(M-495) Single-cell analysis of stem cell-derived microfluidic amniotic sac embryoid

Introduction:: Despite its clinical and fundamental importance, our understanding of early human development remains limited. We previously developed a microfluidic post-implantation amniotic sac embryoid (μPASE) system using human pluripotent stem cells (hPSCs), which recapitulates key landmarks of the early post-implantation human development. Herein, transcriptome at the single-cell level of μPASEs was profiled at different time points. Molecular maps of lineage diversifications were constructed and compared with in vivo data, revealing previously unknown insights about early human embryonic development. Our results also demonstrate the critical role of NODAL signaling in human mesoderm and primordial germ cell specification. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Materials and Methods:: The microfluidic device contains three parallel channels, partitioned by evenly spaced supporting posts. The central gel channel is preloaded with Geltrex. Geltrex contraction during gelation leads to formation of concave gel pockets between supporting posts. Single hPSCs settle into gel pockets and subsequently cluster. Culture medium was then switched to a basal medium with additional BMP4 supplemented only in the cell loading channel to induce μPASE formation (Fig.1A,B). NODAL-knockout hPSC line was generated using CRISPR/Cas9. Single-cell RNA sequencing (scRNA-seq) was conducted using 10X Genomics Chromium system. Analysis of scRNA-seq data and integration of scRNA-seq datasets were performed using Seurat R package. Bam files generated by the Cell Ranger pipeline were used for RNA velocity analysis. Diffusion maps were obtained by the R package Density. To validate cell annotations, the scRNA-seq dataset of the CS7 human gastrula was integrated with μPASE scRNA-seq datasets.

Results, Conclusions, and Discussions:: Single-cell transcriptome analyses reveal that hPSCs in μPASEs develop progressively from a pluripotent epiblast-like cell (EpiLC) stage into three distinct cell lineages: amniotic ectoderm-like cells (AMLCs), mesoderm-like cells (MeLCs), and primordial germ cell-like cells (PGCLCs) (Fig.1C,D). The lineage annotations were validated through the transcriptomic comparison between μPASEs and the CS7 human gastrula. Based on the diffusion map, GABRP, IGFBP3, and WNT6 were identified as novel markers for human amniotic ectoderm. Diffusion map and RNA velocity analyses suggest that AMLCs and PGCLCs share a common progenitor (Fig.1E). μPASEs generated from NODAL-knockout hPSCs show “hyper” AMLC development at the expense of MeLCs (Fig.1F). Based on sequencing data, we conclude that the transcriptome of BMP4-treated primed hPSCs is similar to that of amnion, whereas the transcriptome of trophoblast-like cells derived from naive hPSCs is consistent with that of blastocyst trophoblast (Fig.1G).

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