scRNA-seq of induced pluripotent stem cell derived decidual natural killer cells

Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-placental interface in early gestation. The exploration of dNK cell heterogeneity and function is currently an active area of research, particularly as these differ across gestation and different regions of the placenta. We applied proteomic and transcriptomic single cell definitions of dNK cells and subtypes to characterize these cells at the chorioamniotic membranes (CAM) and basal plate (BP, maternal surface) regions of the placenta at term. We found that there is reduced abundance of low-effector-function dNK1 and increased abundance of high-effector-function dNK3 in term decidual compartments. At term, in comparison to BP-dNK, CAM-dNK had greater abundance of moderate-effector-function dNK2, decreased abundance of dNK3, and lower expression of inhibitory receptor CD9. At the same time, using our knowledge of dNK heterogeneity, we developed a protocol to differentiate induced pluripotent stem cells (iPSC) into dNK cells. In this manuscript, we detail a protocol to differentiate iPSC into dNK-like cells as characterized by scRNA-seq analyses and CD45+CD56brightCD16- protein expression. We further developed the protocol to enrich for dNK2 – the most abundant dNK cell type in first trimester and term CAM – while inducing expression of dNK marker proteins CD9 and CD103 with the addition of TGFß. This study reveals the shift in dNK subtypes underlying functional changes in dNK cells between first trimester and term and detail a protocol with which we can methodically differentiate iPSC into dNK cells and specifically induce the dNK2 subtype for mechanistic perturbation. Overall design: iPSC-dNK cells grown in suspension culture were collected, filtered, counted and loaded on the 10x Genomics chip.

蜕膜自然杀伤（decidual natural killer, dNK）细胞是妊娠早期母胎界面中丰度最高的免疫细胞。目前，针对dNK细胞异质性与功能的研究是领域内的热点方向，这类细胞的特征会随妊娠进程以及胎盘不同区域产生差异。本研究采用基于蛋白质组学与转录组学的单细胞分型方法，对足月妊娠胎盘的绒羊膜膜（chorioamniotic membranes, CAM）与底板（basal plate, BP，母体侧表面）区域的dNK细胞及其亚型开展系统表征。研究发现，在足月蜕膜区域中，低效应功能的dNK1亚型丰度降低，高效应功能的dNK3亚型丰度升高。足月妊娠时，与底板来源的dNK（BP-dNK）相比，绒羊膜膜来源的dNK（CAM-dNK）中，中效应功能的dNK2亚型丰度更高，dNK3亚型丰度更低，且抑制性受体CD9的表达水平显著下调。与此同时，基于对dNK细胞异质性的认知，本团队开发了一套将诱导多能干细胞（induced pluripotent stem cells, iPSC）定向分化为dNK细胞的实验方案。本文详细描述了一套通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析与CD45+CD56brightCD16-蛋白表达特征鉴定的、将iPSC分化为类dNK细胞的实验方案。本团队进一步优化了该方案，可富集得到第一孕期及足月绒羊膜膜中丰度最高的dNK细胞亚型——dNK2，并通过添加转化生长因子β（transforming growth factor-β, TGFβ）诱导dNK细胞标志性蛋白CD9与CD103的表达。本研究揭示了从第一孕期到足月妊娠期间，dNK细胞亚型的动态变化是其功能改变的核心基础，并详细描述了一套可系统性将iPSC分化为dNK细胞、且可特异性诱导dNK2亚型以开展机制性干预的实验方案。实验整体设计：收集悬浮培养的iPSC-dNK细胞，经过滤、计数后，上样至10x Genomics测序芯片。