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A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness

Nature Immunology volume 22, pages 723–734 (2021)Cite this article

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Abstract

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

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Fig. 1: INKA1 and H4K16ac show inverse abundance and visualize heterogeneity in HSC subsets.
Fig. 2: Single-cell transcriptome and protein analysis validates that INKA1 versus its targets PAK4 and H4K16ac are on opposing ends of cell cycle priming and progression.
Fig. 3: PAK4 inhibition induces G0 arrest in vitro, maintaining stem cell regenerative capacity.
Fig. 4: INKA1-OE LT-HSCs resist activation in vitro.
Fig. 5: INKA1 overexpression and PAK4 knockdown preserve long-term stem cell regenerative potential under regeneration-mediated stress.
Fig. 6: INKA1–PAK4 interactome identifies SIRT1 as the link between INKA1 and H4K16ac, and CD112 as a HSPC marker.
Fig. 7: CD112 surface expression delineates alternatively quiescent states of LT-HSCs.
Fig. 8: CD112hi LT-HSCs are primed, whereas CD112lo demarcates latent LT-HSCs.

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Data availability

The RNA-seq and ATAC–seq data generated for this paper are available from the Gene Expression Omnibus under accession number GSE148885, and previously published RNA-seq data are available under accession number GSE125345. Raw RNA-seq and ATAC–seq data are available on The European Genome-phenome Archive (EGA) under accession number EGAD00001006541 (bulk RNA-seq: EGAS00001004768; scRNA-seq: EGAS00001004769; ATAC–seq: EGAS00001005121). For GSEA, GOBP gene sets were retrieved from http://baderlab.org/GeneSets/ (version February 2020), the Hallmark gene set v.7.0 from ftp://ftp.broadinstitute.org://pub/gsea/gene_sets/h.all.v7.0.symbols.gmt, retrieved through GSEA 4.0.3, and Eppert HSC-R from http://www.gsea-msigdb.org/gsea/msigdb/cards/EPPERT_HSC_R.html. Source data are provided with this paper.

Code availability

Code generated during this study is available at https://github.com/andygxzeng/HSC_Pseudotime.

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Acknowledgements

We thank the obstetrics units of Trillium Health and William Osler Health Partners for CB, the UHN-SickKids Flow cytometry facility for cell sorting, and M. Minden and the Leukemia Tissue Bank at Princess Margaret Cancer Centre/UHN for providing primary mPB samples. We thank E. Laurenti and all members of the laboratory of J.E.D., in particular J. C. Y. Wang, for critical feedback. This research was supported by the Deutsche Forschungsgemeinschaft (K.B.K.) and is part of the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. Work in the laboratory of J.E.D. is supported by funds from the Princess Margaret Cancer Centre Foundation, the Canadian Institutes of Health Research (Foundation no. 154293 (to J.E.D), operating grant nos. 154293 and 89932 (to J.E.D), International Development Research Centre, Canadian Cancer Society (grant no. 703212 (to J.E.D)), Terry Fox Research Institute Program Project grant, Ontario Institute for Cancer Research through funding provided by the Government of Ontario, a Canada Research Chair and the Ontario Ministry of Health and Long Term Care.

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Authors and Affiliations

  1. Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

    Kerstin B. Kaufmann, Andy G. X. Zeng, Etienne Coyaud, Laura Garcia-Prat, Alex Murison, Estelle M. N. Laurent, Michelle Chan-Seng-Yue, Olga I. Gan, Kristele Pan, Jessica McLeod, Héléna Boutzen, Sasan Zandi, Shin-ichiro Takayanagi, Brian Raught, Stephanie Z. Xie & John E. Dick

  2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Andy G. X. Zeng & John E. Dick

  3. New York Genome Center, New York, NY, USA

    Efthymia Papalexi & Rahul Satija

  4. Center for Genomics and Systems Biology, New York University, New York, NY, USA

    Efthymia Papalexi & Rahul Satija

  5. PanCuRx Translational Research Initiative, Ontario Institute for Cancer Research, Toronto, Ontario, Canada

    Michelle Chan-Seng-Yue

  6. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

    Sasan Zandi

  7. Cell Therapy Project, R&D Division, Kirin Holdings, Tokyo, Japan

    Shin-ichiro Takayanagi

  8. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Brian Raught

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Contributions

K.B.K. conceived the study, performed research, analyzed data and wrote the paper. E.M.N.L., O.I.G., K.P., J.M., H.B., S.Z. and S.T. performed research. R.S. and B.R. supervised specific experiments. A.G.X.Z., O.I.G. and S.Z.X. edited the paper. S.Z.X. provided conceptual input. J.E.D. wrote the paper, secured funding and supervised the study.

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Correspondence to John E. Dick.

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Competing interests

J.E.D. served on the Scientific Advisory Board at Trillium Therapeutics, reports receiving a commercial research grant from Celgene, and has ownership interest (including patents) in Trillium Therapeutics. S.T. is employed by Kirin Holdings. All other authors declare no competing interests.

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Peer review information Nature Immunology thanks Gay Crooks and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 INKA1 and H4K16ac are mutually exclusive.

a, Representative sorting scheme to obtain LT-, ST-, IT90+- IT90- HSCs or CD34 + CD38- and CD34 + CD38 + cells from lineage depleted CB. b, Confocal images of 2 individual ST-HSC to visualize heterogeneity and representative for n = 3 independent CB pools analyzed. Scale bar = 2 μm. c, Confocal images of 4 individual, uncultured LT-HSC next to thereof derived images for INKA1 and H4K16ac channels after thresholding and binary conversion using ImageJ to visualize intracellular invers staining pattern in double dim cells of the heterogenous cell pool and representative for n = 3 independent CB. Scale bar = 4 μm. d, Percentage INKA1+ cells in HSC subsets according to INKA1+ cell gate in Fig. 1d (immunostaining, ~50 cells/population and CB, n = 3 independent CB pools). Mean ± SD. e, Confocal images and quantification (MFI) of prospectively isolated LT- and ST-HSC stained for INKA1 and CDK6 and representative for n = 3 independent CB as summarized in right panel. Gating as used for Fig. 1f is indicated. Scale bar = 2 μm.

Source data

Extended Data Fig. 2 Mobilized PB single cell RNA pseudotime analysis.

a, Cell cycle analysis (Ki-67, Hoechst, CDK6) of prospectively isolated HSC subsets (LT-, IT90+-, IT90−, ST-HSC) from mPB (n = 3) by flow cytometry. CB HSPC (CD34+CD38−) and Progenitors (Prog, CD34+CD38+) served as gating controls. Mean ± SD; two-sided paired t-test (**p = 0.002). b-e, Single cell RNA-seq data from CD34+CD38−CD45RA− index-sorted mPB HSC (n = 3, also stained and indexed for CD49f, CD90) visualized by diffusion mapping overlaid with (b) cell cycle score (Tirosh et al.22) and indicated transcript expression; (c) donor origin, (d) ITGA (CD49f) transcript expression, (e) CD49f and CD90 indexed protein surface expression and (f) retrospectively gated HSC subsets (LT-, IT90+, IT90−, ST-HSC).

Source data

Extended Data Fig. 3 Protein single cell pseudotime analysis.

a, Gating strategy for cell cycle phases of 2d with 20 hr EdU pulse cultured CB Lin-. b,c, CB LT- and ST-HSC analyzed by immunostaining for Ki-67, H4K16ac, PAK4 and DAPI after 72 hrs of culture; plots in (h) show data combined from 3 independent CB pools (~50 cells/population and sample). Scale bar = 4 µm. d, Longitudinal confocal imaging of LT-HSC harvested at 5 timepoints of culture in high-cytokine conditions. Two individual LT-HSC are shown per timepoint. Scale bar = 4 μm. e, Individual cell fluorescence intensities (IntDen) of nuclear CDK6, H4K16ac, PAK4 and DAPI across pseudotime for HSC subsets (n = 2913). Curve fit was performed with nonlinear regression excluding 1-2% of outliers (fifth order polynomial). f, Pseudotime was split in early, mid and late fractions to visualize distribution of HSC subsets and Progenitors and marker levels across these fractions for all 3442 cells (histograms) or per population and CB (n = 2-3, bar plots). Single cell distribution for each population across pseudotime is also shown in horizontal violin plot. Mean ± SD, two sided t-test (compared to “early”; *p < 0.05; **p < 0.01; ***p < 0.001).

Source data

Extended Data Fig. 4 Pharmacological PAK4 inhibition.

a, Brightfield images of HSPC (CD34+CD38−) and Progenitors (CD34+CD38+) after 5 d treatment with either DMSO or PF-3758309, reproducible across all independent repetitions (n > 9). scale bar = 100 µm. b-e, CB Lin− cells were treated after 2d of prestimulation with either DMSO or PF-3758309 (PF). Concentration as indicated. After 5d of treatment cells were counted (c, n = 2), cell cycle (b, d) combined with CD34 analysis was performed (pool of n = 2). f-g, CB Lin− cells treated after 2 days prestimulation for 5 d with 0.25 uM PF-3758309 or DMSO equivalent were subjected to AnnexinV and CD34 staining (n = 3-4). Mean ± SD; two-sided ratio paired t-test. h-j, CB HSPC and Progenitors treated for 4 and 6 d with 0.25 uM PF-3758309 or DMSO equivalent were subjected to cell cycle analysis (h), counting (i: HSPC; j: Progenitors). Mean ± SD, two-sided paired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). k, Cell counts of treated HSPC on day of injection (d5 treatment, d7 culture). Mean ± SD. l, Secondary LDA (8 wk) in NSG and NSG-SGM3 mice of human CD45+ cells from CB Lin- cells transplanted NSG grafts (24 wk).

Source data

Extended Data Fig. 5 INKA1-OE mediated effects in vitro.

a-c, CTRL and INKA1-OE HSPC were sorted (BFP + ) d6 post transduction and subjected to immunostaining (n = 2) and INKA1 levels were determined (a); two-sided t-test. Colocalization was quantified in (b) by Coloc2/ImageJ and Spearman’s rank correlation values for single cells with Costes’ P-value = 1.00 (**p = 0.0041); and in (c) by proximity ligation assay (PLA) with background set according to negative controls (IgG1: anti-PAK4 + rabbit IgG; IgG2 anti-INKA1 + mouse IgG); two-sided Mann-Whitney-test (b,c; ***p < 0.0001). d, Cell viability of transduced HSPC was determined via dye exclusion (PI, 7AAD; n = 9). e, CTRL and INKA1-OE LT- and ST-HSC (n = 4-5) were sorted (BFP+) d6 post transduction and subjected to cell cycle analysis by flow cytometry. f, Distribution of PAK4 protein levels in nucleus vs. cytoplasm according to confocal analysis of CTRL and INKA1-OE LT- and ST-HSC; two-sided Mann-Whitney-test (***p = 0.0005). g, Differentially expressed genes with adjusted p-value<0.01 (according to DESeq2, **p = 0.003, ***p-value range: <10-4-10-26) in either LT- or ST-HSC transduced with INKA1-OE vs CTRL (log2FC).

Source data

Extended Data Fig. 6 INKA1-OE mediated effects in in vitro differentiation assays.

a, CB LT- and ST-HSC were cultured, transduced (OE) and on day 3 ptd BFP+ cells were sorted into methylcellulose for colony formation, scored and harvested 12 days later and replated (right) for 12 additional days (n = 4). Colony type composition of primary and secondary colony formation assays (CFU-C) is shown in the lower panels; Mean ± SD, two-sided paired t-test. b, Total single cell colony output (d15) from stromal coculture assays per CB (n = 3 independent pools, Mean ± SD) and subfraction of CTRL and INKA1-OE transduced HSC subsets as indicated. My = myeloid (CD33+), E = erythroid (CD45-GlyA+/CD71+), Mk = megakaryocytic (CD41+), NK = CD56+cells. Upper right panel shows proliferative potential and lower panel total colony output per assay, population and condition as assessed by flow cytometry; two-sided paired t-test.

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Extended Data Fig. 7 INKA1-OE, shINKA1 and shPAK4 in xenograft assays.

a, Gating strategy for BM analysis from xenografts. b, Human CD45 chimerism in injected femur (right, RF) and non-injected bone marrow of CB HSPC transduced with CTRL or INKA1-OE at various timepoints post-transplant (5 independent CB pools). Each cell represents individual mouse. c, Relative engraftment in non-injected BM of transduced cells represented as log2 ratios (%BFP+ of CD45+ in vivo output /%BFP+ in vitro input) of 5 independent experiments, two-sided t-test with Welch’s correction (**p = 0.006 and p = 0.008). d, Lineage composition within BFP+ xenografts at 20 wk post xenotransplantation of CTRL and INKA1-OE HSPC (5 experiments with 4-9 mice/group each). e, Flow cytometric analysis of 20 wk BM grafts gated on CD45+BFP+CD34+CD19-CD38- population; Mean ± SD. f, Individual data point representation related to Fig. 5e; mice per group:10/9(4w) - 5/5/5/5 (12w)- 9/7/9/8 (20w). g, Human CD45+ cell counts from injected femurs from NSG mice transplanted with CTRL or INKA1-OE HSPC at indicated timepoints and after single dose PBS or 5-FU treatment at 4 wk; Mean ± SEM, two-sided Mann-Whitney-test. h, Fold change (FC) of CD45+ counts in injected femur at 12 wk relative to 4wk of CTRL and INKA1-OE HSPC transplanted and PBS/5FU treated mice; Mean ± SD, two-sided t-test (*p = 0.043). i, Injected femurs (20w) were analyzed for %CD34+ (left) and total pooled bone marrow was subjected to hierarchy analysis at 20w of CTRL and INKA1-OE HSPC after treatment with PBS or 5-FU at 4w. Pie chart represents CD34 compartment and radius is scaled to percentage CD34. Mean ± SD, two-sided t-test. j, Four shRNAs embedded in lentiviral vectors were tested each, for INKA1 and PAK4 knockdown, along with shRenilla as control in MOLM13 cells. Transduced cells were sorted (BFP+) and subjected to qRT-PCR for the targeted transcript. Vector copy number (VCN) was estimated by initial transduction efficiency assuming Poisson distribution to correct for VCN introduced bias in knockdown-efficiency. Highlighted in color are the shRNAs chosen for functional assays; Mean ± SD, 3 technical replicates. k, Summary of secondary LDA analysis in NSG-SGM3 mice at 8w of shRNA transduced primary grafts (20w). Mice were considered engrafted at human CD45 chimerism >0.1. l, Lineage composition of BFP+ NSG grafts from shRNA transduced HSPC as indicated (20 wk). Mean ± SD.

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Extended Data Fig. 8 INKA1 and PAK4 protein interactions identify SIRT1 as INKA1 interactor.

a, RNA expression (GSE125345) of PAK4/INKA1 interactome as identified by BioID across human CB HSPC and Progenitors. b, Pathway (GO processes) enrichment map (FDR < 0.05, selected) of BioID interactome via Cytoscape. c, Proximity ligation assay of HSPC and Progenitors including staining controls (90 cells, 6 variants as in scheme; 3 independent CB pools). Background was set according to controls. Four PLA signal positive cells are shown enlarged. d, Shown are confocal images of two separate immunostainings of extended chromatin fibers generated from CB HSPC (CD34+CD38-) that were either stained for H4K16ac and SIRT1 (left) or SIRT1 and INKA1 (right). Results were reproducible in 2 independent repetitions. Scale bar = 2 µm. e, Cultures of transduced (OE) CB HSPC (CD34+CD38-; n = 3) were treated with DMSO or 25 µM EX-527 starting 3 days after transduction. EdU was added on d4 and cells were analyzed 16 hrs later by flow cytometry for %EdU+ cells and DNA synthesis rate by Edu MFI within transduced/BFP+ cells; Mean ± SD, two-sided paired t-test (*p < 0.05; **p = 0.004).

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Extended Data Fig. 9 INKA1 and PAK4 protein interactions implicate CD112 in HSPC biology.

a,b, Absolute and relative Nectin2 and AFDN expression from GSE125345(a) and AFDN expression in mPB scRNAseq dataset across diffusion pseudotime (b). c, CD112 levels in CB mononuclear cells (MNC) gated on CD34+CD19+ and CD34+CD19— subpopulations and CD112 vs CD19 expression in CD34+ cells from 20w xenografts. d,e, Flow cytometric analysis of CD34 and CD112 surface expression on CB Lin- cells and CB MNC. FMO + IgG = fluorescence minus one staining without anti-CD112-APC but IgG-APC; two-sided t-test (*p < 0.05, **p < 0.01, ***p < 0.001). f-g, Single cell index-sorting (indexed intensities: CD49f, CD90 and CD112) of 2800 CB CD34+CD38-CD45RA- cells onto MS5 stromal layers followed by 18 days in vitro differentiation and analysis via high-throughput flow cytometry (n = 4). LT- and ST-HSCs were retrospectively gated according to initially indexed CD49f and CD90 levels and respective colony output (f) and colony output per individual CB pool is shown in (g). h, Cultured and transduced CB CD34 + CD38- cells were analyzed for CD112 levels 5-6 d post-transduction of subgated cLT-(CD34+CD45RA-CD90+), cST-(CD34+CD45RA-CD90-) HSC, cProgenitors (CD34+CD45RA+) or total cHSPC (CD34+). c = cultured, two-sided paired t-test (*p < 0.05, **p = 0.004). i, Confocal images of BFP + sorted CB LT- and ST-HSC 6d after transduction followed by immunostaining (n = 3; ~45 cells/ sample); two-sided Mann-Whitney-test. j, Confocal images of immunostaining of CB LT- and ST-HSC (total 74 cells from 3 CB) and thereof derived Spearman’s rank correlation values (calculated by Coloc2/ImageJ) for the indicated parameter pairs, only non-nuclear cell area was considered.

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Extended Data Fig. 10 CD112 separates primed from non-primed LT-HSC.

a,b, Post-sort analysis of LT- and ST-HSC sorted for CD112lo and CD112hi either directly after sorting on the same instrument (a) or in parallel with CellRox staining after 30 min cytokine free culture(b; see g,h) on a FACS analyzer. c, Representative plots of flow cytometric analysis of LT- and ST-HSC sorted for CD112lo and CD112high directly or after 42 hrs culture for CDK6 and Ki-67. d, Quantification of flow cytometric analysis of LT- and ST-HSC sorted for CD112lo and CD112hi directly or after 42 hrs culture for CDK6 and Ki-67 (n = 3; paired t-test). e-g, LT- and ST-HSC sorted for CD112lo and CD112hi (n = 4-7) were analyzed directly via immunostaining for INKA1 (e; total of 1587 single cells from 7 independent CB pools, whiskers indicate 10-90 percentile, Kruskal-Wallis test) or for CellROX labeling after 30 min cytokine-free culture and assessed by flow cytometry (f) and fluorescence microscopy (g); box-and-whisker plots with median as centre and either 10-90% percentile (e) or minimal to maximal values are represented by whiskers (f). Two-sided t-test (f,g; *p < 0.05, **p < 0.01, ***p < 0.001) (h,i) LT-HSC sorted for CD112lo (n = 2) and CD112hi (n = 3) were subjected to ATAC-seq. The number of 500 bp windows overlapping called peaks exclusively in two CD112hi or CD112lo replicates but not in the other is shown in (h) and (i) is a heatmap of -log P-Value of enrichment for known motifs enriched in either CD112hi or CD112lo, relative to a background of all called peaks. CD112hi versus CD112lo LT-HSC and INKA1-OE vs CTRL LT-HSC. k, GSEA results for CD112hi versus CD112lo RNAseq data (3472 GO biological processes; gene set size 25-500; FDR < 0.1). nES = normalized enrichment score; *FDR < 0.05; **FDR < 0.01. l, Expression of leading-edge genes from mTORC1 and GOBP gene sets (2-3 independent CB pools). m, Lineage composition of transplanted CD112lo and CD112hi LT-HSC. Each column represents an individual mouse. n, Summary of secondary LDA from 12 wk primary grafts into NSG-SGM3 for additional 8 wk for 3 independent CB pools. Same cell numbers per dose were transplanted from initially transplanted CD112lo and CD112hi LT-HSC per CB but varied for individual CB cohorts.

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Supplementary information

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1, High-confidence interactors of INKA1 and PAK4 according to proximity-dependent BioID assay. Supplementary Table 2, Differentially expressed genes (289) between CD112lo and CD112hi LT-HSCs (statistics calculated by DESeq2). Supplementary Table 3, Details of commercially available reagents and other materials and resources used.

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Kaufmann, K.B., Zeng, A.G.X., Coyaud, E. et al. A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness. Nat Immunol 22, 723–734 (2021). https://doi.org/10.1038/s41590-021-00925-1

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