Vascular Platform to Define Hematopoietic Stem Cell Factors and Enhance Regenerative Hematopoiesis

Abstract

Hematopoietic stem cells (HSCs) inhabit distinct microenvironments within the adult bone marrow (BM), which govern the delicate balance between HSC quiescence, self-renewal, and differentiation. Previous reports have proposed that HSCs localize to the vascular niche, comprised of endothelium and tightly associated perivascular cells. Herein, we examine the capacity of BM endothelial cells (BMECs) to support ex vivo and in vivo hematopoiesis. We demonstrate that AKT1-activated BMECs (BMEC-Akt1) have a unique transcription factor/cytokine profile that supports functional HSCs in lieu of complex serum and cytokine supplementation. Additionally, transplantation of BMEC-Akt1 cells enhanced regenerative hematopoiesis following myeloablative irradiation. These data demonstrate that BMEC-Akt1 cultures can be used as a platform for the discovery of pro-HSC factors and justify the utility of BMECs as a cellular therapy. This technical advance may lead to the development of therapies designed to decrease pancytopenias associated with myeloablative regimens used to treat a wide array of disease states.

Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Generation of BM-Derived Endothelial and Stromal Cells (A) Schematic of BMEC and BMS cell isolations. VECAD+SCA1+VEGFR3− arteriole (white arrowhead) and VECAD+SCA1−VEGFR3+ sinusoidal (red arrowhead) BMECs in vivo (scale bar represents 100 and 50 μm). Phase-contrast images of isolated BMEC-Akt1 and BMS-Akt1 cultures (scale bar represents 200 μm). (B and C) BMEC-Akt1 cultures demonstrate VECAD+SCA1+VEGFR3− arteriole staining (B) (scale bar represents 50 μm) and produce vessels in vivo (C). BMEC-Akt1 (red), but not BMS-Akt1 (blue), co-localize with intravitally labeled vasculature (Isolectin-B4; green) in matrigel plugs (scale bar represents 25 μm). (D and E) BMS-Akt1 cultures display PDGFRA+PRX1+LEPR+ BMS staining (D) (scale bar represents 50 μm) and give rise to osteogenic (Alizarin Red S), adipogenic (Oil Red O), and chondrogenic (Toluidine Blue O) progeny in vitro (E) (scale bar represents 200 μm).

Figure 2

BMEC-Akt1 and BMS-Akt1 Cells Express Pro-hematopoietic Factors and Are Enriched in Pathways Involved in Vascular and Organ Development (A) Heatmaps of relative gene expression from three independently derived BMEC-Akt1 and BMS-Akt1 lines. (B) BMEC-Akt1 and BMS-Akt1 cultures demonstrate enrichment in endothelial and MSC gene expression, respectively. BMEC-Akt1 and BMS-Akt1 cultures show enrichment for pro-hematopoietic factors. Error bars represent mean ± SEM and significance was determined by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Three independently isolated BMEC-Akt1 and BMS-Akt1 lines were analyzed. (C and D) BMEC-Akt1 lines are enriched in angiogenic and vascular development pathways. GO analysis of differentially expressed genes in BMEC-Akt1 and BMS-Akt1 cell lines reveals the enrichment of distinct pathways (−log10 p value = red line; number of genes enriched in target pathway = bar graph).

Figure 3

BMEC-Akt1 Cultures Support HSPCs under Serum-Free Conditions Ex Vivo (A) Schematic of ex vivo co-culture assay. HSPCs were co-cultured for 9 days with 50-ng/ml sKITL in serum-free media. (B) Phase-contrast images of day 9 HSPC co-cultures (scale bar represents 200 μm). (C) BMEC-Akt1 cells support HSPC maintenance ex vivo. Quantification of total CD45+ hematopoietic cells and LKS HSPCs following 9 days of co-culture. Fold expansion is defined as total LKS (day 9)/input LKS (day 0). (D) Schematic of functional assays. Day 9 co-cultured CD45.2+ cells were purified from feeders and plated in methylcellulose and scored for CFUs or transplanted into lethally irradiated CD45.1+ recipients with a CD45.1+ WBM competitive dose. (E) BMEC-Akt1 co-cultures maintain multilineage hematopoietic progenitor activity ex vivo. (F and G) HSPCs cultured on BMEC-Akt1 cells engraft lethally irradiated recipients (F) and demonstrate multilineage potential 16 weeks post-transplant (G). (H) BMEC-Akt1 cultured HSCs engraft secondary recipients (dashed red line) and maintain multilineage potential. n = 10 mice per condition. Error bars represent mean ± SEM and significance was determined by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

BMEC-Akt1 Support HSPCs Ex Vivo in the Absence of Exogenous Cytokines (A) BMEC-Akt1 cells without sKITL maintain HSPCs ex vivo. Quantification of total CD45+ hematopoietic cells and LKS HSPCs following 9 days of co-culture in serum-free conditions. Fold expansion is defined as total LKS (day 9)/input LKS (day 0). (B) Representative contour plots of expanded LKS cells. Plots are gated on lineage− populations (not shown). Frequency of cKIT+SCA1+ populations (red box) are expressed as a percentage of total lineage− cells. (C) sKITL is not required to maintain multi-lineage hematopoietic progenitor activity. CFUs were assayed from total CD45+ cells following co-culture. (D) BMEC-Akt1 maintain long-term repopulating HSCs in the absence of serum and exogenous cytokines. Total CD45.2+ cells co-cultured on BMEC-Akt1 −sKITL (red circle, solid line) or +sKITL (black square, solid line) were transplanted with a standard CD45.1+ WBM rescue dose. Purified LKS cells from BMEC-Akt1 HSPC co-cultures-sKITL (black box, dashed line) were transplanted with a CD45.1+ SCA1-depleted WBM rescue dose. Engraftment was monitored by %CD45.2+ contribution to the peripheral blood. (E) BMEC-Akt1 +sKITL (red), BMEC-Akt1 -sKITL (black), and BMEC-Akt1 -sKITL with Sca1-depleted rescue dose (white) co-cultures demonstrated multilineage engraftment at 16-weeks post-transplant. n = 10 mice per condition. Error bars represent mean ± SEM, and significance was determined by Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s., not significant.

Figure 5

HSPCs Modulate Gene Expression Changes in BMEC-Akt1 and BMS-Akt1 Cells (A) PCA of BMEC-Akt1 and BMS-Akt1 cell lines alone and in co-culture with HSPCs. (B) Venn diagram of differentially expressed (absolute log2 fold change > 2, p value < 0.05, and expression >10 FPKM) genes in the BMEC-Akt1 ± HSPC and BMS-Akt1 ± co-culture comparisons. (C and D) Distributions of gene expression were visualized by volcano plots of the log2 fold change and −log10 p values and heatmaps of BMEC-Akt1 and BMS-Akt1 cells alone or following HSPC co-culture. Differentially expressed genes (absolute log2 fold change > 2, p value < 0.05) are marked as black and green for BMEC-Akt1 and BMS-Akt1, respectively. Representative conserved gene expression changes are noted. (E) Heatmaps of known pro-hematopoietic factors following HSPC co-culture. (F and G) Differentially expressed transcription factors (F) and chemokines and cytokines identified in BMEC-Akt1 and BMS-Akt1 cell lines following co-culture with HSPCs (G). Three independently isolated BMEC-Akt1 and BMS-Akt1 cell lines were analyzed.

Figure 6

Transplantation of BMEC-Akt1 Promotes Survival and Rapid Hematopoietic Recovery Following Myeloablation (A) C57BL/6J mice were subjected to an LD50 dose of TBI (700 Rads), transplanted on four successive days post-irradiation with either 5 × 105 BMEC-Akt1, 5 × 105 BMS-Akt1, or PBS control and assayed for hematopoietic recovery. (B) BMEC-Akt1, but not BMS-Akt1 transplantation, expedites peripheral hematopoietic recovery. (C) BMEC-Akt1 transplantation, but not BMS-Akt1 or PBS controls, absolutely mitigate radiation-induced death. (D) BMEC-Akt1 transplantations protect phenotypic LT-HSCs. LT-HSCs were quantified from WBM using LKS and CD150+CD48− markers. (E) Hematopoietic progenitor activity in BMEC-Akt1, BMS-Akt1, and PBS transplanted mice. CFUs were assayed from CD45+ WBM at day 28 post-irradiation. (F) H&E-stained BM (trabecular region-femur) show cytopenia 28 days post-irradiation in BMS-Akt1 and PBS control mice, while BMEC-Akt1 transplantations display marked recovery. The spleen, including both red (asterisk) and white pulp (arrowhead), and intestine also demonstrate preserved tissue morphology. n = 10 mice per cell transplant; n = 20 mice per PBS transplant. Error bars mean ± SEM and significance was determined by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Survival curve probability was determined using the log-rank test.

Figure 7

Niche-Specific ECs Efficiently Promote Hematopoietic Recovery Following Myeloablation (A) C57BL/6J mice were subjected to an LD50 dose of TBI (700 Rads), transplanted on four successive days post-irradiation with either 5 × 105 BMEC-Akt1, 5 × 105 pulmonary artery ECs, 5 × 105 brain microvascular ECs, 5 × 105 BM EPCs, or PBS control and assayed for hematopoietic recovery. (B) Transplantation of niche-specific BMEC-Akt1 more efficiently promotes peripheral hematopoietic recovery when compared with heterogeneous EC populations. (C) EC transplantation mitigates radiation-induced death. All heterogeneous ECs tested promote survival following irradiation. (D) Quantification of phenotypic LT-HSCs 28-days post-EC transplantation. LT-HSCs were quantified from WBM using LKS and CD150+CD48− markers. (E) Hematopoietic progenitor activity in EC transplanted mice. CFUs were assayed from CD45+ WBM at day 28 post-irradiation. (F) H&E-stained BM (trabecular region-femur) show cytopenia 28-days post-irradiation in EC and PBS control mice, while BMEC-Akt1 transplantations display pronounced recovery. The spleen, including both red (asterisk) and white pulp (arrowhead), and intestine also demonstrate preserved tissue morphology. n = 10 mice per cell transplant; n = 20 mice per PBS transplant. Error bars represent mean ± SEM, and significance was determined by Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Survival curve probability was determined using the log-rank test.