- Academic Editor
†These authors contributed equally.
Background: Adipose tissue-derived stem cells (ADSCs), a type
of mesenchymal stem cell, have been used extensively in clinical trials for the
treatment of multiple conditions, including sepsis. However, increasing evidence
indicates that ADSCs vanish from tissues within days of administration.
Consequently, it would be desirable to establish the mechanisms underlying the
fate of ADSCs following transplantation. Methods: In this study, sepsis
serum from mouse models was used to mimic microenvironmental effects. Healthy
donor-derived human ADSCs were cultured in vitro in the presence of
mouse serum from normal or lipopolysaccharide (LPS)-induced sepsis models for the purposes of
discriminant analysis. The effects of sepsis serum on ADSC surface markers and
cell differentiation were analyzed by flow cytometry, and the proliferation of
ADSCs was assessed using a Cell Counting Kit-8 (CCK-8) assay. Quantitative real-time PCR (qRT-PCR) was applied to assess the degree
of ADSC differentiation. The effects of sepsis serum on the cytokine release and
migration of ADSCs were determined based on ELISA and Transwell assays,
respectively, and ADSC senescence was assessed by
Sepsis is a frequent complication in hospitalized patients characterized by life-threatening organ dysfunctions in response to microbial infection. Annually, some 50 million people worldwide will experience sepsis, among whom at least 11 million will die [1, 2, 3]. Most types of microorganism, including bacteria, fungi, viruses, and parasites, can cause sepsis [4], which is the final common pathway leading to death from most infectious diseases and is becoming more prevalent [5]. This increase in prevalence could be ascribed to multiple factors, among which, the global aging population, evolution of drug-resistant pathogens, and worldwide spread viral infections, such as SARS-CoV-2/COVID-19, are often cited [6, 7, 8].
Despite considerable research efforts, there have been no therapies developed that directly modify the pathophysiology and injury mechanisms underlying sepsis [4, 9, 10]. During the past two decades, most sepsis-related studies have focused on suppressing the early proinflammatory response to sepsis. To date, however, there have been more than 40 unsuccessful clinical trials of agents that have been optimistically proposed to reduce pathogen recognition or block proinflammatory cytokines or inflammation-signaling pathways [11, 12]. In spite of these failures, however, a number of key insights have accrued in recent years from studies that have evaluated the therapeutic effects mesenchymal stem/stromal cells (MSCs) in attenuating proinflammatory responses whilst enhancing host immune function and the reparative capacity of tissues [13, 14]. The findings of multiple preliminary studies from different groups have demonstrated the potential application of MSC therapy in murine models, albeit with varying degrees of efficacy. Plausible factors that might account for these discrepant findings include the use of non-homogenized MSC populations, different donors, different tissue sources, and different passages during in vitro expansion. MSC engraftment to the site of injury and their retention there, at least for a short period, is required for efficacy in preclinical sepsis models [15, 16, 17]. Moreover, the findings of recent studies have also indicated that the cell microenvironment can contribute to regulating the fate of MSCs [18, 19, 20, 21], which effectively means that the same or different batches of MSCs may have differing effects when transplanted into patients at different stages of the disease. These complex issues accordingly warrant further in-depth study to provide a basis for more effective personalized stem cell therapy.
Adipose tissue-derived stem cells (ADSCs) are a type of MSCs that have the capacity of self-renewal and can be induced to develop into osteoblasts, adipocytes, and chondrocytes [22, 23]. Compared with bone marrow-derived MSCS, ADSCs have multiple unique advantages, including the greater ease of access and harvesting, and their use is less constrained by ethical considerations [24]. Given these advantages, ADSCs have been used extensively in clinical trials to evaluate their utility in the treatment of conditions such as diabetes mellitus, liver disease, corneal lesions, and sepsis. The secretory profile of ADSCs includes a wide range of cytokines, chemokines, and growth factors [25]. Studies have revealed that on the basis of their paracrine effects, ADSCs can stimulate angiogenesis, exert antiapoptotic effects, and recruit other cells to the sites of injury [23]. However, there are few studies on the role of pathological environment on MSCs’ function. In the present study, we exposed human ADSCs to sepsis serum to assess the regulation of the fate of these cells.
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-
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-
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
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
Total RNA was extracted using TRIzol (Sigma, St Louis, MO,USA) using a standard
protocol and quantified using a NanoDrop
For an analysis of cell migration, we used Boyden’s chambers (Transwell Assay)
containing Transwell inserts of pore size 4.5
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
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
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad software, San Diego, CA,
USA). Data are presented as the means
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
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
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.
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.
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
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-
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
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.
Respiration assayusing the Agilent Seahorse XF
technology. (A) An analysis of O
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
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
Mesenchymal stem cells are a rarely occurring type of undifferentiated cell that can be isolated from a large range of adult tissues, including bone marrow, adipose tissue, cord blood, amniotic fluid, dental pulp, and skeletal muscle [30, 31, 32, 33]. Several factors indicate the therapeutic potential of MSCs for the treatment of sepsis. For example, it has been established that MSCs can differentiate into a diverse range of cell lineages both in vitro and in vivo, thereby contributing to the healing of damaged tissue or organs. MSCs also produce large quantities of cytokines and extracellular matrix that are conducive to reconstruction of the microenvironment of injured tissues, and these cells have strong immunosuppressive effects by contributing to the reduction of inflammatory cell numbers and inflammatory cytokine production. Nevertheless, despite the considerable amount of research conducted on these cells over the past decade, further studies are needed to develop optimized and stable treatment regimens for the management of sepsis. Therefore, we explored the influence of sepsis microenvironment on ADSCs in present study. We demonstrated that sepsis serum promotes the cytokine and growth factor secretion and migratory capacity of ADSCs. However, given that these findings were based on in vitro studies, more detailed studies of ADSCs in septic animals are required. In the future, we will continue conduct in vitro and in vivo experiments to gain further clarification of the efficacy and safety of ADSCs in the treatment of sepsis.
In recent years, concerns regarding the potential long-term effects of stem cell
therapy have to a larger extent been allayed by the discovery that these cells
tend to disappear from tissues within days of administration. Regulation of stem
cell self-renewal and differentiation depends on the provision of an appropriate
niche, which is the microenvironment comprising the extracellular matrix, growth
factors, morphogenic factors, small molecules, and cytokines secreted by stem
cells and the surrounding cells. Accumulating evidence indicates that an
inflammatory microenvironment can modulate MSCs, and consequently, there is a
pressing need to elucidate the mechanisms underlying the fate of ADSCs after
having been transplanted into the body. In this study, we exposed ADSCs to
simulated microenvironments comprising normal mouse serum or sepsis serum, and
subsequent cell proliferation assays revealed an inhibitory effect of mouse
sepsis serum, along with altered cell differentiation capacities. Mesenchymal
stem cells have high osteogenesis and adipogenesis potential, and the switch
between osteogenetic and adipogenic commitment and differentiation is mediated
through numerous transcription factors and signaling pathways, as well as
metabolic patterns. Here, we showed that the cell differentiation capabilities
were altered in sepsis serum-treated ADSCs. Furthermore, the metabolic analysis
revealed metabolism reprogramming in ADSCs
after sepsis serum administration, along with enhanced OCR and reduced ECAR. ROS
accumulation in MSCs may contribute to the loss of homeostasis, leading to
senescence. Indeed, our findings suggested that ADSCs administrated with mouse
sepsis serum exhibited senescence markers, including delayed proliferation,
increased expression of senescence-associated
The significance of this experiment for clinical research is that it reveals the variabilities of ADSC preparations. ADSCs treated with sepsis serum may exhibit properties that are higher than the therapeutic efficacy of normal ADSCs due to fate reprogramming.
In conclusion, our findings show that mouse sepsis serum seriously affects the growth and cell fate determination of in vitro cultured ADSCs, which hint that the microenvironmental factors of sepsis recipients may regulate the cell fate of ADSCs, leading to different outcomes in the clinical trials under sepsis conditions. This calls for further investigation of the mechanisms of sepsis serum-mediated ADSCs fate alteration and the development of efficient means to reverse it.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JF, RL, YL, HWang and HWei designed and performed the experiments; JW, QL, HL analyzed and interpreted the data; ZZ, LZ collect literatures and drafted manuscript; RP, YC proposed the concept, revise and review the manuscript. All authors contributed to author contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
The animal experimental protocol was approved by the local medical animal experiment ethics committee (ZJCLA, No. ZJCLA- IACUC-20110043). Samples of human adipose tissue were collected using a protocol approved by the Internal Review Board of Zhejiang Provincial People’s Hospital (2018KY977).
Not applicable.
This work was supported by the National Key Research and Development Program of China [grant number 2018YFC2001904]; and the Key Technologies R&D Program of Zhejiang Province [grant number 2019C03041].
Authors LZ and RP were employed by S-Evans Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.