Extra-hematopoietic immunomodulatory role of the guanine exchange factor DOCK-2 [RNA-Seq]

The molecular basis of stromal immunomodulation is still unresolved. Here, we show a novel function for dedicator of cytokinesis 2 (DOCK2) in regulating extra-hematopoietic immune function of three independent stromal cell sources: induced pluripotent stem cells (iPSC)-derived mesodermal stromal cell (iPS-MSC), severe combined immunodeficiency (SCID) patient-derived fibroblasts and CRISPR/Cas9 knockout cells (iPS-MSCDOCK2KO). We first reprogrammed human mesenchymal stromal cells (MSC) into iPSC before differentiating the iPSCs back into MSC. Immature iPS-MSCs lacked immunosuppressive potential. Successive phenotypic maturation facilitated immunomodulatory function, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNA-seq displayed time-dependent immune-related gene expression eventually resembling parental MSCs. SCID patient-derived fibroblasts harboring bi-allelic DOCK2 mutations also showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. CRISPR/Cas9-mediated DOCK2 knockout in healthy iPSCs resulted in significantly reduced immunomodulatory capacity, reduced F-actin stress-fibers, and a disturbed subcellular localization of CDC42 activation. We provide first evidence for extra-hematopoietic immunomodulation by the guanin exchange factor DOCK2. This suggests that persisting immune disease after successful blood stem cell transplantation in SCID patients could in part be due to loss-of-function DOCK2 mutations. RNAseq analysis of the maturation of induced pluripotent stem cells (iPSCs) derived from Mesenchymal stromal cells (bone marrow and unbilical cord derived MSCs) into MSCs compared to their parental MSCs

基质细胞免疫调控的分子机制至今尚未明确。本研究揭示了细胞分裂调控蛋白2（DOCK2）的全新功能，其可调控三种独立来源基质细胞的非造血免疫功能，分别为诱导多能干细胞（iPSC）分化所得的中胚层基质细胞（iPS-MSC）、重症联合免疫缺陷（SCID）患者来源的成纤维细胞以及CRISPR/Cas9敲除细胞（iPS-MSCDOCK2KO）。我们首先将人间充质基质细胞（MSC）重编程为诱导多能干细胞，随后再将这些iPSC定向分化为间充质基质细胞。未成熟的iPS-MSC不具备免疫抑制潜能，经过连续的表型成熟过程后，其免疫调控功能显著增强，同时仍可维持与亲代MSC相当的克隆形成能力。时序性RNA测序结果显示，免疫相关基因的表达随时间动态变化，最终转录谱与亲代MSC趋于一致。携带双等位基因DOCK2突变的SCID患者来源成纤维细胞，其免疫调控能力相较未突变的成纤维细胞显著降低。在健康诱导多能干细胞中通过CRISPR/Cas9介导的DOCK2敲除，可显著削弱其免疫调控能力，减少F-肌动蛋白应力纤维的形成，并干扰CDC42激活的亚细胞定位。本研究首次证实了鸟苷酸交换因子DOCK2可介导非造血免疫调控。该发现提示，SCID患者在成功接受造血干细胞移植后仍持续存在免疫疾病，其原因可能部分源于DOCK2功能丧失性突变。此外，本研究还对骨髓及脐带来源间充质基质细胞（MSC）重编程得到的诱导多能干细胞（iPSC）向MSC分化的成熟过程进行了RNA测序分析，并与对应的亲代MSC进行了对比。