Genetically enhanced human erythrocytes generated from expandable stem cells

 描述(申请人提供)：人类红细胞(通常称为红细胞或红细胞)是由哺乳动物成年骨髓中短暂增殖的红细胞生成的。从成人外周血(PB)或脐带血(CB)中分离出的造血祖细胞(HSPC)无法大量扩增红系或其前体细胞，这阻碍了我们在体外产生输血和其他细胞治疗所需的数万亿红细胞的能力。最近，人类红细胞和其他体细胞类型被重新编程为具有无限扩增和分化潜力的诱导多能干细胞(IPS)。我们最近开发了一种简便的方法，可以从脐带血或成人外周血单个核细胞(MNC)中产生高质量的人iPS细胞系。我们已经使用这种方法从200名成年健康捐赠者和患者身上培养出人的iPS细胞，这些患者包括镰状细胞病、β-地中海贫血、PNH和需要频繁和长期输血的严重再生障碍性贫血患者。此外，我们开发了一种无饲养层和无异种细胞的系统，将人iPS细胞分化为特定的HSPC，然后再分化为红系细胞。在诱导终末分化时，这些红细胞增殖另外3-4个细胞分裂和形成 表达HBB蛋白但不含细胞核的网织红细胞。因此，可以从本身可通过细胞培养扩增的单个iPS细胞系产生数万亿个红细胞。例如，可以从来自异基因免疫的输血依赖患者的iPS细胞系，或来自具有有利或稀有血型抗原类型的捐赠者的iPS细胞系产生自体红细胞。此外，iPS细胞可以通过精确的基因组编辑技术进行基因改造，而不是人类HSPC或红细胞无法广泛扩张。在这个项目中，我们的目标是在体外产生具有增强功能的人红细胞的生理数量。具体地说，我们建议使用选定的人类iPS细胞系，包括那些具有精确基因修饰的细胞系，以高效和经济地从可扩展的人类干细胞体外生产红细胞。人类红细胞的体外可扩展生产寿命更长并具有增强的特性，这将为输血医学和其他形式的细胞疗法提供前所未有的机会。

 DESCRIPTION (provided by applicant): Human erythrocytes (commonly called red blood cells or RBCs) are generated from transiently proliferating erythroblasts in marrow of mammalian adults. The inability to vastly expand erythroblasts or their precursors hematopoietic stem/progenitor cells (HSPCs) isolated from human adult peripheral blood (PB) or cord blood (CB) hinders our ability to generate ex vivo trillions of RBCs necessary for transfusion and other cell therapeutic applications. Recently, human erythroblasts and other somatic cell types were reprogrammed into induced pluripotent stem (iPS) cells that have unlimited expansion and differentiation potential. We recently developed a facile method to generate high-quality human iPS cell lines from CB or adult PB mononuclear cells (MNCs). We have used this method to generate human iPS cells from >200 adult healthy donors and patients including those with sickle cell disease, beta-thalassemia, PNH and severe aplastic anemia who need frequent and chronic blood transfusion. Moreover, we developed a feeder-free and xeno-free system to differentiate human iPS cells into definitive HSPCs and then into erythroblasts. Upon induction of terminal differentiation, these erythroblasts proliferate additional 3-4 cell divisions and form reticulocytes that express the HBB protein and exclude nuclei. Therefore, trillions of erythrocytes can be generated from a single iPS cell line that itself is expandable by cell culture For example, autologous erythrocytes could be generated from an iPS cell line derived from an alloimmunized, transfusion-dependent patient, or from donors with favorable or rare blood group antigen types. In addition, iPS cells can be genetically modified by precise genome editing technology in contrast to human HSPCs or erythroblasts that are unable to expand extensively. In this project, we aim to produce physiologic numbers of human erythrocytes ex vivo with enhanced functionality. Specifically, we propose to use selected human iPS cell lines including those with precise genetic modification for efficient and economical production of erythrocytes derived ex vivo from expandable human stem cells. The scalable ex vivo production of human erythrocytes that live longer and possess enhanced properties will provide unprecedented opportunities for transfusion medicine and other forms of cell therapies.