The antifibrotic adipose-derived stromal cell: Grafted fat enriched with CD74+ adipose-derived stromal cells reduces chronic radiation-induced skin fibrosis

Abstract

Fat grafting can reduce radiation-induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose-derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74- and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF-β3) and decreased levels of TGF-β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74- or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis.

Keywords: FACS; adipose; adipose stem cells; stem cells; transplantation.

FIGURE 1

Isolation of adipose‐derived stromal cell subpopulations and analysis of effects. A, Schematic of overall experimental design used to explore antifibrotic effects of CD74+ ASCs within irradiated soft tissue beds. Mice were grafted with fat (n = 5/group): (1) enriched with CD74+ ASCs; (2) enriched with CD74− ASCs; (3) enriched with US ASCs; or (4) not enriched with ASCs. B, Isolation of CD74+ ASCs. Flow cytometry plots showing gating strategy used to isolate CD74+ ASCs. ASCs were defined as CD34+ live single cells, negative of lineage markers CD45, CD235a, and CD31. CD74+ ASCs comprised 4.5% of all ASCs within SVF. C, Quantitative real‐time reverse transcription polymerase chain reaction‐PCR showing differentiation expression of antifibrotic growth factors—HGF, FGF2, and TGF‐β3—and pro‐fibrotic growth factor TGF‐β1. CD74+ ASCs had significantly increased expression of HGF, FGF2, and TGF‐β3 and decreased TGF‐β1 compared to both CD74− ASCs (*P < .05) and US ASCs (*P < .05). D, Representative Western blots of TGF‐β3 (top) and TGF‐β1 (middle) from US, CD74+, and CD74− ASCs with beta‐Tubulin loading control (bottom). E, Representative Western blots of Procollagen type 1 (top), Collagen type 1 (second from top), and Collagen type 3 (third from top) synthesized by stimulated human dermal fibroblasts (control) and fibroblasts incubated in conditioned media from US, CD74+, and CD74−, ASCs with beta‐Tubulin loading control (bottom). ASC, adipose‐derived stromal cell; FGF2, fibroblast growth factor 2; HGF, hepatocyte growth factor; SVF, stromal vascular fraction; TGF‐β1, transforming growth factor β1; TGF‐β3, transforming growth factor β3; US, unsorted

FIGURE 2

CD74+ ASC‐enrichment improves fat graft quality. A, Average volume retention, expressed as a percentage with respect to baseline volume, measured over 8 weeks. B, Representative reconstructed microcomputed tomography images of enriched fat and fat only at 8‐weeks postgrafting. There was a trend toward greater fat retention in mice receiving grafts of fat enriched with CD74+ ASCs, compared to fat grafts enriched with CD74− or US ASCs and fat alone. C, Representative H&E‐staining of explanted fat grafts 8 weeks after implantation at ×10 magnification. D, Scoring of graft architectural characteristics based on H&E‐stained sections. Explanted fat grafts enriched with CD74+ ASCs had greater integrity (****P < .001), less inflammation (****P < .0001), and were less fibrotic (****P < .001) compared to the fat grafts enriched with CD74− or US ASCs and fat alone. Scale bar = 100 μm. *P < .05; **P < .01; ****P < .0001. ASC, adipose‐derived stromal cell; US, unsorted

FIGURE 3

CD74+ ASC‐enriched fat grafts improve radiation‐injured skin. A, Tensile strength of irradiated skin overlying enriched and unenriched fat grafts with calculated Young's modulus (*P < .05), and, B, Representative stress‐strain curves demonstrating measurement of Young's Modulus showing that skin overlying fat enriched with CD74+ ASCs was less stiff than skin in mice of all other groups. C, Histological assessment of skin stained with H&E (top) and Masson's Trichrome (bottom). Representative images are shown at ×20 magnification. D, Dermal thickness of skin overlying fat grafts enriched with CD74+ ASCs was significantly thinner (****P < .0001) and (E) had significantly less collagen (***P < .001) than the skin overlying fat grafts enriched with CD74− and US ASCs and fat alone at week 8. Scale bar = 100 μm. **P < .01; ***P < .001; ****P < .0001. ASC, adipose‐derived stromal cell; US, unsorted

FIGURE 4

Dermal fibroblast subpopulation composition in irradiated skin overlying fat grafts. There were significantly fewer papillary (top left) (****P < .0001) and reticular (top right) (****P < .0001) fibroblasts, and significantly greater lipofibroblasts (bottom left) (**P < .01) and zigzag (bottom right) (****P < .0001) fibroblasts in skin overlying CD74+ ASC‐enriched fat compared to skin overlying fat enriched with CD74− and US ASCs or fat alone. ASC, adipose‐derived stromal cell; US, unsorted

FIGURE 5

Immunofluorescence staining for elastic fibers. A, Staining for elastin (red, far left column), fibrillin (yellow, second column), and versican (green, third column) highlights how fat grafting alters components of irradiated extracellular matrix. Merged image with 4',6‐diamidino‐2‐phenylindole (DAPI) counterstain (blue) on the far‐right column. B, Pixel positive‐percent quantification of elastin staining, C, fibrillin staining, and, D, versican staining. Note mice grafted with fat enriched with CD74+ ASCs had improvement in staining for fibrillin (***P < .001, **P < .01, *P < .05), with a concomitant decrease of versican (**P < .01, *P < .05). Scale bar = 100 μm. ASC, adipose‐derived stromal cell