In Vivo Tracking of Expression Levels of Human Growth Factors Secreted by Human ADRCs in a Murine Fat Graft Model.

Abstract

Abstract Background: Our previously published data show that Adipose-derived regenerative cells (ADRCs) enhance the long term retention of autologous fat grafting compared to non-cell-enhanced grafts. To further understand the time course of growth factor expression of ADRCs, we developed an in vivo xenogenic fat graft model.Materials and Methods: Male NCR nude mice were used as recipients to avoid immune rejection of human ADRCs. Inguinal fat pads from male C57Bl6 mice were minced and used as donor fat for transplantation. Human ADRCs from three healthy donors were isolated using the automated Celution® 800/CRS System (Cytori Therapeutics, San Diego, USA) and mixed with minced mouse fat at a concentration of 5x10E5/ml. The mixture was grafted into the dorsal subcutaneous space of nude mice. Grafted samples were harvested at day 0, day 1, day 7, day 14, day 28, day 56 and day 84, for a total of seven different time points (N=4 per time point). DNA and RNA was isolated for further human-specific detection of IGF-1 and VEGF-A and human-specific single copy DNA using real time RT-PCR. All data were normalized to time 0 samples as 100%.Results: Both of human IGF-1 and VEGF-A expression were detected in the first two weeks after fat grafting, and each of the human growth factor showed its unique pattern. For IGF-1, the highest expression level of 100±7.5% was detected at time 0 with a decline to 0.9±0.7% that was maintained for two weeks. By day 14 only 0.22% of the original signal was detected and no more signal could be detected after day 28. In contrast, on day 1 VEGF-A expression showed an increase to 212±82.6% of the time 0 value. It further declined to 30±23% on day 7 and 8.7±11.7% on day 14. After day 28 no more VEGF-A expression could be detected. The engraftment of ADRCs, measured by DNA content, declined to 12±10.7% on day 1 and remained at around 1% until day 14. By day 28, only 0.14% of human DNA signal could be detected and no more signal could be found after day 56.Conclusion: These findings demonstrate that this xenogenic approach allows the evaluation of single growth factor expression of human ADRCs in vivo using a mouse background when studying cell-enhanced fat grafting. It also allows quantitative assessment of cell survival. By using this model we find that human ADRCs secrete several growth factors within the grafts and that each growth factor has its own unique profile. Furthermore, it becomes evident that ADRCs are active for several days after implantation before growth factor expression subsides. This is an important safety feature of this adult cell therapy source since continued, high level expression of growth factors would not be desirable in cases of reconstruction after breast cancer removal, when concerns of residual tumor cells exist. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4124.