Song Bing, Fan Yong, He Wenyin, Zhu Detu, Niu Xiaohua, Wang Ding, Ou Zhanhui, Luo Min, Sun Xiaofang
Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University , Guangzhou City, People's Republic of China .
Stem Cells Dev. 2015 May 1;24(9):1053-65. doi: 10.1089/scd.2014.0347. Epub 2015 Feb 5.
The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.
生成β地中海贫血（β-Thal）患者特异性诱导多能干细胞（iPSCs），随后基于同源重组对β珠蛋白基因（HBB）中的致病突变/缺失进行基因校正，并进行其衍生的造血干细胞（HSC）移植，为治疗这种疾病提供了理想的治疗方案。然而，在以往的研究中，基因校正后的β-Thal iPSCs的造血分化效率尚未得到充分评估。在本研究中，我们使用了最新的基因编辑工具——成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）来校正β-Thal iPSCs；基因校正后的细胞表现出正常的核型和完全的多能性，因为人类胚胎干细胞（hESCs）未显示脱靶效应。然后，我们评估了基因校正后的β-Thal iPSCs的分化效率。我们发现，在造血分化过程中，基因校正后的β-Thal iPSCs的胚状体比例和各种造血祖细胞百分比增加。更重要的是，与未校正组相比，基因校正后的β-Thal iPSC系恢复了HBB表达并减少了活性氧的产生。我们的研究表明，一旦通过CRISPR/Cas9系统校正，β-Thal iPSCs的造血分化效率会大大提高，并且我们从研究中获得的信息将极大地促进β-Thal iPSC衍生的HSCs在移植中的临床应用。
