ADSCs Promote Graft Survival in a Mouse Model
A mouse model was established to assess whether ADSCs possess positive effects on the maintenance of transplanted adipose tissues. In this model, bilateral peritesticular fat pads were resected to construct the pelvic cavity, and the intestinal tract was sutured to the retroperitoneal fascia to prevent it from dropping into the formed cavity. Following this, the resected fat pads were returned into the constructed pelvic defect with or without 1 × 106 cells of ADSCs (n = 20 each) suspended in 1 ml of PBS (A). After 21 days, the mice were sacrificed to assess the transplants. In the control mice, atrophic change of the transplant, hemorrhagic ascites formation in the intraperitoneal space, and adhesion of intraperitoneal tissues were observed by abdominal laparotomy. In contrast to control mice, the grafts in ADSC-treated mice showed a vivid image with surface neovascularization and showed no ascites or adhesion (B). Microscopic analysis of the grafts showed hemorrhagic necrosis with infiltration of inflammatory cells in the control group, whereas healthy fat tissue construction was observed in the grafts of the ADSC-treated group (C).
Figure 1. ADSCs maintain intraperitoneal fat grafts in the pelvic dead space mouse model. (A): Bilateral peritesticular fat pads (arrowheads in the left panel) were resected, and the intestinal tract was sutured to the retroperitoneal fascia to form the pelvic (more ...)
To quantify the engraftment efficacy, the mice were sacrificed on days 3, 7, 14, and 21 after transplantation, and the grafts were then weighed (n = 5 in each arm and on each day). The weight ratios (weight of engraft/weight of implanted fat pad) of the transplants were significantly higher in the ADSC-treated group than in the control group on days 14 and 21 (p < .05) (D).
ADSCs Promote Angiogenesis
The grafts in the ADSC-treated group showed higher neovascularization; therefore, we focused on angiogenesis to clarify the reason why ADSCs induce positive effects on transplant maintenance. Immunohistochemical analysis for the endothelial marker CD31 was performed to assess graft vascularity. The number of CD31+ lumen formations was obviously higher in the ADSC-treated group (A). For definite assessments, the transitive number of CD31+ lumen formations was counted on days 3, 7, 14, and 21 after transplantation (n = 5 in each arm and on each day). This value was significantly higher in the ADSC-treated group on days 3, 7, and 14 when compared with that in the control group (p < .05); however, it was not significant on day 21 (p = .78) (B). This may be because of the increased relative number of blood vessels for higher atrophy of the grafts in the control group at 21 days after transplantation. To assess angiogenic activity, VEGF expression was measured by quantitative reverse transcription PCR, which showed that VEGF mRNA was significantly higher in the ADSC-treated group on days 3 and 7 after transplantation (p < .05) (C). In addition, expression of HGF, an angiogenic and wound-healing factor, was also significantly higher in the ADSC-treated group on days 3 and 7 (p < .05) compared with that in the control group (D). Expression of interferon-γ, tumor necrosis factor-α, and interleukin-6 did not differ significantly between the groups (data not shown).
Figure 2. ADSCs differentiate into CD31+ endothelial vascular cells. (A): Immunohistochemical staining for CD31. Sections from ADSC-treated mice contained abundant stromal structures with CD31+ vascular formation (black arrowheads) compared with those from control (more ...)
Next, GFP-positive ADSCs were traced to assess whether ADSCs differentiate into endothelial cells to form vascular structures (n = 9; sacrificed on days 3, 7, and 21; three mice each). In vivo time course analysis of GFP-positive cells revealed that GFP-positive cells formed cellular clusters with CD31 expression on day 7 and then morphologically changed to vascular structures on day 21 (E).
ADSCs Contain Adipogenic and Angiogenic Characterized Cell Fractions
To clarify and characterize the cell fractions that constitute ADSCs, multicolor flow cytometry analysis was performed (A). Analysis with CD45 revealed that ADSCs were divided into CD45+ and CD45− fractions and these were further divided into CD90+ and CD90− fractions. Regardless of CD90 expression, CD45+ cell fractions could not be maintained in vitro (B). CD45− cells were further analyzed with CD34, CD90, and CD31. In the CD45− cell fraction, a small number of CD31+ cells (~25%) was identified, which were suggested to be endothelial cells. Because CD31+ vascular endothelial cells arose from CD31− GFP+ cells in vitro (E) and the CD31+ cell fraction could not be maintained in vitro (data not shown), the CD31− cell fraction was further analyzed with CD34 and CD90 to clarify the origin of CD31+ cells. CD45− and CD31− cells were constructed by CD34+ and CD34− cell components, and the CD34+ cell component was divided into CD34+CD90+ and CD34+CD90− cell fractions (A). To assess cellular proliferative capacity and angiogenic activity, a colony formation assay and Matrigel (BD Biosciences) tube formation assay were performed in isolated CD45−CD31−CD34+CD90− and CD45−CD31−CD34+CD90+ cell fractions. The colony formation assay revealed that both these cell fractions formed colonies and that there was no significant difference between colony formation activity of the cell fractions (B). Interestingly, in contrast to the CD45−CD31− CD34+CD90− cell fraction, which showed low tube formation activity, the Matrigel tube formation assay revealed high tube formation activity in the CD45−CD31−CD34+ CD90+ cell fraction (B).
Figure 3. Adipose-derived stem cells (ADSCs) constructed from angiogenic and adipogenic subsets. (A): Flow cytometry analysis of mouse ADSCs. CD45− cells were analyzed for CD34, CD90, and CD31. The CD45−CD31−CD34+ subpopulations were further (more ...)
To characterize the cellular function of the CD45− CD31−CD34+CD90+ cell fraction, adipose differentiation activity was assessed. Phase contrast microscopy of adipocyte induction showed that the CD45−CD31−CD34+CD90− cell fraction contained a large amount of lipid droplet-like structures compared with the CD45−CD31−CD34+CD90+ cell fraction, which resulted in higher adipose differentiation activity of the CD45−CD31−CD34+CD90− cell fraction (C). Next, to assess stem cell activity, a sphere formation assay was performed. Interestingly, both CD45− CD31−CD34+CD90+ and CD45−CD31− CD34+CD90− cell fractions efficiently formed spheres (C). Taken together, these findings indicate that ADSCs contain at least two characteristic stem cell-like fractions that possess angiogenic and adipogenic properties. These analyses were performed in triplicate from four independently sacrificed mice, and compatible data were obtained.
Human ADSCs Support Transplant Survival
To assess whether human ADSCs share similar properties with mouse ADSCs, human ADSCs were isolated from subcutaneous fat pads and their cellular properties were analyzed. Flow cytometry analysis of five human ADSC samples revealed cell surface marker expression similar to that of mouse ADSCs, which contained CD45+ and CD45− cells. In CD45− cells, the existence of CD34+CD31− (43.40 ± 12.79%), CD34+CD31+ (11.99 ± 5.41%), and CD34−CD45− (25.73 ± 11.52%) cells was confirmed (A). Although the number of CD45−CD34+CD31+ cells was higher in human ADSCs than that in mouse ADSCs (11.99 ± 5.41% in human vs. 4.25 ± 2.41% in mouse), CD45− CD34−CD31+ cells could not actually be identified in human ADSCs (4.12 ± 2.85% in mouse). An in vitro assay indicated that CD45−CD34+ cells but not CD45−CD34− cells formed colonies and spheres (B), suggesting that CD34+ cells in a CD45− fraction possess stem cell activity also in human ADSCs. To assess whether human ADSCs contribute to long-term graft survival, 5 × 105 cells of freshly isolated ADSCs (fADSCs) were added to 1 ml of human lipoaspirate and then transplanted in nude mice (n = 3 from independent patients). PBS was used as a control. The mice inoculated with ADSCs showed efficient engraftment of the transplants with the control mice 6 months after transplantation (C). To assess whether cultured ADSCs (cADSCs) also possess a high maintenance effect on the transplants, each volume of engrafted transplants of fADSCs, cADSCs, and PBS controls was measured 6 months after transplantation. Interestingly, transplant volumes were highly retained in the fADSC-treated mice compared with those in the cADSC-treated and control mice (D). Immunohistochemical staining for human vWF in resected specimens from the control and fADSC-treated mice revealed high angiogenic activity of fADSCs (E).
Figure 4. Freshly isolated human ADSCs sufficiently maintain fat graft. (A): Flow cytometry analysis of human ADSCs. The CD45− cell fraction was further analyzed for CD31 and CD34. (B): Colony formation and sphere formation abilities of freshly isolated (more ...)