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

描述（由申请人提供）：人类胚胎干（ES）和胚胎生殖（EG）细胞系为研究胚胎和成体干细胞的自我更新和分化以及开发新的基于细胞的临床治疗提供了前所未有的机会。新出现的数据表明，人类胚胎干细胞/EG细胞与小鼠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有所不同。因此，直接研究人ES/EG细胞及其分化是十分必要的。我们已经开始使用人类胚胎干细胞/EG细胞及其直接后代胚胎体衍生（EBD）细胞进行造血分化，建立在我们之前对小鼠胚胎干细胞和EG细胞的研究基础上。利用H1 （WA01）人ES （hES）细胞系，我们在3个方面取得了重大进展：1)开发了一种从分化的hES细胞中生成淋巴细胞（B细胞和NK细胞）和髓细胞的方法；2)建立了一种利用慢病毒载体高效、稳定转导hES细胞的方法；3)开发了一种培养系统，在人骨髓基质细胞（hMSCs）上扩增hES细胞，以取代以前需要的小鼠饲养细胞。因此，我们计划进行第二阶段的项目，以完善和阐明从hES细胞和非肿瘤性EBD细胞系的淋巴造血分化机制。总体目标是产生能够移植NOD/SCID小鼠的可移植淋巴造血祖细胞。除了利用hMSCs作为基质外，我们还将利用慢病毒载体在hES细胞中表达VEGF、Notch、HoxB4等关键调控基因，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定义人类造血干细胞发生和自我更新所需的外部/内部信号。随后，我们将直接研究抗原呈递细胞（APCs）的产生和功能，APCs表达高水平的MHC II类复合物并调节T细胞。利用来自hES和EBD细胞的APCs（+/-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。这个研究项目将使我们更好地了解人类淋巴造血和人类干细胞的早期事件。它还将为开发新的ES细胞疗法提供基础，这些疗法需要重建或重新编程患者的血液/免疫系统。

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.