A canonical to non-canonical Wnt signalling switch in haematopoietic stem-cell ageing

Abstract

Many organs with a high cell turnover (for example, skin, intestine and blood) are composed of short-lived cells that require continuous replenishment by somatic stem cells. Ageing results in the inability of these tissues to maintain homeostasis and it is believed that somatic stem-cell ageing is one underlying cause of tissue attrition with age or age-related diseases. Ageing of haematopoietic stem cells (HSCs) is associated with impaired haematopoiesis in the elderly. Despite a large amount of data describing the decline of HSC function on ageing, the molecular mechanisms of this process remain largely unknown, which precludes rational approaches to attenuate stem-cell ageing. Here we report an unexpected shift from canonical to non-canonical Wnt signalling in mice due to elevated expression of Wnt5a in aged HSCs, which causes stem-cell ageing. Wnt5a treatment of young HSCs induces ageing-associated stem-cell apolarity, reduction of regenerative capacity and an ageing-like myeloid-lymphoid differentiation skewing via activation of the small Rho GTPase Cdc42. Conversely, Wnt5a haploinsufficiency attenuates HSC ageing, whereas stem-cell-intrinsic reduction of Wnt5a expression results in functionally rejuvenated aged HSCs. Our data demonstrate a critical role for stem-cell-intrinsic non-canonical Wnt5a signalling in HSC ageing.

Figure 1. Increased expression of Wnt5a in aged LT-HSCs results in a shift from canonical to non-canonical Wnt signalling

a, Wnt transcript levels in 2–3-month-old or 24-month-old LT-HSCs. n = 4, *P < 0.05. b, c, Wnt5a protein levels in low-density bone marrow (LDBM) cells (b) and densitometric score (c). n = 4, *P < 0.05. d, Immunofluorescence z-stack and three-dimensional merged images of tubulin (green) and β-catenin (red) in LT-HSCs. Scale bar, 5 μm. e, LT-HSCs with nuclear β-catenin. n = 3, 200 cells per sample in total. *P < 0.05. f, Axin 2 transcript levels in LT-HSCs. n = 4, *P < 0.05. g, β-catenin mean fluorescence intensity in young, aged and young Wnt5a-treated haematopoietic progenitor/stem cells. n = 3, *P < 0.05 versus young LT-HSC controls. h, β-catenin mean fluorescence intensity in young control LT-HSCs, or young LT-HSCs treated with Wnt5a or with Wnt5a plus MG-132. n = 3, **P < 0.01 versus young controls. i, β-catenin mean fluorescence intensity in young LT-HSCs or young LT-HSCs treated with Wnt5a. n = 3, **P < 0.01 versus young controls. A paired Student's t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA were used to compare means among three or more independent groups. Error bars represent s.e.m.

Figure 2. Wnt5a activates Cdc42 inducing ageing-like phenotypes in young LT-HSCs

a, b, Cdc42 activity in young and Wnt5a-treated Lin⁻ bone marrow (BM) cells (a) and densitometric score (b). n = 4, *P < 0.05, **P < 0.01. c, Cdc42 (red) and tubulin (green) in LT-HSCs shown by immunofluorescence. Scale bar, 5 μm. d, Polar distribution (percentage) of Cdc42 and tubulin in LTHSCs. n = 6, 200 LT-HSCs per sample in total. *P < 0.001. e, f, Donor-derived Ly5.2⁺ cells and B220⁺, CD3⁺ and myeloid (Gr1⁺, Mac1⁺, Gr1⁺Mac1⁺) cells among Ly5.2⁺ cells in peripheral blood (PB) 24 weeks after transplant. *P < 0.05; n = 10 for casin and Wnt5a plus casin, n = 25 for control and Wnt5a. A paired Student's t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA were used to compare means among three or more independent groups. Error bars represent s.e.m.

Figure 3. Wnt5a haploinsufficient mice present with attenuated HSC ageing

a–d, Wnt5a (a) and Cdc42 activity levels (c) in young and aged Wnt5a^+/+ and Wnt5a^+/− LDBM cells and densitometric analysis (b, d). n = 4, *P < 0.05; **P < 0.01. e, Distribution of Cdc42, tubulin and Wnt5a in young and aged Wnt5a^+/+ and Wnt5a^+/− LT-HSCs. Scale bar, 5 μm. f, g, Percentage of B220⁺, CD3⁺ and myeloid cells among white blood cells in peripheral blood (f) and percentage of LT-HSCs, ST-HSCs and LMPPs among LSKs (g) in Wnt5a^+/+ and Wnt5a^+/− young and aged mice. *P < 0.05, **P < 0.01, ***P < 0.001; n = 5 for aged Wnt5a^+/+ and Wnt5a^+/− mice; n = 7 for young Wnt5a^+/+ and Wnt5a^+/− mice. A paired Student's t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA were used to compare means among three or more independent groups. Error bars represent s.e.m.

Figure 4. Reducing Wnt5a expression in aged LT-HSCs rejuvenates their function in vivo

a, Experimental set-up. b, B220⁺, CD3⁺ and myeloid cells among donor-derived cells in bone marrow. *P < 0.05; n = 10. c, LT-HSCs, ST-HSCs and LMPPs among donor-derived LSKs. *P < 0.05; **P < 0.01. d, e, Cdc42 activity in aged donor-derived low-density bone-marrow (LDBM) cells transduced with non-targeting (NT) shRNA, Wnt5a shRNA (Wnt5a^KD) and untransduced control (d) and densitometric score (e). n = 4, *P < 0.05. f, Cdc42 and tubulin in donor-derived LT-HSCs from aged untransduced or non-targeting shRNA or Wnt5a^KD recipient mice 24 weeks after transplant shown by immunofluorescence. Scale bar, 5 μm. g, Immunofluorescence z-stack and three-dimensional merged images of tubulin (green) and β-catenin (red) localization in aged non-targeting shRNA or Wnt5a^KD LT-HSCs. Scale bar, 5 μm. h, Aged donor-derived non-targeting shRNA or Wnt5a^KD LT-HSCs exhibiting nuclear accumulation of β-catenin. n = 3, *P < 0.05. A paired Student's t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA were used to compare means among three or more independent groups. Error bars represent s.e.m.

Extended Data Figure 1. Increased expression of Wnt5a in aged LT-HSCs results in a shift from canonical to non-canonical Wnt signalling

a, Reverse transcriptase real-time PCR analysis of Wnt5a transcript levels in young (10-week-old), middle-aged (10-month-old) and aged (24-month-old) LT-HSCs (Lin⁻c-kit⁺Sca-1⁺Flk2⁻CD34⁻ bone marrow cells) sorted from C57BL/6 mice. Data are expressed as fold increased compared to the lowest expressed transcript arbitrarily set to 1. Wnt5a mRNA is barely detectable in young LT-HSCs and is markedly upregulated in middle-aged and aged LT-HSCs. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic scale. Bars are mean + 1 s.e.; n = 3, *P < 0.05. b, Reverse transcriptase real-time PCR analysis of Wnt5a transcript levels in young (10-week-old), middle-aged (10-month-old) and aged (24-month-old) Lin⁻ bone marrow cells from C57BL/6 mice. Data are expressed as fold increased compared to the lowest expressed transcript arbitrarily set to 1. Wnt5a mRNA is barely detectable in young Lin⁻ cells and is upregulated in middle-aged and aged LT-HSCs. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic scale. Scale bars represent results of one set of samples. The experiment was repeated twice with similar results. c, Reverse transcriptase real-time PCR analysis of Wnt5a transcript levels in young (10-week-old) and aged (24-month-old) LT-HSCs (Lin⁻c-Kit⁺Sca-1⁺Flk2⁻CD34⁻ bone marrow cells) sorted from C57BL/6 and DBA/2 mice. Data are expressed as fold increased compared to the lowest expressed transcript arbitrarily set to 1. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic scale. Error bars are mean + 1 s.e.; n = 3, *P < 0.05, **P < 0.01. d, Representative three-dimensional confocal picture of Wnt5a distribution in an aged LT-HSC. The nucleus is stained with DAPI. Three-dimensional localization of Wnt5a was analysed by scanning the cells along the z-axis and acquiring a picture of the xy-plane every 0.7 μm. Three-dimensional images were then reconstructed by using Volocity v6.0 software. e, Representative immunofluorescence picture of Wnt5a (green) membrane distribution (non-permeabilized cells) in young and aged LT-HSCs. Immunofluorescence pictures are shown as overlap with the phase contrast image. Scale bar, 5 μm. f, Representative immunofluorescence picture of Wnt5a (green) and clathrin (red) localization in young and aged LT-HSCs. Pictures are shown on a dark background and as overlap with DAPI (staining nuclei). Scale bar, 5 μm. g, Representative expression of Wnt5a in MEFs (mouse embryonic fibroblasts) and aged LT-HSCs from Wnt5a^+/+ mice determined by immunofluorescence. Wnt5a fluorescence signal is not detected when MEFs from Wnt5a^−/− mice are stained with the same procedure. Wnt5a pictures are shown on a dark background and as overlap with DAPI (blue, staining nuclei) and phase contrast images. Scale bar, 10 μm. h, Representative FACS dot plots of LT-HSCs (Lin⁻c-Kit⁺Sca-1⁺Flk2⁻CD34⁻), ST-HSCs (Lin⁻c-Kit⁺Sca-1⁺Flk2⁻CD34⁺), LMPPs (Lin⁻c-Kit⁺Sca-1⁺Flk2⁺CD34⁺), LSKs (Lin⁻c-Kit⁺Sca-1⁺) and LKs (Lin⁻c-Kit⁺Sca-1⁻) gating strategy of young and aged lineage-depleted bone marrow cells. i, Representative FACS histograms of β-catenin expression in young, aged and young Wnt5a-treated LT-HSCs, ST-HSCs, LMPPs and LSKs.

Extended Data Figure 2. Wnt5a activates Cdc42 inducing ageing-like phenotypes in young LT-HSCs

a, Representative distribution of Cdc42, tubulin and Per2 (staining the centrosome) in young control, young Wnt5a-treated (100 ng ml⁻¹) and young Wnt5a (100 ng ml⁻¹) 1 casin (5 μM)-treated LT-HSCs determined by immunofluorescence. Scale bar, 5 μm. Shown are also representative fluorescence intensity plots obtained by collecting pixel intensity through the section of the cell as indicated by the dotted line in the corresponding merge picture. b, Representative distribution of Cdc42, tubulin and NCam2 (membrane protein) in young control, young Wnt5a-treated (100 ng ml⁻¹) and aged LT-HSCs determined by immunofluorescence. Scale bar, 5 μm. c, Graph of the percentage of young control, young Wnt5a-treated (100 ng ml⁻¹) and aged LT-HSCs with a polar distribution of NCam2. Shown are mean + 1 s.e., n = 4; ~200–300 LT-HSCs scored per sample in total. *P < 0.001. d, Reverse transcriptase real-time PCR analysis of Cdc42, Rhou, Rac1, Rac2, Rhoa, Rhoj, Rhov and Rhoq transcript levels in young, aged and young Wnt5a-treated (100 ng ml⁻¹, 16 h treatment) LT-HSCs. Rhou and Rhov transcripts were below detection limits in all the assayed samples (ND). Data are expressed as fold difference compared to the expression of Cdc42 mRNA in young LT-HSCs arbitrarily set to 1. Data were analysed with the 2^−ΔΔCt method and plotted on a linear scale. Bars are mean + 1 s.e.; n = 3, *P < 0.05. e, Schematic representation of the experimental set-up for the transplantation. Recipient mice were analysed 24 weeks after transplant.

Extended Data Figure 3. Wnt5a haploinsufficient mice present with attenuated HSC ageing

a–d, White blood (WB) cell count (a), red blood (RB) cell count (b), haemoglobin (Hb) dosage (c) and lymphocyte cell count (d) in peripheral blood of young and aged Wnt5a^+/− and Wnt5a^+/+ mice. *P < 0.05; shown are mean + 1 s.e., n = 5. e, Percentage of LSKs among Lin⁻ cells in bone marrow of young and aged Wnt5a^+/− and Wnt5a^+/+ mice. *P < 0.05; shown are mean + 1 s.e., n = 5. f, Reverse transcriptase real-time PCR analysis of Wnt5a transcript levels in young and aged LT-HSCs and young and aged collagenase-digested and sorted CD45⁻ cells (stroma cells). Data are expressed as fold difference compared to the expression in young LT-HSCs arbitrarily set to 1. Wnt5a mRNA shows significantly increased expression in stroma CD45⁻ cells when compared to young and aged LT-HSCs. In contrast to the situation in LT-HSCs, young stroma CD45⁻ cells express higher levels of Wnt5a mRNA than aged stroma CD45⁻ cells. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic scale. Error bars are mean + 1 s.e.; n = 4, *P < 0.05. g, Schematic representation of the experimental set-up for transplantation. Young donor (Ly5.1⁺) bone marrow cells were transplanted into recipient (Ly5.2⁺) young Wnt5a^+/− and Wnt5a^+/+ mice. Recipient mice were killed and analysed 20 months after transplant. h, i, Percentage of engrafted cells, B220⁺, CD3⁺ and myeloid cells among donor-derived Ly5.1⁺ cells in peripheral blood (h) and bone marrow (i) 20 months after transplants. Columns are mean values + 1 s.e., n = 5. j, Percentage of donor-derived LSKs among donor-derived Lin⁻ cells in bone marrow of Wnt5a^+/− and Wnt5a^+/+ recipient mice 20 months after transplant. Columns show mean + 1 s.e.m., n = 5. k, Percentage of donor-derived LT-HSCs, ST-HSCs and LMPPs among donor-derived LSKs in Wnt5a^+/− and Wnt5a^+/+ recipient mice 20 months after transplant. Columns are mean values + 1 s.e. n = 5. l, m, Percentage of LT-HSCs polarized for Cdc42 (l) and tubulin (m) in young and aged Wnt5a^+/+ and Wnt5a^+/− mice. Shown are mean + 1 s.e., n = 4 and 200 cells scored per sample in total. *P < 0.01 versus young Wnt5a^+/+ and P < 0.05 versus young Wnt5a^+/− and aged Wnt5a^+/− mice.

Extended Data Figure 4. Validation of the knockdown efficiency in 3T3 fibroblast cells and aged and young Lin⁻ bone marrow cells

a, Transduced fibroblast cells were sorted and analysed by western blot for Wnt5a protein levels. Wnt5a protein levels were normalized on actin. Three different Wnt5a knockdown vectors (3a-GFP⁺, 4a-GFP⁺, 5b-GFP⁺) were tested and Wnt5a protein levels were compared to non-targeting transduced fibroblasts (NT-GFP⁺) and to untransduced cells sorted as GFP⁻ from the initial mixed culture (3a-GFP⁻, 4a-GFP⁻, 5b-GFP⁻). b, Transduced fibroblast cells were sorted and analysed by reverse transcriptase real-time PCR for Wnt5a mRNA levels. Wnt5a mRNA levels are normalized to actin mRNA levels. Three different Wnt5a knockdown vectors (3a-GFP⁺, 4a-GFP⁺, 5b-GFP⁺) were tested and Wnt5a transcript levels were compared to non-targeting transduced fibroblasts (NT-GFP⁺). c, Not pre-stimulated transduced Wnt5a^KD or non-targeted aged Lin⁻ bone marrow cells were sorted and analysed by reverse transcriptase real-time PCR for Wnt5a mRNA levels. Wnt5a mRNA levels are normalized on Gapdh mRNA levels. d, Percentage of B220⁺, CD3⁺ and myeloid cells among donor-derived cells in peripheral blood 24 weeks after transplant. *P < 0.05; shown are mean values + 1 s.e. Mice were considered as engrafted when the percentage of Ly5.2⁺ cells in peripheral blood was higher than 1.0 and contribution was detected for all peripheral blood lineages. Data are based on two different lentiviral infection/transplant experiments with 5–7 recipient mice per group (for example, n = 10). e, Schematic representation of the experimental set-up for transplantation of Wnt5a knock down (Wnt5a^KD), Wnt5a non-targeting (Wnt5a-NT) and untransduced young haematopoietic progenitor/stem cells. Young donor (Ly5.2⁺) lineage-negative (Lin⁻) bone marrow cells were infected with the indicated lentiviral vectors or left untransduced. Infected cells were sorted based on GFP expression. Cells (1–3 × 10⁵ Ly5.2⁺) were transplanted into recipients (Ly5.1⁺). Recipient mice were analysed 12–16 weeks after transplant. f, Percentage of engrafted donor-derived cells in peripheral blood 12–16 weeks after transplant. Shown are mean values + 1 s.e. Mice were considered as engrafted when the percentage of Ly5.2⁺ cells in peripheral blood was higher than 1.0 and contribution was detected for all peripheral blood lineages. Data are based on two different lentiviral infection/transplant experiments with 3 recipient mice per group (for example, n = 3 for Wnt5a^KD and Wnt5a-NT mice and n = 6 for untransduced mice). g, Percentage of B220⁺, CD3⁺ and myeloid cells among donor-derived cells in peripheral blood 24 weeks after transplant. *P < 0.05; shown are mean values + 1 s.e. Mice were considered as engrafted when the percentage of Ly5.2⁺ cells in peripheral blood was higher than 1.0 and contribution was detected for all peripheral blood lineages. Data are based on two different lentiviral infection/transplant experiments with 5–7 recipient mice per group (for example, n = 10). h, Ratio of the densitometric score of the total Cdc42 expression as shown in Fig. 4d. The experiment was repeated four times with mice (1 mouse for 1 sample) from different lentiviral infection/ transplant experiments. Shown are mean + 1 s.e., n = 4, *P < 0.05. i, j, Percentage of donor-derived LT-HSCs polarized for Cdc42 (i) and tubulin (j) 24 weeks after transplant. Shown are mean values + 1 s.e., n = 4, ~200 cells scored per sample in total. *P < 0.05. k, Representative immunofluorescence z-stack pictures of tubulin (green) and β-catenin (red) localization in aged Wnt5a-NT (Ly5.2⁺GFP⁺) or aged Wnt5a^KD (Ly5.2⁺GFP⁺) LT-HSCs. Nuclei are stained with DAPI (blue). Shown is also the final three-dimensional reconstructed merged image. Scale bar, 5 μm.

Extended Data Figure 5. Wnt pathways in HSCs and ageing

a–g, Reverse transcriptase real-time PCR analysis of Notch1, Notch2, Jag1, Jag2, Dll1 (delta like1), Dll4 (delta like 4) and Hes1 transcript levels in young, aged and young Wnt5a-treated (16 h treatment) LT-HSCs. Notch3, Notch4 and Dll3 (delta like 3) transcripts were below detection limits in all the assayed samples. Data are expressed as fold difference compared to the expression in young LT-HSCs arbitrarily set to 1. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic or linear scale. Bars are mean + 1 s.e.; n = 3, *P < 0.05. h, Representative immunofluorescence picture of p-CamKII (green) expression and localization in young control and young Wnt5a-treated LT-HSCs. Pictures are shown on a dark background and as overlap with DAPI (staining nuclei). Scale bar, 5 μm. i, Relative expression of p-CamKII in young LT-HSCs and on Wnt5a treatment, determined by integration of pixel intensity. *P < 0.05. j, Representative immunofluorescence picture of NFATc (green) expression and localization in young control and young Wnt5a-treated LT-HSCs. Pictures are shown on a dark background and as overlap with DAPI (staining nuclei). Scale bar, 5 μm. k, Relative expression of NAFTc in young LT-HSCs and on Wnt5a treatment, determined by integration of pixel intensity. *P < 0.05. l, Changes in intracellular Ca²⁺ concentrations in ST-HSCs and LT-HSCs in response to stimulation with Wnt5a as determined by flow cytometry. m, Reverse transcriptase real-time PCR analysis of p57 and p27 transcript levels in young, aged and young Wnt5a-treated (100 ng ml⁻¹, 16 h treatment) LT-HSCs. Data are expressed as fold difference compared to the expression in young LT-HSCs arbitrarily set to 1. Data were analysed with the 2^−ΔΔCt method and plotted on a logarithmic or linear scale. Bars are mean + 1 s.e.; n = 3, *P < 0.05.

Extended Data Figure 6. Mechanisms of haematopoietic stem-cell ageing

Cartoon scheme summarizing the main phenotypic and functional differences between young and aged LT-HSCs.