Allogeneic adipose-derived stem cells mitigate acute radiation syndrome by the rescue of damaged bone marrow cells from apoptosis

Abstract

Acute radiation syndrome (ARS) is the radiation toxicity that can affect the hematopoietic, gastrointestinal, and nervous systems upon accidental radiation exposure within a short time. Currently, there are no effective and safe approaches to treat mass population exposure to ARS. Our study aimed to evaluate the therapeutic potential of allogeneic adipose-derived stem cells (ASCs) for total body irradiation (TBI)-induced ARS and understand the underlying mitigation mechanism. We employed 9.25 Gy TBI dose to C57BL/6 mice and studied the effect of allogeneic ASCs on mice survival and regeneration of the hematopoietic system. Our results indicate that intraperitoneal-injected ASCs migrated to the bone marrow, rescued hematopoiesis, and improved the survival of irradiated mice. Our transwell coculture results confirmed the migration of ASCs to irradiated bone marrow and rescue hematopoietic activity. Furthermore, contact coculture of ASCs improved the survival and hematopoiesis of irradiated bone marrow in vitro. Irradiation results in DNA damage, upregulation of inflammatory signals, and apoptosis in bone marrow cells, while coculture with ASCs reduces apoptosis via activation of DNA repair and the antioxidation system. Upon exposure to irradiated bone marrow cells, ASCs secrete prosurvival and hematopoietic factors, such as GM-CSF, MIP1α, MIP1β, LIX, KC, 1P-10, Rantes, IL-17, MCSF, TNFα, Eotaxin, and IP-10, which reduces oxidative stress and rescues damaged bone marrow cells from apoptosis. Our findings suggest that allogeneic ASCs therapy is effective in mitigating TBI-induced ARS in mice and may be beneficial for clinical adaptation to treat TBI-induced toxicities. Further studies will help to advocate the scale-up and adaptation of allogeneic ASCs as the radiation countermeasure.

Keywords: acute radiation syndrome; adipose stem cells; cytokines; hematopoiesis; intraperitoneal injection; total body irradiation.

FIGURE 1

Characterization of allogeneic adipose‐derived stem cells (ASCs). Phase‐contrast microscopic images represent the morphology of ASCs derived from adipose tissues of C57BL/6‐GFP and FVB‐GFP (A). The microscopic fluorescence images of GFP positive ASCs fixed and stained with TRITC‐phalloidin for F‐actin (red) and Hoechst to visualize cell nuclei (blue) (B). Scale bars = 100 μm. Magnification 10x. C, Representative flow cytometry histograms. Expression of CD34, CD29, CD90, CD105, CD26, and CD45 was analyzed. D, Isolated ASCs were subjected to adipocyte differentiation for 14 days using the adipocyte differentiation medium. Oil‐Red‐O staining to confirm differentiation was performed and shown

FIGURE 2

Allogeneic and autologous allogeneic adipose‐derived stem cells (ASCs) transplant improve the survival of total body irradiated mice. Schematic representation of autologous ASCs (A) and allogeneic ASCs (C) transplanted to irradiated C57 BL/6 recipients. (B) The survival curve of irradiated C57BL/6 mice injected with autologous ASCs or phosphate‐buffered saline (PBS) as control is shown. Survival was monitored for 30 days (n = 20/group). D, Survival curve of irradiated C57BL/6 mice injected with allogeneic ASCs or PBS as control is shown. Survival was monitored for 30 days (n = 20/group). E, White blood count analyses at day 14 post‐ASCs injection. The results are presented as mean ± SD; *P < .05. F,G, Schematic representation of autologous ASCs (F) and allogeneic ASCs (G) transplanted to irradiated FVB recipients. H, Survival curve of irradiated FVB mice injected with either autologous ASCs, allogeneic ASCs, or PBS. Survival was monitored for 30 days (n = 10/group). I, Survival curve of 9.25 Gy irradiated C57BL/6 mice injected with either C57BL/6 ASCs CM, FVB ASCs CM, control culture media, or PBS. Survival was monitored for 10 days (n = 10/group)

FIGURE 3

Transplanted allogeneic adipose‐derived stem cells (ASCs) migrate to the bone marrow of irradiated mice. A, Bone marrow from day 35 postirradiation surviving mice was isolated and cultured. Day 24 postculture bone marrow cells were fixed and stained with TRITC‐phalloidin for F‐actin (red) and Hoechst to visualize cell nuclei (blue). B, Day 52 postculture images for green fluorescent protein (GFP) positive cells. C, Schematic representation of transwell migration experiment. D,E, Fluorescence microscopy images of the ASCs isolated from C57BL/6‐GFP (D) and FVB‐GFP (E) mice cocultured with irradiated and nonirradiated bone marrow. The cells were fixed with 4% paraformaldehyde and stained with Phalloidin‐TRITC for F‐actin (red). Hoechst was used for staining nuclei (blue). ASCs: GFP⁺, Phalloidin‐TRITC⁺, and Hoechst⁺. Bone marrow cells: GFP⁻, Phalloidin‐TRITC⁺, and Hoechst⁺. Scale bars = 100 μm. Magnification 10x. F, Bone marrow from 24 hours postirradiated mice, or nonirradiated mice were cocultured with either allogeneic or autologous ASCs for 2 weeks. ASCs were seeded in the top chamber, and bone marrow cells from irradiated and nonirradiated were seeded in the lower chamber. Phase‐contrast microscopic images of cobblestone formation during coculture. G, Ex vivo real‐time quantitative gene expression analysis of irradiated bone marrow cells 24 hours postirradiation. The results are presented as mean ± SD; **P < .01, *P < .05

FIGURE 4

Allogeneic adipose‐derived stem cells (ASCs) migrate through the transwell and protect irradiated bone marrow from apoptosis. A,C, Migration of C57BL/6 ASCs across the transwell membrane toward irradiated bone marrow (A) and nonirradiated bone marrow (C) was analyzed by FACS. The percentage of GFP negative (bone marrow cells) and GFP positive (ASCs) as determined. B,D, Apoptosis of cocultured irradiated (B) and nonirradiated (D) bone marrow cells was analyzed by Annexin V and PI staining by FACS. E,G, Migration of FVB ASCs across the transwell membrane toward irradiated bone marrow (E) and nonirradiated bone marrow (G) was analyzed by FACS. The percentage of GFP negative (bone marrow cells) and GFP positive (ASCs) was determined. F,H, Apoptosis of cocultured irradiated (F) and nonirradiated (H) bone marrow cells was analyzed by Annexin V and PI staining by FACS. I, Real‐time quantitative gene expression analysis of sorted cocultured irradiated bone marrow cells. The results are presented as mean ± SD; **P < .01, *P < .05

FIGURE 5

The contact coculture of allogeneic adipose‐derived stem cells (ASCs) improves the survival of the irradiated bone marrow. A, Morphology of cocultured irradiated bone marrow with or without ASCs. Scale bars = 100 μm. Magnification 10x. B, Irradiated bone marrow cocultured with or without ASCs were stained with Annexin‐V‐APC/Propidium iodide and analyzed by FACS. Flow cytometry histograms of non‐GFP positive gated bone marrow cells apoptosis. C, The percentage of apoptosis during contact coculture. The results are presented as mean ± SD; **P < .01. D, Phase‐contrast images of irradiated and nonirradiated bone marrow cultured in ASCs conditioned media or fresh media. Scale bars = 50 μm. Magnification 10x

FIGURE 6

Allogeneic adipose‐derived stem cells (ASCs) secrete prohematopoietic factors to support the recovery of damaged bone marrow cells. A‐L, Luminex assay readout of the factors released by monoculture and cocultured irradiated and nonirradiated bone marrow cells with ASCs (n = 3). The results are presented as mean ± SD; *P < .05