Transplantability of induced pluripotent stem cells for skeletal tissues

描述(由申请人提供)：本申请是为响应美国国立卫生研究院探索/发展研究资助计划(家长R21)而提出的试探性R21资助申请：PA-09-164。本探索性应用的目的是评估诱导多能干细胞(IPSC)来源的细胞在全身移植后向骨骼组织的迁移、植入和分化。在全身注射后，祖细胞迁移到骨骼组织的能力对于治疗全身性骨骼疾病至关重要。在动物模型中评估MSCs治疗成骨不全的可移植性的研究产生了好坏参半的结果。在应用MSCs治疗OI方面，大多数争议可能是由于不同研究人员用于移植的细胞类型所致。胚胎干细胞(ESC)来源于胚泡内细胞团，可分化为体内任何类型的细胞，并可无限扩增而不丧失其多能性，因此在包括全身性骨骼疾病在内的各种疾病的细胞治疗中具有更大的应用潜力。然而，由于伦理方面的担忧，在利用这些细胞的力量方面进展甚微。最近，有研究表明，通过引入四种转录因子的组合：Oct-3/4、Sox2、c-Myc和Klf4，可以将小鼠和人类成纤维细胞重新编程为ESC样状态。被称为诱导多能干细胞(IPSC)的重新编程细胞为产生用于治疗目的和药物筛选的患者特有的干细胞提供了机会。作为了解IPSC在全身性骨骼疾病治疗中应用前景的前奏，目前的探索性应用建议评估IPSC来源的MSCs在移植到成骨不全小鼠模型后的迁移、植入和分化。为了实现上述任务，将采用以下目标：1)通过对小鼠尾尖成纤维细胞进行重编程获得IPSC，以获得MSCs；2)评估IPSC来源的MSC样细胞在OI模型小鼠骨骼组织中的可移植性、迁移、植入和分化。初步数据显示，我们可以通过对小鼠尾尖成纤维细胞进行重新编程来产生IPSC，并且细胞呈现ESC样状态。我们将培育携带胶原突变的杂合子小鼠，以产生3种小鼠基因类型(野生型、杂合子和纯合子)。用于重编程的成纤维细胞将从野生型小鼠中制备出来，同基因杂合子和纯合子小鼠将成为细胞接受者。初步数据表明，IPSC短暂暴露于转化生长因子-21可产生表现出间充质干细胞特征的细胞。我们将使用这种方法产生用于移植的细胞。细胞在体内的迁移、植入和分化将通过生物成像、组织学和基因表达分析来评估。这一探索性应用的结果将为进一步研究IPSC细胞在肌肉骨骼组织修复和再生中的应用提供平台。 公共卫生相关性：目前探索性应用的目标是评估系统移植后诱导多能干细胞(IPSC)来源的细胞向骨骼组织的迁移、植入和分化。这一探索性应用的结果将为IPSC细胞在肌肉骨骼组织修复和再生中的未来研究提供平台

DESCRIPTION (provided by applicant): The present application is an exploratory R21 grant application in response to NIH Exploratory/ Developmental Research Grant Program (Parent R21): PA-09-164. The goals of the present exploratory application are to assess migration, engraftment and differentiation of cells derived from induced pluripotent stem cells (iPSC) into skeletal tissues following systemic transplantation. The ability of progenitors to migrate to skeletal tissues following systemic injection is critical for the treatment of generalized skeletal diseases. Studies in animal models assessing transplantability of MSCs to treat osteogenesis imperfecta have generated mixed results. Most of the controversies in the application of MSCs to treat OI may be due to the types of cells used for transplantation by different investigators. Embryonic stem cells (ESC) derived from the inner cell mass of the blastocyst can give rise to any cell type of the body and can be expanded indefinitely without losing their pluripotency, thus these possess a greater potential for application in cell therapies for various diseases including generalized skeletal diseases. Because of ethical concerns however, little progress has been made in harnessing the power of these cells. Recently, it has been demonstrated that mouse and human fibroblasts can be reprogrammed into an ESC-like state by introducing combinations of four transcription factors; Oct-3/4, Sox2, c-Myc and Klf4. The reprogrammed cells referred to as induced pluripotent stem cells (iPSC) offer opportunities for generating patient specific stem cells for therapeutic purposes and drug screening. As a prelude to understanding the future application of iPSC for generalized skeletal disease treatment, the present exploratory application proposes to assess migration, engraftment and differentiation of MSCs derived from iPSC following transplantation into a mouse model of osteogenesis imperfecta. The following aims will be employed to achieve the above tasks; 1) generate MSCs from iPSC created by reprogramming mouse tail tip fibroblasts 2) Assess transplantability, migration, engraftment and differentiation of MSC like cells derived from iPSCs into the skeletal tissues of a mouse model of OI. Preliminary data show that we can generate iPSC by reprogramming murine tail tip fibroblasts and that the cells exhibit ESC like state. We will breed heterozygous mice that carry a collagen mutation to generate 3 mice genotypes (wildtype, heterozygous and homozygous). Fibroblasts for reprogramming will be prepared from wildtype mice and the syngeneic heterozygous and homozygous mice will be the cell recipients. Preliminary data indicate that brief exposure of iPSC to TGF-21 generates cells that exhibit MSCs characteristics. We will use this approach to generate cells for transplantation. Migration, engraftment and differentiation of the cells in vivo will be assessed by bioimaging, histology and gene expression analysis. The results from this exploratory application will provide a platform for future investigations in the application of iPSC cells for musculoskeletal tissue repair and regeneration. PUBLIC HEALTH RELEVANCE: The goals of the present exploratory application are to assess migration, engraftment and differentiation of cells derived from induced pluripotent stem cells (iPSC) into skeletal tissues following systemic transplantation. The results from this exploratory application will provide a platform for future investigations of iPSC cells in musculoskeletal tissue repair and regeneration