Human adipose-derived stem cells enriched with VEGF-modified mRNA promote angiogenesis and long-term graft survival in a fat graft transplantation model

Abstract

Background: Fat grafting, as a standard treatment for numerous soft tissue defects, remains unpredictable and technique-dependent. Human adipose-derived stem cells (hADSCs) are promising candidates for cell-assisted therapy to improve graft survival. As free-living fat requires nutritional and respiratory sources to thrive, insufficient and unstable vascularization still impedes hADSC-assisted therapy. Recently, cytotherapy combined with modified mRNA (modRNA) encoding vascular endothelial growth factor (VEGF) has been applied for the treatment of ischemia-related diseases. Herein, we hypothesized that VEGF modRNA (modVEGF)-engineered hADSCs could robustly enhance fat survival in a fat graft transplantation model.

Methods: hADSCs were acquired from lipoaspiration and transfected with modRNAs. Transfection efficiency and expression kinetics of modRNAs in hADSCs were first evaluated in vitro. Next, we applied an in vivo Matrigel plug assay to assess the viability and angiogenic potential of modVEGF-engineered hADSCs at 1 week post-implantation. Finally, modVEGF-engineered hADSCs were co-transplanted with human fat in a murine model to analyze the survival rate, re-vascularization, proliferation, fibrosis, apoptosis, and necrosis of fat grafts over long-term follow-up.

Results: Transfections of modVEGF in hADSCs were highly tolerable as the modVEGF-engineered hADSCs facilitated burst-like protein production of VEGF in both our in vitro and in vivo models. modVEGF-engineered hADSCs induced increased levels of cellular proliferation and proangiogenesis when compared to untreated hADSCs in both ex vivo and in vivo assays. In a fat graft transplantation model, we provided evidence that modVEGF-engineered hADSCs promote the optimal potency to preserve adipocytes, especially in the long-term post-transplantation phase. Detailed histological analysis of fat grafts harvested at 15, 30, and 90 days following in vivo grafting suggested the release of VEGF protein from modVEGF-engineered hADSCs significantly improved neo-angiogenesis, vascular maturity, and cell proliferation. The modVEGF-engineered hADSCs also significantly mitigated the presence of fibrosis, apoptosis, and necrosis of grafts when compared to the control groups. Moreover, modVEGF-engineered hADSCs promoted graft survival and cell differentiation abilities, which also induced an increase in vessel formation and the number of surviving adipocytes after transplantation.

Conclusion: This current study demonstrates the employment of modVEGF-engineered hADSCs as an advanced alternative to the clinical treatment involving soft-tissue reconstruction and rejuvenation.

Keywords: Angiogenesis; Fat transplantation; Graft survival; hADSCs; modVEGF.

Fig. 1

Efficacy and kinetics of modRNA transfection in hADSCs. a–c Transfection efficiency and the expression kinetics of modGFP in hADSCs. a Representative images depicting GFP signal in hADSCs at 4, 8, 16, 24, and 48 h post-transfection. b Flow cytometry analysis of transfection efficiency at 4, 8, 16, 24, and 48 h post-transfection. c Flow cytometry analysis of mean fluorescence signal intensity at 4, 8, 16, 24, and 48 h post-transfection. d–f Expression levels of d VEGF mRNA and e–f VEGF protein at 24 h post-transfection. g–h Kinetics of g newly produced and h cumulative VEGF protein concentrations periodically monitored for several days following transfection of modVEGF in hADSCs. Scale bar = 100 μm. Error bars showed means ± SD. (n = 3; *p† < 0.05, **p† < 0.01, ***p† < 0.001, ****p† < 0.0001)

Fig. 2

Multipotent differentiation potential and characterization of isolated and mRNA-engineered hADSCs in vitro. a The proliferation capability of hADSCs was assessed using cell count kit, and the percentage of optical density values relative to the control was calculated. (n = 6; *p† < 0.05, **p† < 0.01). b, c The migration potential of hADSCs was evaluated using transwell assay, and the number of migrated cells dyed with crystal violet (at 12 h post-seeding) was quantified. d Cell surface marker expression of hADSCs was analyzed at 24 h post-transfection. e The multipotent capacity of hADSCs following modRNA transfections was tested by inducing adipogenic, osteogenic, and chondrogenic differentiations. Successful differentiations of the lineages were detected and analyzed using Oil Red O, von Kossa, and Alcian Blue stainings, respectively. Scale bars = 50 μm. Error bars showed means ± SD. (n = 3; *p† < 0.05)

Fig. 3

hADSCs engineered with modVEGF promotes angiogenesis in an in vivo Matrigel plug assay. Matrigel plugs harvested at 1-week post-implantation were assessed. a Representative gross morphological assessment (left) and microphotographs (right) of hematoxylin & eosin (H&E) stainings of extracted plugs. Yellow arrows indicate new blood vessel formation within the plug. b Representative photomicrographs of Matrigel plugs stained for VEGF detection. c The percentage of VEGF expression per area was calculated. d Representative photomicrographs of Matrigel plugs stained for human nuclei antibody (HNA) and CD31. White arrows partially indicate the localization of hADSCs detected with HNA. Yellow arrows partially indicate CD31⁺ blood vessels. e, f Ratio of HNA⁺ cells (e) and the number of CD31⁺ capillaries were quantified (f). g Representative images depicting levels of proliferating hADSCs in plugs using Ki67 stainings. h The ratio of Ki67⁺ cells was quantified. i Representative TUNEL stainings depict levels of apoptotic hADSCs within the Matrigel plugs. j The ratio of apoptotic cells was quantified. Error bars showed means ± SD. Scale bar = 20 μm. (n = 3, *p† < 0.05, **p† < 0.01, ***p† < 0.001, ****p† < 0.0001)

Fig. 4

Preconditioning hADSCs with modVEGF improves fat graft survival in vivo. a Schematic illustration and design of the animal study. b, c Quantification of b VEGF-A in vivo produced protein and c HIF1α protein levels in fat grafts at days 1, 2, 3, 4, 5, and 6 post-transplantation. (n = 3) d Representative gross morphological images of fat grafts at 15, 30, and 90 days post-transplantation. e–g Weights of the fat grafts at 15, 30, and 90 days post-transplantation. (n = 6) Error bars showed means ± SD. (*p† < 0.05, **p† < 0.01, ***p† < 0.001, ****p† < 0.0001)

Fig. 5

Histological analysis of viable adipocytes in fat grafts treated with native hADSCs and modRNA-engineered hADSCs. a Representative H&E stainings of fat grafts harvested at 15, 30, and 90 days post-transplantation. b Analysis of the percentage of vacuoles in the grafted fat. c Fat grafts stained with perilipin identifies viable adipocytes at 15, 30, and 90 days post-transplantation. d The percentage of the viable fat area was analyzed and quantified at 15, 30, and 90 days post-engraftment. Scale bars = 40 μm. Error bars showed means ± SD. (*p < 0.05, ***p† < 0.001)

Fig. 6

Histological evaluation of angiogenesis and cell proliferation in fat grafts. a CD31⁺ capillaries were stained and identified at 15, 30, and 90 days post-transplantation. Black arrowheads indicate blood vessels. b, c Quantitative analysis of b vessel diameters and c capillary densities among the different treatment groups. Note: Significantly larger vascular diameters and increased vessel densities were shown in the hADSC^modVEGF group. d Representative Ki67 stainings on paraffin sections of fat grafts at 15, 30, and 90 days post-transplantation. e Analysis of cell proliferation activity using Ki67⁺ stainings. Scale bars = 20 μm. Error bars showed means ± SD. (*p† < 0.05, **p† < 0.01, ***p† < 0.001)

Fig. 7

modVEGF-engineered hADSCs reduce levels of fibrosis, apoptosis, and necrosis in fat grafts. Fat grafts were harvested at 90 days after grafting for detailed molecular assessment. a Representative Masson’s trichrome staining of fat grafts. b Quantification of collagen (% area). Fibrotic areas are highlighted in blue with regions of extensive collagen deposition. c Representative immunostainings of α-SMA and CD31 shows mature blood vessels. d Analysis of α-SMA⁺ and CD31⁻ adipocytes (%). e Analysis of α-SMA⁺ and CD31⁺ vessels (%). Note: the hADSC^modVEGF group presented the least fibrotic tissue and gave rise to the most SMA⁺ capillaries, an indication of mature blood vessel formation. f Visualization of TUNEL⁺ cells indicates apoptotic activity. g Quantification of apoptotic cells. h Representative photomicrographs of TNF-α⁺ stainings. Black arrowheads practically indicate necrotic cells. i Analysis of TNF-α⁺ area (%). Scale bars = 20 μm. Error bars showed means ± SD. (*p† < 0.05, **p† < 0.01, ***p† < 0.001)

Fig. 8

modVEGF-engineered hADSCs retain higher implantation viability and differentiation capacity to support a long-term survival of fat grafts. hADSCs engineered with modLuc or modVEGF were labeled with CM-DiI before mixing with fat and undergoing transplantation. Fat grafts were harvested at 90 days after grafting for histological evaluation. a Representative images of CM-DiI-labeled hADSCs in fat grafts. b Ratio of CM-DiI-labeled hADSCs. c Representative immunostainings of CD31- and CM-DiI-labeled cells identify vascular endothelial cells differentiated from hADSCs. d Percentage of CD31⁺ and CM-DiI⁺ cells in total CM-DiI⁺ cells (%). e Visualization of perilipin⁺ and CM-DiI⁺ cells indicates adipocytes differentiation activity from hADSCs. f Percentage of perilipin⁺ and CM-DiI+ cells in total CM-DiI⁺ cells (%). Note: the hADSC^modVEGF group presented with more viable hADSCs in fat grafts and gave rise to more capillaries and differentiated adipocytes. Scale bar = 20 μm. Error bars showed means ± SD. (*p† < 0.05, **p† < 0.01)

Fig. 9

A novel combined model of hADSCs engineered with modRNAs could be considered as a potential treatment to promote angiogenesis and graft survival for clinical soft-tissue augmentation and reconstruction