A microRNA-mediated approach towards generating functional hematopoietic stem cells from human pluripotent stem cells in vitro

项目摘要 目前，体外生产可移植造血干细胞（HSC）可以使数千名患者受益 无法接受血液系统疾病的治疗。长期目标是能够在临床上产生 从人多能干细胞（PSC）体外培养活的HSC。这一成就将彻底改变 许多血液疾病可以治疗，理论上可以减轻病人不得不等待发现 骨髓移植“匹配”，因为已经有一个完善的临床路线图用于HSC的使用 在一个治疗环境中。我们的中心假设是基于我们的战略转录谱分析， 遗传上相同的体内HSC（功能性的）和体外衍生的“前-HSC”（非功能性的）。通过消除 遗传背景作为变异性的来源，以确定功能差异的关键分子差异 缺乏存在于体外产生的HSC，我们能够发现新的差异，在调控 由miRNAs调控的通路。在强有力的初步数据的指导下，本申请的总体目标是 在体外分化过程中调节关键的miRNAs，从而提供一种获得功能性miRNAs的新方法， 来自人PSC的HSC。这一目标将在两个具体目标下实现：（1）鉴定miRNA的变化 在人PSC介导的造血分化过程中。在这个目标中，关键的miRNA表达水平将是 在PSC-造血分化测定期间分析;和（2）为了检查miRNA在造血细胞分化测定中的潜力， 影响体外和体内造血潜能。在这个目标中，关键的miRNA表达水平将被改变， 在PSC-造血分化测定期间，并分析它们成功产生完全分化的能力。 功能性HSC的体外培养。我们期望我们的战略方法将产生积极的转化影响 通过研究一种新的体外生成HSC的途径， 能力，同时也为造血领域提供了急需的综合数据集资源， 整个朝着临床上可行的HSC在体外的成功衍生。

PROJECT SUMMARY Producing transplantable hematopoietic stem cells (HSCs) in vitro could benefit thousands of patients currently unable to receive treatment for hematological disorders. The long-term goal is to be able to generate clinically viable HSCs in vitro from human pluripotent stem cells (PSCs). This achievement would revolutionize the way numerous blood diseases could be treated, and could theoretically alleviate patients from having to wait to find a bone marrow transplant ‘match’, since a well-established clinical roadmap already exists for the use of HSCs in a therapeutic setting. Our central hypothesis is based on our strategic transcriptional profiling analysis of genetically identical in vivo HSCs (functional) and in vitro-derived ‘pre-HSCs’ (not functional). By eliminating genetic background as a source of variability, to identify the key molecular differences that underlie the functional deficiencies present in in vitro-generated HSCs, we were able to discover novel differences in regulatory pathways modulated by miRNAs. Guided by strong preliminary data, the overall objective of this application is to modulate key miRNAs during in vitro differentiation to thus provide a novel approach to obtaining functional HSCs from human PSCs. This objective will be pursued under two specific aims: (1) To identify miRNA changes during human PSC-mediated hematopoietic differentiation. In this aim, key miRNA expression levels will be analyzed during PSC-hematopoietic differentiation assays; and (2) To examine the potential of miRNAs to influence hematopoietic potential in vitro and in vivo. In this aim, key miRNA expression levels will be modified during PSC-hematopoietic differentiation assays, and analyzed for their ability to successfully generate fully functional HSCs in vitro. It is our expectation that our strategic approach will have a positive translational impact by examining a novel pathway to generate HSCs in vitro that leads to long-term hematopoietic transplantation capability, while also providing a much-needed comprehensive dataset resource for the hematopoietic field as a whole towards the successful derivation of clinically viable HSCs in vitro.