Cardiovascular and Therapeutic Potential of Reprogrammed Human Fibroblasts

描述（由申请人提供）：内细胞团的细胞分化为形成包含活生物体的复杂组织所需的特化细胞，传统上被视为单向过程，胚胎中的细胞逐渐定型为特定的细胞类型。然而，体细胞核移植实验已经证明，卵母细胞可以将成体分化细胞的细胞核返回到多能胚胎样状态。虽然很少有人知道的因素，诱导这一过程中，最近的几份报告已经描述了四个转录因子的逆转录病毒过表达的能力，使诱导小鼠成纤维细胞的多能状态。多能性相关POU结构域5类转录因子1（Oct 3/4）、含SRY盒的基因2（Sox 2）、原癌基因myc（c-Myc）和Kruppel样因子4（Klf 4）的同时过表达导致诱导多能干（iPS）细胞的产生，所述诱导多能干（iPS）细胞表现出与胚胎干（ES）细胞相似的形态和生长特性，所述胚胎干（ES）细胞能够形成生殖系嵌合体。最近，研究人员已经从成人细胞中创造了iPS细胞，使用类似于小鼠系统的因素组合。这些人iPS细胞具有正常的核型，表达端粒酶活性，细胞表面标志物和代表人ES细胞的基因，并保持分化为所有三个初级胚层的高级衍生物的发育潜力。将分化的人类体细胞成功重编程为多能状态不仅可以消除在研究应用中有争议地使用人类ES细胞的需要，还提供了一种潜在地产生定制的患者特异性多能细胞的方法，用于再生医学工作，包括心血管组织工程。然而，这并不意味着需要批判性地研究iPS细胞的分化行为，因为定向分化方案对于这些基于干细胞的疗法成为临床现实至关重要。我们的初步结果表明，鼠iPS细胞可以分化成心血管和造血谱系的细胞，并且有可能从具有分化成心血管谱系的所有三种细胞类型的能力的分化iPS细胞中分离Flk 1阳性祖细胞。直接重编程体细胞以产生患者匹配的多能干细胞，这些干细胞可以作为自体材料的无限来源，这可能会彻底改变心脏病的治疗;然而，iPS细胞的分化和治疗能力仍然是未知的。该应用将建立在我们已经取得的进展的基础上，并进一步探索iPS衍生的心血管祖细胞的生物学和治疗潜力。公共卫生相关性：尽管医学进步，心血管疾病仍然是死亡和发病的主要原因。因此，恢复正常功能的再生心血管疗法将产生巨大的社会和经济影响。将分化的人类体细胞重编程为多能状态不仅可以消除对人类ES细胞有争议的使用的需要，而且还可以提供一种机制来生成定制的、患者特异性的多能细胞，用于包括心血管组织工程在内的再生医学工作。

DESCRIPTION (provided by applicant): Differentiation of the cells of the inner cell mass into the specialized cells required for forming the complex tissues that comprise living organisms has traditionally been viewed as a unidirectional process, with cells in the embryo becoming gradually committed to a specific cell type. However, somatic cell nuclear transfer experiments have demonstrated that the oocyte can return the nucleus of an adult differentiated cell into a pluripotent embryonic-like state. While little is known about the factors that induce this process, several recent reports have described the ability of four transcription factors whose retroviral overexpression enabled the induction of a pluripotent state in murine fibroblasts. Simultaneous overexpression of the pluripotency-associated POU domain class 5 transcription factor 1 (Oct3/4), SRY-box containing gene 2 (Sox2), proto-oncogene myc (c-Myc), and Kruppel-like factor 4 (Klf4) led to the generation of induced pluripotent stem (iPS) cells that exhibited morphology and growth properties similar to embryonic stem (ES) cells that were competent for formation of germline chimera. Rrecently investigators have created iPS cells from adult human cells using either a combination of factors similar to the mouse system. These human iPS cells had normal karyotypes, expressed telomerase activity, cell surface markers and genes that typify human ES cells, and maintained the developmental potential to differentiate into advanced derivatives of all three primary germ layers. The successful reprogramming of differentiated human somatic cells into a pluripotent state may not only eliminate the need of controversial use of human ES cells in research applications, it also provides a method to potentially generate customized, patient- specific pluripotent cells for regenerative medicine efforts including cardiovascular tissue engineering. However, this does not obviate the need to critically study the differentiation behavior of iPS cells as directed differentiation protocols will be essential for these stem cell-based therapies to become clinical reality. Our preliminary results suggest that murine iPS cells can be differentiated into cells of the cardiovascular and hematopoietic lineage, and that it is possible to isolate a Flk1-positive progenitor cell from differentiating iPS cells that possesses the ability to differentiate into all three cell types of the cardiovascular lineage. Direct reprogramming of somatic cells to generate patient-matched pluripotent stem cells that could serve as unlimited source of autologous material could revolutionize the treatment of heart disease; however, the differentiation and therapeutic capacity of iPS cells is still unknown. This application will build on the progress we have already made and further explore the biology and therapeutic potential of iPS-derived cardiovascular progenitor cells. PUBLIC HEALTH RELEVANCE: Despite medical advances, cardiovascular disease remains a leading cause of mortality and morbidity. Thus, regenerative cardiovascular therapies that restore normal function would have an enormous societal and financial impact. Reprogramming of differentiated human somatic cells into a pluripotent state may not only eliminate the need of controversial use of human ES cells but it would also provide a mechanism to generate customized, patient-specific pluripotent cells for regenerative medicine efforts including cardiovascular tissue engineering.