Endothelial transplantation rejuvenates aged hematopoietic stem cell function

Abstract

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Aged BM vasculature displays functional alterations in vivo.

(A) Representative longitudinal and inset images of femurs intravitally labeled with a vascular-specific VECAD antibody (red), showing morphological alterations in aged vasculature (white line demarcates cortical bone). Scale bars: 100 μm (longitudinal images) and 50 μm (insets). (B and C) Analysis of BM vascular leakiness in young and aged femurs. (B) Quantification of Evans blue dye extravasation (n = 5 mice/cohort). (C) Representative femurs injected with Evans blue dye. Noninjected controls were used to determine baselines (n = 5 mice/cohort). (D and E) Frequency of recoverable (D) VECAD+CD31+CD45–TER119– BM ECs and (E) VECAD–CD31–CD45–TER119– stroma in young and aged femurs (n = 5 mice/cohort). (F) Quantification of mean fluorescence intensity (MFI) and representative histogram of ROS in VECAD+CD31+CD45–TER119– ECs from young and aged femurs showing an increase in ROS in aged ECs (n = 3 mice/cohort). (G) MFI quantification and representative histogram of pimonidazole adducts as detected by an anti-pimonidazole antibody (HypoxyProbe) in VECAD+CD31+CD45–TER119– ECs from young and aged femurs, demonstrating an increased hypoxia state in aged ECs (n = 3 mice/cohort). (H) Representative immunofluorescence images of HypoxyProbe-stained young and aged femurs, showing local changes in hypoxia (white line demarcates cortical bone). Scale bar: 50 μm. Error bars represent the sample mean ± SEM. *P < 0.05 and ***P < 0.001, by unpaired, 2-tailed Student’s t test.

Figure 2. Characterization of cultured ECs from aged mice.

(A) Representative phase-contrast and immunofluorescence images of cultured BM-derived ECs from young and aged mice. Scale bars: 200 μm (phase-contrast) and 50 μm (immunofluorescence). (B) Representative flow plots of cultured ECs stained for VECAD+CD31+ demonstrating highly purified EC populations. (C–E) AFM analysis of elasticity in cultured young and aged ECs showing an increase in aged EC stiffness. (C) Representative reconstructed images of EC monolayers. (D) Box plots of the median stiffness in cultured young and aged ECs (n = 3 biological replicates). (E) Normalized relative EC stiffness (n = 3 biological replicates). (F and G) Proliferation status of cultured young and aged ECs. (F) Representative histograms of Edu incorporation following cell-cycle synchronization. (G) Quantification of Edu incorporation demonstrating an early inhibition of cell-cycle entry into the S phase in aged ECs that was resolved by 24 hours (n = 3 biological replicates). (H) Quantification of SA β-gal activity in young and aged ECs (n = 3 biological replicates). (I and J) In vitro scratch wound-healing assay showing a functional delay in cell migration in aged ECs. (I) Representative phase-contrast images (dashed lines demarcate the initial scratch wound). Scale bar: 400 μm. (J) Quantification of EC wound healing (n = 3 biological replicates). (K) Normalized gene expression in cultured young and aged ECs (n = 3 biological replicates). *P < 0.05, **P < 0.01, and ***P < 0.001. Significance was determined using an unpaired, 2-tailed Student’s t test, with error bars representing the sample mean ± SEM. A nonparametric, 1-sided Wilcoxon rank-sum test was used to compare median endothelial stiffness in D. Data are presented as box plots, with whiskers representing an IQR of ± 1.5. Relative endothelial stiffness in E was normalized to young ECs and presented as a 95% CI.

Figure 3. Aged ECs are sufficient to induce aged hematopoietic phenotypes.

(A and B) Quantification of hematopoietic expansion by flow cytometry. (A) Total phenotypic CD45+ hematopoietic cells and (B) CD45+lineage–cKIT+SCA1+ HSPCs (n = 3 independent cocultures). (C) Quantification of CD45.2+ donor chimerism in PB 4 months after transplantation (Tpx), as measured by flow cytometry (n = 5 mice/cohort). Results show the ability of young ECs to restore hematopoietic engraftment of HSPCs following coculture, while aged ECs impaired young hematopoietic engraftment relative to that seen in age-matched coculture controls. Unmanipulated pre-expansion WBM cells from young or aged mice were competitively transplanted into lethally irradiated recipients to confirm age-dependent hematopoietic reconstitution phenotypes (n = 5 mice/cohort). (D–F) Quantification of donor-derived lineage+ hematopoietic repopulation 4 months after transplantation. Frequencies of (D) CD11B+GR1+ myeloid cells, (E) B220+CD19+ B cells, and (F) CD8+ (black)/CD4+ (gray) T cell populations in PB were determined by flow cytometry. Young HSPCs cocultured with aged ECs acquired myeloid-biased engraftment at the expense of lymphopoiesis, while young ECs were unable to reverse the myeloid bias in aged HSPC expansions. Error bars represent the sample mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test for like groupings.

Figure 4. Infusion of young endothelium promotes hematopoietic recovery in aged recipients following myelosuppressive injury.

(A) Schematic of the EC infusion strategy. (B and C) Time course of PB recovery of (B) young and (C) aged mice following irradiation (6.50 Gy) and infusion of either young ECs, aged ECs, or PBS vehicle control (n = 5 mice/cohort). The results demonstrated the myeloprotective effect of young EC transplantation following hematopoietic insult in both young and aged recipients, while the result with aged EC transplantation was indistinguishable from that observed in the PBS vehicle-infused controls. (D–F) Quantification of CD45.2+ donor chimerism and multilineage engraftment in PB 4 months after donor WBM transplantation as measured by flow cytometry (n = 5 mice/cohort). (D) Unmanipulated steady-state young and aged WBM was competitively transplanted to confirm reduced CD45.2+ hematopoietic engraftment and phenotypic CD11B+/GR1+ myeloid bias in the aged WBM transplantation cohort (n = 5 mice/cohort). (E) Young and (F) aged donors infused with young ECs demonstrated an increase in hematopoietic engraftment, while supporting an increase in B220+ and CD3+ lymphoid reconstitution. Error bars represent the sample mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed, Student’s t test for comparisons at individual time points. Steady-state and PBS controls were included as recovery reference points and were not included in the statistical analysis.

Figure 5. Coinfusion of young endothelium enhances BM transplantation.

(A and B) Survival curves for mice transplanted with 105 WBM cells from either (A) young or (B) aged animals, showing an increase in overall survival in WBM cohorts coinfused with ECs (n = 10 mice/cohort). Note: Data in A and B share the same steady-state, PBS, and EC-alone controls. The survival curve significance between the WBM and WBM-plus-EC cohorts was calculated using a log-rank test. (C and D) Time course of hematopoietic recovery in the PB of recipient mice coinfused with 105 young or aged WBM cells, with or without young ECs. EC-coinfused animals had a significant increase in hematopoietic recovery (n = 10 mice/cohort). (E and F) Quantification of CFU-S in mice transplanted with 105 WBM cells from (E) young or (F) aged donors, with or without young ECs, demonstrating an increase in hematopoietic progenitor activity in cohorts coinfused with ECs. CFU-S numbers were scored 8 days after irradiation (n ≥ 5 mice/cohort). (G and H) Log-fraction plot of limiting dilution analysis showing the frequency of long-term multilineage repopulation of WBM from (G) young or (H) aged mice transplanted into lethally irradiated recipients and coinfused or not with young ECs (n = 10 mice/cohort). Dashed lines indicate 95% CIs. Stem cell frequency and significance were determined using ELDA. Error bars represent the sample mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test. Steady-state, PBS, and EC-alone controls were reference points and were not included in the statistical analysis.

Figure 6. Young EC coinfusion radioprotects the BM microenvironment.

Lethally irradiated (9.50 Gy) mice were coinfused with either 105 young or aged WBM cells and 5 × 105 young BM ECs. (A) Representative H&E-stained longitudinal femur sections from coinfused mice 7 days after irradiation (n = 10 mice/cohort). Original magnification, ×100. (B) Representative images of damaged VEGFR3+ femoral vessels, including type I hemorrhagic (asterisk), type I discontinuous (red arrow), and type II regressed (blue arrow), 7 days after irradiation, demonstrating radioprotection of the vascular niche (n = 10 mice/cohort). Sections were counterstained with hematoxylin. Original magnification, ×200. (C and D) Quantification of total BM CD45+ cells showing mitigation of panhematopoietic injury in cohorts coinfused with young or aged WBM and young ECs (n = 10 mice/cohort; data are related to A). (E and F) Quantification of type I/II damaged VEGFR3+ sinusoidal vessels in cohorts coinfused with young or aged WBM and young ECs (n = 10 mice/cohort; data are related to B). Error bars represent the sample mean ± SEM. ***P < 0.001, by unpaired, 2-tailed Student’s t test. Steady-state controls were used as a reference point and were not included in the statistical analysis.

Figure 7. Young EC coinfusion mitigates myeloablative hematopoietic injury.

(A and B) Representative H&E-stained longitudinal femur time course after irradiation (9.50 Gy) for mice coinfused with (A) young and (B) aged WBM plus ECs (n = 10 mice/cohort). Original magnification, ×100. Steady-state control H&E-stained sections are shown in the inset. (C and D) Representative contour plots of BM GM frequency in (C) young and (D) aged WBM-EC coinfusions after irradiation. (E and F) Time course of BM GM counts in (E) young and (F) aged WBM-EC coinfusions 7 days after irradiation. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test, for comparisons of WBM with WBM-EC coinfusion cohorts at individual time points. Steady-state controls were used as a reference point and were not included in the statistical analysis. FSC, forward scatter; SSC, side scatter.