Single cell atlas of amnion development of the first trimester of human pregnancy

The amnion is an essential extraembryonic tissue that maintains the intrauterine environment during pregnancy, yet its development remains enigmatic given the scarcity of samples from early-stage pregnancies. Here, we performed single-cell transcriptomic analysis of the amnion across three stages in the first trimester of pregnancy. We identified 13 distinct cell types, including amnion epithelial stem cells (AESCs), amnion mesenchymal stem cells (AMSCs), and several known and novel cell types. Epithelial lineages predominate in the early amnion, with mesenchymal and ectodermal, ectodermal cells arising later. We identify a population of bipotent progenitors that generates both AESCs and AMSCs. Trajectory and RNA velocity analyses of amniotic cells revealed epithelial, mesenchymal, and ectodermal trajectories. Moreover, the expression of secreted ligands uncovered four signaling patterns that change as development progresses. Finally, we detected mixed fetal and maternal blood and immune cells within the amnion. This cellular map highlights the landscape and complexity of the human amnion and serves as a resource for further studies of amnion development, physiology, and therapeutic applications. All tissues for sequencing and spatial work were collected in HypoThermosol FRS preservation solution (H4416-100ML Merck) and stored at 4 °C until processing. Tissue dissociation was conducted within 24 h of tissue retrieval. Tissues were cut into <1 mm3 segments before being digested with Trypsin/EDTA 0.25% phenol red for 10-15 min at 37 °C with intermittent shaking. Digested tissue was passed through a 100 µm filter and cells collected by centrifugation (500g for 5 min at 4 °C). Cells were washed with PBS and then resuspended in PBS 0.04% BSA before cell counting. cells were loaded according to the manufacturer’s protocol for the Chromium Next GEM Single Cell 5 v2 (Dual index) kit for CS17 amnion and Chromium Next GEM Single Cell 3 v3.1 (Dual index) kit for CS19 and CS22 amnion from 10X Genomics to attain 7,000 cells per reaction. Library preparation was carried out according to the manufacturer’s protocol. Libraries were sequenced, aiming at a minimum coverage of 20,000 raw reads per cell, on the Illumina HiSeq4000 or Novaseq 6000 systems using the sequencing format: read 1, 26 cycles; i7 index, 8 cycles, i5 index, 0 cycles; read 2, 98 cycles