Osteoblast Derived Exosomes Alleviate Radiation- Induced Hematopoietic Injury

Abstract

As hematopoietic stem cells can differentiate into all hematopoietic lineages, mitigating the damage to hematopoietic stem cells is important for recovery from overdose radiation injury. Cells in bone marrow microenvironment are essential for hematopoietic stem cells maintenance and protection, and many of the paracrine mediators have been discovered in shaping hematopoietic function. Several recent reports support exosomes as effective regulators of hematopoietic stem cells, but the role of osteoblast derived exosomes in hematopoietic stem cells protection is less understood. Here, we investigated that osteoblast derived exosomes could alleviate radiation damage to hematopoietic stem cells. We show that intravenous injection of osteoblast derived exosomes promoted WBC, lymphocyte, monocyte and hematopoietic stem cells recovery after irradiation significantly. By sequencing osteoblast derived exosomes derived miRNAs and verified in vitro, we identified miR-21 is involved in hematopoietic stem cells protection via targeting PDCD4. Collectively, our data demonstrate that osteoblast derived exosomes derived miR-21 is a resultful regulator to radio-protection of hematopoietic stem cells and provide a new strategy for reducing radiation induced hematopoietic injury.

Keywords: apoptosis resistance; hematopoietic injury; irradiation; miR-21; osteoblast derived exosomes.

FIGURE 1

Osteoblast derived exosomes promote white blood cell, lymphocyte and monocyte recovery after irradiation. (A) Detection of protein levels of Alix, HSP70, TSG101, TFIIB and Lamin A/C in OB-exosomes and parental cell lysates by western blot. (B) Nanoparticle tracking the size distribution of OB-exosomes (representative of five independent measurements). (C) Representative transmission electron microscope (TEM) images of OB-exosomes. Scale bar, 200 nm. (D) The mice were injected with PBS or OB-exosomes immediately after 5 Gy TBI at 1st, 3rd, 5th day after irradiation. (E) white blood cell counts, (F) lymphocytes counts, (G) monocyte counts, (H) neutrophils counts, (I) red blood cell counts, (J) platelet counts and (K) hemoglobin level were measured by a hematology analyzer. Statistical analysis of peripheral blood cell number in irradiated mice injected with PBS (n = 8) and osteoblast-exosomes (*p < 0.05, mean ± sem, n = 6).

FIGURE 2

Osteoblast derived exosomes mitigate irradiation injury to hematopoietic stem and progenitor cells. (A) The mice were injected with PBS or OB-exosomes immediately after 5 Gy TBI at 1st, 3rd, 5th day after irradiation. (B) Representative FACS plots of LSK cells, MMP, LT-HSC and ST-HSC were detected by flow cytometry. (C) The LSK frequency, (D) LT-HSC frequency (E) ST-HSC frequency, (F) MPP frequency, (G) BM numbers, (H) LSK numbers, (I) LT-HSC numbers, (J) ST-HSC numbers, (K) MPP numbers were analyzed 21 days after 5 Gy TBI. Statistical analysis of the hematopoietic stem cell number in irradiated mice injected with PBS (n = 9) and osteoblast-exosomes (*p < 0.05, **p < 0.01, mean ± sem, n = 7).

FIGURE 3

Osteoblast derived exosomes inhibit irradiation induced apoptosis of FDC-P1. (A,B) Representative imaging showed the fluorescence intensity signal detected by flow cytometry. Colocalization of OB-exosomes with FDC-P1 cells using confocal microscopy imaging. Exosomes were labeled by 3′-dioctadecyloxacarbocyanine perchlorate (Dio, green) and cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. (C) FDC-P1 cells were irradiated with 2 Gy before treated with OB-exosomes, and apoptosis was measured by flow cytometry. n = 3/group (D) Statistical analysis of the difference between control, irradiation and co-cultured with OB-exosomes after irradiation groups (***p < 0.001, mean ± sem, n = 3/group). (E) Detection of protein levels of Cleaved PARP and BAX in irradiated FDC-P1 by western blot.

FIGURE 4

The osteoblast derived exosomes miRNA analysis. (A) The heat map of miRNA in OB-exosomes, the red color represents high miRNA expression level, and the blue color represents low miRNA expression level. (B) qRT-PCR analysis of expression of miRNA levels in OB-exosomes. (C) qRT-PCR analysis of expression of miRNA levels in FDC-P1 treated with or without OB-exosomes for 6 h. The relative expression of miR-21 was normalized to U6. (D) qRT-PCR analysis of expression of miRNA levels in bone marrow cells of irradiated mice injected with or without OB-exosomes. (mean ± sem, n = 3). (E) Venn diagram of top 10 miRNA in OB-exosomes, top 5 miRNA in FDC-P1 and top 5 miRNA in bone marrow.

FIGURE 5

MiR-21 in osteoblast derived exosomes alleviated irradiation induced FDC-P1 apoptosis. (A) qRT-PCR analysis of expression of miR-21 levels in osteoblast transfected with NC or miR-21 mimics. (B) qRT-PCR analysis of expression of miR-21 levels in OB-exosomes transfected with NC or miR-21 mimics. (C) qRT-PCR analysis of expression of miR-21 levels in FDC-P1 which were cocultured with OB-exosomes transfected with NC or miR-21 mimics. (D) FDC-P1 cells were irradiated with 2 Gy before co-cultured with OB-exosomes (NC/miR-21 mimics), and apoptosis was measured by flow cytometry. (E) Statistical analysis of the difference between control, irradiation and co-cultured with OB-exosomes (NC/miR-21 mimics) after irradiation groups. (mean ± sem, n = 3/group). (F) FDC-P1 cells were irradiated with 2 Gy before co-cultured with OB-exosomes (NC/miR-21 mimics), and protein levels of Cleaved PARP and BAX was measured by western blot.

FIGURE 6

Osteoblast derived exosomes inhibit radiation-induced damage to hematopoietic stem cell. OB-exosomes led to a recovery of hematopoietic stem and progenitor cells numbers in vivo which decreased after total body irradiation, and significant improvement was observed in radiated mice injected with OB-exosomes. The miR-21 of OB-exosomes was identified as the target mediated the effect on the apoptosis of HSC.