Graduate School of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Sıhhiye, 06100, Ankara, Turkey.
Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Sıhhiye, 06100, Ankara, Turkey.
Stem Cell Res Ther. 2021 May 13;12(1):287. doi: 10.1186/s13287-021-02364-z.
Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2.
Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media.
All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage.
Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.
格雷西利综合征 2 型（GS-2）是一种罕见的常染色体隐性免疫缺陷综合征，由 RAB27A 基因突变引起，导致参与囊泡运输的蛋白质缺失，从而导致细胞毒性 T 细胞和 NK 细胞功能丧失。诱导多能干细胞（iPSC）表达与多能性相关的基因，具有无限扩增的能力，并能分化为来自三个胚层的细胞。它们可以通过整合或非整合系统诱导，以转移 Oct4、Sox2、Klf4 和 cMyc（OSKM）转录因子。为了更好地理解 GS-2 的病理生理学，并测试新的治疗方案，需要建立 GS-2 的体外模型。
本研究使用慢病毒载体从 3 名 GS-2 患者中生成 iPSC。使用流式细胞术和 RT-PCR 对 iPSC 进行鉴定，并检测多能性标志物的表达。通过畸胎瘤试验检测体内向三个胚层细胞的分化情况。使用 Op9 饲养层和特定培养基，在体外将 GS-2 iPSC 分化为造血干/祖细胞。
所有 GS-2 iPSC 克隆均显示正常核型（46XX 或 46XY），并证明存在原始体细胞供体细胞中存在的相同 RAB27A 基因突变。GS-2 iPSC 表达 SSEA1、SSEA4、TRA-1-60、TRA-1-81 和 OCT4 蛋白，通过 RT-PCR 证实 SOX2、NANOG 和 OCT4 的表达。通过畸胎瘤试验证实了向三个胚层细胞分化的能力。GS-2 iPSC 显示出向造血谱系细胞分化的能力。
通过慢病毒转染 OSKM，我们能够从 3 名 GS-2 患者中生成不同的 iPSC 克隆。这些细胞可用于未来的研究，以开发新的治疗方案，并研究 GS-2 疾病的病理生理学。
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