Supplementation of Fat Grafts with Adipose-Derived Rengenerative Cells Improves Long-Term Graft Retention

Min Zhu, MD, MS, Zhengyu Zhou, BS, Yan Chen, MD, MS, Ronda Schreiber, PhD, John T. Ransom, PhD, John K. Fraser, PhD, Marc H. Hedrick, MD, Kai Pinkernell, MD, and Hai-Chien Kuo, PhD.

Introduction

The use of adult stem and progenitor cells is emerging as a novel therapeutic option for many diseases: adipose-derived regenerative cells (ADRCs) include several types of stem and regenerative cells, inculding adipose-derived stem cells (ADSCs). ADSCs have the capacity to differentiate into multiple cell lineages and secrete factors that are angiogenic, antiapoptotic, and pro-adipogenic. The objective of this study was to determine whether ADRC supplementation could improve the long-term graft retention and survival in a mouse fat transplantation model.

Materials and Methods

Murine ADRCs were isolated from the fat pads of ROSA26 mice after 6-12 weeks, through washing, mincing, digestion with collagenase I, and centrifugation. Total RNA was isolated from ADRCs and analyzed using RT-PCR for genes of interest, including mouse VEGFA, HGF, IGF-I, and GAPDH. Inguinal fat pads were isolated, mixed with either saline or a solution of ADRCs, and injected subcutaneously into the skulls of female mice. 6 and 9 months after transplantation, the animals were killed and the grafts were weighed and prepared for hematoxylin and eosin staining, beta-gal staining, and immunohistochemistry. Morphometric analyses were performed to evaluate intact and nucleated adipocytes, cysts and vacuoles, fibrosis, and capillary density. LacZ-postive ADRCs were identified using X-gal. Immunohistochemistry and immunocytochemistry were performed using antibodies directed against CD31, CD34, and CD144.

Results

ADRCs formed extensive tubule-like structures after reaching confluence when plated. These structures showed the presence of for endothelial cell markers, suggesting that ADRCs include cells producing pro-angigogenic factors and extracellular matrix. PCR analysis showed high levels of expression in angiogenesis-related genes.

FIGURE 1. ADRCs form an extensive tubule-like network in vitro. ADRCs form tubule-like structures in DMEM+ 10% FBS in the absence of coated extracellular matrix and additional growth factors. These capillary-like structures were positively stained for endothelial cell markers: CD31 (A), CD34 (B), and CD144 (C). All the images were taken at 100× magnification.

FIGURE 2. ADRCs express angiogenesis-related genes. A comparison between ADRCs and BM-MNCs shows ADRCs highly express many angiogenesis-related genes as compared with BM-MNCs. A relative gene expression ratio above 1 indicates a given gene is expressed more in ADRCs than in BM-MNCs; actual values are noted above each bar. Gene array comparisons were performed twice, showing a similar pattern of relative gene expression, one of which is shown here (A). A comparison of ADRCs to fibroblasts, BM-MNCs, and PBMNCs using real time RT-PCR confirms that ADRCs abundantly express VEGFA, HGF, and IGF-1. For the ease of data presentation, gene expression in each cell type is compared with fibroblasts here. The number above or below each bar indicates the fold of gene expression change in each cell type relative to fibroblasts with a positive value indicating gene up-regulation and a negative value indicating gene down-regulation (B).

Mice transplanted with fat and ADRCs showed greater fat retentention on the skull, with the weight of grafts with ADRCs being about twice that of grafts with only fat at both 6 and 9 months. Histologic examination showed that grafts that included ADRCs had significantly more intact and nucleated adipocytes and fewer cysts and vacuoles than those with only fat. These grafts also had a higher capillary density, as shown by the immunostaining of CD31.

FIGURE 5. ADRCs improve the quality of fat grafts and increase capillary densities within the grafts. Representative H&E sections of fat grafts harvested at 6 months showed that the grafts in the Fat+ADRCs group (B) contained more adipocytes and fewer cysts/vacuoles than those in the Fat-only group (A). Immunostaining of CD31 showed that the grafts in the Fat+ADRCs group (D) had a higher capillary density than those in the Fat-only group (C). CD31⁺ endothelia cells exhibit a brown color, whereas the blue color is due to nuclear staining with methylene blue. All the images were taken at 200× magnification.

FIGURE 6. Incorporation of ADRCs into vessel walls in fat grafts. A representative histologic section of fat grafts harvested at 6 months. Immunofluorescence staining of CD31: CD31⁺ endothelial cells were stained in red (A). [beta]-gal staining:LacZ⁺ cells were stained in blue (B). LacZ⁺ cells were found in vessel walls (arrow). The 2 pictures were taken from the same tissue section. All the images were taken at 200× magnification.

Discussion

Supplementation of fat grafts with ADRCs can improve the long-term retention and quality of grafts, mediated at least in part by the ability of ADRCs to increase neovascularization within the grafts. The presence of ADSCs in vessel walls and the presence of pro-angiogenic and anti-apoptotic factors suggested that ADRCs promote angiogenesis through paracrine signaling. A cooperative interaction between multiple types of stem and regenerative cells in ADRCs may enhance graft survival and quality through several mechanisms.

The clinical utility of ADRC-enhanced fat grafting has been tested in cosmetic breast augmentation and breast reconstruction with positive results. The breat reconstruction trials showed a statistically significant improvement in mean tissue thickness measurement and no significant loss of benefit within twelve months. Previous results also showed that ADRCs did not promote tumor formation when coimplanted with different types of breast cancer cells in SCID mice. Overall, these studies suggest that ADRC therapy is feasible and likely to be safe.

Limitations of this study include the the harshness of the environment in which the fat grafts were placed (the skull of the mice), which led to a low baseline level of graft retention. Another limitation is that the study cannot distinguish between fat graft retention and endogenous adipose regeneration. In conclusion, this model shows that the supplementation of fat grafts with ADRCs can improve the long-term retention and quality of the transplant, an effect mediated by multiple mechanisms that include angiogenesis, with implications for clinical breast augmentation and reconstruction procedures.

Critique

It seemed that authors pushed analysis in the direction of angiogenesis and vasculogenesis as the main mechanism through which the presence of ADRCs improves graft retention. The results did not seem to be very strong regarding the presence of adipose-derived stem cells in vessel walls in fat grafts, and in fact the authors describe it as a “rare event.” This point was addressed in the discussion of the paper, as the authors admitted that there were multiple mechanisms taking place and that the low presence of ADSCs suggested angiogenesis as opposed to vasculogenesis. Effectively, I was given the impression by the way this paper was written that when the authors did not find data that strongly backed up their assumptions, they were not very willing to reconsider these assumptions.

The presentation of data regarding histologic staining was also slightly difficult to read. An explanation in tabular format of the scores used as measurements, as opposed to written explanations, would have been more clear.

Overall, this study succeeds at building upon previous research in tissue engineering to further the possibilities for clinical application of stem cells and progenitor cells. It is clear that the authors are focused on addressing issues related to tissue engineering, such as immunogenicity (solved in the approach of using autologous cells) and the possibility of tumor formation (a different study conducted by the same authors and referred to in the discussion).