的形成，并测试它们在移植后分化为骨和软骨的能力。这笔拨款开发的试剂将对使用胚胎干细胞修复受损软骨和骨骼的治疗方法产生重要影响。

DESCRIPTION (provided by applicant): The central goal of our work is to use embryonic stem (ES) cells as a source from which to derive therapeutically relevant skeletal progenitor cells to treat conditions associated with bone and cartilage damage. The field of cell based therapeutics is largely at its infancy and enormous challenges lie ahead, including the ability to isolate specific cell types, maintain cells appropriately in culture, differentiate cells down a restricted lineage, and assess engrafted cell function after transplantation. There is an absolute need within this field to generate the necessary research tools to aid investigators in resolving these challenges. The process of differentiating ES cells into cartilage and bone, like embryonic development, will involve several intermediate differentiation steps and currently it is unknown at what point in the differentiation process will the ideal progenitor cell population(s) exist. Therefore, it will be critical to test and compare different progenitor cell populations that transiently form within a lineage for their capacity to differentiate into chondrocytes and osteoblasts upon transplantation. The aims of this proposal are focused on identifying and testing progenitor cell types of the axial skeleton lineage. To carry this out, we propose to generate fluorescent protein reporter ES cell lines that will mark progenitor and mature skeletal cell types. Aim1 focuses on generating a three fluorescent protein reporter ES cell line to distinctly identify cell types for early mesoderm, presomitic paraxial mesoderm, and sclerotome allowing us to study axial skeletal progenitor cell formation. Aim2 will generate ES cell lines that will couple different axial skeletal progenitor markers with a chondrocyte/osteoblast ES cell line thus enabling us to test and compare the ability to differentiate progenitor cell types to differentiate into cartilage and bone cells upon transplantation. PUBLIC HEALTH RELEVANCE: The aims of this application are focused on the study and testing of embryonic stem (ES) cell derived axial skeletal progenitor cells. We propose to generate novel ES cell lines that use fluorescent protein reporters to aid in the identification of progenitor and mature skeletal cell types of the cartilage and bone lineage. These ES cell lines will allow us to study the formation of axial skeletal progenitor cells and test their ability to differentiate into bone and cartilage upon transplantation. The reagents developed from this grant will have an important impact on therapeutic approaches involving the use ES cells to repair damaged cartilage and bone.