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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE302146 | GEO | 2026/01/09

REPOSITORIES: GEO

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Project description:Previously we have shown that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were restricted by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450K array). The array identified 444 differentially methylated CpG loci in dNK-treated EVT compared to medium control (P<0.05). The represented genes from these loci had critical biological roles in cellular development, cellular organization and maintenance by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltranferase IV) were chosen for follow-up studies because of the biological relevance found in the research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced, and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by siRNA reduced trophoblast invasion, possibly through the altered MMP-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduce protein expressions. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility. This studied analyzed 17 samples of placental villous explant cultre exposed to different growth conditions. 6 Control samples (biological replicates) were cultured and unexposed to any other cells. 5 samples (biological replicates and from the same placentas as the controls) were cultured and exposed to conditions containing decidua natural killer cells trapped inside hollow fibres (thus only the factors secreted by the cells could interact with the explant culture and not the cells themselves. 6 samples (biological replicates and from the same placentas as the controls) were cultured and exposed to media containing IL-15. The DNA from the villous explants was isolated, bisulfite converted and run on the array.

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

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