Induced pluripotent stem cells (iPSCs) from patients with genetic disorders are a valuable source for in vitro disease models, which enable drug testing and validation of gene and cell therapies. We generated iPSCs from a severe congenital neutropenia (SCN) patient, who presented with a nonsense mutation in the glucose-6-phosphatase catalytic subunit 3 (G6PC3) gene causing profound defects in granulopoiesis, associated with increased susceptibility of neutrophils to apoptosis. Generated SCN iPSC clones exhibited the capacity to differentiate into hematopoietic cells of the myeloid lineage and we identified two cytokine conditions, i.e., using granulocyte-colony stimulating factor or granulocyte-macrophage colony stimulating factor in combination with interleukin-3, to model the SCN phenotype in vitro. Reduced numbers of granulocytes were produced by SCN iPSCs compared with control iPSCs in both settings, which reflected the phenotype in patients. Interestingly, our model showed increased monocyte/macrophage production from the SCN iPSCs. Most importantly, lentiviral genetic correction of SCN iPSCs with a codon-optimized G6PC3 transgene restored granulopoiesis and reduced apoptosis of in vitro differentiated myeloid cells. Moreover, addition of vitamin B3 clearly induced granulocytic differentiation of SCN iPSCs and increased the number of neutrophils to levels comparable with those obtained from healthy control iPSCs. In summary, we established an iPSC-derived in vitro disease model, which will serve as a tool to test the potency of alternative treatment options for SCN patients, such as small molecules and gene therapeutic vectors.

中文翻译：

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慢病毒基因治疗和维生素 B3 治疗使 G6PC3 缺陷型诱导多能干细胞的粒细胞分化成为可能。

来自遗传疾病患者的诱导多能干细胞 (iPSC) 是体外疾病模型的宝贵来源，可用于药物测试和基因和细胞疗法的验证。我们从一名严重的先天性中性粒细胞减少症 (SCN) 患者中生成 iPSC，该患者在葡萄糖-6-磷酸酶催化亚基 3 (G6PC3) 基因中出现无义突变，导致粒细胞生成严重缺陷，与中性粒细胞对细胞凋亡的易感性增加有关。生成的 SCN iPSC 克隆表现出分化为骨髓谱系造血细胞的能力，我们确定了两种细胞因子条件，即使用粒细胞集落刺激因子或粒细胞 - 巨噬细胞集落刺激因子与白细胞介素 3 组合来模拟 SCN 表型体外。与两种环境中的对照 iPSC 相比，SCN iPSC 产生的粒细胞数量减少，这反映了患者的表型。有趣的是，我们的模型显示 SCN iPSC 的单核细胞/巨噬细胞产量增加。最重要的是，使用密码子优化的 G6PC3 转基因对 SCN iPSC 进行慢病毒遗传校正可恢复粒细胞生成并减少体外分化骨髓细胞的凋亡。此外，添加维生素 B3 明显诱导 SCN iPSCs 的粒细胞分化，并将中性粒细胞的数量增加到与从健康对照 iPSCs 获得的水平相当的水平。总之，我们建立了一个 iPSC 衍生的体外疾病模型，它将作为一种工具来测试 SCN 患者替代治疗方案的效力，例如小分子和基因治疗载体。