Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

The human genome (GRCh38) sequence data used in this study is available through the Ensembl genome browser: http://ftp.ensembl.org/pub/release-104/fasta/ homo_sapiens/dna/ The mouse genome (GRCm38) sequence data used in this study is available through the Ensembl genome browser: http://ftp.ensembl.org/pub/release-104/fasta/ mus_musculus/dna/ Source data are provided with this paper.
No sample size calculation was performed. CITE-seq analysis was performed on one fetal liver sample as the cost was prohibitive to profile several biological repeats at this resolution. The observed expression patterns were validated across 5 additional fetal liver samples using an orthogonal approach (multi-parameter cytometry).
During CITE-seq analysis and quality control, cells with more than 25% of reads mapping to mitochondrial genes were filtered out. A cluster representing cells with high mitochondrial content and mixed lineage identities was also excluded from downstream analysis. This is indicated in the methods. The exclusion criteria were not pre-established and reflect what is commonly done in the field in terms of quality control.
Multi-dimensional flow cytometry characterization confirmed the expression patterns observed based on antibody-derived tag sequencing in the CITE-seq experiment and validated these findings across five biologically distinct fetal liver samples A list of the antibodies used is provided below. These are all commercially available antibodies and have been validated by the manufacturer for their target and application. Validation information, recommended dilution and references are all available on the manufacturer's website and can be accessed through the links provided in this antibody list as well as by using the catalog numbers listed in Supplementary tables 1 and 5.
Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.
All cell lines used tested negative for mycoplasma.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

nature research | reporting summary
April 2020

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals

Wild animals
Field-collected samples

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
The study did not involve samples collected from the field.
All mouse research complied with the Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital (Protocol #2009N000136) This study was reviewed by the Mass General Brigham Institutional Review Board (IRB Protocol #2016P001106) and was determined not to constitute human subjects research given its use of de-identified, discarded material. All human donor tissues were obtained under the approved protocol from participants who consented in writing to the use of donations for research. Participants received no compensation. FL cells were thawed and allowed to recover at 37C for an hour prior to staining. Cells were blocked with TruStain FcX (BioLegend, 422301) and stained with TotalSeq A antibody mix containing 1ug of each TotalSeq A antibody per condition (BioLegend, see Table S1 for a list of antibodies). Anti-human CD235a-APC antibody (BD Biosciences, 551336) was added to the CD34-fraction. The CD34+ fraction was stained with anti-human CD34-APC antibody (BD Biosciences, 555824) and antihuman GPI-80-PE antibody (MBL International, D087-5). All samples were stained with calcein blue (Invitrogen, C34853) for live/dead exclusion prior to sorting. Cells and single stain controls were analyzed on a 5-laser Aurora spectral flow cytometry (Cytek Biosciences) and raw fluorescence data from 64 channels were unmixed using ordinary least square algorithm in Spectraflo v2.2.1 (Cytek Biosciences). For cell sorting, a Beckman Coulter MoFlo Astrios device was used with Summit v6.3.1 software. Transplantation experiments were read out on a Stratedign S1300 with CellQuest v.4.1 software.