Autologous fat grafting has become a common technique for treating volume and contour deficiencies in reconstructive and cosmetic procedures for hemifacial atrophy, lipodystrophy, and breast reconstruction.1,2 However, one major caveat of fat grafting is its absorption over time. With the development of various techniques to assist fat transfer,2,3 fat graft retention has become more predictable but is still less than ideal.4,5 Currently, cell-assisted lipotransfer is the most frequently used technique to increase fat graft retention.6–8 Given their expansive pluripotent capacities and secretion of abundant growth factors and cytokines, it is now widely accepted that mesenchymal stem cells—in particular, adipose-derived stem cells—increase fat graft retention through promotion of angiogenesis and tissue regeneration.9–14
The therapeutic effects of mesenchymal stem cells were originally thought to derive from their engrafting and differentiation capacities. However, more recent studies found that these cells mediate tissue repair and regeneration predominantly in an indirect manner by means of their paracrine factors.15–17 In addition, cell therapies in general have been limited by several factors, including uncontrolled differentiation, undesirable long-term side effects, senescence-induced genetic instability or loss of function, and limited cell survival.18,19 At the same time, immune compatibility issues may likely prevent their allogenic use, thus further limiting their commercial and clinical potential.20,21 Furthermore, the current regulatory environment severely restricts the use of cell therapies.22 Therefore, a cell-free therapy potentially could be an ideal alternative to cell-based therapies.
In addition to soluble proteins, mesenchymal stem cells release numerous extracellular vesicles, a large and heterogeneous family of membrane-enclosed embodiments differing in size and biogenesis.23 Among subtypes of extracellular vesicles, exosomes are considered important mediators of cell-to-cell communication in both physical and pathologic processes. They range from 30 to 150 nm in size, are released from many cell types, and are ubiquitously distributed in various body fluids.24–26 By transferring their contained proteins, mRNAs and microRNAs, exosomes act as vehicles of information believed to play novel roles in tissue repair and regeneration.27,28 Exosomes released by mesenchymal stem cells and adipose-derived stem cells have recently been identified as the principal agents mediating the therapeutic efficacy of their source cells in several disease or tissue regeneration scenarios, such as myocardial ischemia/reperfusion injury and tissue regeneration.29–33 Therefore, it is highly impactful, as potentially allogeneic exosomes could be used across hosts in a variety of therapeutic applications.34
In this study, we used lipotransfer as a surrogate to investigate the effects of adipose-derived stem cell–derived exosomes in fat graft retention. We hypothesized that these exosomes would enhance graft retention by improving angiogenesis and regulating inflammation, similar to their adipose-derived stem cell source cells, thus establishing a theoretical basis for a future cell-free therapeutic approach.
MATERIALS AND METHODS
Culturing of Adipose-Derived Stem Cells
Adipose-derived stem cells were prepared and cultured as described in previous studies,35 with inguinal fat pads collected from C57/BL6 mice. The phenotype of cultured adipose-derived stem cells at passage 4 was characterized by immunocytochemistry as being positive for CD44 and CD90 but not for CD34 and CD45 (BioLegend, San Diego, Calif.).
Preparation of Adipose-Derived Stem Cell–Derived Exosomes
Low passage (fewer than five passages) adipose-derived stem cells were grown to 70 to 80 percent confluence, the culture medium was replaced with basal medium (Dulbecco’s Modified Eagle Medium supplemented with 2 mM l-glutamine and 100 U penicillin/100 U streptomycin, but no fetal bovine serum), and the cells were cultured for another 48 hours. The conditioned medium was centrifuged to remove cell debris and passed through a 0.22-μm filter. The clarified conditioned medium was then size-fractionated and concentrated 50× by tangential flow filtration using a membrane with a molecular weight cutoff of 100 kDa (Sartorius, Goettingen, Germany), and then passed through a 100-nm filter. The resultant exosomes were stored in a −20°C freezer until use. The protein content of exosomes was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher, Rockford, Ill.).
Identification of Adipose-Derived Stem Cell–Derived Exosomes
The size of adipose-derived stem cell–derived exosomes was determined by NanoSight LM10 (Amesbury, United Kingdom). The morphology of adipose-derived stem cell–derived exosomes was analyzed by JEOL 1200EX (JEOL, Tokyo, Japan) transmission electron microscopy. Western blot analysis was performed with anti-CD9 (Thermo Fisher), HSP 70 (R&D Systems, Minneapolis, Minn.), and β-actin (Abclonal, Woburn, Mass.).
Adipogenic Differentiation Assays
A total of 1 × 105 adipose-derived stem cells at passage 4 were seeded into each well of a six-well plate. Adipose-derived stem cells were cultured in complete medium overnight, then rinsed with phosphate-buffered saline, and then incubated with one of three different culture media for up to 7 days. The media used for adipogenic differentiation assay were as follows: (1) Adipogenesis Differentiation Medium (Cell Applications, San Diego, Calif.) supplemented with 40 μg/ml of adipose-derived stem cell–derived exosomes (n = 3); (2) Adipogenesis Differentiation Medium supplemented with phosphate-buffered saline at the same volume as exosomes (n = 3); (3) complete medium supplemented with phosphate-buffered saline at the same volume as exosomes (control, n = 3). The medium was replaced every 2 days. The cells were harvested at day 7 with TRIzol Reagent (Thermo Fisher) for quantitative polymerase chain reaction analysis. Adipogenic differentiation was determined by oil red O (Sigma-Aldrich, St. Louis, Mo.) staining.36
Animals and Treatment
Eight-week-old male C57/BL6 mice were obtained from the Jackson Laboratory (Bar Harbor, Me.). All experiments were approved by the Lahey Animal Ethics Committee.
A total of 48 mice were anesthetized with isoflurane inhalation anesthesia. Fat tissue was harvested from the inguinal fat pads of C57/BL6 mice and gently dissected into small pieces, similar to the size of aspirated fat tissue used for clinical fat injection in humans. The weight of 200 μl of prepared adipose tissue served as the wet weight baseline for fat grafts. Two hundred microliters of minced fat with 1 × 104 cells of adipose-derived stem cells in 20 μl of phosphate-buffered saline (cell-assisted lipotransfer, n = 16), 40 μg of adipose-derived stem cell–derived exosomes in 20 μl of phosphate-buffered saline (n = 16), or 20 μl of phosphate-buffered saline (n = 16), as a bolus, were injected bilaterally under the skin of the lower back of each mouse using 16-gauge needles. At day 7 and day 14, each transplanted fat bolus of the exosome group was injected with another 40 μg of adipose-derived stem cell–derived exosomes in 20 μl of phosphate-buffered saline, whereas the cell-assisted lipotransfer and phosphate-buffered saline groups were injected with the same volume of phosphate-buffered saline. At 3 and 10 weeks after lipotransfer, eight mice of each group were euthanized and the fat grafts on both sides of the mice were harvested, weighed, and analyzed.
Paraffin-embedded tissue sections were stained with hematoxylin and eosin for histologic structure analysis. Overall integrity, oil cysts, and fibrosis in fat grafts were assessed by three blinded observers.9
CD31 immunohistochemistry staining (Abcam, Cambridge, Mass.) was performed for angiogenesis analysis. Immunofluorescent staining of Mac-2 (Cedarlane Corp., Burlington, Ontario, Canada), perilipin (Progen, Heidelberg, Germany), and collagen VI (Abcam) was performed for further histologic analysis.
Quantitative Reverse Transcription Polymerase Chain Reaction
Fat tissue was excised, snap-frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted and analyzed using published methodologies.37,38 All primers designed for this study were determined through established GenBank sequences, synthesized by the Massachusetts General Hospital DNA Core facility. (See Table, Supplemental Digital Content 1, which shows primer sequences for real-time reverse transcription polymerase chain reaction, http://links.lww.com/PRS/D748.) The amount of each target gene was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, Calif.). All data were expressed as mean ± SEM, with the results in the three groups compared by one-way analysis of variance for three groups and t test for two groups, with a value of p < 0.05 considered significant.
Characterization of Adipose-Derived Stem Cell–Derived Exosomes
The adipose-derived stem cell–derived exosomes were characterized by independent methods. Under transmission electron microscopy, the isolated adipose-derived stem cell–derived exosomes appeared round or cup-like, with double-membrane structures (Fig. 1, above, left). The size distribution as measured by NanoSight revealed that adipose-derived stem cell–derived exosomes were homogeneous with a peak at 123 nm (Fig. 1, above, right). The characteristics of adipose-derived stem cell–derived exosomes were further validated with Western blotting for expression of CD9 and β-actin. The results showed that CD9, a tetraspanin characteristically expressed on exosomes, was specifically enriched in exosomes, whereas the cellular marker β-actin was detected exclusive in cell lysates (Fig. 1, below). The vesicles were thus characterized as exosomes, as they possessed the published defining criteria.39
Evaluation of Fat Graft Retention Rate
Fat grafts receiving either adipose-derived stem cells or exosomes were covered with a thin, well-vascularized fibrous capsule, whereas the control group had poor surface vascularization (Fig. 2, left). Their survival was assessed as retention rates (final weight/initial weight) at 3 and 10 weeks after lipotransfer. At week 3, no significant difference in graft retention was observed across the three groups. At week 10, retention rate in the cell-assisted lipotransfer group was significantly better than that of the phosphate-buffered saline group (50.9 ± 2.4 percent versus 40.7 ± 4.7 percent; p = 0.03). Exosome treatment also significantly improved the retention rate [56.4 ± 1.6 percent versus 40.7 ± 4.7 percent (control); p < 0.001]. No significant difference was observed between the exosome and cell-assisted lipotransfer groups (p > 0.05) (Fig. 2, right).
Histologic Assessment of the Transferred Fat
Histologic characteristics were examined at 3 and 10 weeks after fat transfer. [See Figure, Supplemental Digital Content 2, which shows histologic appearance of fat grafts in the three groups (hematoxylin and eosin; original magnification, × 100). PBS, phosphate-buffered saline; CAL, cell-assisted lipotransfer; Exo, exosome, http://links.lww.com/PRS/D749.] Oil cysts and fibrosis could be found in all three groups, but the grafts in the phosphate-buffered saline group had more and larger oil cysts. However, the grafts in both the exosome and the cell-assisted lipotransfer groups showed better fat integrity, fewer oil cysts, and reduced fibrosis at week 10 (versus the phosphate-buffered saline group; p < 0.05) (Fig. 3).
Inflammation Level in the Transferred Fat
Immunohistochemistry staining of Mac-2 and perilipin showed the transferred adipose tissue structure in the three groups at week 3. [See Figure, Supplemental Digital Content 3, which shows representative immunofluorescent staining of Mac-2 and perilipin in the transferred fat (original magnification, × 100). PBS, phosphate-buffered saline; CAL, cell-assisted lipotransfer; Exo, exosome; DAPI, 4′,6-diamidino-2-phenylindole, http://links.lww.com/PRS/D750.] Mac-2–positive crown-like structures appeared in the transferred fat in all three groups. Larger crown-like structures and fewer perilipin-positive adipocytes could be found in the phosphate-buffered saline group. However, many more perilipin-positive adipocytes and some small Mac-2–positive structures appeared in both the exosome and cell-assisted lipotransfer groups. To evaluate the inflammation level of the transferred fat, the inflammatory cytokines were assessed by quantitative reverse transcription polymerase chain reaction. Interleukin-1β, a proinflammatory cytokine, had higher expression in the exosome and cell-assisted lipotransfer groups; the expression of interleukin-10, an antiinflammatory cytokine, was elevated in the exosome and cell-assisted lipotransfer groups (versus the phosphate-buffered saline group; p < 0.05) (Fig. 4, left). The two chemokines that regulate migration and infiltration of monocytes/macrophages, the macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 were also elevated in the exosome and cell-assisted lipotransfer groups (versus the phosphate-buffered saline group; p < 0.05, respectively) (Fig. 4, right).
Vessel Density in the Transferred Fat
Angiogenesis in the transferred fat 3 weeks after transplantation was evaluated by immunostaining with antibody to CD31. [See Figure, Supplemental Digital Content 4, which shows representative immunohistochemistry staining of CD31 (arrows) in the transferred fat (original magnification, ×200). PBS, phosphate-buffered saline; CAL, cell-assisted lipotransfer; Exo, exosome, http://links.lww.com/PRS/D751.] The level of angiogenesis, shown as the number of capillaries stained by CD31 per high optical field, was higher in the exosome and cell-assisted lipotransfer groups than in the phosphate-buffered saline group (p < 0.05) (Fig. 5, left), but comparable between the exosome and cell-assisted lipotransfer groups. Angiogenic factors (hepatocyte growth factor and basic fibroblast growth factor) were assessed by quantitative real-time polymerase chain reaction. Results showed that hepatocyte growth factor mRNA expression was significantly higher in the exosome and cell-assisted lipotransfer groups. Interestingly, compared with the cell-assisted lipotransfer group, the expression of hepatocyte growth factor in the exosome group was also significantly increased (p < 0.05 and p < 0.05) (Fig. 5, right). A higher level of basic fibroblast growth factor expression was also observed in the exosome and cell-assisted lipotransfer groups (p < 0.05) (Fig. 5, right).
Immunostaining of the perilipin showed tissue structure of the transferred adipose in the three groups at week 10 (Fig. 6, above). The transferred fat in the exosome and cell-assisted lipotransfer groups had more complete perilipin-positive adipose structure, whereas the phosphate-buffered saline group had fewer perilipin-positive adipocytes. To evaluate the adipogenesis level in the transferred fat at weeks 3 and 10, the expression level of adipogenic genes (peroxisome proliferator-activated receptor–gamma and lipoprotein lipase) in the fat grafts was tested by quantitative polymerase chain reaction analysis. Results showed no significant difference of peroxisome proliferator-activated receptor–gamma expression among the three groups at both time points after lipotransfer (Fig. 6, second row, left). However, at weeks 3 and 10 after transplantation, grafts in both exosome and cell-assisted lipotransfer groups had significantly higher expression of lipoprotein lipase than the phosphate-buffered saline group (p < 0.05); however, levels were comparable between the exosome and cell-assisted lipotransfer groups (Fig. 6, second row, right).
The effects of adipose-derived stem cell–derived exosomes on adipogenesis were investigated in vitro. After 7 days, lipid droplets could be observed in both phosphate-buffered saline and exosome groups by oil red O staining (Fig. 6, third row). The absorbance of eluted oil red O was expectedly higher in exosome group than in the phosphate-buffered saline group (p = 0.002) (Fig. 6, below, left), suggestive of additional differentiation effects on adipose-derived stem cells by means of those exosomes. Quantitative polymerase chain reaction analysis assay showed that adipogenic relative genes, peroxisome proliferator-activated receptor–gamma and lipoprotein lipase, of both adipogenesis medium treated groups were significantly higher than in the control group, and the expression of both genes in the exosome group was significantly higher than in the phosphate-buffered saline group. Compared with the control group, the CCAAT/enhancer-binding protein beta expression of both adipogenesis groups was increased but with no significant difference (p < 0.05, p < 0.01, and p < 0.001) (Fig. 6, below, right).
Extracellular Matrix Remodeling in the Transferred Fat
The immunofluorescence staining of collagen VI showed that, at week 10, the transferred fat in the exosome and cell-assisted lipotransfer groups had more complete collagen VI–positive fibers around the adipocytes, whereas the phosphate-buffered saline group had fewer collagen VI–positive fibers. [See Figure, Supplemental Digital Content 5, which shows representative immunofluorescence staining of collagen VI in the transferred fat (original magnification, ×100). PBS, phosphate-buffered saline; CAL, cell-assisted lipotransfer; Exo, exosome; Col-VI, collagen VI; DAPI, 4′,6-diamidino-2-phenylindole, http://links.lww.com/PRS/D752.] Compared with the phosphate-buffered saline control groups, collagen III α1 mRNA expression, as assessed by quantitative polymerase chain reaction analysis, was significantly higher in the exosome and cell-assisted lipotransfer groups (p < 0.05) (Fig. 7). Meanwhile, a higher level of collagen VI α3 expression was also observed in the exosome and cell-assisted lipotransfer groups (p < 0.05) (Fig. 7).
The novelty of our finding is that adipose-derived stem cell–derived exosomes produced results similar to those of their source cell-assisted lipotransfer regarding graft retention. This finding is significant in confirming the behaviors of exosomes in general and in exploring a cell-free alternative in allogeneic tissue regeneration in particular.
Exosomes are believed to function primarily as one-way conveyors of cellular signals to regulate downstream activities.40 Exosomes are considered a distinct type of microvesicles derived from several cells by their size in the range of 30 to 150 nm.24–26 However, adipose-derived stem cell–derived exosomes were also reported to have a peak size distribution of 150 to 200 nm, indicating that the size of exosomes may differ, depending on the secreting cell types.41 Exosomes carry a complex cargo that includes nucleic acids, proteins, and lipids.42–45 The exosomal proteins and microRNAs are thus functionally complex and are implicated in many diverse biochemical and cellular processes, such as communication, structure and mechanics, inflammation, exosome biogenesis, tissue repair and regeneration, and metabolism.43,44 Although it is not entirely clear how a specific cargo load is determined by the source cells, it is conceivable that the load may include excessive molecular products derived from the active functions of those source cells,46–48 which could therefore explain why the exosomal functions may mimic those of their source cells (Fig. 8). Adipose-derived stem cell–derived exosomes probably function in a similar fashion and may possess the versatility and capacity to interact with multiple cell types within the immediate vicinity and remote areas to elicit appropriate cellular responses.49
In our study, we proved that adipose-derived stem cell–derived exosomes successfully improved angiogenesis in the transferred fat by up-regulating the expression of hepatocyte growth factor and basic fibroblast growth factor in the fat grafts. The adipose-derived stem cell–derived exosome treatment showed proangiogenic effects similar to those of the adipose-derived stem cell treatment itself. It has been well demonstrated that exosomes derived from different cell types play a significant role in angiogenesis through delivery of angiogenic factors.50–57 Exosomes containing MFG-E8, carbonic anhydrase 9, and other proteins could increase the expression of vascular endothelial growth factor, endothelin-1, or hypoxia-inducible factor-1, which in turn induce angiogenesis.53,54 Using a Matrigel (Corning Life Sciences, Corning, N.Y.) angiogenesis model and a severe combined immunodeficiency mouse model, Deregibus and colleagues confirmed that the angiogenic effects of endothelial progenitor cells are ascribed to the microvesicles transferring of mRNA.57 Besides abundant angiogenic growth factors and mRNAs, exosomes also contain plenty of proangiogenic miRNAs.58,59
In parallel fashion, we have shown that exosomes up-regulated the expression of macrophage inflammatory protein-1α and monocyte chemotactic protein-1 in the transferred fat (at a somewhat greater level than their source stem cells), leading to early macrophage infiltration at 3 weeks after lipotransfer. It is reported that macrophage activation represents the first step in the revascularization of transferred fat through chemotactic effects on vascular stem cells.60 They also play a key role in the extracellular matrix reconstruction process by regulating the expression of collagen proteins and matrix metalloproteinases.61 An appropriately controlled inflammation profile primarily orchestrated by interleukin-1, a proinflammatory cytokine, and interleukin-10, an antiinflammatory cytokine, profoundly influences the fat graft take. In our study, both interleukin-1 and interleukin-10 were significantly up-regulated on exosome treatment at 3 weeks after lipotransfer, suggestive of active inflammatory regulations in the transferred fat. Interestingly, those exosome-induced increased chemokine levels appeared even more robust than those induced by their source stem cells at the early stage after lipotransfer.
It has been reported that exosomes can induce adipogenesis of adipose-derived stem cells and adipose tissue regeneration.62,63 Exosomes derived from adipose tissue are also known to promote adipogenic differentiation of adipose-derived stem cells by conveying miR-450a-5p, a proadipogenic miRNA.64 In our study, a better graft retention rate and a correspondingly more complete adipose tissue structure were observed in the exosome group compared with the phosphate-buffered saline control, on par with those achieved using the source adipose-derived stem cells. Increased perilipin-positive adipocytes and enhanced lipoprotein lipase expression level in the transferred fat were also achieved with the treatment of exosomes, indicative of a proadipogenesis effect of exosomes, similar to adipose-derived stem cells. Our results of adipogenesis-inducing culture also showed that exosomes may have increased adipogenesis of adipose-derived stem cells through early up-regulation of peroxisome proliferator-activated receptor–gamma (Fig. 6, below, right), suggestive of a potential autocrine pathway of adipose-derived stem cell differentiation.
An intact extracellular matrix scaffold is essential to support normal cellular function.65 Fat mass extension in humans is also correlated with collagen deposition.66,67 In the present study, we further investigated the collagen level in the transferred fat to assess the tissue integrity. An intact and increased adipose extracellular matrix structure could be found in the exosome-assisted transferred fat, as evidenced by structured collagen VI staining. Higher expression of collagen VI α3 and collagen III α1 was further verified by quantitative polymerase chain reaction analysis. By both measures, exosomes were shown to be at least comparable to their source adipose-derived stem cells in promoting extracellular matrix deposition. Together with the increased perilipin-positive adipocytes and enhanced lipoprotein lipase expression level in the transferred fat of the exosome group, a more complete extracellular matrix structure may have also contributed to the higher graft retention rate.
Several studies have shown that cell-assisted fat transplantation improves graft retention.8,9 However, limitations of cell-based therapies have constrained their use, including uncontrolled differentiation, unwanted long-term side effects, allogenic incompatibility, and regulatory hurdles.19,22 With the current study, we have demonstrated that adipose-derived stem cell–derived exosomes are more than comparable to their source cells in their ability to increase graft volume retention. This suggests that, as a cell-free and potentially “off-the-shelf” strategy, adipose-derived stem cell–derived exosomes could be an effective and appealing alternative to the cell-assisted lipotransfer technique in lipotransfer. Furthermore, similar cell-free exosomal approaches may be adoptable to other tissue regenerative applications such as enhancement of angiogenesis, wound healing, and nerve regeneration.
This work is partially funded by the Robert E. Weiss Foundation at Lahey Hospital & Medical Center.
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