Defining BMP-responsive lncRNA for bone regeneration

描述(申请人提供)：多能细胞具有分化为体内几乎任何细胞的能力，是再生医学和修复骨骼等缺陷组织的强大工具。然而，它们的临床应用仍然面临一些挑战，其中最主要的挑战是它们形成肿瘤的倾向。在植入诱导多能细胞或胚胎干细胞后，经常可以观察到自发畸胎瘤的形成。虽然已经描述了一些方法，如FACS清除SSEA-5+细胞，加入自杀基因对更昔洛韦的反应，以及使用小分子抑制剂，但没有一种方法被证明是完全成功的。最近的研究表明，表观遗传格局在指定细胞命运方面发挥了关键作用，了解这一点对于促进安全和有效地使用多能细胞来替代骨等缺乏组织的治疗策略至关重要。新出现的数据描述了一类新的非编码RNA，其范围从200个核苷酸到超过10千个碱基，在给定的细胞类型中大致与蛋白质编码的RNA一样多样化。这些长的非编码RNA被发现与组蛋白甲基化的小分子调节器相互作用，针对特定的区域进行转录活动或基因沉默。非编码RNA被控制了多长时间，它们在调节分化中的确切作用仍未确定，但随着对其更好的了解，有可能改变多能细胞的发育过程。这将是我们提案的重点。我们将通过定义整个转录组的反应和染色质景观的变化来评估诱导的多能干细胞和胚胎干细胞对BMP-2的反应。综合两个细胞系的数据，我们将识别对BMP-2有反应的新的长非编码转录本。我们将寻求操纵这些新的长非编码RNA转录本，并评估多能性状态和骨分化潜力的变化。最后，我们还将评估多能细胞在操纵新的长非编码RNA后的体内反应。具体地说，将评估临界大小的颅骨缺损模型中的骨再生能力和畸胎瘤的形成潜力。我们期待这些创新研究的结果能够确定一种突破性的方法来理解和调控细胞命运的决定，从而允许开发使用多能细胞的安全的基于细胞的疗法。

DESCRIPTION (provided by applicant): With the ability to differentiate into almost any cell in the body, pluripotent cells represent a powerful tool for regenerative medicine and repair of deficient tissues such as bone. Several challenges to their clinical application remain, however, chief among which is their propensity to form tumors. Spontaneous teratoma formation has been frequently observed following implantation of induced pluripotent cells or embryonic stem cells. While approaches such as immunodepletion of SSEA-5+ cells by FACS, incorporation of suicide genes responsive to ganciclovir, and use of small molecular inhibitors have been described, none have proven completely successful. Recent studies have now shown the epigenetic landscape to play a key role in the specification of cell fate, and understanding how this may be regulated would be critial to facilitate safe and efficient use of pluripotent cells in therapeutic strategies to replace deficient tissues such as bone. Emerging data have described a novel class of noncoding RNAs ranging from 200 nucleotides to over 10 kilobases that are roughly as diverse in a given cell type as protein---coding mRNA. These long noncoding RNAs have been found to interact with small molecule regulators of histone methylation, targeting specific domain for transcriptional activity or gene silencing. How long noncoding RNAs are controlled and their precise role in regulating differentiation remains undefined, but with better understanding, the potential exists to redirect the developmental process in pluripotent cells. This will be the focus of our proposal. We will evaluate how induced pluripotent stem cells and embryonic stem cells respond to BMP-2 with definition of whole transcriptome response and changes to the chromatin landscape. Integrating data from both cell lines, we will then identify novel long noncoding transcripts responsive to BMP-2. We wil look to manipulate these novel long noncoding RNA transcripts and evaluate for changes to the pluripotent state and bone differentiation potential. Finally, we will also evaluae the in vivo response of pluripotent cells following manipulation of novel long noncoding RNAs. Specifically, the ability for both bone regeneration in a critical-sized calvaril defect model and the teratoma formation potential will be evaluated. We expect result of these innovative studies to identify a ground---breaking approach to understand and regulate cell fate determination, allowing for development of safe cell---based therapies employing pluripotent cells.