Blood and immune development in human fetal bone marrow and Down syndrome

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 11-12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. B-lymphocytes expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window. There are no restrictions on data availability for new data presented in this study. FASTQ and raw count matrices for droplet-based scRNA-seq data for FBM from fetuses with Down syndrome and fetuses without Down syndrome have been deposited at EMBL-EBI ArrayExpress and at the European Nucleotide Archive (ENA), with accession codes as follows: E-MTAB-9389 (FBM from fetuses with Down syndrome and FBM from fetuses without Down syndrome), E-MTAB-10042 (FBM from fetuses with Down syndrome) and ERP125305 (FBM from fetuses without Down syndrome). FASTQ and raw count matrices for all other new data in this study have been deposited at EMBL-EBI ArrayExpress and at the Gene Expression Omnibus (GEO) with accession codes E-MTAB-9801 (FBM Smart-seq2 scRNA-seq); E-MTAB-9389 (BCR- or TCR-enriched VDJ FBM scRNA-seq; FASTQs only); GSE166895 (CD34+ FBM, fetal liver and cord blood CITE-seq) and GSE166895 (FBM total CITE-seq). The following data are also available to download as Scanpy h5ad objects with transformed counts through our interactive webportal: https://fbm.cellatlas.io/: (i) FBM scRNA-seq (fetuses with Down syndrome); (ii) FBM scRNA-seq (fetuses without Down syndrome); (iii) CD34+ FBM, fetal liver and cord blood CITE-seq; and (iv) FBM total CITE-seq. All source data are available in the accompanying source data file, unless manuscript or figure legend refers to a Supplementary Table. External datasets incorporated into this study include: (i) human fetal liver and yolk sac scRNA-seq data4 (E-MTAB-7407); (ii) human blood monocyte–DC scRNA-seq data10 (GSE94820); (iii) mouse BM scRNA-seq data19 (GSE122467); (iv) fetal and paediatric thymus scRNA-seq data3 (E-MTAB-8581); and (v) adult BM and cord blood scRNA-seq data from the Human Cell Atlas Data Coordination Portal ‘Census of Immune Cells’ project (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79). At the time of submission, there are no known accessibility restrictions on these external datasets. Source data are provided with the paper. Cell count: Total post-QC is 164,709: - 119,971 for 10x scRNA-seq (103,228 from fetal bone marrow; 16,743 from Down syndrome fetal bone marrow) - 486 for Smart-seq2 scRNA-seq (from fetal bone marrow) - 44,252 for CITE-seq (35,273 from CD34+ fetal bone marrow, fetal liver, and cord blood; 8,978 from non-FACs sorted fetal bone marrow)

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