The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient’s age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term “wound repair priming.” Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat
The adipose-derived stromal vascular fraction (SVF) is a cell population derived from enzymatic digestion of adipose tissue [
Here, we show that surgical treatments for adipose tissue reproducibly increase and reorganize adipose-derived SVFs. We term this phenomenon “wound repair priming” and developed a novel method to reproducibly obtain SVFs with increased number and good quality. The primed SVFs not only have increased cell numbers but also have enhanced angiogenesis capacity during the regeneration of ischemic limbs. Both injury-associated stimuli and ischemia are needed for the priming, and immune cells are heavily involved through heterogeneous cell-cell communications. We investigated the boosted performance of primed SVFs over nonprimed SVFs to promote angiogenesis and regeneration in a mouse limb ischemia model.
All animal experiments adhered to the Guidelines for Animal Experimentation of Dokkyo Medical University, with all effort taken to minimize animal numbers and suffering. Inbred male BALB/c mice (CLEA Japan, Inc., Shizuoka, Japan) aged 9–12 weeks were anesthetized with an intraperitoneal injection of 90 mg/kg ketamine hydrochloride (Ketalar; Daiichi Sankyo Propharma Co., Ltd., Tokyo, Japan) and 10 mg/kg xylazine hydrochloride (Celactal; Bayer Yakuhin, Ltd., Osaka, Japan). SVFs were isolated from subcutaneous inguinal fat in BALB/c mice and transplanted into a syngeneic limb ischemia model as described previously [
In a preliminary experiment, we observed that the number of SVFs was dramatically increased when a mouse received skin injury by fighting with a littermate (data not shown). When we transplanted the SVFs (adjusted to the same number) of a donor mouse receiving a skin injury to the ischemic limbs of a syngeneic mouse, the capacity of angiogenesis was greatly enhanced (data not shown). We found that the transplantation of SVFs from an injured mouse displayed much faster blood flow recovery than that from an uninjured mouse. Therefore, we hypothesized that adipose tissue injury substantially increases the number and quality of SVFs.
We then designed the present study to reproduce this phenomenon in a controlled condition and to investigate the underlying mechanisms. A few days before isolating SVFs, adipose tissue was surgically damaged. The priming models were classified into 3 groups based on the following procedures: mincing fat parenchyma (injury), ligating the subcutaneous fat-feeding artery (ischemia), and both (injury+ischemia group). A sham operation group, which underwent cutting and suturing of the epidermis but with no surgical damage to fat tissues, was also used.
SVFs were isolated from inbred BALB/c mice with wound repair priming (injury only, ischemia only, and injury+ischemia) or sham operation (nonprimed SVFs) as previously described [
Total RNA was purified from SVFs after the priming procedure using phenol-chloroform extraction (RNAiso Plus, Takara Bio Inc., Shiga, Japan). For comparison, three time points were taken (0, 1, or 3 days after). Three individual mice were used for each time point for statistical analysis. After confirming the quality of RNA (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA), gene expression levels were measured by microarray analysis (Affymetrix GeneChip Expression Array, Mouse430_2, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Differentially expressed genes were selected using Affymetrix Transcriptome Analysis Console (TAC, Ver3.1.0.5) software. Filtering criteria were as follows: (1) the expression ratio was less than half or more than double, and (2) the
SVFs with or without priming were isolated, and whole-cell proteins were denatured with a solubilizer (7 M urea, 2 M thiourea, and 4% CHAPS, Thermo Fisher). Protein concentrations were measured using the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and plate reader (Infinite F200 PRO, Tecan, Zurich, Switzerland). Proteins were subsequently diluted in lithium dodecyl sulfate (NuPAGE Sample Buffer, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at the concentration of 2 mg/mL and reduced in 50 mM dithiothreitol. The entangled genomic DNA was sheared by pulsed sonication for 30 min at 4°C (Bioruptor® II, Sonic Bio Inc., Kanagawa, Japan). Proteins were size-fractionated by electrophoresis in 12% sodium dodecyl sulfate polyacrylamide gel, then wet-transferred to a PVDF membrane (Immobilon-P, Merck Millipore, Billerica, MA, USA). After blocking with 2% skim milk TBST (Tris/HCl pH 7.4, 150 mM NaCl, and 0.1% Tween 20), the membranes were incubated with VEGF-A antibody (#AB1876-I, Merck Millipore) or GAPDH antibody (#5174, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Nonspecific binding was washed away with TBST, and the protein-antibody complex was visualized using horseradish peroxidase-conjugated secondary antibody (#7074, Cell Signaling Technology) and the luminescence reaction (#WSE-7120 or 7110, Atto, Tokyo, Japan). Images were taken with a CCD camera (LuminoGraph I, Atto).
Subcutaneous fat prepared from the inguinal region of the mice was sampled on days 1, 3, and 7 after priming (injury+ischemia). These samples were minced into small pieces and digested with collagenase in phosphate-buffered saline (PBS) with Ca2+ and Mg2+ containing 2% bovine serum albumin (BSA) at 37°C for 45 min using a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The samples were passed through a 100
Flow cytometry was used to determine cell surface marker presentations of SVFs [
Mice underwent ligation of the right external iliac artery and hindlimb vein to produce right hindlimb ischemia [
Hindlimb blood flow was analyzed using a laser Doppler blood perfusion imager (PeriScan PIM III; Perimed AB, Stockholm, Sweden) on postoperative day 0 (within 1 h of the operation) and on days 3, 7, and 14. A depilatory cream was used to remove excess limb hair before imaging. Mice were placed on a heating plate at 38°C to minimize temperature variation during imaging. Blood perfusion was calculated on the scanned images as perfusion units (PU), and serial changes in the ischemic (right) hindlimb were compared among the groups. Changes in blood perfusion for the contralateral nonischemic (left) hindlimb blood perfusion were also assessed to avoid variation bias resulting from ambient light and temperature.
Paraffin-embedded sections (5
Data are shown as
We counted the cell number of surgically treated (injury+ischemia) SVFs to evaluate the impact of wound repair priming. The live cell number of primed SVFs increased on day 7 compared with the baseline, whereas the live cell number of nonprimed SVFs did not change on day 1, 3, or 7. The total live cell number of primed SVFs was 1.72-fold higher (
Surgical injury increased the live cell number of SVFs. The number of SVFs per tissue weight. Primed SVFs were created by both mincing fat parenchyma (injury) and ligating the subcutaneous fat-feeding artery (injury+ischemia). The error bars represent the standard deviation of measurements from 16 samples.
To gain biological insight into SVF priming, we performed microarray and pathway analyses. We compared the gene expression profile of primed SVFs with that of nonprimed SVFs (i.e., day 1 or day 3 vs. day 0 after priming). Canonical pathway analysis clearly highlighted innate immunity-related signals such as triggering receptor expressed on myeloid cells (TREM), genes related to the acute phase response, and Toll-like receptor signaling, on both day 1 and day 3 after priming (Figure
Canonical pathways and upstream-downstream effects involved in SVF priming. Gene expression profiles were compared between primed SVFs and nonprimed SVFs (i.e., 0 vs. 1 day or 0 vs. 3 days after priming), and the lists of differentially expressed genes were imported into Ingenuity Pathway Analysis software. (a) Canonical pathways were clustered according to the activation
Stabilization of HIF-1 alpha with or without surgical treatments in cutaneous fat
Change in cellular compositions of SVFs by priming. Flow cytometry analysis of surface antigens in primed (injury+ischemia) and nonprimed SVFs. (a) CD45-positive mononuclear (left) and CD31-CD45-double negative (right) mesenchymal cell fractions on days 1, 3, and 7 after priming. (b) Scatterplots of CD45-positive and CD31-CD45-double negative cells are shown. Note that the primed and nonprimed SVFs were clearly segregated on day 7 after priming. (c) Mesenchymal-gated CD34-positive adipose progenitors, mesenchymal-gated CD140A-positive/CD9-high preadipocytes, or mesenchymal-gated CD140A-positive/CD9-low prefibroblasts on days 1, 3, and 7 after priming. (d) F4/80-positive macrophages, F4/80-positive/CD11c-positive/CD206-negative M1 macrophages, and F4/80-positive/CD11c-negative/CD206-positive M2 macrophages on days 1, 3, and 7 after priming.
A combination of surgical injury and ischemia was necessary for efficient blood flow recovery during limb ischemia. Blood flow recovery following injection of primed SVFs. (a) Representative laser Doppler blood perfusion imaging. Compared to the sham group (without SVF transplantation), the surgical injury-only group or the ischemia-only group did not show improved blood flow recovery on days 3, 7, and 14 after SVF injection. In contrast, efficient blood flow recovery was achieved in the injury+ischemia group. (b) Quantitative assessment of serial changes in blood perfusion. There was no significant difference in blood perfusion among the sham group, the injury-only group, and the ischemia-only group. The injury+ischemia group showed significantly higher blood perfusion than the sham group, the injury-only group, and the ischemia-only group on days 7 and 14 after injection. (c) Representative appearance of the ischemic (left) and nonischemic (right) hindlimbs. Note that the ischemic limb treated with both injury and ischemia retained an almost intact appearance.
A combination of surgical injury and ischemia was necessary for capillary angiogenesis during limb ischemia. Angiogenesis was visualized by immunohistochemical staining against CD31. (a) Representative microscopic images of CD31-positive capillaries located in ischemic hindlimb muscles. Compared with the sham group (without SVF transplantation), the injury+ischemia group showed increased CD31-positive capillary density on day 14 after injection. The arrows indicate capillaries in the hindlimb muscles.
Protein synthesis of VEGF-A was induced by a combination of surgical injury and ischemia in SVFs. 20
As SVFs consist of a heterogeneous cell population, we evaluated the percentage of CD45-positive mononuclear cells, CD31-CD45-double negative mesenchymal cells [
It is known that the CD31-CD45-double negative fraction include CD34-positive cells with great variability, from 3.5% [
The percentage of the F4/80-positive fraction (macrophages) was
To quantify the angiogenetic capacity of the primed SVFs, we transplanted SVFs into syngeneic hindlimb ischemia mice. We prepared four different experimental groups to see the essential factors for the priming. After ligation of the right external iliac artery (day 0), ischemic hindlimb blood perfusion was significantly lower than that of the contralateral control hindlimbs in all groups (Figure
Finally, we determined the angiogenetic capacity of SVFs by measuring capillary density. Figure
Injured tissues release various wound-related factors that affect repair and remodeling [
We incidentally discovered that adipose-derived SVFs in severely injured mice are increased in number and have enhanced angiogenetic capacity through syngeneic transplantation experiments. We subsequently developed a method to reproduce this phenomenon and investigated its mechanisms. The wound repair priming of SVFs requires two surgical procedures: mincing fat parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). When either one (injury or ischemia) was lacking, the enhancement of angiogenesis in limb ischemia was limited. In the presence of both injury and ischemia, however, we observed enhanced angiogenesis and regeneration of ischemic limbs. After the priming, the cell number of SVFs increased, and microarray analysis showed remarkable changes in the gene expression profile. Thereafter, we performed subsequent experiments to elucidate the molecular and cellular basis of wound repair priming. Microarray data clearly indicated the extensive upregulation of innate immunity signals, suggesting the involvement of the acute inflammatory response. Damage-associated molecular patterning is a possible trigger that induces the inflammatory response [
In the present study, flow cytometry analysis demonstrated that the cellular composition of primed SVFs changed over time. After priming, the CD45-positive mononuclear cell fraction decreased, but the CD31-CD45-double negative mesenchymal cell fraction increased on day 7 (Figures
The processing of human adipose-derived SVFs has been automated for angiogenesis therapy, and autologous transplantation of adipose-derived SVFs is now performed in various clinical settings. However, concerns over individual variations in cell number and quality remain an unsolved issue. Although we have provided a unique concept known as “wound repair priming” and have generated a reproducible surgical procedure, adopting this methodology for humans is unlikely to be feasible. In the future, the priming procedure should be replaced by chemical or pharmacological approaches, the effect of which is equivalent to surgical priming. For example, priming with chemical compounds that stimulate innate immune signals would be a promising alternative. Such chemical or pharmacological priming might contribute to the development of reliable and effective angiogenic therapy for ischemic cardiovascular diseases such as critical limb ischemia.
Surgical priming with injury+ischemia of adipose tissue results in increased cell numbers and better quality of adipose-derived SVFs. The primed SVFs rapidly reorganize their cell components during wound repair. Residential stromal cells and mobilized immune cells collaborate to achieve effective angiogenesis in ischemic tissues.
The microarray data obtained in this study have been deposited in the Gene Expression Omnibus (accession number: GSE 134613). Other data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflict of interest.
Kishimoto S and Inoue K contributed equally to this work.
We thank Ms. N. Ohshima (Center for Research Collaboration and Support, Dokkyo Medical University), Mr. H. Hirata, Mr. Y. Machida, and Dr. M. Terada (Research Center for Laboratory Animal, Dokkyo Medical University), and Ms. S. Satoh (Research Center for Advanced Medical Science) for their technical support. This study was supported by JSPS KAKENHI (Grant Number 17K09559).