Comprehensive Human Embryogenesis Reference Tool using Single-Cell RNA-Sequencing Data

Stem cell-based embryo models offer unprecedented experimental tools for studying early human development. The usefulness of embryo models hinges on their molecular, cellular and structural fidelities to their in vivo counterparts. To authenticate human embryo models, single-cell RNA-sequencing has been utilised for unbiased transcriptional profiling of human embryo models. However, a well-organised and integrated human single-cell RNA-sequencing dataset, serving as a universal reference tool for benchmarking human embryo models, remains unavailable. Herein, we developed such a reference, through integration of six published human datasets covering developmental stages from the zygote to the gastrula. Lineage annotations are contrasted and validated with available human and non-human primate datasets. Using stabilised UMAP we constructed a web tool, where query datasets can be projected on the reference and annotated with predicted cell identities. Using this reference tool, we examined several recent human embryo models, highlighting the risk of misannotation when relevant references are lacking. Primed hPSC growing on hrLN-521 (10 μg/mL, Biolamina, LN521-02) and NutriStem hPSC XF (Biological Industries, 05-100-1A) were enzymatically dissociated and seeded onto new plates also coated with hrLN-521 at a cell density of 1,84x104 cells/cm2. 24h after seeding, hPSC were moved to a 5% CO2/5% O2 incubator and were differentiated into trophoblastic cells using NutriStem hPSC XF without bFGF and TGFb that was supplemented with 10ng/mL BMP4 (Biotechne), 1µM A83-01 (Biotechne) and 0,1µM PD173074 (Biotechne) (BAP treated).