背景:人诱导多能干细胞(hiPSCs)可以产生构成人体的所有细胞,理论上。因此,hiPSCs被认为是再生医学干细胞的候选来源。同种异体hiPSC来源的细胞产物的主要挑战是它们的免疫原性。低免疫原性细胞策略是不使用免疫抑制剂的同种异体细胞疗法。基因工程技术的进步现在允许产生低免疫原性细胞以避免同种异体免疫排斥。在这项研究中,我们产生了低免疫原性hiPSC(HyPSC)克隆,该克隆减少了人白细胞抗原(HLA)Ia类和II类的表达,并表达了免疫检查点分子和安全开关.
方法:首先,我们产生了HLAIa类和II类双敲除(HLAIa/II类DKO)hiPSCs。然后,通过引入外源β-2-微球蛋白(B2M)产生HyPSC克隆,HLA-G,PD-L1和PD-L2基因,和雷帕霉素激活的半胱天冬酶9(RapaCasp9)为基础的自杀基因作为安全开关进入HLAIa/II类DKOhiPSC。分析了HyPSC及其衍生物的特性和免疫原性。
结果:我们发现,通过将外源HLA-G基因与B2M基因一起引入HLAIa/II类DKOhiPSCs,可以增强细胞表面HLA-G的表达。HyPSC保留正常核型并具有多能干细胞的特征。此外,HyPSC可以分化为所有三个胚层谱系的细胞,包括CD45造血祖细胞(HPCs),功能性内皮细胞,和肝细胞。HyPSC衍生的HPCs表现出逃避先天和适应性免疫的能力。Further,我们证明RapaCasp9可以在体外和体内用作安全开关。
结论:携带HLA-G的HLAIa/II类DKOhiPSC,PD-L1,PD-L2和RapaCasp9分子是同种异体移植干细胞的潜在来源。
BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch.
METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous β-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed.
RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo.
CONCLUSIONS: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.