Samples of human adipose tissue were collected from the abdominal wall of healthy donors (20–35 years old healthy females with standard body mass index, no history of diabetes or other metabolic diseases) undergoing cosmetic surgery, using a protocol approved by the Internal Review Board of Zhejiang Provincial People’s Hospital. The samples were washed with pre-cooled phosphate-buffered saline (PBS) containing a 1% antibiotic–antimycotic solution (Beyotime, Shanghai, China), diced into small pieces using surgical blades, and then digested with 0.2% Type I collagenase (Sigma, St Louis, MO, USA) in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG; Cellgro, Herndon, VA, USA) at 37 °C shaking at 100 rpm. Cell suspensions were harvested after incubating for 30 min, filtered through an 80-$\mu$m mesh, and centrifuged for 10 min at 250 $\times$ g. The cell pellets thus obtained were washed twice with pre-cooled PBS, resuspended with DMEM-LG supplemented with 10% fetal bovine serum (FBS: Gibco), and seeded in 10-cm tissue culture-treated Petri dishes (Sigma-Aldrich, St Louis, MO, USA) at a density of 1 $\times$ 10${}^3$ cells/cm${}^2$. The cultures were incubated at 37 °C in a 5% CO${}_2$ atmosphere at 95% humidity. Non-adherent cells were removed by renewing the medium after 24 h, and the adherent cells were thereafter continuously cultured.

Sterile cloning cylinders and 0.25% trypsin were subsequently used to obtain single colony-derived ADSC populations. Care was taken to select well-separated colonies, thereby ensuring that each cloning cylinder contained only a single colony. The isolated ADSC colonies were then cultured in the aforementioned growth medium and passaged via 0.25% trypsin-EDTA digestion at a 1:5 ratio. Cells of passage five were used in subsequent experiments.

Eight-week-old Balb/c mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and maintained in the specific pathogen-free laboratory of a local animal facility. The experimental protocol was approved by the local medical animal experiment ethics committee (ZJCLA, No. ZJCLA-IACUC-20110043). Lipopolysaccharide-induced sepsis was stimulated as described by other groups [26]. Briefly, lipopolysaccharide (5 mg/kg dosage) was intraperitoneally injected into mice to induce experimental conditions, and survival was recorded at 2 and 20 h post-administration. Thereafter, surviving mice were anesthetized, and blood was collected from the retro-orbital venous plexus. Serum samples were harvested, mixed, and stored at –80 °C. Prior to use for cell culture, the mouse sepsis serum was thawed, added to the DMEM-LG, and passed through a 0.22-$\mu$m filter.

The phenotype of ADSCs was examined based on flow cytometric analysis. Briefly, cells were trypsinized, washed, and incubated for 1 h at room temperature with appropriate dilutions of the following fluorescently conjugated antibodies (BD Bioscience, San Jose, CA, USA): anti-CD73 (550257), anti-CD90 (555596), anti-CD105 (560839), anti-CD14 (555397), anti-CD19 (555413), anti-CD34 (555822), anti-CD45 (555483), anti-HLA-DR (555811). Corresponding isotype-matched antibodies were used as the negative controls. Cells were washed twice with PBS and analyzed using a Becton Dickinson Calibur flow cytometer (BD, East Rutherford, NJ, USA).

To evaluate the multilineage differentiation capacity of ADSCs, osteogenic, adipogenic, and chondrogenic inductions were assessed in vitro as previously described [27]. Briefly, ADSCs derived from Stromal Vascular Fraction (SVF) were cultured in $\alpha$-MEM (Invitrogen, Carlsbad, CA, USA) medium containing 10% FBS, 10${}^{-7}$ M dexamethasone, 10 mM $\beta$-glycerol phosphate, and 50 $\mu$M ascorbate-2-phosphate for osteogenic differentiation and stained with Alizarin Red S. ADSCs were incubated for 2 weeks in an adipogenic induction medium (10${}^{-6}$ M dexamethasone, 0.5 $\mu$M isobutylmethylxanthine, and 10 ng/mL insulin) for adipogenic differentiation, fixed, and stained with Oil Red O solution (Cyagen, Santa Clara, CA, USA). ADSCs were centrifuged to form a pelleted micromass and maintained in high-glucose DMEM containing 10${}^{-7}$ M dexamethasone, 1% insulin–transferrin–sodium selenite, 50 $\mu$M ascorbate-2-phosphate, 1 mM sodium pyruvate, 50 $\mu$g/mL proline, and 20 ng/mL TGF-$\beta$3 for chondrogenic differentiation and stained with Alcian blue.

Cell proliferation assays were conducted using a Cell Counting Kit-8 (CCK-8) cell viability kit (Yeasen) according to the manufacturer’s protocol. Briefly, ADSCs at the log-phase of growth were trypsinized, resuspended, and seeded at a density of 2 $\times$ 10${}^3$ cells/well in 96-well plates, to which 10 $\mu$L of CCK-8 reagent was added at the indicated time points. Following incubation for 1 h at 37 °C, the absorbance the well contents was measured at 450 nm using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). For population doubling time analysis, 1 $\times$ 10${}^5$ cells were seeded in 35-mm dishes (Corning Incorporated, Corning, NY, USA) and cultured for 120 h. The medium was changed at 2-day intervals, and the number of viable cells in each plate was estimated based on hemocytometer counts. The population doubling time after 120 h was determined using the software (http://www.doubling-time.com/ Doubling Time, Mathieu, France).

Total RNA was extracted using TRIzol (Sigma, St Louis, MO,USA) using a standard protocol and quantified using a NanoDrop${}^{\text{TM}}$ 2000 spectrophotometer. Reverse transcription reactions were performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) reactions were conducted using gene-specific primers (Supplementary Table 1) in an Applied Biosystems 7900 thermocycler (Waltham, MA, USA).

For an analysis of cell migration, we used Boyden’s chambers (Transwell Assay) containing Transwell inserts of pore size 4.5 $\mu$m. Briefly, ADSCs at the log-phase of growth were harvested, resuspended in DMEM-LG supplemented with 1% FBS, and seeded at 2 $\times$ 10${}^4$ cells in the upper chamber of the Transwell apparatus. To the lower chamber, we added 2 mL of DMEM-LG containing 10% FBS to create a chemotactic gradient. The quantification of migrated cells was performed after incubating for 18 h. The transwell insert from the plate was removed and a cotton-tipped applicator was used carefully to remove the media and remaining cells on the top of the membrane without damaging it. Then, the transwell insert was placed in methanol for 10 minutes to allow cell fixation and stain with 0.2% crystal violet for 5 minutes. Images were captured under an inverted light microscope. The number of migrating cells in different fields of view was counted and the mean value was calculated.

ADSCs were exposed to either 5% FBS + 2.5% normal mouse serum (NS) or 5% FBS + 2.5% sepsis mouse serum (SS) media for 72 h. Then, the cells were harvested and seeded on 96-well plates at a density of 6700 cells/well. Cells were cultured for 48 h in 200 $\mu$L DMEM-LG 5% FBS/well before the culture medium was collected. To determine the levels of cytokines released into culture supernatants, we used a Quantikine® QuicKitTM Enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer’s instructions, with absorbance being measured at 450 nm using a SpectraMax Plus384 microplate reader (Molecular Devices, Wokingham, UK).

Whole-cell lysates were run on 10% polyacrylamide gels and the separated proteins were transferred to Hybond-P membranes. The membranes were blocked with 5% skim milk and immunoblotted overnight at 4 °C with the following primary antibodies: P53 (Beyotime, Shanghai, China; AF1162), P21 (Beyotime, Shanghai, China; AP021) and Lamin B (Proteintech, Rosemont, IL, USA; 12987-1). As a loading control, we used Rabbit anti-human GAPDH (Beyotime, Shanghai, China; AF1186). Following primary antibody incubation, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature, and were subsequently developed using an ECL Chemiluminescence Kit, with quantification being performed using ImageJ software 2.0 (NIH, Bethesda, MD, USA).

Changes in the rates of extracellular acidification (ECAR) and oxygen consumption (OCR) in ADSCs were determined in real-time using a Seahorse XF96 Flux Analyzer (Seahorse, Agilent, MA, USA) in conjunction with a Seahorse XF Glycolytic Rate Assay Kit (Seahorse Bioscience, USA) and a Seahorse XF Mitochondrial Respiration Assay Kit (Seahorse Bioscience, USA), respectively, following the manufacturers’ instructions.

The levels of cellular ATP and reactive oxygen species (ROS) produced by ADSCs were measured using standard assay Kits purchased from Beyotime according to the manufacturer’s instructions. The data presented are the average of three independent experiments.

ADSC senescence was determined microscopically based on $\beta$-galactosidase staining using a dedicated detection kit (Cell Signal Technology, VIC, AUS).

Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad software, San Diego, CA, USA). Data are presented as the means $\pm$ standard deviations [28] of the results of at least three independent replicates. Student’s t-tests were used to identify differences between groups. p-values 0.05 were considered to be statistically significant.

To investigate the effects of sepsis serum on ADSCs, we monitored the growth of these cells. Rapidly growing passage 5 ADSCs were harvested and seeded for one of the following four treatments: standard culture (10% FBS), 5% FBS culture, 5% FBS + 2.5% NS, and 5% FBS + 2.5% SS. CCK-8 analysis indicated that among these culture conditions, the 10% FBS standard culture was the most effective in terms of supporting ADSC growth. Compared with the 5% FBS medium, the addition of normal mouse serum was found to enhance cell growth, whereas the addition of mouse sepsis serum had the opposite effect, with significantly inhibited ADSC growth being detected at 48 and 72 h (p 0.05, Fig. 1A). We subsequently repeated the aforementioned assessments using passage 1 and 3 ADSCs, and calculated the cell doubling times. Cells cultured in the presence of sepsis serum were observed to be characterized by a prolonged time to doubling, and the effects were more pronounced with successive in vitro passages (Fig. 1B). To further evaluate the effects of sepsis serum on ADSCs, in subsequent experiments we systematically compare the fates of cells exposed to the 5% FBS + 2.5% NS and 5% FBS + 2.5% SS media.

Fig. 1.

Sepsis serum treatment inhibit cell proliferation of ADSCs. (A) Cell proliferation was assessed using CCK-8 assays. (B) The population doubling time of various passage ADSCs with different culture mediums. Data are presented as the mean of four experiments. * p 0.05.

As shown in Fig. 2, the results of flow cytometry analysis revealed that ADSCs cultured in both the 5% FBS + 2.5% NS and 5% FBS + 2.5% SS media exhibited a typical cell surface phenotype characteristic of multipotent mesenchymal stem cells, as defined by the International Society for Cellular Therapy guidelines published in 2006 [29]. These cells were established to be positive for the markers CD73, CD90, and CD105, and negative for CD14, CD19, CD34, CD45, and HLA-DR. In addition, we found that the cells can successfully differentiate into classical mesenchymal derivatives, including osteoblasts, adipocytes, and chondrocytes, albeit with differing differentiation efficiency. Histochemical staining results and qRT-PCR data for lineage-specific markers indicated that ADSCs exposed to sepsis serum were characterized by an increased capacity to differentiate into osteoblasts but reduced capacity to undergo adipogenic and chondrogenic differentiation (Fig. 3). These findings would thus tend to indicate that sepsis serum may regulate factors determining the fate of ADSCs.

Fig. 2.

Representative flow cytometry analysis of cell surface markers in ADSCs. (A) ADSCs cultured in DMEM-LG supplemented with 5% FBS + 2.5% NS. (B) ADSCs cultured in DMEM-LG supplemented with 5% FBS + 2.5% SS.

Fig. 3.

Sepsis serum treatment influence the differentiation bias of ADSCs. (A) ADSCs cultured in 5% FBS + 2.5% NS and 5% FBS + 2.5% SS were induced to differentiate along adipogenic, osteogenic, and chondrogenic lineages. (B) The differentiation degrees were evaluated by qRT-PCR analysis of lineage marker gene expression. GAPDH was used as a housekeeping gene. All measures were performed in triplicate. * p 0.05.

To further to assess the impact of sepsis serum on ADSC behavior, we determined the cytokine secretion profiles of these cells. We accordingly found that the expression of a majority of these factors were increased in ADSCs cultured in the 5% FBS + 2.5% SS medium. Among which, we detected approximately 2.3-, 3.3-, 1.6-, 3.2-, 2.1-fold significant increases in the secretion of IFN-$\gamma$, IL6, SDF1a, TNF-a, and VEGF, respectively (Fig. 4A). These responses were subsequently verified by qRT-PCR analysis of the mRNA expression of these cytokines in ADSCs (Fig. 4B). Furthermore, Transwell migration analysis revealed a significant increase the proportion of migratory ADSCs among those exposed to 5% FBS + 2.5% SS compared with cells pre-cultured in DMEM-LG supplemented with 5% FBS + 2.5% NS (Fig. 4C,D).

Fig. 4.

Cell behavior. (A) Cytokine and growth factor secretion of ADSCs exposed to normal mouse serum and sepsis serum were determined. (B) qRT-PCR verification of mRNA expression of representative cytokines and growth factors in two groups of ADSCs. (C) Transwell assay evaluated ADSCs migration after 24 h of incubation. (D) Data are presented as means $\pm$ SEM of triplicate experiments. Three independent experiments were performed in each group. * p 0.05.

To evaluate whether sepsis serum treatment alters the cellular bioenergetics of ADSCs, we examined the rates of oxygen consumption and extracellular acidification in these cells. OCR was measured following sequential addition of the inhibitors oligomycin, FCCP, rotenone, and antimycin (Fig. 5A). The OCR curves of the two groups of cells showed minimal overlap, thereby tending to indicate a significant change in oxidative phosphorylation. ADSCs exposed to sepsis serum were characterized by significant increases in basal OCR, maximal OCR, and reserve capacity, whereas in contrast, we detected no significant difference with respect to non-mitochondrial OCR. ECAR analysis revealed that in response to sepsis serum treatment, the glycolysis and glycolytic capacity of ADSCs were reduced relative to the mean values measured in control cells.

Fig. 5.

Respiration assayusing the Agilent Seahorse XF technology. (A) An analysis of O${}_2$ consumption in two groups of cells using different inhibitors. (B) Representative graph of the ECAR determination curves reveals the differences in glycolysis.

We subsequently examined whether changes in ADSC metabolism were associated with altered mitochondrial function. In line with expectations, cells exposed to sepsis serum were found to have significantly higher levels of ATP (p 0.05, Fig. 6A). Furthermore, we detected significant increases in the generation of ROS in cells exposed to sepsis serum (p 0.05, Fig. 6B). Similarly, ADSCs exposed to sepsis serum exhibited significantly higher $\beta$-galactosidase activity and flattened morphology in (Fig. 6C). In addition, western blotting analysis indicated that the expression of P53 and P21 were increased, while Lamin B was reduced in the sepsis serum-treated ADSCs (Fig. 6D). Collectively, these findings tend to indicate that sepsis serum promote the senescence of ADSCs, which may partially responsible for the altered differentiation bias.

Fig. 6.

ADSCs exposed to sepsis serum are more senescent than controls. (A) Average rates of ATP level in mitochondria. (B) Relative levels of ROS production. (C) Representative microscopies of $\beta$-galactosidase staining of the two groups of ADSCs. Scale bars = 50 $\mu$m. (D) Expression levels of the P53, P21 and Lamin B1 were determined using western blot and normalized to GAPDH. * p 0.05, ** p 0.01.