Genetically enhanced human erythrocytes generated from expandable stem cells

由可扩增干细胞产生的基因增强的人类红细胞

• 批准号: 9142351
• 负责人: Linzhao Cheng
• 金额: $ 49.39万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-10 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9142351
• 关键词: Adult; Alloimmunization; Antigens; Aplastic Anemia; Autologous; Blood Group Antigens; Blood Transfusion; Blood typing procedure; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Therapy; Cell division; Cells; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Economics; Erythroblasts; Erythrocytes; Gene Deletion; Gene Expression; Gene Mutation; Generations; Genes; Genetic; Genetic Engineering; Guide RNA; Health; Hematopoietic; Hemoglobin; Human; Individual; Knock-out; Laboratories; Laboratory Research; Life; Malaria; Marrow; Mediating; Medicine; Methods; Modification; Parasites; Patients; Peptides; Peripheral Blood Mononuclear Cell; Physiological; Plasmid Cloning Vector; Plasmodium; Population; Production; Proliferating; Property; Proteins; Regulatory Element; Reporter; Research; Resistance; Reticulocytes; Sickle Cell Anemia; Somatic Cell; Stem cells; System; Technology; Thalassemia; Therapeutic; Transfusion; Transgenes; Umbilical Cord Blood; beta Thalassemia; cell type; cost; cytokine; fetal; genome editing; human embryonic stem cell; human stem cells; improved; in vivo; induced pluripotent stem cell; novel; novel strategies; overexpression; peripheral blood; precursor cell; protein expression; screening; small molecule; stem; transgene expression

 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.

 描述（由申请方提供）：人红细胞（通常称为红细胞或RBC）由哺乳动物成体骨髓中短暂增殖的成红细胞生成。不能极大地扩增从成人外周血（PB）或脐带血（CB）分离的成红细胞或其前体造血干/祖细胞（HSPC）阻碍了我们离体产生输血和其他细胞治疗应用所需的数万亿RBC的能力。最近，人类成红细胞和其他体细胞类型被重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，从CB或成人PB单核细胞（MNC）产生高质量的人iPS细胞系。我们已经使用这种方法从>200名成年健康供体和患者中产生了人iPS细胞，这些患者包括患有镰状细胞病、β-地中海贫血、PNH和严重再生障碍性贫血的患者，他们需要频繁和长期输血。此外，我们开发了一种无饲养层和无异种的系统来将人iPS细胞分化为永久HSPC，然后分化为成红细胞。在诱导终末分化时，这些成红细胞增殖另外的3-4次细胞分裂并形成 表达HBB蛋白且不含细胞核的网织红细胞。因此，可从自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，自体红细胞可从来源于同种免疫的输血依赖性患者或来源于具有有利或罕见血型抗原类型的供体的iPS细胞系产生。此外，iPS细胞可以通过精确的基因组编辑技术进行遗传修饰，这与不能广泛扩增的人HSPC或成红细胞相反。在这个项目中，我们的目标是生产生理数量的人红细胞体外增强功能。具体地，我们提出使用选定的人iPS细胞系，包括具有精确遗传修饰的那些，用于从可扩增的人干细胞高效且经济地生产离体衍生的红细胞。活得更长并具有增强特性的人红细胞的可扩展离体生产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。