Human Adipose Mesenchymal Stromal/Stem Cells Improve Fat Transplantation Performance

Abstract

The resorption rate of autologous fat transfer (AFT) is 40-60% of the implanted tissue, requiring new surgical strategies for tissue reconstruction. We previously demonstrated in a rabbit model that AFT may be empowered by adipose-derived mesenchymal stromal/stem cells (AD-MSCs), which improve graft persistence by exerting proangiogenic/anti-inflammatory effects. However, their fate after implantation requires more investigation. We report a xenograft model of adipose tissue engineering in which NOD/SCID mice underwent AFT with/without human autologous AD-MSCs and were monitored for 180 days (d). The effect of AD-MSCs on AFT grafting was also monitored by evaluating the expression of CD31 and F4/80 markers. Green fluorescent protein-positive AD-MSCs (AD-MSC-GFP) were detected in fibroblastoid cells 7 days after transplantation and in mature adipocytes at 60 days, indicating both persistence and differentiation of the implanted cells. This evidence also correlated with the persistence of a higher graft weight in AFT-AD-MSC compared to AFT alone treated mice. An observation up to 180 d revealed a lower resorption rate and reduced lipidic cyst formation in the AFT-AD-MSC group, suggesting a long-term action of AD-MSCs in support of AFT performance and an anti-inflammatory/proangiogenic activity. Together, these data indicate the protective role of adipose progenitors in autologous AFT tissue resorption.

Keywords: adipogenesis; adipose stem cells; mesenchymal stem cells (MSCs); tissue engineering.

Figure 1

Human lipoaspirate originate performing AD-MSCs in vitro: (a) photomicrograph of fibroblastoid elements adhering to plastic after adipose tissue digestion (Ph1, scale bar = 100 μm); (b) AD-MSC cumulative population doubling after passage 3 (P3); (c) representative immunophenotypical characterizations of AD-MSCs, with cells positive for CD90, CD105, and CD73 and negative for CD14, CD45, HLA-DR, CD31, and CD146 (isotype control in gray; on the right, the table shows the mean values ± SEM of antigen expressed on AD-MSCs by FACS; (d) differentiation assays on expanded cells after passage P3; osteogenic differentiation visualized by Alizarin Red staining after 14 days of induction and a corresponding noninduced control (upper panels), adipogenic differentiation after 10 days of induction visualized by Oil Red O staining and a corresponding noninduced control (middle panel), and three-dimensional chondrogenic differentiation after 21 days of induction in pellet culture stained with Alcian blue and a corresponding noninduced control (lower panel), scale bar = 100 μm; (e) AD-MSCs expressed GFP after retroviral transduction visualized by both GFP filter fluorescence (upper panel) and ph1 (lower panel), scale bar = 100 μm.

Figure 2

Transplantation approach: Engraftment in vivo was performed according to the autologous fat transfer (AFT) method with/without cells. The AD-MSCs were isolated and expanded from a first lipoaspirate and combined with autologous fat (second lipoaspirate) obtained from the same patient; for the in vivo engraft for cell tracking studies, AD-MSCs were genetically modified by GFP viral vector (AD-MSC-GFP); AFT alone was performed as a control by implantation of only lipoaspirate derived from the same donor, and hyaluronic acid was added as a carrier.

Figure 3

Xenotransplantation did not affect the NOD SCID mice’s health: (a) the average weight of the mice (left graph) and food intake (right graph) were similar between the two groups; (b) serological CREJ2 increased in the AFT group compared to the AFT-ADMSC-GFP group, while the ASTL levels were similar; (c) the hematological parameter results were similar for both groups; (d) the organ weight results were comparable between the AFT and AFT-ADMSC-GFP groups at 7 days (graph left) and at 60 days (right graph); (e) the grafts had similar weights at 7 days post-transplantation, while at 60 days, AFT-ADMSC-GFP had a statistically significant increase compared to AFT. * p < 0.05, n = 7; grey = AFT group; black = AFT-ADMSC-GFP group.

Figure 4

AD-MSC-GFP were tracked in the graft at 7 and 60 days after transplantation. (a,b) H&E-stained representative photomicrographs of AFT (upper panel and inset) and an AFT-ADMSC–GFP specimens (bottom panel and inset) at 7 days post-engraftment and 60 days post-engraftment, respectively. (c) Immunohistochemistry of GFP staining of specimen (upper panel and inset) and AFT-ADMSC-GFP specimens (bottom panel and inset) at 7 days post-engraftment; the AFT samples were negative for GFP staining. On the contrary, the AFT-ADMSC-GFP positive cells were tracked in the thin layer of connective tissue among the adipocytes cluster. (d) Immunohistochemistry GFP staining of AFT (upper panel and inset) and AFT-ADMSC-GFP specimens (bottom panel and inset) at 60 days post-engraftment. The AFT samples were negative for GFP staining. On the contrary, the AFT-ADMSC-GFP specimens were positive. Scale bar = 100 µm.

Figure 5

Proangiogenic and pro-inflammatory characterization of AFT-ADMSC-GFP: (a,b) immunohistochemistry for CD31 in the AFT samples (upper panel and inset) and in the AFT-ADMSC-GFP grafts (bottom panel and inset) at 7 days and 60 days after transplantation; (c,d) quantitative measurement of CD31-positive vessels for the AFT and AFT-ADMSC-GFP specimens, * p < 0.05; (e,f) immunohistochemistry for F4/80 in the AFT samples (upper panel and inset) and in the AFT-ADMSC-GFP grafts (bottom panel and inset) at 7 days and 60 days after transplantation; (g,h) quantification of the F4/80-positive area/graft, respectively, at 7 days and 60 days post-engraftment performed using Image J software (NIH), * p < 0.05; n = 7. Scale bar = 100 µm.

Figure 6

Wild-type ADMSCs exerted proangiogenic and anti-inflammatory effects on the grafts: (a–c) histology analysis of the graft at 7 days after transplantation of AFT (upper panel and inset) and AFT-ADMSC (bottom panel and inset) for (a) H&E, (b) immunohistochemistry for CD31, and (c) immunohistochemistry for F4/80; (d–f) histology analysis of the graft at 180 days after transplantation of AFT (upper panel and inset) and AFT-ADMSC (bottom panel and inset) for (d) H&E, (e) immunohistochemistry for CD31, and (f) immunohistochemistry for F4/80, scale bar = 100 µm; (g) quantitative measurement of CD31-positive vessels for AFT and AFT-ADMSC (left graph) and F4/80-positive area/graft (right graph) at 7 days post-transplantation; (h) quantitative measurement of CD31-positive vessels for AFT and AFT-ADMSC (left graph) and F4/80-positive area/graft (right graph) at 180 days post-transplantation. Quantification was performed using Image J software; * p < 0.05, n = 7.