Human embryonic stem (ES) and embroyonic germ (EG) cells to blood and immune cell

描述(申请人提供)：人类胚胎干细胞(ES)和胚胎生殖细胞(EG)细胞系提供了前所未有的机会来研究胚胎和成体干细胞的自我更新和分化，并开发基于细胞的新型临床疗法。新出现的数据显示，人类ES/EG细胞与小鼠的细胞一样具有多能性，但在细胞增殖和分化的许多关键特性上也不同。因此，有必要对人ES/EG细胞及其分化进行直接研究。我们已经开始使用人类ES/EG细胞及其直接后代，即类胚体衍生(EBD)细胞，在我们之前对小鼠ES和EG细胞的研究基础上，用于造血分化。对于H1(WA01)人ES(HES)细胞系，我们在3个方面取得了重大进展：1)建立了从分化的HES细胞同时生成淋巴(B和NK)和髓系细胞的方法；2)开发了一种通过慢病毒载体高效、稳定地转导HES细胞的方法；3)开发了一种在人骨髓基质细胞(HMSCs)上扩增HES细胞的培养体系，以取代以前需要的小鼠饲养细胞。因此，我们计划了一个第二阶段的项目，以改进和阐明HES细胞和非肿瘤EBD细胞系的淋巴-造血分化机制。总体目标是制造能够移植到NOD/SCID小鼠体内的可移植的淋巴造血祖细胞。除了使用hMSCs作为基质外，我们还将使用慢病毒载体在HES细胞中表达关键调控基因，如VEGF、Notch和HOXB4，以进一步促进生成的淋巴造血祖细胞的自我更新和植入。这些方法将使我们能够确定人类造血干细胞发生和自我更新所需的外部/内部信号。稍后，我们将直接研究抗原提呈细胞(APC)的生成和功能，APC表达高水平的MHC II类复合体，并调节T细胞。使用来自HES和EBD细胞的APC(/-基因修饰)，我们将尝试灭活同种异体反应性T细胞，并探索最终导致免疫耐受诱导的策略。这一研究项目将使我们能够更好地了解人类淋巴造血和人类干细胞的早期事件。它还将为开发基于ES细胞的新型疗法提供基础，这些疗法需要重建患者的血液/免疫系统或对其重新编程。

DESCRIPTION (provided by applicant): Human embryonic stem (ES) and embryonic germ (EG) cell lines provide unprecedented opportunities to study self-renewal and differentiation of embryonic and adult stem cells, and to develop novel cell-based clinical therapies. Emerging data has revealed that human ES/EG cells are pluripotent like their mouse counterparts, but also distinct in many critical properties of cell proliferation and differentiation. Therefore, it is necessary to study directly human ES/EG cells and their differentiation. We have begun to use human ES/EG cells and their immediate progeny, embryoid body-derived (EBD) cells, for hematopoietic differentiation, building upon our previous studies with mouse ES and EG cells. With the H1 (WA01) human ES (hES) cell line, we have made significant progress in 3 areas: 1) developed a method to generate both lymphoid (B and NK) and myeloid cells from differentiated hES cells; 2) developed a method to efficiently and stably transduce hES cells by lentiviral vectors; 3) developed a culture system to expand hES cells on human marrow stromal cells (hMSCs) in replacing previously required mouse feeder cells. Therefore, we planned a 2nd stage project to improve and elucidate mechanisms of lympho-hematopoietic differentiation from hES cells and non-tumorgeneic EBD cell lines. The overall goal is to produce transplantable lympho-hematopoietic progenitors capable of engrafting in NOD/SCID mice. In addition to using hMSCs as stroma, we will also use lentiviral vectors to express key regulatory genes such as VEGF, Notch as well as HoxB4 in hES cells to further facilitate self-renewal and engraftment of the generated lympho-hematopoietic progenitors. These approaches will allow us to define external/internal signals required for the genesis and self-renewal of human hematopoietic stem cells. Later on, we will directly examine the generation and functionality of antigen-presenting cells (APCs) which express high levels of MHC class II complex and regulate T cells. Using APCs derived from hES and EBD cells (+/- gene modification), we will attempt to inactivate alloreactive T cells and explore strategies ultimately leading to immune tolerance induction. This research project will allow us to better understand early events of human lympho-hematopoiesis and human stem cells. It will also provide a foundation for developing novel ES cell-based therapies that require reconstituting or re-programming patient's blood/immune systems.