Adipocyte Stem Cells Ameliorate Total Body Irradiation-Induced Hematopoietic Syndrome and Late Radiation Fibrosis

Abstract

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at day 14 post irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell coculture revealed significant down regulation of profibrotic genes (TGF β, and Col 1-4) in irradiated cells. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Irradiated HFF revealed high expression of HGF receptor c-Met. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson’s Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation-induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. A single 1 x 106 ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.