Implantation of Dedifferentiated Fat Cells Ameliorated ANCA Glomerulonephritis by Immunosuppression and Increases in TSG-6

We examined the effects of implantation of dedifferentiated fat (DFAT) cells on renal function, proteinuria and glomerulonephritis in SCG mice as a preclinical study of DFAT cell therapy for antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis and investigated mechanisms underlying the immunosuppressive effects of the implantation of DFAT cells. After their intravenous infusion, almost all DFAT cells were trapped in the lung and not delivered into the kidney. Implantation of DFAT cells in SCG mice suppressed glomerular crescent formation, decreased urinary protein excretions, and increased expression of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA, protein and immunostaining in kidney from these mice. Implantation of DFAT cells increased the expression of microRNA 23b-3p in plasma, kidney and lung in SCG mice and decreased the expression of CD44 mRNA and increased the expression of prostaglandin E2 and interleukin-10 mRNAs in kidney from these mice. Implantation of DFAT cells increased expression of TSG-6 protein and decreased expression of tumor necrosis factor-a protein in kidney from SCG mice. Further, implantation of DFAT cells increased the expression of C-C motif chemokine ligand 17 protein, a chemokine for M2 macrophages, and decreased the expression of MCP-1 protein, a chemokine for M1 macrophages, in kidney from SCG mice. Survival rates were higher in SCG mice with implantation of DFAT cells than in SCG mice without implantation. These results indicate that implantation of DFAT cells suppressed renal injury including glomerular crescent formation in kidney from SCG mice while increasing the expression of TSG-6 without delivery of DFAT cells directly into kidney. Mechanisms underlying the effects of improvement of ANCA glomerulonephritis are associated with immunosuppressive effects by TSG-6 and the transition of M1 to M2 macrophages. These ndings suggest that implantation of DFAT cells may become a cell therapy for ANCA glomerulonephritis.


Introduction
Implantation of mesenchymal stem cells (MSCs) has recently been reported to repair tissue injuries through anti-in ammatory and immunosuppressive effects [1,2]. We established dedifferentiated fat (DFAT) cells that show identical characteristics to MSCs [3,4]. Systematic infusion of MSCs has been reported to suppress graft rejection in animal models [5], and the implantation of MSCs has been investigated in clinical studies in which MSCs were reported to effectively inhibit graft-versus-host disease [6].
We have shown that DFAT cells have potential immunosuppressive effects. The systematic implantation of DFAT cells effectively ameliorated antibody-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage in ltration, and it increased the production of serum and renal tumor necrosis factor-stimulated gene-6 (TSG-6), which improved antibody-induced renal degeneration. These ndings suggest that DFAT cells can potentially be a suitable cell source for the treatment of immunological progressive renal diseases [7]. Systematic implantation of DFAT cells effectively ameliorated monoclonal antibody (mAb) 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage in ltration and expression of interleukin (IL)-6, IL-10 and IL12β, and increased production of serum and renal TSG-6, which improved the mAb 1-22-3-induced renal degeneration, through its immunosuppressive effects. Thus, DFAT cells may be a suitable cell source for the treatment of immunological progressive renal diseases.
Autoimmune-associated kidney diseases such as antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis and lupus nephritis have been refractory diseases in the clinical eld. ANCA glomerulonephritis is a nephropathy among the ANCA-associated vasculitides that commonly involve the glomerulus, including microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis [8]. ANCA is classi ed into a perinuclear pattern (P-ANCA) and cytoplasmic pattern (C-ANCA) by indirect immuno uorescence ndings. The antigen for P-ANCA is mainly myeloperoxidase (MPO), which is almost positive in microscopic polyangiitis [9]. A typical renal pathological nding is that of glomerular necrotic crescent formation. Mild lesions of ANCA glomerulonephritis shows segmental necrotic glomerulonephritis, and almost sever lesion shows glomerular necrotic crescent formation [10].
The spontaneous crescentic glomerulonephritis-forming (SCG) mouse as a model for ANCA glomerulonephritis is a hybrid inbred strain established by brother-sister inbreeding of the BXSB mouse inducing crescent forming glomerulonephritis and the MRL/lpr mouse inducing ANCA-associated vasculitis. Thus, the SCG mouse is a genetic model mouse with the autoimmune promoting gene lpr [11].
The SCG mouse shows proteinuria and lymph node swelling, and crescent forming glomerulonephritis along with increases in MPO-ANCA and TNF-α from 8 weeks of age. In addition, SCG mice show systematic vasculitis of small vessels initially that gradually spreads to large vessels and each organ. Half of SCG mice show proteinuria by 90 days after birth, have an average survival period of around 120 days, and die from renal failure [12].
The number of patients with rapidly progressive glomerulonephritis has recently begun to rapidly increase in Japan as a cause of dialysis-introduced primary disease. ANCA glomerulonephritis is the most frequent disease leading to rapidly progressive glomerulonephritis and has a poor life prognosis because repeat relapses of this disease occur after transient improvement with steroid therapy [8].
In the present study, we examined the effects of the implantation of DFAT cells on renal function, proteinuria and glomerulonephritis in SCG mice as a preclinical study of DFAT cell therapy for ANCA glomerulonephritis and investigated mechanisms of the immunosuppressive effects of the implantation of DFAT cells.

Ethics and animals
This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, 1996). The ethics committee of the Nihon University School of Medicine approved this study (approval no.: AP18MED021-1). Male SCG/Kj mice were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition and were bred with female ICR mice (Charles River Laboratory Japan, Yokohama, Japan).
Preparation of DFAT cells from adipose tissue Around 1 g of epididymal adipose tissue from six male ICR mice was treated with collagenase and centrifuged. Adipocytes were isolated from the top layer. More than 99% of the isolated cells were mature lipid-lled adipocytes. The mature adipocytes oating on top of the culture medium attached to the upper surface of the culture asks within a few days. Approximately 10-20% of the adherent cells attened out by day 3 and changed into a spindle-shaped morphology by day 7. The cells subsequently entered a proliferative log-phase upon inversion of the asks and changing of the media and reached con uence by day 14. During this stage, the cells completely lose their lipid droplets and exhibit the broblast-like morphology of DFAT cells. Six different DFAT cells from six ICR mice, and combined them as allogenic DFAT cells.

Distribution of DFAT cells after implantation
DFAT cells from the ICR mice were labeled with PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma Chemical, St. Louis, MO, USA). In total, 10 5 labeled DFAT cells were infused through the posterior orbital venous plexus of ICR mice. At 1 hour, 24 hours, and 1 week after the injection, kidney, aorta, liver and lungs were removed and xed in 3% formalin in phosphate-buffered saline (PBS) and embedded in para n. Sections were observed under a uorescence microscope (IX73, Olympus, Tokyo, Japan), and images were obtained with a digital imaging system. Figure 2 shows the protocols used for the implantations of DFAT cells. ICR mice without implantation of DFAT cells were the control for SCG mice. SCG mice without implantation of DFAT cells were the control for the implantations of DFAT cells. Protocol 1 indicates SCG mice with single implantation of DFAT cells at 8 weeks of age. Protocol 2 indicates SCG mice with triple implantation of DFAT cells at 8, 9 and 10 weeks of age. Each mouse was sacri ced at 12 weeks of age, blood was sampled, and kidneys were removed. Protocol 3 indicates SCG mice with single implantation of DFAT cells at 8 weeks of age. Urine was collected from the mice at 12 to 17 weeks of age in metabolic cages, and urinary protein was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Serum blood urea nitrogen (BUN) and creatinine were measured by SRL, Inc. (Wako, Saitama, Japan). The MPO-ANCA titer was determined by an ELISA kit (MBL Laboratory, Tokyo, Japan).

Determination of renal injury
The 3-mm para n-embedded sections of removed renal cortex were stained with hematoxylin and eosin.

Immunohistochemical analysis of TSG-6
For immunohistochemical analysis of TSG-6, rabbit polyclonal anti-TSG-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:500 was used as a rst antibody and incubated for 30 min. Then, ImmPRESS Reagent (VECTOR LABORATORIES, Burlingame, CA, USA) was used as secondary antibodies. Counterstaining was then performed before the sections were examined under a light microscope.
Ampli cations were done at 95°C for 15 s, then 60°C for 60 s, and 95°C for 15 s with a GeneAmp PCR System 2700 (Applied Biosystems) for 55 cycles. After we determined the threshold cycle (Ct), we used the comparative Ct method to calculate the relative quanti cation of mRNA expression of the marker gene. The quality and concentration of the ampli ed PCR products were determined using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Table 1 shows the sequences of these primers.
Quanti cation of miRNA Peripheral blood was collected by cardiac blood samplings from three SCG mice each with or without implantation of DFAT cells and mixed with an equal volume of PBE buffer solution (containing 10% fetal bovine serum, 2 mM ethylenediaminetetraacetic acid and PBS. Peripheral blood mononuclear cells were isolated by the Percoll method. Kidneys and lungs from three SCG mice each with or without implantation of DFAT cells were homogenized and mixed in PBE buffer solution. Total RNA was isolated from the PBEmixed samples from kidney and lung with TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. Total RNA was puri ed and ultimately eluted into 20 µL of RNase-free water. RNA quantity was assessed with a NanoDrop system (NanoDrop Products, Wilmington, DL, USA).
Sequencing libraries were constructed using the QIAseq™ miRNA Library Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The quality of the libraries was assessed with an Agilent Bioanalyzer using a High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA, USA). The pooled libraries of the samples were sequenced using NextSeq 500 (Illumina, Inc., San Diego, CA, USA) in 76base pair single-end reads.
The QIAseq microRNA (miRNA) library kit adopts the Unique Molecular Indexes (UMI) system, enabling unbiased and accurate quanti cation of mature miRNAs. Original FASTQ les generated by NextSeq were uploaded to the Qiagen GeneGlobe Data Analysis Center (https://geneglobe.qiagen.com) and aligned to the miRBase v21 (http://www.mirbase.org) and piRNABank (http://pirnabank.ibab.ac.in/). All reads assigned to a particular miRNA or piRNA were counted, and the associated UMI were aggregated to count unique molecules. A matrix of the UMI counts of miRNA or piRNA was subjected to downstream analyses using StrandNGS 3.4 software (Agilent Technologies). The UMI counts were quanti ed using a Trimmed Mean of M-value method [13]. miRNAs were annotated against miRBase.

Western blot analysis
Renal medulla from mice were disrupted with lysis buffer (50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 µg/mL phenylmethylsulfonyl uoride, 1 µg/mL aprotinin and 1% Triton X-100). Total Determination of regulatory T cell population Spleen was removed and homogenized 4 weeks after implantation of 10 6 DFAT cells in SCG mice. Cells from spleen were labelled with uorogenic antibodies CD4-APC-Cy7, CD25-AlexaFluor® 647 and FOXP3-PerCP5.5 to evaluate the proportion of CD4 + CD25 + FOXP3 + regulatory T cells. Cells were xed and permeabilized with a FOXP3 Staining Buffer Set according to the manufacturer's instructions and included the blocking step with 2% rat serum. Flow cytometry was performed with a FACSAria system, and data were analyzed using FlowJo 7.6.5 software.

Statistics
Values are reported as the mean ± SEM. Two-way ANOVA with the Bonferroni/Dunn procedure as a posttest was also used. A value of P < 0.05 was considered to be statistically signi cant.

Distribution of implanted DFAT cells
In total, 10 5 PKH26-labeled DFAT cells were infused through the posterior orbital venous plexus in ICR mice. One hour after injection, these DFAT cells were trapped in the lung (Fig. 1) and were not delivered into the kidney, aorta, or liver. The PKH26-positive cells were trapped for one day and had disappeared from the lung by 1 week after implantation (data not shown).
Comparison of effects of single and triple implantations of DFAT cells on renal function, MPO-ANCA and TSG-6 We examined the effects of single and triple implantations of DFAT cells on renal function, plasma MPO-ANCA titer and expression of TSG-6 mRNA in kidney. Single and triple implantations of DFAT cells did not affect increased serum creatinine levels in SCG mice (Fig. 3A). Single implantations of DFAT cells signi cantly (P < 0.05) suppressed increased BUN levels in SCG mice, but triple implantations of DFAT cells had no signi cant effect on BUN levels (Fig. 3B). Single implantation of DFAT cells signi cantly (P < 0.05) suppressed increased plasma MPO-ANCA titer in SCG mice, whereas triple implantations of DFAT cells did not affect the plasma titer (Fig. 3C). Figure 3D shows the effects of single and triple implantations of DFAT cells on the expression of TSG-6 mRNA in kidney from SCG mice. Single implantations of DFAT cells signi cantly (P < 0.05) increased the amount of TSG-6 mRNA in kidney from SCG mice, whereas triple implantations of DFAT cells had no signi cant effect on TSG-6 mRNA expression. Thus, the single implantations of DFAT cells rather effectively improved renal function, decreased the MPO-ANCA titer, and increased the expression of TSG-6 in kidney in SCG mice compared with the effects from triple implantations of DFAT cells. Therefore, we examined single implantations of DFAT cells in SCG mice in the following experiments.

Effects of implantation of DFAT cells on renal injury in SCG mice
The glomerulus from 12-week-old SCG mice showed cellular crescent formation (arrowheads in Fig. 4A). Implantation of DFAT cells suppressed glomerular cellular crescent formation. The GIS and TIS were signi cantly (P < 0.05) higher in kidney from SCG mice than those from ICR mice. Implantation of DFAT cells did not signi cantly affect the increased GIS and TIS in SCG mice (Figs. 4B, C). Expression of TSG-6 in kidney from SCG mice after implantation of DFAT cells Figure 6 shows immunostaining of TSG-6 in SCG mice without and with implantation of DFAT cells. TSG-6 was positively stained in the glomerular mesangium but not in the nephrotubulus. After implantation of DFAT cells, immunostaining of TSG-6 was obviously increased as shown by the positive staining in the glomerular mesangium and nephrotubulus of kidney from SCG mice.

Effects of implantation of DFAT cells on urinary protein excretion in SCG mice
Expression of miRNA in plasma, kidney and lung Effects of implantation of DFAT cells on expression of immune regulator mRNAs in kidney from SCG mice Implantation of DFAT cells signi cantly (P < 0.05) decreased the abundance of CD44 mRNA (Fig. 7A) and signi cantly (P < 0.05) increased that of PGE2 mRNA (Fig. 7B) in kidney from SCG mice. Implantation of DFAT cells increased the abundance of IL-10 mRNA but not statistically signi cantly (Fig. 7C).

Effects of implantation of DFAT cells on expression of immune regulator proteins in kidney from SCG
mice Implantation of DFAT cells signi cantly (P < 0.05) increased the amount of TSG-6 protein in kidney from SCG mice (Fig. 8A), whereas it signi cantly (P < 0.05) decreased the amount of TNF-α protein in kidney from SCG mice (Fig. 8B). Implantation of DFAT cells signi cantly (P < 0.05) increased the amount of CCL-17 protein, a chemokine for M2 macrophage, in kidney from SCG mice (Fig. 9A), but it signi cantly (P < 0.05) decreased the amount of MCP-1 protein, a chemokine for M1 macrophage, in kidney from these mice (Fig. 9B). Fig. 1 shows the survival rates in SCG mice with and without implantation of DFAT cells. Seven weeks after the implantation of DFAT cells, the survival rate was 89% in SCG mice, whereas it was 67% in SCG mice without the implantation of DFAT cells. Figure 10 shows ow cytometric analyses used to evaluate the proportion of CD4 + CD25 + FOXP3 + regulatory T cells in spleen from SCG mice with or without implantation of DFAT cells. There was no signi cant difference in the analyses between SCG mice with or without implantation of DFAT cells.

Discussion
In the present experiments, we rst evaluated delivery of DFAT cells intravenously infused in SCG mice. Almost all of the DFAT cells were trapped in the lung and did not reach the kidney. Despite the nondelivery of DFAT cells into the kidney, the implantation of DFAT cells improved renal function (decrease in BUN), suppressed the expression of in ammatory cytokine MCP-1, and decreased urinary excretion of protein in SCG mice. We also previously showed that DFAT cells intravenously infused were trapped mainly in the lungs without reaching the kidneys, and implantation of DFAT cells reduced proteinuria and improved glomerulosclerosis and interstitial brosis in mAb 1-22-3-induced glomerulonephritis in rats. Moreover, the systematic implantation of DFAT cells through the vein was more effective in improving mAb 1-22-3-induced glomerulonephritis than direct implantation of DFAT cells through the renal artery [7].
Thus, it is surmised that the DFAT cells trapped in lungs released substrates that may have then reached the kidney to improve ANCA glomerulonephritis in the SCG mice. Mechanisms underlying the immunosuppressive effects of the MSCs trapped in the lungs after intravenous implantation have been reported to be associated with exosomes including cytokines, miRNAs and peptides that improve acute graft-versus-host disease and immune-induced acute kidney injury [14,15]. In terms of the mechanisms underlying the induction of immunosuppressive and anti-in ammatory effects by lung-trapped MSCs and DFAT cells, these effects of the implantation of MSCs are associated with the secretion of soluble factors with paracrine actions that are mediated by exosomes. Exosomes are predominantly released from the endosomal compartment and contain miRNA, cytokines, and proteins from MSCs. Recent studies in animal models suggest that exosomes have signi cant potential as a novel alternative to whole-cell therapies [16]. Bruno et al. [14] showed that exosomes derived from MSCs improve acute tubular injury. Chaubey et al. [17] reported that implantation of MSCs improved experimental bronchopulmonary dysplasia in part via exosome-associated factor TSG-6. Thus, DFAT cell-derived exosomes may improve ANCA glomerulonephritis by increasing TSG-6 in kidney. It is thought that the direct infusion of exosomes from ex vivo-cultured DFAT cells will be effective immunosuppressive therapy for ANCA glomerulonephritis.
In the present experiments, the single implantation of DFAT cells was rather more effective in improving renal function, decreasing MPO-ANCA titer, and increasing the expression of TSG-6 in the kidney of SCG mice than the triple implantations of DFAT cells. Recently, single implantation has been also reported to show rather effective immunosuppressive effects on autoimmune-induced colitis in mice [19] and osteoarthritis in a clinical study [20]. Thus, the single implantation of DFAT cells effectively induced the immunosuppression of ANCA glomerulonephritis, which may be associated with exosome release from the lungs.
We con rmed the ANCA glomerulonephritis as cellular crescent formation in kidney from the 12-week-old SCG mice. Neumann et al. [20] showed that the number of glomerular crescent formations increased along with aging. In addition, plasma levels of MPO-ANCA concentrations in SCG mice were higher than those in ICR mice. Implantation of DFAT cells decreased the GIS but did not affect the TIS of kidney from ICR mice. These results indicate that implantation of DFAT cells mainly improved the glomerular injury whereas their implantation did not appreciably affect nephrotubular degeneration.
Concerning the immunosuppressive effects of MSCs, two mechanisms have been considered to regulate immunoactivity [21]. One mechanism has been reported in which MCSs release TSG-6 that suppresses adhesion molecule CD44 on T cells to inhibit T-cell activity and cell in ltrations, by which the regulatory T cells are increased to obtain immune tolerance [22]. In the other reported mechanism, MSCs release PGE2 that induces the transition of M1 to M2 macrophages [24].
In our previous study, we observed that the systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage in ltration and expression of IL-6, IL-10 and IL-12β, and increased the production of serum and renal TSG-6, which improved the mAb 1-22-3-induced renal degeneration through the immunosuppressive effects of TSG-6 [7].
In the present experiments, the implantation of DFAT cells markedly increased the expression of TSG-6 mRNA and proteins in kidney from SCG mice, even though no DFAT cells were delivered into the kidney. The implantation of DFAT cells did not affect the number of regulatory T cells in spleen from SCG mice. However, implantation of DFAT cells decreased the expression of CD44 mRNA in kidney from these mice, thus suggesting that DFAT cells may regulate immunoactivity by suppression of the activity and invasion of T cells.
Moreover, expression of PGE2 and IL-10 mRNAs in the present experiments was increased in kidney from SCG mice after the implantation of DFAT cells. After the implantation of DFAT cells, the expression of MCP-1 as M1 macrophage chemokines was decreased in kidney of SCG mice, and that of CCL17 as a chemokine for M2 macrophages was increased in kidney of SCG mice. These results indicate that mechanisms underlying the immunoregulatory effects of DFAT cells appear to be associated with induction of the transition of M1 to M2 macrophages with production of in ammatory cytokine IL-10.
Because the implantation of DFAT cells may improve ANCA glomerulonephritis through increases in TSG-6 in kidney, which may be mediated by exosomes, we analyzed the expression of miRNAs in plasma, kidney and lungs in SCG mice after the implantation of DFAT cells. Expression of miR23b-3p was obviously higher in plasma, kidney and lung from these mice with implantation of DFAT cells compared to those in plasma, kidney and lung from SCG mice without implantation of DFAT cells. These results indicate that the increases in TSG-6 in kidney from SCG mice may be mediated by miR23b-3p delivered by the exosomes.
In the present experiments, the survival rate was higher in SCG mice with rather than without the implantation of DFAT cells. Longer-term investigations of the survival rate and side effects such as tumor genesis are needed for application of the implantation of DFAT cells for ANCA glomerulonephritis as the average survival period of SCG mice is only 120 days.

Conclusions
In conclusion, the intravenous implantation of DFAT cells in SCG mice suppressed renal injury including glomerular cellular crescent formation in kidney and increased the expression of TSG-6 without the delivery of DFAT cells directly into kidney. Mechanisms underlying the improvement of ANCA glomerulonephritis are associated with the immunosuppressive effects of TSG-6 and the transition of M1 to M2 macrophages. These ndings suggest that implantation of DFAT cells can be a potential cell therapy for ANCA glomerulonephritis.   Figure 1 Distribution of implanted DFAT cells. In total, 105 of PKH26-labeled DFAT cells were infused through the posterior orbital venous plexus in ICR mice. One hour, 24 hours, and one week after the injection, kidney, aorta, liver and lungs were removed and xed in 3% formalin in PBS and embedded in para n.
Arrowheads indicate trapped DFAT cells. Bar = 50 μm. DFAT dedifferentiated fat Figure 2 Experimental protocols for the implantations of DFAT cells in SCG mice. Protocol 1: SCG mice with single implantation of DFAT cells at 8 weeks of age. Protocol 2: SCG mice with triple implantation of DFAT cells at 8, 9 and 10 weeks of age. Each mouse was sacri ced at 12 weeks of age, its blood sampled, and its kidney removed. Protocol 3: Excreted urinary protein was collected from SCG mice at 12 to 17 weeks of age in metabolic cages. DFAT dedifferentiated fat Effects of implantation of DFAT cells on urinary protein excretion in SCG mice. SCG mice were infused with 105 DFAT cells through the posterior orbital venous plexus once at 8 weeks of age. Excreted urinary protein was collected from the mice at 12 to 17 weeks of age in metabolic cages. Data are the mean ± SEM (n = 6). *P < 0.05 and **P < 0.01 between with and without implantation of DFAT cells. DFAT dedifferentiated fat; SEM standard error of the mean Figure 6 Expression of TSG-6 in kidney from SCG mice after implantation of DFAT cells. SCG mice were infused with 105 DFAT cells through the posterior orbital venous plexus once at 8 weeks of age. Mice were sacri ced at 12 weeks of age, and kidneys were removed. The 3-mm para n sections of the removed renal cortex were stained with rabbit polyclonal anti-TSG-6 antibody. Bar = 50 μm. DFAT dedifferentiated fat; TSG-6 tumor necrosis factor-stimulated gene-6 Effects of implantation of DFAT cells on expression of TSG-6 and TNF-α proteins in kidney from SCG mice. SCG mice were infused with 105 DFAT cells through the posterior orbital venous plexus once at 8 weeks of age. Mice were sacri ced at 12 weeks of age, and kidneys were removed. Protein samples were subjected to electrophoresis on polyacrylamide gels and then transblotted to nitrocellulose membranes.
Blots were incubated with anti-TSG-6 polyclonal antibody and anti-TNF-α polyclonal antibody, and antibeta actin antibody in TBST solution. Data are the mean ± SEM (n = 4). *P < 0.05 between with and without implantation of DFAT cells. DFAT dedifferentiated fat; SEM standard error of the mean; TNF, tumor necrosis factor; TSG-6 tumor necrosis factor-stimulated gene-6