Vascular Regeneration with Direct Reprogramming and Engineering Strategies

项目摘要 缺血性心血管疾病是发病率和死亡率的主要原因。底层 这些疾病的病理生理学与血管的损失或功能障碍和/或受损的新生血管相关。 血管形成（新血管形成）。虽然细胞疗法已成为一种有前途的选择， 血管，成人干细胞的影响是不确定的，胚胎或诱导多能干细胞 (ESCs/iPSC）具有潜在的致瘤性。为了避免这些问题，开发了一种新方法， 用于体细胞直接转化或重编程的谱系或细胞类型特异性转录因子（TF） 转化为其他谱系细胞。我们已经尝试了这种直接重编程向内皮细胞（EC），使用 我们首次发现ETV 2单独足以转化人成纤维细胞， 到EC。然而，由于我们使用了慢病毒载体，这些重编程的EC（rEC）具有有限的临床应用前景。 适用性 直接重编程方法允许两种治疗策略：基于细胞的治疗或直接体内重编程。 重新编程对于临床应用，这两种方法都需要更安全的递送载体，以最小化 基因组整合的可能性。因此，我们开发了腺病毒-ETV 2（Ad-ETV 2）载体并产生了 rEC（Adeno-rEC）。细胞治疗的另一个重要障碍是移植细胞的短期存活。到 为了克服这个问题，我们一直在研究生物工程细胞疗法。 在这项研究中，首先，我们将产生一个最佳的结构，结合这些腺rEC与新的 生物材料我们开发了一种由明胶和聚甘油组成的新型可生物降解杂化共聚物 癸二酸酯（PGS），其进一步用藻酸盐制成微珠形式。我们将这种共聚物称为 AlGPM。这种混合聚合物是可生物降解的，并消除最小的炎症反应。而且其 微珠形式促进注射后包囊细胞的广泛分布。AlGPM的组成将 优化以促进细胞存活并最大化rEC的功能。然后我们将确定 选择的AlGPM微珠包封rEC的新血管形成和治疗效果， 缺血动物模型。第二，我们将确定局部注射ETV 2病毒颗粒到动物体内是否 模型可以直接将体细胞重编程为内皮细胞， 在体内修复。而且，通过使用各种转基因小鼠，我们将从基因上追踪体细胞的命运 对体内EC的影响。总之，该项目的目标是开发临床适用的血管再生 使用直接重编程方法和生物工程技术的治疗。

Project Summary Ischemic cardiovascular diseases are the leading cause of morbidity and mortality. Underlying pathophysiology of these diseases is associated with loss or dysfunction of blood vessels and/or impaired new vessel formation (neovascularization). While cell therapy has emerged as a promising option to form blood vessels, the effects of adult stem cells are uncertain and embryonic or induced pluripotent stem cells (ESCs/iPSCs) are potentially tumorigenic. To avoid these problems, a new approach has been developed using lineage- or cell-type specific transcription factors (TFs) for direct conversion or reprogramming of somatic cells into other lineage cells. We have attempted this direct reprogramming toward endothelial cells (ECs) using combinations of seven TFs and found for the first time that ETV2, alone, is sufficient to convert human fibroblasts into ECs. However, since we used a lentiviral vector, these reprogrammed ECs (rECs) have limited clinical applicability. The direct reprogramming approach allows two therapeutic strategies: cell-based therapy or direct in vivo reprogramming. For clinical application, both approaches require a safer delivery vector to minimize the possibility of genomic integration. Thus, we developed an adenoviral-ETV2 (Ad-ETV2) vector and generated rECs (Adeno-rECs). Another important barrier for cell therapy is short-term survival of the transplanted cells. To overcome this problem, we have been investigating bioengineered cell therapy. In this study, first, we will generate an optimal construct combining these Adeno-rECs with novel biomaterials. We have developed a novel biodegradable hybrid copolymer consisting of gelatin and poly glycerol sebacate (PGS), which was further made into a microbead form with alginate. We refer to this co-polymer as AlGPM. This hybrid polymer is biodegradable and elicits minimal inflammatory response. Furthermore, its microbead form promotes wide distribution of encapsulated cells after injection. The composition of AlGPM will be optimized to promote cell survival and maximize function of rECs. We will then determine the neovascularization and therapeutic effects of the selected AlGPM microbeads encapsulating rECs using ischemic animal models. Second, we will determine whether local injection of viral particles of ETV2 into animal models can directly reprogram somatic cells into endothelial cells and promote vascular regeneration and tissue repair in vivo. Moreover, by using various transgenic mice, we will genetically track the fate of somatic cells toward ECs in vivo. Together, the goal of this project is to develop clinically applicable vascular regenerative therapy using direct reprogramming approaches and bioengineering technologies.