Engineering of functional smooth muscle cells from gastrointestinal myofibroblast

 描述(申请人提供)：平滑肌细胞(SMCs)功能障碍与SM疾病包括胃肠道(GI)、肠梗阻和先天性巨结肠的肥大、增殖和纤维化有关。有缺陷的SMC是失去收缩表型的去分化细胞。近50年前，在血管损伤中已经发现了SMC向无功能去分化细胞的表型转变，但转化细胞的表型和遗传特性尚不清楚。我们发现在肥大的小肠中转化的细胞是弱表达血小板衍生生长因子受体α和β的肌成纤维样细胞(称为PDGFRalow？高细胞)。我们从结肠和空肠分离了分化的PDGFRaHigh细胞和SMC，并获得了基因组水平的基因表达谱。通过对转录本的分析，我们鉴定了SMC特异的转录因子，包括SRF和MYOCD。此外，我们还发现SMC的表型受SRF和表观遗传调节子DNA甲基转移酶1和3a(DNMT1/3a)的调控。本项目旨在揭示细胞和遗传学机制，以了解SMC在肠SM肥大发展过程中如何去分化，以及去分化细胞如何在恢复过程中重新分化为SMC。此外，该项目旨在分离去分化细胞，建立培养中的细胞系，并使用SMC特异性转录因子和表观遗传调控因子DNMT1/3a对该细胞系中的SMC进行基因工程。为了实现这些目标，我们组建了一个合作的多机构研究团队，包括Seungil Ro博士(PhD，PI)，Moon Young Lee博士(MD/PhD，研究肥大条件下SMC的可塑性)，应聘博士后(PhD，研究PDGRFalow细胞的SMC工程)，Douglas Redelman(PhD，细胞分离)，尕布藏Lee(PhD/DVM，细胞系建立)，Terence Smith(博士/PhD，组织供应和咨询)，Kent Sasse(MD，组织供应)，Joseph Miano和Kenton Sanders(博士，组织供应和咨询)研究工具供应和项目咨询)。这些研究将极大地促进我们在转录组水平上对SMC表型转变的理解，并可能为临床修复在GI SM疾病中发现的受损或无功能的SMC提供一个人类SMC系。

 DESCRIPTION (provided by applicant): Dysfunctions of smooth muscle cells (SMCs) are associated with hypertrophy, hyperplasia and fibrosis found in SM diseases including gastrointestinal (GI) bowel obstruction and Hirschprung's disease. Defective SMCs are dedifferentiated cells that have lost their contractile phenotype. The phenotypic change of SMCs to non- functional dedifferentiated cells has been discovered in vascular injuries ~50 years ago, but the phenotypic and genetic identity of the transformed cells are still unknown. We have found that the transformed cells in the hypertrophic small intestine are myofibroblast like cells that weakly express platelet-derived growth factor receptor alpha and beta (PDGFRa/ß) (called "PDGFRalowßhigh cells"). We have isolated differentiated PDGFRahigh cells and SMCs from colon and jejunum, and obtained genome scale gene expression profiles. By analyzing the transcriptomes, we identified SMC-specific transcription factors including SRF and MYOCD. Moreover, we found that the phenotype of SMCs is regulated by SRF and an epigenetic modulator DNA methyltransferase 1 and 3a (DNMT1/3a). The present project seeks to uncover a cellar and genetic mechanism for understanding how SMCs are dedifferentiated during the development of intestinal SM hypertrophy and how dedifferentiated cells are redifferentiated into SMCs during the recovery process. Furthermore, this project aims to isolate dedifferentiated cells, establish a cell line in culture, and genetically engineer SMCs from the cell line using SMC-specific transcription factors and the epigenetic regulator DNMT1/3a. To achieve these aims, we have formed a collaborative, multi-institutional research team with Drs. Seungil Ro (PhD, PI), Moon young Lee (MD/PhD, studying the plasticity of SMCs in the hypertrophic condition), a to-be-hired postdoc (PhD, studying the engineering of SMCs from PDGRFalow cells), Douglas Redelman (PhD, cell isolation), Gabsang Lee (PhD/DVM, cell line establishment), Terence Smith (PhD, calcium imaging), Laren Becker (MD/PhD, tissue supply and consulting), Kent Sasse (MD, tissue supply), and Joseph Miano and Kenton Sanders (PhDs, research tool supply and project consulting). These studies will significantly advance our understanding on the phenotypic transition of SMCs at the transcriptome level and could offer a human SMC line for clinical use to repair damaged or non-functional SMCs found in GI SM diseases.