Intraportally delivered stem cell spheroids localize in the liver and protect hepatocytes against GalN/LPS-induced fulminant hepatic toxicity
Systemic inflammatory response syndrome (SIRS) is common in severe fulminant hepatic failure (FHF) and has a high mortality rate (20–50%) due to irreversible cerebral edema or sepsis. Stem cell-based treatment has emerged as a promising alternative therapeutic strategy to prolong the survival of patients suffering from FHF via the inhibition of SIRS due to their immunomodulatory effects.
3D spheroids of adipose-derived mesenchymal stem cells (3D-ADSC) were prepared by the hanging drop method. The efficacy of the 3D-ADSC to rescue FHF was evaluated in a d-galactosamine/lipopolysaccharide (GalN/LPS)-induced mouse model of FHF via intraportal transplantation of the spheroids.
Intraportally delivered 3D-ADSC better engrafted and localized into the damaged livers compared to 2D-cultured adipose-derived mesenchymal stem cells (2D-ADSC). Transplantation of 3D-ADSC rescued 50% of mice from FHF-induced lethality, whereas only 20% of mice survived when 2D-ADSC were transplanted. The improved transplantation outcomes correlated with the enhanced immunomodulatory effect of 3D-ADSC in the liver microenvironment.
The study shows that the transplantation of optimized 3D-ADSC can efficiently ameliorate GalN/LPS-induced FHF due to improved viability, resistance to exogenous ROS, and enhanced immunomodulatory effects of 3D-ADSC.
KeywordsMesenchymal stem cell 2D-cultured cells Spheroids Intraportal delivery Fulminant hepatic failure
2D-cultured adipose-derived mesenchymal stem cells
3D spheroids of adipose-derived mesenchymal stem cells
Adipose-derived mesenchymal stem cells
Acute liver failure
Analysis of variance
Cell Counting Kit-8
C-C motif chemokine ligand 2
C-X-C chemokine receptor type 4
Enzyme-linked immunosorbent assay
Fulminant hepatic failure
Glutamic oxaloacetic transaminase
Glutamic pyruvic transaminase
Intercellular Adhesion Molecule 1
Indoleamine 2,3 dioxygenase
- IL-1 α
Interleukin 1 alpha
Interleukin 1 beta
Mesenchymal stem cells
Polymerase chain reaction
Reactive nitrogen species
Reactive oxygen species
Systemic inflammatory response syndrome
Superoxide dismutase 2
Tumor necrosis factor alpha
Tumor necrosis factor-inducible gene 6
Vascular cell adhesion protein 1
Toxins, drugs, viral infections, and genetic and metabolic disorders cause liver dysfunction requiring medical intervention . During fulminant hepatic failure (FHF; also known as acute liver failure (ALF)), bacterial toxins or other hepatotoxins cause massive hepatocellular damage [2, 3]. In addition, systemic inflammatory response syndrome (SIRS) mediated by massive stimulation of innate and/or adaptive immune system is frequently observed in severe cases of FHF. In fact, SIRS is the leading cause of fatality in FHF and 20–50% of FHF patients succumb to irreversible cerebral edema or sepsis with multiple organ dysfunction . Thus, suppression of excessive immune responses during liver dysfunction may reduce the risk of hepatic failure-induced mortality.
Liver transplantation is the only life-saving treatment for permanent liver failure; however, various obstacles, such as limited donor issue, risk of fungal infection during the procedures, immune rejection response against the graft, and long-term immunosuppressant-associated complications decrease the success rate [5, 6]. To overcome the potential risks of whole organ transplantation, non-invasive cell-based therapy using primary hepatocytes is of keen interest for the treatment of liver diseases [7, 8]. However, many aspects of hepatocyte culture, including their isolation, expansion, and preservation, have proven to be difficult, and poor engraftment efficacy limits the hepatocyte transplantation [9, 10]. In this context, mesenchymal stem cells (MSC) are promising candidates for cell therapy because of their multipotent differentiation ability and immunomodulatory activity [11, 12, 13]. Furthermore, MSC have been reported to rescue impaired hepatocytes by differentiating into hepatocyte-like cells and/or by improving the inflammatory, fibrotic microenvironment through paracrine effects  and preventing further liver damage during FHF . Notably, earlier MSC-based therapies were mainly dependent on the multi-lineage differentiation ability, whereas recent studies have focused on the paracrine and autocrine functions of MSC-derived therapeutic factors [16, 17, 18]. Previous reports have revealed that the aggregation of MSC into 3D spheroids causes enhancement in the secretion of paracrine factors and proangiogenic cytokines [19, 20] and protection of the stemness [21, 22]. Furthermore, 3D culture methods have been reported to enhance stem cell survival in vitro as well as in vivo and provide better engraftment efficiency [23, 24]. Thus, 3D culture methods offer a means of improving the efficacy of stem cell therapies and eliminate the need for genetic modification and growth factor delivery.
In cell therapy, the route of cell delivery must be considered deliberately to maximize therapeutic efficacy. In most cases, MSC are administrated via intravenous injection [25, 26, 27], which results in non-specific distributions of cells. The cells are primarily accumulated in the lungs, and only a small number of cells localize into the liver despite their homing ability . Although various injection routes have been considered, including delivery into targeted areas, no report has been issued on maximizing the localization of the transplanted cells in models of liver injury. In the current study, we aimed to evaluate the therapeutic potential of adipose-derived mesenchymal stem cells (ADSC) in a d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced murine model of hepatic injury, because it closely resembles FHF [2, 3]. To enhance ADSC engraftment and localization in the liver, we delivered the 3D spheroids derived from ADSC (3D-ADSC) via the portal vein. The liver-entrapped 3D-ADSC helped in the modulation of inflammatory hepatic microenvironment through the secretion of anti-inflammatory cytokines/growth factors and improved outcomes of mesenchymal stem cell therapy in FHF.
Material and methods
Human adipose-derived mesenchymal stem cells (ADSC) were purchased from Zenbio (Morrisville, NC, USA). ADSC were cultured in alpha-MEM (Hyclone, South Logan, UT, USA) containing 10% v/v fetal bovine serum (Hyclone, South Logan, UT, USA) and 1% v/v antibiotics/antimycotics (GenDEPOT, Barker, TX, USA). Cells from passages 4–8 were used in this study.
Preparation and optimization of 3D-ADSC
A previously described hanging drop technique was used to prepare 3D-ADSC . The differently sized spheroids, i.e., 3D-ADSC500, 3D-ADSC1000, 3D-ADSC2000, and 3D-ADSC4000 containing 500, 1000, 2000, and 4000 ADSC per spheroid, respectively, were prepared. The viability and size of 3D-ADSC were considered for the optimization of size of the 3D-ADSC for in vivo studies. The viability of differently sized 3D-ADSC was assessed using a live/dead assay kit (Molecular Probes, Eugene, OR, USA), and Western blot analysis as reported previously .
Induction of FHF
Balb/c nude mice (7–8 weeks old) purchased from Orient Biosciences (Seoul, Republic of Korea) were used in this study. Animals were housed in the animal facility of Yeungnam University. Animal experimental protocols were approved by the regulations of Institutional Animal Care and Use Committee (IACUC) of Yeungnam University (IACUC: YL 2018-028). For the determination of the optimal dose required to induce FHF, mixtures of GalN (Carbosynth, Old Station Business Pk, Compton RG20 6NE, UK) (1000, 1500, 2000, or 3000 mg/kg) and LPS (Sigma, St. Louis, MO, USA) (20 μg/kg) were administered intraperitoneally (i.p.). The anti-inflammatory effect of stem cells was evaluated using an optimized GalN/LPS dose (single injection; 1500 mg/kg GalN and 20 μg/kg LPS). Mice were divided randomly into different groups after the induction of ALF.
Intraportal delivery of ADSC
The therapeutic efficacy of ADSC was determined by infusing cells via the portal vein 5 h after GalN/LPS injection. One thousand 3D-ADSC1000 or 1 × 106 2D-ADSC were suspended in 100 μL PBS and injected into the portal vein using an insulin syringe. Sham group received an intraportal injection of 100 μL PBS. Subsequently, mice were monitored 6 hourly and blood samples were obtained at 1, 3, 5, and 7 days after transplantation. Serum glutamate-pyruvate transaminase (GPT) and serum glutamic oxaloacetic transaminase (GOT) levels were measured via the colorimetric method using a chemistry analyzer (DRI-CHEM 4000i, FUJIFILM Corporation, Tokyo, Japan).
DNA extraction and real-time PCR
At 10 h after MSC transplantation, the livers, lungs, and hearts were isolated to investigate the presence of the transplanted cells in these organs. Depending on the weight of the organ, lysis buffer was added and homogenized. Afterwards, DNA was extracted using a genomic DNA extraction kit (Bioneer Corp, Daejeon, South Korea) and quantified using a spectrophotometer (Nanodrop; TECAN). Real-time PCR (RT-PCR) was performed using 25 ng DNA, 5 μL SYBER green (Thermo Scientific, Waltham, MA, USA), 37.5 nM ALU primer, and 450 nM GAPDH primer. The primer sequences used were as follows: ALU, 5′-GTCAGGAGATCGAGACCATCCC-3′ (F) and 5′-TCCTGCCTCAGCCTCCCAAG-3′ (R), and GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (F) and 5′-TCCACCACCCTGTTGCTGTA-3′ (R). Calibration curve was prepared using crossing point (Cp) values obtained with various amounts of human DNA (16%, 8%, 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.0156%, 0.0078%, and 0.0039%).
Levels of ROS and ROS-related enzymes in 3D-ADSC1000
To understand the mechanism responsible for the effects of 3D-ADSC1000 transplantation, amounts of ROS produced by 2D-ADSC and 3D-ADSC1000 were evaluated by the fluorescence method using 20 μM of 2′,7′-dichlorofluorescein diacetate (DCFDA) (Sigma-Aldrich, St. Louis, MO, USA) and normalized with the DNA contents measured using PicoGreen Kit (Molecular Probes, Eugene, OR, USA). Similarly, levels of the antioxidant enzymes superoxide dismutase 2 (SOD2), catalase (CAT), and hemeoxygenase-1 (HO-1) were determined by the Western blot analysis using antibodies against SOD2 (Cell Signaling Technology, Danvers, MA, USA), CAT (Cell Signaling), and HO-1 (Cell Signaling).
Viability of 3D-ADSC1000 under oxidative stress condition
In order to examine cell viability in a highly oxidative environment that resembles the FHF liver, 3 × 104 monolayer-cultured MSCs or 30 spheroids (each spheroid containing 1000 MSCs) were cultured with different concentrations of TBHP (tert-butyl hydroperoxide) (Tokyo Chemical Industry, Nihonbashi-honcho, Chuo-ku, Tokyo, Japan) (200 and 400 μM) for 24 h in a 96-well plate. Viability was assayed by using live/dead imaging and Cell Counting Kit-8 (CCK-8) as described previously .
Anti-inflammatory effects of 3D-ADSC1000 and 2D-ADSC
The levels of prostaglandin E2 (PGE2) secreted in supernatants after 3 days of culture of 2D-ADSC and 3D-ADSC1000 were measured using a PGE2 ELISA kit (R & D Systems, Minneapolis, MN, USA). Similarly, in vitro anti-inflammatory effects of 3D-ADSC1000 and 2D-ADSC were assessed by culturing them with activated macrophages. Briefly, RAW 264.7 macrophages were cultured at a density of 4 × 104 per well in a 96-well plate for 24 h and activated by adding 1 mg/mL of LPS for 90 min. Ten 3D-ADSC1000 or 1 × 104 2D-ADSC were added to each well, and 10 h later, supernatants were collected, centrifuged, and stored at − 70 °C until analysis. Levels of IL-10 and TNF-α were measured by ELISA using a Mouse IL-10 ELISA kit (R & D Systems, Minneapolis, MN, USA) or a Mouse TNF-α kit (Elabscience, Bethesda, MD, USA), respectively. Finally, levels of ROS in activated macrophages were measured by the fluorescence method using DCFDA.
In vivo assessment of the anti-inflammatory effect of 3D-ADSC1000 and 2D-ADSC
For the in vivo assessment of the effect of 3D-ADSC1000 and 2D-ADSC in attenuating inflammation in the FHF model, polarization of macrophage in the liver was evaluated using flow cytometry. After 7 h of cell transplantation, isolation of monocytes from the liver was conducted as described previously . The isolated cells were stained with anti-mouse PE-Cy7-conjugated CD11b (Biolegend, San Diego, CA, USA), PE-conjugated F4/80 (Biolegend), APC-Cy7-conjugated CD86 (Biolegend), and PE-Cy7-conjugated CD206 (Biolegend) and analyzed by BD FACS Verse flow cytometer (BD Biosciences, San Jose, CA, USA). To check M1 and M2 phenotypes of macrophage, F4/80+ population was selected, and the expression of CD86 (M1 surface marker) and CD206 (M2 surface marker) was evaluated. Median fluorescence intensity (MFI) was calculated using FlowJo Software (FlowJo LLC, OR, USA). In addition, the concentration of IL-10 in serum was also evaluated by ELISA.
3D-ADSC1000- and 2D-ADSC-injected mice were sacrificed 7 h after injections, and the livers were surgically removed, fixed in 4% paraformaldehyde solution, and embedded in paraffin. Samples were then sectioned at 5 μm, deparaffinized in xylene, rehydrated using an alcohol series, and stained with hematoxylin and eosin (H&E).
Ex vivo imaging
Cells were stained with VivoTrack680 (pernElmer, Boston, MA, USA) according to the manufacturer’s protocol. Then, 1 × 106 monolayer cells or 1000 3D-ADSC1000 were injected into the portal vein via a surgical process. At 7 h post-transplantation, different organs were isolated to observe the fluorescence using in vivo imaging system (FOBI; NeoScience, Suwon, Korea).
Results are expressed as mean ± SEM. Data were analyzed using GraphPad Prism software version 5 (GraphPad Software, La Jolla, CA, USA). Unless otherwise stated, unpaired t test was performed to calculate the statistical significance values. Statistical significance was accepted for p < 0.05.
Optimization of 3D-ADSC
Intraportal delivery of 3D-ADSC1000 ameliorated FHF
Serum levels of GOT and GPT in the sham, 2D-ADSC, and 3D-ADSC1000 groups 5 h after GaLN/LPS injection, were significantly higher than those of untreated mice (untreated mice; GOT 110 ± 20 and GPT 32 ± 9) (Additional file 1: Figure S2). Notably, no significant difference was observed in GPT and GOT levels among all the experimental groups at the time of transplantation. However, at 19 h after cell administration, the levels of serum GPT and GOT were markedly lower in the 3D-ADSC1000-treated group compared to the 2D-ADSC-treated group (GPT: p < 0.01, GOT: p < 0.001) or the sham group (GPT: p < 0.001, GOT: p < 0.01), indicating a reduction in the severity of liver injury after spheroid transplantation. Similarly, at day 3 post-transplantation, serum GPT levels in the 3D-ADSC1000 group were significantly lower compared to those in the 2D-ADSC (p < 0.01) and sham (p < 0.05) groups (Fig. 2c, d).
Intraportally transplanted 3D-ADMSC1000 localized in the liver
Histological analysis revealed the localization of transplanted 3D-ADSC1000 in the portal triad. In addition, H&E staining showed an intact morphology of 3D-ADSC1000 at 7 h of transplantation (Fig. 3a). Similarly, ex vivo imaging of the liver and lungs also demonstrated the localization of ADSC in the liver and minimal migration into the lungs in the 3D-ADSC1000-transplanted group (Fig. 3b). Further, to quantify total human ADSC after transplantation, the expression of human-specific ALU sequence was evaluated in the liver, lungs, and heart. A calibration curve was generated using different percentages of human DNA mixed with mouse DNA (Fig. 3c). This equation was further used to calculate the total percent of human DNA in mouse tissue. Results revealed a higher number of cells distributed in the liver of the 3D-ADSC1000-transplanted group compared to the 2D-ADSC-transplanted group. In contrast, the percentage of human DNA in the lungs of the 3D-ADSC1000-transplanted group was significantly lower than that of the 2D-ADSC-transplanted group, suggesting a minimal migration of ADSC from the spheroids to extrahepatic tissues after transplantation. No significant migration of MSC was observed in the heart of both groups (Fig. 3b, d).
3D-ADSC1000 were resistant to oxidative stress
3D-ADSC1000 transplantation exerted improved immunomodulation
Stem cell therapy is based on cell replacement by differentiation into desired cell types or on rearrangement of the microenvironment in a manner that promotes tissue repair . MSC have been reported to be useful in FHF due to their ability to differentiate into hepatocytes . Furthermore, MSC have the ability to improve the liver microenvironment and promote the regeneration of hepatocytes by regulating inflammatory cells and cytokines [14, 36]. Although MSC have been demonstrated to be beneficial in various disease conditions, their effectiveness is compromised by limited survival and engraftment after transplantation . 3D-ADSC have been reported to enhance cell viability possibly due to the phosphorylation of AKT or the upregulations of hypoxia-induced survival factors and nutrient deprivation-induced factors [23, 38, 39, 40]. Recently, we have developed extracellular matrix-modified scaffolds to enhance viability and therapeutic effectiveness in inflammatory diseases . In the current study, transplantation of viability-strengthened MSC spheroids via the portal vein alleviated ALF by exerting a local immunomodulatory function.
Delivery of cells via the portal vein might increase risks of venous thrombosis and embolism. In a recent study, thrombosis and infarction were more prominent after intraportal delivery of 2D-ADSC compared to the 3D-ADSC-delivered group , which may be due to higher expressions of thrombogenic proteins, including tissue factor (TF) and chemokine (C-C motif) ligand 2 (CCL2) in 2D-ADSC compared to that of the 3D-ADSC. Furthermore, 3D-ADSC containing 10,000 cells were reported to aggravate thrombosis and infarction in the livers , which indicates a necessity of control over spheroid size to protect the cells against hypoxia or nutrient deficiency after transplantation. The size-controlled MSC spheroids used for in vivo experiments in the current study survived well in a highly oxidative environment under in vitro conditions. This resembles the environment of an inflamed liver during FHF. Interestingly, no signs of hepatic thrombosis were observed in the 3D-ADSC-transplanted group.
GalN/LPS-induced FHF induces the activation of liver macrophage and the recruitment of neutrophils, monocytes, and natural killer cells in the liver to provoke inflammation . Moreover, these activated immune cells and secreted inflammatory cytokines are responsible for the production of reactive oxygen/nitrogen species (ROS/RNS) [43, 44, 45] and hepatocyte death [46, 47]. Previous studies have adopted different strategies, such as treatment with antioxidants [48, 49], overexpression of anti-apoptotic Bcl-2 , HO-1 [51, 52], or AKT , and hypoxic preconditioning , to improve MSC survival. In the present study, we used 3D-ADSC1000 which had enhanced expression of Bcl-2 and HO-1 and antioxidant proteins which led the MSCs to thrive well in the oxidative stressful environment after in vivo transplantation.
MSC may actively home to tissues via leucocyte-like cell adhesion and transmigration . Alternatively, they may be passively entrapped into small-diameter blood vessels . We delivered 3D-ADSC1000 via the portal vein to ensure passive entrapment by liver sinusoids and portal triads. During systemic infusion, a large fraction of MSC are typically trapped in lung capillaries , and this retention often results in poor homing . Thus, local delivery of stem cells to injured sites probably promotes MSC entrapment in damaged organs [57, 59]. Although the mechanisms responsible for MSC engraftment and homing at sites of injury are unknown, various homing and adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, integrins, and CXCR4, have been reported to be involved . Long-term culture of 2D-MSC in vitro has been reported to decrease the expressions of these adhesion and homing molecules that compromise homing and engraftment efficiencies at sites of injury . In a recent study, it was reported that the upregulation of CXCR4 expression in 3D aggregates promotes the adhesion of MSC spheroids with the endothelial cells , which would aid the localization of transplanted 3D spheroids at the site of injury. In another study, when bone marrow-derived mononuclear cell spheroids were delivered intraportally, they were found to be entrapped in the liver, whereas spheroid-derived single cells delivered intraportally drained into the systemic circulation .
During inflammation, high concentrations of the pro-inflammatory cytokines, such as TNF-α and IFN-γ, activate MSC to secrete soluble immunomodulatory factors, such as IDO, PGE2, and NO , which results in macrophage polarization to the anti-inflammatory phenotype (M2) and T cell switching to the regulatory phenotype . Interestingly, 3D culture results in the upregulation of various pro-inflammatory cytokines, including TNF-α, IL-1α, IL-1β, and IL-8, which creates an early inflammatory microenvironment within the 3D spheroids that activates MSC in spheroids to produce immunoregulatory factors like TNF-α-stimulated gene/protein 6 (TSG6), stanniocalcin-1 (STC-1) , and PGE2  and leads to the attenuation of inflammation or repairment of damaged tissues [64, 65]. In the present study, we observed higher COX2 and PGE2 expressions in 3D-ADSC1000 compared to 2D-ADSC, which resulted in more IL-10 secretion but less TNF-α secretion from macrophages co-cultured with 3D-ADSC1000 compared to the macrophages co-cultured with 2D-ADSC. Furthermore, an increase in M2 and a decrease in M1 macrophages in the liver suggested the better immunomodulatory role of 3D-ADSC spheroids. In our previous study, MSC were stimulated to secrete PGE2 by pretreating with muramyl dipeptide, which upregulates COX2 expression. The results reported an enhanced immunomodulatory effect in experimental colitis . Similarly, IL-1β-primed MSC were reported to be more efficient at suppressing inflammation due to the upregulation of COX2 and PGE2 [67, 68]. Therefore, increased COX2 expression was correlated with increased anti-inflammatory effects. In the current study, 3D-ADSC effectively ameliorated ALF compared to 2D-ADSC, at least, in part, due to increased resistance to oxidative stress via the upregulation of various intracellular antioxidant mechanisms and enhanced polarization of macrophages to anti-inflammatory macrophage. Further studies should reveal the effectiveness of MSC spheroids in other inflammatory diseases.
Intraportal delivery of 3D-ADSC led to the MSC engraftment into the liver and enhanced therapeutic outcomes in a mouse model of FHF. These promising results were observed due to an upregulation of antioxidant defense mechanism during the formation of 3D spheroids from monolayer-cultured ADSC. This resulted in higher cell viability after intraportal transplantation. Furthermore, the enhanced immunomodulatory activity of 3D-ADSC led to the effective polarization of the macrophage from pro- to anti-inflammatory phenotype resulting in the attenuation of inflammation in GalN/LPS-induced hepatic toxicity. Recently, Chondrosphere® has been approved in Europe for the treatment of cartilage defects in human. Several studies including ours have revealed the superiority of 3D MSC spheroids compared to the monolayer MSC. 3D MSC spheroids have a great potential to improve the therapeutic effectiveness of MSC-based therapy. However, further studies on optimization of technique for large-scale manufacturing of clinical-grade 3D spheroids and mechanism behind the improvement of MSC effect are required for clinical translation.
SR, SP, HSK, and JJH participated in the research design. SR and SP participated in the performance of the experiment. SR, SP, TPT, and TTN participated in the data analysis. SR participated in writing the manuscript. JHS, SY, JOK, CSY, IC, KOD, PHP, JBP, BKK, DML, IJM, and YS provided technical advice during the experiment and manuscript preparation. All authors read and approved the final manuscript.
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (grant no. 2015R1A5A2009124 and 2018R1A5A2023879) and funded by the Ministry of Education (grant no. 2017R1D1A1B03027831); by the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI) and the Korean Ministry of Health and Welfare (grant no. HI18C0453); and by the Creative Economy Leading Technology Development Program through the Gyeongsangbuk-Do and Gyeongbuk Science and Technology Promotion Center of Korea (SF316001A).
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The authors declare that they have no competing interests.
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