Secondary Logo

Journal Logo

Exosomes Are Comparable to Source Adipose Stem Cells in Fat Graft Retention with Up-Regulating Early Inflammation and Angiogenesis

Chen, Bin M.D., Ph.D.; Cai, Junrong M.D., Ph.D.; Wei, Yating M.D., Ph.D.; Jiang, Zhaohua M.D., Ph.D.; Desjardins, Haley E. B.S.; Adams, Alexandra E. B.S.; Li, Shengli M.D., Ph.D.; Kao, Huang-Kai M.D.; Guo, Lifei M.D., Ph.D.

Plastic and Reconstructive Surgery: November 2019 - Volume 144 - Issue 5 - p 816e-827e
doi: 10.1097/PRS.0000000000006175
Experimental
Free
SDC

Background: Exosomes derived from mesenchymal stem cells possess functional properties similar to those of their parent cells, suggesting that they could play a pivotal role in tissue repair and regeneration.

Methods: Using lipotransfer as a surrogate, exosomes were isolated from mouse adipose-derived stem cell–conditioned medium and characterized. Minced fat tissue mixed with exosomes, source cells (cell-assisted lipotransfer), or saline was implanted subcutaneously in the lower back of C57/BL mice bilaterally (n = 16 each). Transferred fat tissues were harvested and analyzed at 3 and 10 weeks.

Results: At 3 and 10 weeks after the transfer, fat grafts in groups of exosomes and cell-assisted lipotransfer showed better fat integrity, fewer oil cysts, and reduced fibrosis. At week 10, graft retention rates in cell-assisted lipotransfer (50.9 ± 2.4 percent; p = 0.03) and exosome groups (56.4 ± 1.6 percent; p < 0.001) were significantly higher than in the saline group (40.7 ± 4.7 percent). Further investigations of macrophage infiltration, inflammatory factors, angiogenic factors, adipogenic factors, and extracellular matrix revealed that those exosomes promoted angiogenesis and up-regulated early inflammation, whereas during mid to late stages of fat grafting, they exerted a proadipogenic effect and also increased collagen synthesis level similarly to their source cells.

Conclusions: The adipose-derived stem cell–derived exosomes demonstrated effects comparable to those of their source cells in achieving improved graft retention by up-regulating early inflammation and augmenting angiogenesis. These features may enable exosomes to be an attractive cell-free alternative in therapeutic regenerative medicine.

Burlington and Boston, Mass.; Guangzhou, Hong Kong, and Shanghai, People’s Republic of China; and Taoyuan, Taiwan

From the Laboratory of Tissue Regeneration, Division of Plastic Surgery, Lahey Hospital & Medical Center; the Department of Plastic Surgery, Nanfang Hospital, Southern Medical University; the Department of Rehabilitation Sciences, Hong Kong Polytechnic University; the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering; Tufts University School of Medicine; and the Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung University.

Received for publication November 29, 2018; accepted April 12, 2019.

The first two authors contributed equally to this work.

Disclosure:The authors have no financial interest to declare in relation to the content of this article.

Related digital media are available in the full-text version of the article on www.PRSJournal.com.

Lifei Guo, M.D., Ph.D., 41 Mall Road, Burlington, Mass. 01805, lifei.guo@lahey.org, Bin Chen, M.D., Ph.D., 41 Mall Road, Burlington, Mass. 01805, bin.chen@lahey.org

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.

Back to Top | Article Outline

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.).

Back to Top | Article Outline

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.).

Back to Top | Article Outline

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.).

Back to Top | Article Outline

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

Back to Top | Article Outline

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.

Back to Top | Article Outline

Histologic Analysis

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.

Back to Top | Article Outline

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).

Back to Top | Article Outline

Statistical Analysis

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.

Back to Top | Article Outline

RESULTS

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

Fig. 1.

Fig. 1.

Back to Top | Article Outline

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).

Fig. 2.

Fig. 2.

Back to Top | Article Outline

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).

Fig. 3.

Fig. 3.

Back to Top | Article Outline

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).

Fig. 4.

Fig. 4.

Back to Top | Article Outline

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).

Fig. 5.

Fig. 5.

Back to Top | Article Outline

Adipogenesis Evaluation

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).

Fig. 6.

Fig. 6.

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).

Back to Top | Article Outline

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).

Fig. 7.

Fig. 7.

Back to Top | Article Outline

DISCUSSION

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

Fig. 8.

Fig. 8.

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.

Back to Top | Article Outline

ACKNOWLEDGMENT

This work is partially funded by the Robert E. Weiss Foundation at Lahey Hospital & Medical Center.

Back to Top | Article Outline

REFERENCES

1. Strong AL, Cederna PS, Rubin JP, Coleman SR, Levi B. The current state of fat grafting: A review of harvesting, processing, and injection techniques. Plast Reconstr Surg. 2015;136:897–912.
2. Khouri RK Jr, Khouri RK. Current clinical applications of fat grafting. Plast Reconstr Surg. 2017;140:466e–486e.
3. Cheriyan T, Kao HK, Qiao X, Guo L. Low harvest pressure enhances autologous fat graft viability. Plast Reconstr Surg. 2014;133:1365–1368.
4. Yu NZ, Huang JZ, Zhang H, et al. A systemic review of autologous fat grafting survival rate and related severe complications. Chin Med J (Engl.) 2015;128:1245–1251.
5. Mineda K, Kuno S, Kato H, et al. Chronic inflammation and progressive calcification as a result of fat necrosis: The worst outcome in fat grafting. Plast Reconstr Surg. 2014;133:1064–1072.
6. Toyserkani NM, Quaade ML, Sørensen JA. Cell-assisted lipotransfer: A systematic review of its efficacy. Aesthetic Plast Surg. 2016;40:309–318.
7. Laloze J, Varin A, Bertheuil N, Grolleau JL, Vaysse C, Chaput B. Cell-assisted lipotransfer: Current concepts. Ann Chir Plast Esthet. 2017;62:609–616.
8. Luan A, Duscher D, Whittam AJ, et al. Cell-assisted lipotransfer improves volume retention in irradiated recipient sites and rescues radiation-induced skin changes. Stem Cells 2016;34:668–673.
9. Paik KJ, Zielins ER, Atashroo DA, et al. Studies in fat grafting: Part V. Cell-assisted lipotransfer to enhance fat graft retention is dose dependent. Plast Reconstr Surg. 2015;136:67–75.
10. Hong KY, Yim S, Kim HJ, et al. The fate of the adipose-derived stromal cells during angiogenesis and adipogenesis after cell-assisted lipotransfer. Plast Reconstr Surg. 2018;141:365–375.
11. Wang S, Qu X, Zhao RC. Mesenchymal stem cells hold promise for regenerative medicine. Front Med. 2011;5:372–378.
12. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
13. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100:1249–1260.
14. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279–4295.
15. Maguire G. Stem cell therapy without the cells. Commun Integr Biol. 2013;6:e26631.
16. Yáñez-Mó M, Siljander PR, Andreu Z, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 2015;4:27066.
17. Doorn J, Moll G, Le Blanc K, van Blitterswijk C, de Boer J. Therapeutic applications of mesenchymal stromal cells: Paracrine effects and potential improvements. Tissue Eng Part B Rev. 2012;18:101–115.
18. Lim P, Patel SA, Rameshwar P. Gorodetsky R, Schäfer R. Effective tissue repair and immunomodulation by mesenchymal stem cells within a milieu of cytokines. In: Stem Cell-Based Tissue Repair. 2011:Cambridge: RSC Publications; 346–365.
19. Haarer J, Johnson CL, Soeder Y, Dahlke MH. Caveats of mesenchymal stem cell therapy in solid organ transplantation. Transpl Int. 2015;28:1–9.
20. Aronowitz JA, Lockhart RA, Hakakian CS, Hicok KC. Clinical safety of stromal vascular fraction separation at the point of care. Ann Plast Surg. 2015;75:666–671.
21. Banyard DA, Salibian AA, Widgerow AD, Evans GR. Implications for human adipose-derived stem cells in plastic surgery. J Cell Mol Med. 2015;19:21–30.
22. Poulos J. The limited application of stem cells in medicine: A review. Stem Cell Res Ther. 2018;9:1.
23. Raposo G, Stoorvogel W. Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol. 2013;200:373–383.
24. Rani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell-derived extracellular vesicles: Toward cell-free therapeutic applications. Mol Ther. 2015;23:812–823.
25. Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255–289.
26. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012;64:676–705.
27. Schorey JS, Bhatnagar S. Exosome function: From tumor immunology to pathogen biology. Traffic 2008;9:871–881.
28. De Jong OG, Van Balkom BW, Schiffelers RM, Bouten CV, Verhaar MC. Extracellular vesicles: Potential roles in regenerative medicine. Front Immunol. 2014;5:608.
29. Sun B, Peng J, Wang S, et al. Applications of stem cell-derived exosomes in tissue engineering and neurological diseases. Rev Neurosci. 2018;29:531–586.
30. Lankford KL, Arroyo EJ, Nazimek K, Bryniarski K, Askenase PW, Kocsis JD. Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord. PLoS One 2018;13:e0190358.
31. Dougherty JA, Mergaye M, Kumar N, Chen CA, Angelos MG, Khan M. Potential role of exosomes in mending a broken heart: Nanoshuttles propelling future clinical therapeutics forward. Stem Cells Int. 2017;2017:5785436.
32. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci. 2014;15:4142–4157.
33. Lai RC, Arslan F, Lee MM, et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res. 2010;4:214–222.
34. Kordelas L, Rebmann V, Ludwig AK, et al. MSC-derived exosomes: A novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 2014;28:970–973.
35. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng. 2001;7:211–228.
36. Kraus NA, Ehebauer F, Zapp B, Rudolphi B, Kraus BJ, Kraus D. Quantitative assessment of adipocyte differentiation in cell culture. Adipocyte 2016;5:351–358.
37. Chen B, Kao HK, Dong Z, Jiang Z, Guo L. Complementary effects of negative-pressure wound therapy and pulsed radiofrequency energy on cutaneous wound healing in diabetic mice. Plast Reconstr Surg. 2017;139:105–117.
38. Kao HK, Chen B, Murphy GF, Li Q, Orgill DP, Guo L. Peripheral blood fibrocytes: Enhancement of wound healing by cell proliferation, re-epithelialization, contraction, and angiogenesis. Ann Surg. 2011;254:1066–1074.
39. Chen TS, Lai RC, Lee MM, Choo AB, Lee CN, Lim SK. Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res. 2010;38:215–224.
40. Zhang B, Wang M, Gong A, et al. HucMSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing. Stem Cells 2015;33:2158–2168.
41. Katsuda T, Tsuchiya R, Kosaka N, et al. Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes. Sci Rep. 2013;3:1197.
42. Lai RC, Yeo RW, Lim SK. Mesenchymal stem cell exosomes. Semin Cell Dev Biol. 2015;40:82–88.
43. He C, Zheng S, Luo Y, Wang B. Exosome theranostics: Biology and translational medicine. Theranostics 2018;8:237–255.
44. Lai RC, Tan SS, Teh BJ, et al. Proteolytic potential of the MSC exosome proteome: Implications for an exosome-mediated delivery of therapeutic proteasome. Int J Proteomics 2012;2012:971907.
45. Baglio SR, Rooijers K, Koppers-Lalic D, et al. Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res Ther. 2015;6:127.
46. Cabral J, Ryan AE, Griffin MD, Ritter T. Extracellular vesicles as modulators of wound healing. Adv Drug Deliv Rev. 2018;129:394–406.
47. Guay C, Regazzi R. Exosomes as new players in metabolic organ cross-talk. Diabetes Obes Metab. 2017;19(Suppl 1):137–146.
48. Phinney DG, Pittenger MF. Concise review: MSC-derived exosomes for cell-free therapy. Stem Cells 2017;35:851–858.
49. Zhang Y, Yu M, Tian W. Physiological and pathological impact of exosomes of adipose tissue. Cell Prolif. 2016;49:3–13.
50. Todorova D, Simoncini S, Lacroix R, Sabatier F, Dignat-George F. Extracellular vesicles in angiogenesis. Circ Res. 2017;120:1658–1673.
51. Merino-González C, Zuñiga FA, Escudero C, et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: Potential clinical application. Front Physiol. 2016;7:24.
52. Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H. Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J Mol Med (Berl.) 2014;92:387–397.
53. Horie K, Kawakami K, Fujita Y, et al. Exosomes expressing carbonic anhydrase 9 promote angiogenesis. Biochem Biophys Res Commun. 2017;492:356–361.
54. Hu Y, Rao SS, Wang ZX, et al. Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function. Theranostics 2018;8:169–184.
55. Gong M, Yu B, Wang J, et al. Mesenchymal stem cells release exosomes that transfer miRNAs to endothelial cells and promote angiogenesis. Oncotarget 2017;8:45200–45212.
56. Shabbir A, Cox A, Rodriguez-Menocal L, Salgado M, Van Badiavas E. Mesenchymal stem cell exosomes induce proliferation and migration of normal and chronic wound fibroblasts, and enhance angiogenesis in vitro. Stem Cells Dev. 2015;24:1635–1647.
57. Deregibus MC, Cantaluppi V, Calogero R, et al. Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 2007;110:2440–2448.
58. Das S, Halushka MK. Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc Pathol. 2015;24:199–206.
59. Chen Q, Shou P, Zheng C, et al. Fate decision of mesenchymal stem cells: Adipocytes or osteoblasts? Cell Death Differ. 2016;23:1128–1139.
60. Zhang Y, Yu M, Dai M, et al. miR-450a-5p within rat adipose tissue exosome-like vesicles promotes adipogenic differentiation by targeting WISP2. J Cell Sci. 2017;130:1158–1168.
61. Cai J, Feng J, Liu K, Zhou S, Lu F. Early macrophage infiltration improves fat graft survival by inducing angiogenesis and hematopoietic stem cell recruitment. Plast Reconstr Surg. 2018;141:376–386.
62. Dai M, Yu M, Zhang Y, Tian W. Exosome-like vesicles derived from adipose tissue provide biochemical cues for adipose tissue regeneration. Tissue Eng Part A 2017;23:1221–1230.
63. Shoshani O, Livne E, Armoni M, et al. The effect of interleukin-8 on the viability of injected adipose tissue in nude mice. Plast Reconstr Surg. 2005;115:853–859.
64. Brown SA, Levi B, Lequeux C, et al. Basic science review on adipose tissue for clinicians. Plast Reconstr Surg. 2010;126:1936–1946.
65. Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol. 2011;3:a005058.
66. Divoux A, Tordjman J, Lacasa D, et al. Fibrosis in human adipose tissue: Composition, distribution, and link with lipid metabolism and fat mass loss. Diabetes 2010;59:2817–2825.
67. Spencer M, Yao-Borengasser A, Unal R, et al. Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation. Am J Physiol Endocrinol Metab. 2010;299:E1016–E1027.

Supplemental Digital Content

Back to Top | Article Outline
Copyright © 2019 by the American Society of Plastic Surgeons