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Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone

Nature Biomedical Engineering volume 9, pages 754–771 (2025)Cite this article

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Abstract

The engraftment of haematopoietic stem and progenitor cells (HSPCs), particularly in cord-blood transplants, remains challenging. Here we report the role of the corticotropin-releasing hormone (CRH) in enhancing the homing and engraftment of human-cord-blood HSPCs in bone marrow through mechanical remodelling. By using microfluidics, intravital two-photon imaging and long-term-engraftment assays, we show that treatment with CRH substantially enhances HSPC adhesion, motility and mechanical remodelling, ultimately leading to improved bone-marrow homing and engraftment in immunodeficient mice. CRH induces Ras homologue gene family member A (RhoA)-dependent nuclear translocation of the yes-associated protein (YAP), which upregulates the expression of genes encoding extracellular-matrix proteins (notably, thrombospondin-2 (THBS2)). This process guides the mechanical remodelling of HSPCs via modulation of the actin cytoskeleton and the extracellular matrix, with THBS2 interacting with the integrin αvβ3 and coordinating the nuclear translocation of YAP upon CRH/CRH-receptor-1 (CRH/CRHR1) signalling. Overall, the CRH/CRHR1/RhoA/YAP/THBS2/αvβ3 axis has a central role in modulating HSPC behaviour via a mechanical feedback loop involving THBS2, αvβ3, the actin cytoskeleton and YAP signalling. Our findings may suggest avenues for optimizing the transplantation of HSPCs.

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Fig. 1: CRH treatment enhances HSPC adhesion, motility and mechanical remodelling.
Fig. 2: Intravital two-photon imaging reveals enhanced HSPC motility and homing to bone marrow in NSG mice after CRH treatment.
Fig. 3: CRH promotes HSPC homing and long-term engraftment in NSG mice.
Fig. 4: Bulk RNA-seq analysis reveals CRH-induced upregulation of ECM remodelling genes, particularly THBS2.
Fig. 5: CRH-induced RhoA activation and YAP nuclear translocation in HSPCs lead to THBS2 upregulation.
Fig. 6: THBS2–integrin αvβ3 interaction coordinates YAP nuclear translocation on CRH/CRHR1 signalling.
Fig. 7: CRH modulates HSPC biomechanics and homing via CRHR1/RhoA/YAP/THBS2/integrin αvβ3 signalling.

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Data availability

The RNA-seq data generated in this study are available from the Gene Expression Omnibus under accession number GSE240116. The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available from the corresponding authors on reasonable request. Source data for the figures are provided with this paper.

Code availability

Codes for data visualization are available in GitHub at https://github.com/Tility/CRH_RNAseq (ref. 67).

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Acknowledgements

We acknowledge support from the National Key Research and Development Program of China (2019YFA0801800), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0940201), the Natural Science Foundation of China (32070916, 82370159, 12025207 and 11872357), the Extension Grant of the Anhui Provincial Distinguished Young Scholars Science Foundation (2408085JX012), Research Funds of the Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20220007) and the USTC Research Funds of the Double First-Class Initiative (YD2090002024).

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Author notes

  1. These authors contributed equally: Mingming Wu, Haoxiang Yang, Senquan Liu.

Authors and Affiliations

  1. Department of Hematology, The First Affiliated Hospital of USTC, Key Laboratory of Immune Response and Immunotherapy, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Institute of Blood and Cell Therapy and Anhui Provincial Key Laboratory of Blood Research and Applications, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Mingming Wu, Senquan Liu, Tingting Liang, Yan Wang, Mingming Zhu, Xian Song, Xiaoyu Zhu, Linzhao Cheng & Fang Ni

  2. Institute of Immunology, The CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Mingming Wu, Tingting Liang, Yan Wang, Mingming Zhu, Xian Song & Fang Ni

  3. The CAS Key Laboratory of Mechanical Behavior and Design of Materials, University of Science and Technology of China, Hefei, China

    Haoxiang Yang, Hao Liu, Jinghao Shen, Shuangzi Wang & Hongyuan Jiang

  4. Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Lai Jiang

  5. Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Winship Cancer Institute, Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA

    Cheng-Kui Qu

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Contributions

F.N., H.J. and L.C. conceived and conducted the project. F.N., H.J., M.W. and H.Y. designed the experiments; M.W., H.Y. and S.L. conducted the research and summarized the data. T.L. analysed the bulk RNA sequencing data. Y.W., M.Z., X.S., H.L., J.S. and S.W. provided critical advice on experimental designs and interpretation of the data, and edited the manuscript. L.J. and S.L. collected human samples. X.Z. provided comments and suggestions. C.K.Q. discussed the work, provided advice, and edited the manuscript. M.W., H.Y., H.J. and F.N. wrote the manuscript. F.N. supervised the project.

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Correspondence to Linzhao Cheng, Hongyuan Jiang or Fang Ni.

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Extended data

Extended Data Fig. 1 CRH enhances HSPC adhesion and migration via CRHR1.

a, Representative fluorescent images of CRHR1 and CRHR2 expression in CB CD34+ cells. b, Statistical analysis of the adhesion of CB CD34+ cells to HUVECs (n = 3 independent experiments). c, d, Measurement of relative sizes of non-treated and CRH-treated CB CD34+ cells (1 × 105) by FACS, based on forward scatter (FSA) (c), and Leica microscopy (d), (n = 3 independent experiments). e, Representative images of the cell cycle of non-treated and CRH-treated CB CD34+ cells, and statistical analysis of the percentage of cells in G0, G1, and S/G2/M phases (n = 4 independent experiments). f, Fold-change in total cell numbers after culture of 2 ×104 CB CD34+ cells from non-treated or CRH-treated groups for 7 days (n = 5 independent experiments). g, CFU assay results from analysis of 500 CB CD34+ cells from non-treated or CRH-treated groups (n = 5 independent experiments). h, Statistical analysis of CB CD34+ cell migration speed in the non-treated, CRH-treated, and NBI pretreated before CRH treatment groups in a 3D HSPC mobility assay (n = 3 independent experiments). i, Immunofluorescence images of F-actin (green), myosin II (red) and nuclei (DAPI, blue) in non-treated and CRH-treated CB CD34+ cells, with or without 2.5 µM Lat A pretreatment, and statistical analysis of F-actin and myosin II intensity at the cell cortex (n = 3 independent experiments). j, Mechanical stiffness measurements of CB CD34+ cells in the non-treated, CRH-treated, and 2.5 µM Lat A pretreated before CRH treatment groups by Atomic-force microscopy (AFM) (n = 45–68 individual cells). k, Immunofluorescence images of F-actin (green), myosin II (red) and nuclei (DAPI, blue) in non-treated and CRH-treated CB CD34+ cells seeded on 10 μg/ml FN, with or without 2.5 µM Lat A pretreatment, and statistical analysis of F-actin and myosin II intensity at the protrusions (n = 3 independent experiments). l, Protrusion lengths were analyzed in non-treated and CRH-treated CB CD34+ cells, with or without 2.5 µM Lat A after seeding on 10 µg/ml FN for 4 h (n = 80–89 individual cells). m–o, Real-time time-lapse images (m), statistical analysis of the velocity (n), and protrusion extension frequency (o), of CB CD34+ cells seeded on culture dishes pre-coated with 10 μg/ml FN, observed using the Leica imaging system (n = 90 individual cells per group). For e–g, parametric data were analyzed using two-tailed Student’s t-test. For b and h, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For d, nonparametric data were analyzed using the Mann‒Whitney test. For i–l, n and o, nonparametric data were analyzed using Kruskal‒Wallis test with correction for multiple comparisons by Dunn’s test. Data are mean ± s.d. values.

Source data

Extended Data Fig. 2 Intravital two-photon imaging reveals enhanced HSPC motility and bone marrow homing in NSG mice following CRH treatment.

a, Picture of tail vein cannulation used for CB CD34+ cell infusion. b, c, Pictures of NSG mouse skull imaging (b), and head fixation (c), device installation. d, Photo of the observation window for two-photon imaging of the skull of a surgically operated NSG mouse. e, Representative fluorescent images of in vivo imaging of HSPCs in the skulls of NSG mice and analysis of the position and ratio of CB CD34+ cells both inside and outside vessels at 6 and 16 h after transplantation (n = 7 mice per group). Blood vessels were indicated by dashed white lines. Red, CD31+ vascular endothelial cells. Green, CB CD34+ cells. White arrows, CB CD34+ cells outside vessels; blue arrows, CB CD34+ cells inside vessels. f–i, Representative fluorescent images of non-treated and CRH-treated CB CD34+ cells in the skull bones of NSG mice at 0–2 (f), 6 (g), 16 (h), and 24 (i) h after transplantation. Red, CD31+ vascular endothelial cells. Green, CB CD34+ cells (n = 7 mice per group). j, Representative flow cytometry dot plots showing CMFDA+ cells in NSG mouse skull bone marrow and statistical analysis of homing efficiency in non-treated and CRH-treated groups (n = 7 mice per group; data were normalized to the control group). Data are mean ± s.d. values of at least three independent experiments, two-tailed t-test.

Source data

Extended Data Fig. 3 CRH enhances HSPC homing, without affecting homing of other mature blood cells.

a, Representative dot plots of FACS analysis of HSPCs before and after MACS enrichment. b, Representative gating strategy employed in FACS analysis for HSPC homing assays. c, Flow cytometry dot plots showing human CD45+ cells in the femur bone marrow of NSG mice and statistical analysis of the homing efficiency in the uncultured, vehicle-treated (Control) and CRH-treated cultured groups (n = 5 mice per group). d, Schematic diagram of the experimental design of the cord blood mononuclear cells (CBMCs) homing experiment. e, f, Gating strategy for CBMCs homing assay (e), and statistical analysis (f), of CBMCs in the bone marrow of NSG mice. g, Flow cytometry dot plots showing human CD45+ cells in NSG mouse femur bone marrow and statistical analysis of homing efficiency of HSPCs from mobilized peripheral blood (mPB) in the non-treated and CRH-treated groups (n = 5 mice per group). h, Representative immunofluorescence images and statistical analysis of CRHR1 expression levels in CB CD34+ cells compared to those in mPB CD34+ cells (n = 3 independent experiments). i, Statistical analysis of the homing efficency in CB CD34+ cells compared to mPB CD34+ cells (n = 5–12 mice per group). For c, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For f, g and i, parametric data were analyzed using two-tailed Student’s t-test. For h, nonparametric data were analyzed using the Mann‒Whitney test. Data are mean ± s.d. values.

Source data

Extended Data Fig. 4 CRH promotes long-term engraftment of human HSPCs in NSG mice.

a, Representative gating strategy employed in FACS analyses to assess total human engraftment in bone marrow of primary NSG recipients at 16 weeks after transplantation. b, Flow cytometry dot plots illustrating the percentages of human CD45+ cells in the peripheral blood and bone marrow of primary NSG recipients. c, Flow cytometry dot plots of the percentages and statistical analyses of human CD45+ cell chimerism in the BM of primary NSG recipients 16 weeks post-transplantation, in the uncultured, vehicle-treated (Control) and CRH-treated cultured groups (n = 4–5 mice per group). d, Representative confocal images and statistical analysis of CRHR1 expression in CRHR1 shRNA-expressing CB CD34+ cells (shCRHR1) compared to negative shRNA (NC) (n = 3 independent experiments). e, Flow cytometry dot plots of the percentages and statistical analysis of human CD45+ cell chimerism in the BM of primary NSG recipients 16 weeks after transplanting CB CD34+ cells, including cells transduced with negative shRNA without CRH treatment (Control), cells with negative shRNA treated with CRH (CRH), and cells with CRHR1 shRNA treated with CRH (CRH+shCRHR1) (n = 4–5 mice per group). f, g, Experimental strategy (f), and summary table (g), of human SRCs among cultured CB CD34+ cells with or without CRH treatment. h, Statistical analysis of human myeloid (hCD33+), T lymphoid (hCD3+), B lymphoid (hCD19+), and NK (hCD56+ hCD3−) cell populations in the bone marrow of secondary NSG recipients (n = 5–6 mice per group). For c and e, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For d, nonparametric data were analyzed using the Mann‒Whitney test. For h, parametric data were analyzed using two-tailed Student’s t-test. Data are mean ± s.d. values.

Source data

Extended Data Fig. 5 THBS2 treatment enhances the adhesion, motility, mechanical remodeling, and homing of HSPCs.

a, Flow cytometry analysis of surface THBS2 expression in non-treated or CRH-treated CB CD34+ cells (n = 6 independent experiments). b, Immunoblot showing THBS2 expression in HSPCs after CRH treatment (n = 2 independent experiments). c, Immunoblot showing THBS2 presence in culture supernatants from non-treated or CRH-treated CB CD34+ cells, with or without 2 µM Brefeldin A (BFA). d, Representative immunofluorescence images of CB CD34+ cells on FN, with nuclei stained by DAPI, F-actin in green, and Vinculin in red. e, Representative immunofluorescence images and statistical analysis of the mature number and average area of FAs in CB CD34+ cells cultured on the indicated FN coatings, with nuclei stained by DAPI, F-actin in green, and Vinculin in red (n = 30 cells measured/condition). f, Cell migration velocity on small islands on the indicated FN coatings (n = 30 cells measured/condition). g, Statistical analysis of the mature number and average area of FAs in CB CD34+ cells cultured on 10 μg/ml FN under the indicated conditions (n = 30 cells measured/condition). h, Cell migration velocity on small islands under the indicated conditions (n = 30 cells measured/condition). i, j, Immunoblot (i), and representative immunofluorescence images (j), showing THBS2 protein expression in CB CD34+ cells transduced with Neg shRNA (NC) or THBS2 shRNA (shTHBS2) (n = 3 independent experiments). k, l, Statistical analysis of CB CD34+ cell adhesion to collagen (k), or FN (l), with or without 400 ng/ml rhTHBS2 treatment (n = 3 independent experiments). m, n, Statistical analysis of cell area (m), and average area of FAs per cell (n = 3 independent experiments) (n). o, Statistical analysis of the results of transwell assays of CB CD34+ cells response to SDF-1α (100 ng/ml) with or without 400 ng/ml rhTHBS2 treatment (n = 3 independent experiments). p, Statistical analysis of migration speed of CB CD34+ cells in upper layers in 3D HSPC mobility assay, with or without 400 ng/ml rhTHBS2 treatment (n = 4 independent experiments). q, Left: immunofluorescence staining images of F-actin (green) in non-treated and rhTHBS2-treated CB CD34+ cells; nuclei counterstained using DAPI (blue). Right: analysis of the cell cortex intensity of F-actin (n = 3 independent experiments); r, s, Statistical analysis of the transit time (r), and pass ratio (s), of CB CD34+ cells in the chemotactic-mediated microfluidic movement assay (n = 4 independent experiments). t, Statistical analysis of the migration velocity of CB CD34+ cells in microfluidic assays (n = 3 independent experiments). u, Dot plot showing human CB CD34+ cells in NSG femur bone marrow and statistical analysis of homing efficiency in the non-treated and rhTHBS2-treated groups (n = 5 mice per group). v, Flow cytometry dot plots of the percentages and statistical analyses of human CD45+ cell chimerism in the BM of primary NSG recipients 16 weeks post-transplantation, in the non-treated and rhTHBS2-treated groups (n = 3 mice per group). w, Flow cytometry dot plots of the percentages and statistical analysis of human CD45+ cell chimerism in the BM of primary NSG recipients 16 weeks after transplanting CB CD34+ cells, including cells transduced with negative shRNA without CRH treatment (Control), cells with negative shRNA treated with CRH (CRH), and cells with THBS2 shRNA treated with CRH (CRH+shTHBS2) (n = 4–5 mice per group). For a, k, l, m, o, p, q, s, u and v, parametric data were analyzed using two-tailed Student’s t-test. For j, n, r and t, nonparametric data were analyzed using the Mann‒Whitney test. For w, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For e and g, nonparametric data were analyzed using the Kruskal‒Wallis test with correction for multiple comparisons by Dunn’s test. Except for f, h, data are mean ± SEM, for all other figures data are mean ± s.d. values.

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Extended Data Fig. 6 CRH induces RhoA activation and YAP nuclear translocation in HSPCs.

a, Gene set enrichment analysis of RNA-seq profiles from non-treated and CRH-treated CB CD34+ cells, demonstrating enrichment of transcripts associated with positive regulation of small GTPase-mediated signal transduction. b, c, Cdc42 (b), and Rac1 (c), activity measured by ELISA (n = 5, data normalized to control). d, Relative levels of activated RhoA in CB CD34+ cells induced by CRH treatment were blocked by NBI (n = 4, data normalized to control). e, Representative immunofluorescence images and statistical analysis of YAP (red) Nuc/Cyto ratio and F-actin (green) fluorescence intensity in CB CD34 cells from non-treated, CRH-treated, and VP pretreated before CRH treatment groups. Nuclei were stained with DAPI (blue) (n = 3 independent experiments). f, Representative immunofluorescence images and statistical analysis of THBS2 (red) and F-actin (green) fluorescence intensity in CB CD34+ cells from non-treated, CRH-treated, and peptide 17 pretreated before CRH treatment groups (n = 3 independent experiments). For b and c, nonparametric data were analyzed using the Mann‒Whitney test. For d, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For e and f, nonparametric data were analyzed using the Kruskal‒Wallis test with correction for multiple comparisons by Dunn’s test. Data are mean ± s.d. values.

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Extended Data Fig. 7 THBS2-Integrin αvβ3 interaction mediates YAP1 nuclear translocation via CRH/CRHR1 signaling pathway.

a–c, Representative fluorescence images showing THBS2 (a–c), CD47 (a), CD36 (b), and integrin α9β1 (c), in CB CD34+ cells. d, Representative immunofluorescence images and statistical analysis of the co-localization of THBS2 (green) and integrin αvβ3 (red) in CB CD34+ cells with nuclei stained with DAPI (blue) under the indicated conditions (n = 3 independent experiments). e–g, Representative immunofluorescence images (e), and statistical analysis of F-actin (f), and YAP Nuc/Cyto ratio (g), in CB CD34+ cells under the indicated conditions (n = 3 independent experiments). h–l, Representative immunofluorescence images under the indicated conditions (h, i), and quantification of YAP Nuc/Cyto ratio in CB CD34+ (2 × 105) cells cultured in dishes pre-coated with rhTHBS2 (j), intensity of F-actin (k), and cell migration speed (l), were captured using a Leica microscope and analyzed (n = 3 independent experiments). For d, f, g, j (right), k and l, nonparametric data were analyzed using the Kruskal‒Wallis test with correction for multiple comparisons by Dunn’s test. For j (left), parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. Data are mean ± s.d. values.

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Extended Data Fig. 8 CRH modulates HSPC biomechanics via CRHR1/RhoA/YAP/THBS2/integrin αvβ3 signaling.

a–d, Statistical analysis of the results of cell adhesion assays of CB CD34+ cells seeded on collagen (a), or FN (b), (n = 4–5 independent experiments). Representative immunofluorescence images of F-actin (green), vinculin (red), and nuclei (DAPI, blue) in CB CD34+ cells adhered to FN (c), and statistical analysis of the average area of FAs and the number of mature FAs per cell (d), (n = 3 independent experiments). e, Transwell assay of CB CD34+ cells (n = 4 independent experiments). f, g, Quantification of CB CD34+ cell migration speed, in a microfluidic channel (f) (n = 3 independent experiments), and chemotactic-mediated microfluidic assay to measure the transit time (g) of CB CD34+ cells traversing the microfluidic channel; CB CD34+ cells from the non-treated, CRH-treated, NBI-pretreated before CRH treatment, and Cyclo(-RGDfK)-pretreated before CRH treatment groups were included in these analyses. h, i, Transwell assay of CB CD34+ cells (n = 5 independent experiments) (h), and chemotactic-mediated microfluidic assay to measure transit time (i) of CB CD34+ cells traversing the microfluidic channel; CB CD34+ cells from non-treated, CRH-treated, and Rhosin-pretreated before CRH treatment groups were analyzed. j, k, Transwell assay of CB CD34+ cells (n = 5 independent experiments) (j), and chemotactic-mediated microfluidic assay to measure the transit time (k) of CB CD34+ cells traversing the microfluidic channel; CB CD34+ cells from non-treated, CRH-treated, and VP-pretreated before CRH treatment groups were analyzed. l, Representative fluorescence images and quantification of intensity of F-actin (green) under indicated conditions. m–p, Statistical analysis of adhesion of CB CD34+ cells seeded on collagen (m), or FN (n) (n = 3–4 independent experiments). Representative immunofluorescence images of F-actin (green), vinculin (red), and nuclei (DAPI, blue) in CB CD34+ cells adhered to FN (o), and statistical analysis of the average area of FAs and the number of mature FAs per cell (p), (n = 3; data normalized to control). q, Transwell assay of CB CD34+ cells (n = 3 independent experiments). r, s, Quantification of CB CD34+ cell migration speed in a microfluidic channel (r) (n = 3 independent experiments), and chemotactic-mediated microfluidic assay to measure transit time (s) of CB CD34+ cells traversing the microfluidic channel; CB CD34+ cells from the non-treated, rhTHBS2-treated, and Cyclo(-RGDfK)-pretreated before rhTHBS2 treatment groups were included in these analyses. For a, b, e, h, j, m, n and q, parametric data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For d, f, g, i, k, l, p, r and s, nonparametric data were analyzed using the Kruskal‒Wallis test with correction for multiple comparisons by Dunn’s test. Data are mean ± s.d. values.

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Reporting Summary

Video 1

Movement HSPCs cultured in a restricted environment with a height of 5 μm, tracked in real time using a Leica microscope.

Video 2

Cells passing through a restricted microfluidic pathway.

Video 3

Live z-stack video, imaged from the skull of an NSG mouse.

Video 4

Motion representation of CMFDA-labelled HSPCs in the bone marrow of the skull of NSG mice.

Video 5

HSPCs traversed vascular endothelial cells in the bone marrow of the skull of NSG mice.

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Wu, M., Yang, H., Liu, S. et al. Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone. Nat. Biomed. Eng 9, 754–771 (2025). https://doi.org/10.1038/s41551-024-01316-1

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