Goh, Botting, Webb and Rose et al., 2022, 'Yolk sac cell atlas reveals multiorgan functions during human early development'
This repository contains all code relating to the anaylsis of the Human Cell Atlas: Fetal Cell Atlas - Yolk Sac project.
In this study, we profile in detail the human yolk sac (YS) from 3-8 post-conception weeks (~169k cells) and matched embryonic liver using scRNA-seq, CITE-seq, spatial validation using RNAscope and tissue context of near whole YS using 3D light sheet microscopy. We contextualised human YS haematopoiesis and metabolic functions through comparison with datasets from matched embryonic liver, human and mouse gastrulas, fetal bone marrow, embryonic skin and brain and inducible pluripotent stem cell-derived myeloid cells.
Multiorgan functions of the human yolk sac We characterized functions of the developing human YS, combining scRNA-seq and CITE-seq, with 2D and 3D imaging techniques providing spatial context and validation. Our findings revealed YS contributions to metabolic and nutritional support, and early hematopoiesis. We characterized myeloid-bias in early hematopoiesis, distinct myeloid differentiation trajectories, evolutionary divergence in initial erythropoiesis, and YS contributions to developing tissue macrophages.
The yolk sac (YS) is an extraembryonic structure that generates the first blood and immune cells, and provides nutritional and metabolic support to the developing embryo. Current knowledge of these functions is based on pivotal studies in model systems, and insights from human studies are limited. Single cell genomics technologies have facilitated the interrogation of human developmental tissues at unprecedented resolution. Atlases of blood and immune cells from multiple organs have been greatly enhanced by focused, time-resolved analyses of specific tissues.
To characterize the functions of human YS, we performed single cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes (CITE-seq) on YS and paired embryonic liver. After integration with external datasets, our reference comprised 169,798 cells from 10 samples spanning 4-8 post-conception weeks (PCW) or Carnegie Stages (CS) 10-23, from which we identified 43 distinct cell states. A repertoire of 2D and 3D imaging techniques provided spatial context and validation. We benchmarked the cellular products and differentiation pathways in two hematopoietic inducible pluripotent stem cell (iPSC) culture protocols, compared against our reference.
We identified that YS metabolic and nutritional support originates in the endoderm, and discovered that endoderm produced coagulation proteins and growth factors that modulate hematopoiesis—erythropoietin (EPO) and thrombopoietin (THPO). While metabolic and coagulation protein production was conserved between human, mouse, and rabbit, EPO and THPO production was observed in human and rabbit only.
We reconstructed trajectories from the transient YS hemogenic endothelium to early hematopoietic stem/ progenitor cells (HSPCs). Using transcriptomic signatures of early and definitive hematopoiesis, we were able to parse YS HSPCs into myeloid-biased early HSPCs, rapidly outnumbered by lymphoid and megakaryocyte-biased definitive HSPCs around CS14, when hematopoietic cells first emerge from the aorta–gonad–mesonephros (AGM) region. Human embryonic liver remained macroscopically pale prior to CS14, and tracking hemoglobin subtypes led us to conclude that initial erythropoiesis is YS-restricted. This differed from the situation in the mouse, where Hb subtypes suggested two waves of pre-AGM erythropoiesis, including maturation in the macroscopically red embryonic liver.
Distinct myeloid cell types and differentiation pathways were observed pre– and post– AGM. Before CS14, monocytes were absent and macrophages originated from HPSCs via a pre-macrophage cell state. After CS14, monocytes emerged and a second, monocyte-dependent differentiation trajectory was reconstructed. A rare subset of TREM2+ macrophages, with a microglia-like transcriptomic signature, was present after CS14. Assembling a 12-organ reference of developing macrophages, we revealed that TREM2+ macrophages reside in skin, gonads, brain and AGM, but not in bone marrow, liver, kidney, thymus, mesenteric lymph nodes or gut. Using a transcriptomic signature of pre-AGM macrophages to predict the contribution of YS macrophages to developing tissue macrophage populations, we found retention of this signature in gonad, liver, skin, and AGM macrophages. The iPSC system optimized for macrophage production recapitulated the two routes to macrophage differentiation, but did not generate the diversity of macrophages (including TREM2+ macrophages) observed in developing tissues.
Our study illuminates a previously obscure phase of human development, where vital functions are delivered by the YS– a transient extraembryonic organ. Our comprehensive single cell atlas will provide a valuable resource for studying the cellular differentiation pathways unique to early life, and leveraging these for tissue engineering and cellular therapy.
Single cell datasets used in this study include: 10X data (YS, Embryonic Liver, FBM, AGM, Liver, Brain, Skin, Kidney, Thymus, Gut, MLN, Spleen, Myeloid iPSC culture and mouse gastrulation), SS2 data (YS), and CITE-seq data (CD34+/- YS and Embryonic Liver).
There are no restrictions on data availability for novel data presented in this study.All novel raw sequencing data from this study are made publicly available at ArrayExpress as FASTQs and count matrices as follows:
- i) Human embryonic liver and yolk sac 10x scRNA-seq (E-MTAB-10552)
- ii) Human embryonic yolk sac Smart-seq2 scRNA-seq (E-MTAB-10888)
- iii) Human embryonic yolk sac CITE-seq (E-MTAB-11549)
- iv) Human embryonic liver CITE-seq (E-MTAB-11618)
Please refer to our manuscript supplementary table 3 for accessions of all published data used in this manuscript. The following data are also available to download as Scanpy h5ad objects with transformed counts via our interactive webportal: https://developmental.cellatlas.io/yolk-sac:
- i) Yolk sac 10x scRNA-seq data
- ii) Yolk sac and embryonic liver 10x scRNA-seq data combined
- iii) Human gastrulation data (SS2) integrated with yolk sac scRNA-seq combined (10x)
- iv) Mouse gastrulation data (10x) - haematopoietic cells only
- v) iPSC data - macrophage lineage only
- vi) Yolk sac CITE-seq data (RNA/protein)
- vii) Embryonic liver CITE-seq (RNA/protein) Our webportal also contains a searchable database of genes implicated in inherited blood and immune cell disorders. All source data are available in the accompanying source data file, unless manuscript or figure legend refers to a Supplementary Table.
Metadata for single cell datasets described above are provided in manuscript Supplementary Tables (see manuscript Supplementary Information guide), with an overview provided in Supplementary Table 1. Source data for graphs in main and extended figures are provided as excel files. T
Please refer to our manuscript supplementary table 6 & 7 for accessions of all published data used in this manuscript. There are no known accessibility restrictions on these external datasets.
Fetal: Organs include: yolk sac (n=10,k=169494), aorta-gonad-mesonephros (AGM) (n=4,k=12248), skin (n=13, k =178563), brain (n=72, k=2.16million), gonads (n=44, k=14244), thymus (n=11, k=104251), gut (n= 5, k= 79435), kidneys (n= 4, k=26372), liver (n=14, k=210549), spleen (n=10, k=127186), bone marrow (n=8, k=93677) and mesenteric lymph node (MLN) (n=2, k=6039).
Adult: Organs include: bladder (n=3, k=2831), bone marrow (n=10, k=9408), brain (n=7, k=832), fat (n=2, k=1667), gut (n=13, k=1678), heart (n=7, k=486), kidney (n=9, k=358), liver (n=12, k=4823), lung (n=19, k=46805), lymph node (n=,15 k=2507), muscle (n=13, k=2553), pancreas (n=2, k=1030), omentum (n=4, k=103), prostate (n=2, k=356), skin (n=7, k=1088), spleen (n=15, k=13643), thymus (n=3, k=508), trachea (n=6, k=1376), uterus (n=6, k=632), vasculature (n=2, k=1602)
Cellranger count matrix files for FBM 10X data were loaded into one object as described in the Haniffa Lab Github for downstream analysis.
Generalisable analysis scripts are saved in the 'pipelines' directory. Please refer to pipelines/readme for further information on the methods used for each pipeline. The figure panels created through use of each pipeline are detailed in readme.
Custom scripts for each figure (that do not fall under 'generalisable' pipeline scripts) are saved in the 'figures' directory, where you will see all analysis performed on raw count matrix files in order to produce each figure panel for manuscript, with exception of those detailed in 'pipelines' directory. Please refer to methods section of fetal YS manuscript for further information on methods used in each custom script.
The authors of custom scripts (whether novel approaches or adapted from published workflows) are noted by initials appended to script filename as follows: Issac Goh (IG),Antony Rose) (AR), Mariana Quiroga Londono (MQL), [Simone Webb (SW),