Embryonic Stem Cell Models to Study the Axial Skeletal Lineage

描述(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，从中获得具有治疗意义的骨骼祖细胞，用于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还处于初级阶段，面临着巨大的挑战，包括分离特定类型的细胞、在培养中适当地保持细胞、向有限的谱系分化细胞，以及评估移植后的移植细胞功能。这一领域绝对需要产生必要的研究工具，以帮助调查人员解决这些挑战。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，目前尚不清楚理想的祖细胞群体(S)将存在于分化过程的哪个时间点。因此，测试和比较同一谱系中瞬时形成的不同祖细胞群体在移植后分化为软骨细胞和成骨细胞的能力至关重要。这项建议的目的集中在鉴定和测试轴向骨骼谱系的祖细胞类型。为了实现这一点，我们建议建立荧光蛋白报告ES细胞系，以标记祖细胞和成熟骨骼细胞类型。AIM1致力于建立三种荧光蛋白报告ES细胞系，以明确鉴定早期中胚层、卵裂期前轴旁中胚层和硬化体的细胞类型，从而使我们能够研究轴向骨祖细胞的形成。AIM2将产生ES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ES细胞系偶联，从而使我们能够测试和比较在移植后分化为软骨和骨细胞的祖细胞类型的能力。公共卫生相关性：这项应用的目的集中在胚胎干细胞来源的轴向骨骼祖细胞的研究和测试上。我们建议建立新的ES细胞系，使用荧光蛋白报告器来帮助鉴定软骨和骨系的祖细胞和成熟骨骼细胞类型。这些ES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。从这笔赠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.