Cardiovascular and Therapeutic Potential of Reprogrammed Human Fibroblasts

描述（由申请人提供）：内部细胞群的细胞分化为构成生物体的复杂组织所需的特化细胞，传统上被认为是一个单向的过程，胚胎中的细胞逐渐变成特定的细胞类型。然而，体细胞核移植实验表明，卵母细胞可以将成年分化细胞的细胞核转化为多能胚胎样状态。虽然对诱导这一过程的因素知之甚少，但最近的一些报道描述了四种转录因子的能力，这些转录因子的逆转录病毒过表达能够诱导小鼠成纤维细胞的多能状态。同时过表达多能性相关的POU结构域5类转录因子1 （Oct3/4）、含有SRY-box基因2 （Sox2）、原癌基因myc （c-Myc）和kruppel样因子4 (Klf4)，导致诱导多能干细胞（iPS）的产生，其形态和生长特性与胚胎干细胞（ES）细胞相似，能够形成种系嵌合体。最近，研究人员利用类似于小鼠系统的多种因素组合，从成人细胞中培育出了iPS细胞。这些人类iPS细胞具有正常的核型，表达端粒酶活性、细胞表面标记和人类胚胎干细胞的基因，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。成功地将分化的人类体细胞重编程为多能状态，不仅可以消除在研究应用中使用人类胚胎干细胞的争议，而且还提供了一种潜在的方法，可以为包括心血管组织工程在内的再生医学工作产生定制的、患者特异性的多能细胞。然而，这并不能排除对iPS细胞分化行为进行批判性研究的必要性，因为定向分化方案对于这些基于干细胞的治疗成为临床现实至关重要。我们的初步结果表明，小鼠iPS细胞可以分化为心血管和造血谱系的细胞，并且有可能从分化的iPS细胞中分离出flk1阳性的祖细胞，该细胞具有分化为心血管谱系的所有三种细胞类型的能力。直接对体细胞进行重编程以产生与患者匹配的多能干细胞，这种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。这项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，心血管疾病仍然是导致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，不仅可以消除使用人类胚胎干细胞的争议，而且还可以为包括心血管组织工程在内的再生医学工作提供一种生成定制的、针对患者的多能细胞的机制。

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.