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Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors

Nature Cell Biology volume 24, pages 1475–1486 (2022)Cite this article

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Abstract

Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.

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Fig. 1: Life-long blood progenitor expansion in the FL is modest.
Fig. 2: Validation of the utility of the ROSA26rtTa/rtTApTRE-H2BGFP+/GFP model for tracking cell divisions.
Fig. 3: Phenotypic FL-HSCs undergo many cell divisions.
Fig. 4: E14.5 FL-HSC progenitors are biased to differentiate.
Fig. 5: E14.5 FL-HSC progenitors are biased to differentiate.
Fig. 6: E16.5 FL-HSCs are transcriptionally different from adult HSCs. Updated model of the role of the FL niche in HSC ontogeny.

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Data availability

Source data for Supplementary Table 1, Figs. 1c,d, 2d, 3c,d, 4b, 4e, 4f(ii), 5b, 5c and 6a,b and Extended Data Figs. 2, 3, 4b,c and 5d,e are provided in Source Data Figs. 1–6, Source Data ED Figs. 2–5 and Source Data Supplementary Table 1. Previously published E16.5 FL-HSC and adult HSC data that were re-analysed here are available in the Gene Expression Omnibus under accession code GSE128761. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank W. Clements, the McKinney-Freeman laboratory and Department of Hematology at St. Jude Children’s Research Hospital (St. Jude) for critical discussions and reading of the manuscript; D. Ashmun, S. Schwemberger and J. Laxton for FACS support; C. Davis-Goodrum, K. Millican, A. Reap and C. Savage for help with transplants. This work was supported by the American Society of Hematology (S.M.-F.), the Hartwell Foundation (S.M.-F.), the NIDDK (K01DK080846 and R01DK104028, S.M.-F.), the American Lebanese Syrian Associated Charities (ALSAC) (S.M.-F.). M.G. is funded by the American Society of Hematology (Global Research Award), Barts Charity, Leukaemia UK (John Goldman Fellowship, 2020/JGF/001) and Medical Research Council (MRC Career Development Award, MR/V009222/1). E.O. is supported by an Edward P. Evans Foundation Discovery Research Grant, an American Society of Hematology Scholar Award and a Gabrielle’s Angel Foundation Medical Research Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Miguel Ganuza, Trent Hall, Chris Nevitt, Ashley Chabot, Emilia Kooienga, Claire Caprio & Shannon McKinney-Freeman

  2. Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK

    Miguel Ganuza & Raúl Sánchez-Lanzas

  3. Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Jacquelyn Myers & Esther Obeng

  4. Department of Biostatistics, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Juan Ding & Guolian Kang

  5. Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    David Finkelstein

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Contributions

M.G. designed the study, treated mice, performed single-cell assays, cell division history experiments, BM and FL transplants, collected and analysed data, and wrote the paper. T.H. contributed to experimental design, performed BM and FL transplants, and collected and analysed data. A.C. contributed to experimental design, and collected and analysed Confetti mice. E.K. processed and stained haematopoietic colonies. C.C. contributed mouse colony management. R.S.-L. performed and analysed single-cell assays. C.N. carried out and analysed PVA cultures. J.M. and E.O. performed transcriptional analyses of FL-HSC and adult HSCs. J.D., D.F. and G.K. performed statistical analyses. S.M.-F. designed the study, analysed data and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Miguel Ganuza or Shannon McKinney-Freeman.

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Extended data

Extended Data Fig. 1 Schematic of Confetti-allele based approach to estimate clonal complexity, flow cytometry gating and summary of Confetti labelling in blood and bone marrow.

A. Confetti-allele approach to estimate cell numbers. Ai. Schematic of Confetti allele. Aii. Mouse-to-Mouse Variance in the distribution of Confetti colors (MtMV) inversely correlates with the number of initiating events. B. Representative Confetti gating. B-cells from a TAM-treated ROSA26+/ConfettiUbiq+/ERT2-Cre mouse and a ROSA26+/ConfettiUbiq+/+ negative control are shown. C. Flow cytometry gating strategy of PB. (Ci) B-cells (B), T-cells (T) and myeloid cells (M), as well as BM compartments (Cii).

Extended Data Fig. 2 Summary of Confetti labelling in blood and bone marrow.

A. Average total PB Confetti label of mice at (Ai) two (n ≥ 8: E8-10, n = 16; E12-14, n = 18; P1, n = 11; P8-9, n = 18; P14-15, n = 13; P21-22, n = 8) and (Aii) six months of age (n ≥ 4: E8-10, n = 5; E12-14, n = 9; P1, n = 11; P8-9, n = 18; P14-15, n = 13; P21-22, n = 4). B. Average total Confetti labeling in the BM at six months of age (n ≥ 4: E8-10, n = 5; E12-14, n = 9; P1, n = 11; P8-9, n = 18; P14-15, n = 13; P21-22, n = 4). A-B. Related to Fig. 1C-D and Extended Data Fig. 3. A-B. Means are shown. Error bars indicate standard deviation. Individual data points are shown in black.

Source data

Extended Data Fig. 3 Numbers of fetal liver hematopoietic progenitors contributing to specific adult blood compartments.

MtMV-based estimates of numbers of progenitors contributing to PB at P60 (n ≥ 8: E8-10, n = 16; E12-14, n = 18; P1, n = 11; P8-9, n = 18; P14-15, n = 13; P21-22, n = 8) (A) and P180 (n ≥ 4: E8-10, n = 5; E12-14, n = 9; P1, n = 11; P8-9, n = 18; P14-15, n = 13; P21-22, n = 4) (B) labeled at distinct windows of ontogeny are shown. Total white blood cells (WBC), B-cells (B), T-cells (T) and myeloid cells (M). Related to Fig. 1C. Error bars indicate the 95% confidence intervals.

Source data

Extended Data Fig. 4 Window of active labelling of DOX in vivo.

A. Experimental schematic. ROSA26 rtTa/+ Col1a1tetO-H2B-GFP/+ CD45.2+ donor BM68 was pooled from five donors and transplanted into CD45.1+/CD45.2+ recipient mice previously treated with DOX on day three (-3), two (-2) or one (-1) before transplantation or on the same of transplant (day 0) (n = 3 recipients per group). B. %GFP+ BM of recipients 4, 7, or 11 days post-transplant (Trx). C. To corroborate the ability of non-labelled transplanted CD45.2+ cells to respond to DOX, CD45.2+ c-Kit+ sorted cells from each mouse cohort were cultured in vitro in the presence or absence of DOX showing that cells were responsive to DOX in vitro.

Source data

Extended Data Fig. 5 Fetal liver factor ANGPTL3 is not able to promote self-renewing expansion of E14.5 FL-HSCs.

A. Gating strategy on E12.5-CD45+c-Kit+ cells and E14.5-c-Kit+ and E14.5-HSC cells. Related to Fig. 3B-C & 4. B-D. LSK CD150+CD48−C57Bl/6 HSCs were isolated from E14.5 FL or adult BM and cultured in PVA cultures tailored for self-renewing expansion either with or without addition of ANGPTL3, for 2 weeks. Immunophenotypic HSC expansion was then quantified. Due to known immunophenotypic shifts during PVA culture, HSC were defined as LSK CD150+EPCR+ after culture. B. Experimental schematic. C. Gating strategy of LSK CD150+EPCR+ HSC after culture. D. Proportion of wells expanding, defined as containing at least 100 immunophenotypic LT-HSCs. E. For those wells showing cell expansion, HSC expansion as a ratio of output/input is shown. For each condition, 5 biological replicates and 2 independent experiments with 10 wells/replicate. Each circle/square represents an individual biological replicate. Means and standard deviations are depicted. For (D), the Holm-Sidak method (2-tailed) was used to calculate statistical significance and correct for multiple comparisons. Exact p-values are shown in Figure.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1. Estimates of initiating cell numbers contributing to adult haematopoiesis. Supplementary Table 2. Antibodies, key chemicals, cell lines, experimental models, oligonucleotides and software.

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Ganuza, M., Hall, T., Myers, J. et al. Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors. Nat Cell Biol 24, 1475–1486 (2022). https://doi.org/10.1038/s41556-022-00999-5

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