ORIGINAL ARTICLE

Quantitative evaluation of human bone mesenchymalstem cells rescuing fulminant hepatic failure in pigsDongyan Shi,1 Jianing Zhang,2 Qian Zhou,1 Jiaojiao Xin,1 Jing Jiang,1 Longyan Jiang,1

Tianzhou Wu,1 Jiang Li,1 Wenchao Ding,1 Jun Li,3 Suwan Sun,1 Jianzhou Li,1

Ning Zhou,1 Liyuan Zhang,1 Linfeng Jin,1 Shaorui Hao,1 Pengcheng Chen,2

Hongcui Cao,1 Mingding Li,1 Lanjuan Li,1 Xin Chen,2,4 Jun Li1

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/gutjnl-2015-311146).

For numbered affiliations seeend of article.

Correspondence toProfessor Jun Li, State KeyLaboratory for Diagnosis andTreatment of InfectiousDiseases, CollaborativeInnovation Center for Diagnosisand Treatment of InfectiousDiseases, The First AffiliatedHospital, Zhejiang UniversitySchool of Medicine, 79Qingchun Rd, Hangzhou31000, China; lijun2009@zju.edu.cnProfessor Xin Chen, Institute ofBiochemistry, College ofPharmaceutical Sciences,Zhejiang University, 866Yuhangtang Rd, Hangzhou,310058. China; xinchen@zju.edu.cnProfessor Lanjuan Li, State KeyLaboratory for Diagnosis andTreatment of InfectiousDiseases, CollaborativeInnovation Center for Diagnosisand Treatment of InfectiousDiseases, The First AffiliatedHospital, Zhejiang UniversitySchool of Medicine, 79Qingchun Rd, Hangzhou,310003, China; ljli@zju.edu.cn

Received 22 November 2015Revised 13 January 2016Accepted 25 January 2016

To cite: Shi D, Zhang J,Zhou Q, et al. Gut PublishedOnline First: [please includeDay Month Year]doi:10.1136/gutjnl-2015-311146

ABSTRACTObjective Stem cell transplantation provides apromising alternative for the treatment of fulminanthepatic failure (FHF). However, it lacks fundamentalunderstanding of stem cells’ activities. Our objective wasto clarify stem cell-recipient interactions for overcomingbarriers to clinical application.Design We used an in-house large-animal (pig) modelof FHF rescue by human bone marrow mesenchymalstem cells (hBMSCs) and profiled the cells’ activities. Thecontrol and transplantation groups of pigs (n=15 pergroup) both received a D-galactosamine (D-Gal) injection(1.5 g/kg). The transplantation group received hBMSCsvia intraportal vein infusion (3×106 cells/kg) immediatelyafter D-Gal administration. The stem cell-recipientinteractions were quantitatively evaluated by biochemicalfunction, cytokine array, metabolite profiling,transcriptome sequencing and immunohistochemistry.Results All pigs in the control group died within anaverage of 3.22 days, whereas 13/15 pigs in thetransplantation group lived >14 days. The cytokine arrayand metabolite profiling analyses revealed that hBMSCtransplantation suppressed D-Gal-induced life-threatening cytokine storms and stabilised FHF within7 days, while human-derived hepatocytes constitutedonly ∼4.5% of the pig hepatocytes. The functionalsynergy analysis of the observed profile changesindicated that the implanted hBMSCs altered the pigs’cytokine responses to damage through paracrine effects.Delta-like ligand 4 was validated to assist liverrestoration in both pig and rat FHF models.Conclusions Our results delineated an integratedmodel of the multifaceted interactions between stemcells and recipients, which may open a new avenue tothe discovery of single molecule-based therapeutics thatsimulate stem cell actions.

INTRODUCTIONFulminant hepatic failure (FHF) is a lethal condi-tion characterised by widespread hepatocyte necro-sis, acute deterioration of liver function andsubsequent multiorgan failure.1 2 Orthotopic livertransplantation is currently viewed as one of thebest effective treatment options for FHF, but donororgan shortages, contraindications and irreversiblehepatic encephalopathy result in the death of manypatients awaiting liver transplantation.3 4

Therefore, alternative therapeutic strategies forFHF are greatly needed.5 6

Stem cell transplantation has shown substantialpotential in treating many lethal diseases.7–9

Small-animal and preliminary human studies haveindicated overall improvement in the regenerationof damaged organs.6 10 11 However, the exactmechanisms through which stem cells assist organrepair are still elusive, and the preliminary resultsof several nonrandomised clinical trials havedemonstrated a level of efficacy insufficient for

Significance of this study

What is already known on this subject?▸ Fulminant hepatic failure (FHF) is a

life-threatening condition for which livertransplantation is an effective treatment option.

▸ Stem cell-based therapy represents a promisingalternative for patients with FHF and hassignificant implications in clinics.

▸ The precise mechanism of stem cell-recipientinteractions after stem cells transplantationremains unclear.

What are the new findings?▸ Human bone marrow mesenchymal stem cell

(hBMSC) transplantation rescued FHF pigsthrough suppressing life-threatening cytokinestorms and stabilised FHF within 7 days.

▸ The proliferation and transdifferentiation of theimplanted hBMSCs replaced only ∼4.5% of thehepatocytes in recipient pig liver.

▸ The implanted hBMSCs altered the recipients’cytokine responses to damage throughparacrine effects.

▸ An important cytokine/biological process,Delta-like ligand 4/Notch, may participate inrestoring the damaged liver in FHF.

How might it impact on clinical practice inthe foreseeable future?▸ Mechanistic understanding of stem

cell-recipient interactions might be helpful inovercoming barriers to clinical translation anddeveloping novel single molecule-basedtherapeutics that simulate stem cell actions.

Shi D, et al. Gut 2016;0:1–10. doi:10.1136/gutjnl-2015-311146 1

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these cells’ application as a first-line therapy.11 Therefore, funda-mental understanding of stem cell-recipient interactions isrequired for the successful development and optimisation ofstem cell-based therapies.12

Stem cell application in FHF treatment is attractive, but manyquestions remain unanswered.13 As we and others havereported, liver repair through stem cell proliferation and trans-differentiation into hepatocytes has been postulated as the majormechanism of this therapy’s action.5 14 Recent studies also indi-cated a substantial role for paracrine effects in delivering overallbenefits, although no specific signalling molecule has yet beenidentified to mediate these paracrine effects.15 16 The relativeimportance of these two mechanisms is also a topic of contro-versy.12 Furthermore, we still do not know the timeframe inwhich stem cells act to stop disease progression. Answers tothese key questions will be the foundation of designing furthertranslational studies on the development and optimisation ofstem cell-based FHF therapy.

To overcome these barriers to clinical translation, in thepresent study, we used our own in-house translational model,namely human bone marrow mesenchymal stem cell(hBMSC)-mediated rescue of D-galactosamine (D-Gal)-inducedFHF in pigs, to delineate stem cells’ activities after transplant-ation. The stem cell-recipient interactions were quantitativelyevaluated by biochemical function, cytokine array, metaboliteprofile, messenger RNA sequencing (mRNA-seq) and immuno-histochemistry (IHC) (figure 1).

MATERIALS AND METHODSIsolation, culture and identification of hBMSCshBMSCs were isolated by bone marrow aspiration from the iliaccrest of healthy male volunteers. More details on the methodsof hBMSC isolation, culture, phenotypic identification and mul-tilineage differentiation are available in online supplementarymaterials and methods. Cells were used for experiments duringpassages 3–7.

FHF modelsAll experimental protocols were approved by the Animal CareEthics Committee of the First Affiliated Hospital, Zhejiang

University, and all animals received humane care according tothe criteria of the ‘Guide for the Care and Use of LaboratoryAnimals’.17 The experimental methods were carried out inaccordance with the approved guidelines. Briefly, male Chineseexperimental miniature pigs (Taihe Biotechnology, Jiangsu,China) weighing 8–10 kg and aged 2.5 months underwent FHFinduction with D-Gal at a dose of 1.5 g/kg body weight viajugular vein catheterisation. Additionally, male Sprague–Dawleyrats (Zhejiang Academy of Medical Sciences, Hangzhou, China)weighing 200–250 g and aged 2 months underwent FHF induc-tion with D-Gal at a dose of 3.0 g/kg body weight via intraperi-toneal injection.14

hBMSC transplantation in pigs with FHFTwo groups (n=15/group, randomised assignment) of pigs withFHF were studied: an intraportal hBMSC transplantation (T)group, in which the animals received a transfusion with 3×106

hBMSCs/kg suspended in 10 mL of normal saline (NS) via theintrahepatic portal vein under B-ultrasound guidance immedi-ately after D-Gal injection, and a control (C) group, in whichthe animals underwent a sham procedure with an equal volumeof NS without cells. No animal received any medical support(eg, infusions, drugs) during the entire experimental period.

Delta-like ligand 4 treatment in both pigs and rats with FHFTwo groups (n=15/group, randomised assignment) of pigs withFHF were studied: a Delta-like ligand 4 (DLL4) treatmentgroup, in which the animals received multiple injections ofDLL4 at a dose of 0.03 mg/kg (2 mL) through the jugular veinat 12, 36, 60 and 84 h after D-Gal induction, and a control(CTL) group, in which the animals underwent a sham procedurewith an equal volume of NS without DLL4. The beneficial roleof DLL4 was also validated in the male SD rat model (200–250 g) with FHF. Two groups (n=50/group, randomised assign-ment) of rats with FHF were also studied: a DLL4 group, inwhich the animals received multiple injections of DLL4 at adose of 0.2 mg/kg (1 mL) through the intraperitoneal route at 8,14, 20, 26, 32, 38 and 44 h after D-Gal induction, and a CTLgroup, in which the animals underwent a sham procedure withan equal volume of NS without DLL4. No animal received any

Figure 1 Scheme of human bonemarrow mesenchymal stem cell(hBMSC) transplantation for rescuingfulminant hepatic failure (FHF) in pigs.To understand the fundamentalinteractions between implanted stemcells and recipient hosts with FHF, wequantitatively profiled stem cells’activities after transplantation using anautomated biochemistry analyser,cytokine arrays, ultra-performanceliquid chromatography tandem massspectrometry (UPLC-MS), messengerRNA sequencing (mRNA-seq) andimmunohistochemistry (IHC).

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medical support (eg, infusions, drugs) during the entire experi-mental period.

Survival curve analysesAnimal survival was monitored for up to 2 weeks after hBMSCtransplantation and DLL4 treatment in pigs and rats with FHF.Survival curves were compared using the non-parametricMantel–Cox test. Animal survival was observed by a blindedexpert with 10 years of experience in animal studies.

Materials, reagents, antibodies and PCR primersThe materials, reagents and antibodies, and quantitative reversetranscription PCR (qRT-PCR) and primers for RT-PCR used inthe study are provided in online supplementary tables S1–S3,respectively.

Sample collectionSerum, peripheral blood mononuclear cells (PBMCs) and livertissues were collected from the experimental animals accordingto the experimental design for a period of 14 days after FHFinduction. The serum samples were used in biochemical tests,ultra-performance liquid chromatography tandem mass spec-trometry (UPLC-MS) analysis, cytokine array detection andELISA. The PBMCs were profiled via mRNA-seq. Finally, theliver tissues were profiled/analysed using UPLC-MS, H&E stain-ing, IHC, mRNA-seq and qRT-PCR. Sequencing reads from ourmRNA-seq experiments are available in the Sequence ReadArchive database (accession number: SRP063325). Detailedmethods are available in online supplementary materials andmethods.

Statistical analysisThe results of the measurements are presented as the mean±SEM, unless otherwise noted. No statistical method was usedto predetermine sample sizes. Comparisons between two groupswith a sample size >3, except the comparisons of the most sig-nificant pig-derived cytokine expression, were performed usingthe non-parametric Mann–Whitney U test, assuming two-taileddistributions. Comparisons between two groups with a samplesize of 3 and the comparisons of the most significant pig-derivedcytokine expression (n=5 per group) were performed with theheteroscedastic Welch’s t test to ensure sensitivity, assuming two-tailed distributions. The normality assumption of Welch’s t testwas validated using the Shapiro–Wilk test. The sample data ineach compared group showed p>0.05. Comparison of metabol-ite profiles, represented by the first two principal components,was performed using the nonparametric MANOVA permutationtest (the R ‘vegan’ Package). False discovery rate (FDR) calcu-lated with the Benjamini–Hochberg procedure was used tocontrol type I error in multiple tests. The criteria ‘p<0.05,FDR<0.25’ was used in detecting biochemical marker changes,‘p<0.05, FDR<0.1’ in detecting serum cytokine changes and‘p<0.05, FDR<0.05’ in detecting transcript changes.Hierarchical clustering was performed using the R ‘stats’Package, involving the Euclidean distance matrix and thePearson product-moment correlation coefficient.

RESULTSHuman BMSC transplantation stabilised FHF in pigs within7 daysPhenotypic analysis by standard flow cytometry showed thatseventh-passage hBMSCs were positive for CD29 and CD90but negative for CD34 and CD45. The results of multipotentialdifferentiation showed that the hBMSCs could differentiate into

osteocytes, adipocytes and hepatocyte-like cells. These findingsindicated that the cells used in this study possessed typicalhBMSC phenotypes and multipotential stem cell characteristics(see online supplementary figure S1). All pigs in the controlgroup died within an average of 3.22 days, whereas 13/15 pigsin the transplantation group lived beyond day 7 (figure 2A), andall of these 13 pigs survived for up to 6 months. The other twopigs died within 4 days, and subsequent autopsy indicated thatFHF pathology resulted in rapid death.

Biochemical assay analysis showed that 21 biomarkers in thecontrol group and 22 biomarkers in the transplantation groupexhibited significant changes at day 3 relative to day 0(figure 2B). Among these biomarkers, 20 were shared betweenthe two groups. Transplantation attenuated the 18 of the 20 bio-marker changes (see online supplementary figure S2). Cytokinearray analysis indicated that hBMSC transplantation altered liverresponses to damage, conferring a protective effect at day 3(detailed later in a dedicated section). UPLC-MS profiling ofthe serum metabolites indicated a significant difference inhepatic metabolic functions between the two groups, whichwas observed as early as at day 1 (p=0.012, figure 2C).These results indicated that implanted hBMSCs produced imme-diate protective effects within the first three days aftertransplantation.

At day 7, most (24/35) of the biochemical markers wererestored to their baseline levels in the transplantation group (seefigure 2D for six representative markers), except two signifi-cantly downregulated biomarkers and nine significantly upregu-lated ones (figure 2B). Similarly, in the transplantation group,no monitored cytokine exhibited a significant differencebetween days 0 and 7 (figure 2E). Consistent with these results,UPLC-MS analysis (figure 2C) of 2768 distinguishable com-pounds in the serum revealed no significant differences betweenthe metabolite profiles at days 0 and 7 in the transplantationgroup (p=0.813). In contrast, these compounds shifted to righton day 3 in the control group (p=0.001). H&E staining(figure 2F) further confirmed that transplantation was notablycoupled with repair of the damaged liver structure at day 7,whereas the deceased control animals showed a typical FHFhistology with extensive hepatic necrosis and haemorrhage.These results indicated that hBMSC transplantation stabilisedFHF within the first seven days after D-Gal administration.

Furthermore, as shown in the online supplementary figure S2,the levels of low, very low and high density lipoprotein, andtotal cholesterol were significantly decreased in both groupsafter D-Gal administration and they restored to baseline on day5–7 after hBMSC transplantation. These findings also indicatedthat the implanted hBMSCs restored hepatic synthetic functionsand stabilised FHF within the first seven days.

Human-derived hepatocytes replaced approximately 4.5%of pig hepatocytesTo understand the interactions between stem cells and recipientpig hosts, we quantified the relative contribution of cell replace-ment to the course of hBMSC-assisted liver restoration. IHCusing human-specific antibodies showed that both undifferenti-ated hBMSCs (CD90+ and CD29+) and human-derived hepato-cytes (albumin (ALB)+ and hepatocyte-specific antibody(HSA)+) were found in the pigs’ liver tissue at day 3, but onlyhuman-derived hepatocytes were found after day 7 (figure 3A,and see positive and negative controls in online supplementaryfigure S3). These data suggested that transdifferentiation of theimplanted hBMSCs was close to completion at day 7.

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The percentage of human-derived hepatocytes in the livertissue was estimated by counting HSA+ cells (figure 3B).Human-derived hepatocytes constituted approximately 4.5%and 4.7% of the liver hepatocytes at days 7 and 14, respectively(see online supplementary figures S4–S7). Consistent with theseresults, mRNA-seq analysis showed that 1.3–1.8% of the totaltranscripts in the liver were human derived (figure 3C, D).UPLC-MS analysis of peptides in the liver tissue and of secretedpeptides in the serum indicated that 3.5–5.1% and 1.2–1.7%,respectively, were human derived (figure 3E). ALB secretion is acharacteristic function of hepatocytes, and UPLC-MS analysis ofthree pairs of peptides that distinguish between human-originated and pig-originated ALB indicated that only 0.4% of

the ALB in the liver tissue was human derived (figure 3F, G).Therefore, proliferation and transdifferentiation of theimplanted hBMSCs replaced ∼4.5% of liver hepatocytes whenFHF was stabilised. However, the percentage of liver functioncompensated for by transplantation-derived cells could belower, as evidenced by the lower levels of human-derived geneexpression and ALB production in the liver tissue.

hBMSCs altered liver responses to damage throughparacrine effectsTo explore the paracrine interactions between hBMSCs andrecipient pig hosts, we measured the changes in the serum levelsof 215 cytokines using cytokine arrays (see online

Figure 2 Human bone marrow mesenchymal stem cell (hBMSC) transplantation stabilises fulminant hepatic failure (FHF) by day 7 (D7).(A) Survival analysis. (B) Differentially regulated biochemical markers. (C) Trajectory of metabolite profiles plotted with their first two principalcomponents (PC1, PC2). (D) Temporal changes in six typical biochemical markers of liver function. (E) Differentially regulated cytokines. (F) H&Estaining of liver tissue. hBMSC transplantation led to notable improvements in the liver tissue structure at D7. C, control group; T, transplantationgroup. D3:D0, day 3 vs day 0; D0-D1, day 0 to day 1. Error bars, SEM. Mann–Whitney U test, **p<0.01, *p<0.05. Scale bar, 100 μm. (A) n=15/group; (B, D) n=5–15/group; (C) n=10–15/group; (E) n=3/group; (F) n=5/group.

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supplementary table S4), and randomly selected six of signifi-cantly changed cytokines for validation using ELISA (see onlinesupplementary figure S8). In the control group at day 3 relativeto day 0 and in the transplantation group at days 3, 7 and 14relative to day 0, cytokine changes were clustered (figure 4A).The D-Gal-induced life-threatening cytokine storms, observedin both groups, were suppressed in the transplantation group atday 7. In the transplantation group, in particular, 46 cytokinesshowed significant changes at one or more time points (seeonline supplementary table S5). Hierarchical clustering analysisrevealed four co-regulated cytokines clusters (see onlinesupplementary figure S9). Most (36/46) of the cytokines exhib-ited a change pattern with a peak at day 3 that returned to anear-baseline level at day 7 and remained stable at day 14 (seeonline supplementary figure S9A, cluster 1). This patternaligned with the progression of FHF and its restoration afterhBMSCs transplantation. Other (10/46) cytokines showed alter-native temporal change patterns (see online supplementaryfigure S9A, clusters 2–4). Functionally, these cytokines wererelated to inflammation, regeneration, apoptosis, immunoregula-tion or metabolism (see online supplementary figure S9B).

In the cytokine storms at day 3, 57 cytokines showed signifi-cant changes in either one or both groups (figure 4B).Surprisingly, specific changes observed in only one group (49changes) were more frequent than common changes observed inboth groups (8 changes), suggesting a disparity in the liverresponses to damage between the two groups. From a functionalperspective, most (48/57) of the altered cytokines had functionsrelated to signal transduction, metabolism, apoptosis/cell death,immunoregulation or development (see online supplementarytable S5). Cytokines from many pleiotropic signal transductionpathways and many metabolic processes were differentially

induced between the two groups, indicating a fundamental dif-ference in the regulation of cell behaviours (see onlinesupplementary figure S9C,D). Interestingly, the altered cytokinesin the control group were more frequently involved in the regu-lation of apoptosis and cell death (5/27 changes in the controlgroup vs 1/38 changes in the transplantation group). By con-trast, the altered cytokines in the transplantation group weremore enriched with functions in immunoregulation (1/27changes in the control group vs 12/38 changes in the transplant-ation group) and developmental control (2/27 changes in thecontrol group vs 5/38 changes in the transplantation group)(figure 4C, and annotation enrichment analysis results in onlinesupplementary table S6). Comparison of liver gene expressionbetween pigs with transplantation (at day 7) and pigs without(at day 3) confirmed this trend of response divergence (seeonline supplementary table S7).

The qRT-PCR using human-specific mRNA further confirmedthat the pigs’ cells produced the three most significant changesin the serum cytokine levels that were specifically observed inthe transplantation group (changes in EpCAM, VEGF-R2 andendoglin levels; figure 4D). These results indicated that theimplanted hBMSCs remodelled the pigs’ liver responses todamage by releasing small amounts of regulatory cytokines.

Profiling analysis indicated DLL4-Notch activationTo identify the important signalling processes through whichthe implanted hBMSCs exerted their paracrine effects, we ana-lysed the gene expression changes in PBMCs. PBMCs respond-ing to soluble factors without the complication of the tissuemicroenvironment may exhibit signalling changes that are moredirectly related to the paracrine effects of implanted hBMSCs.We also identified the differentially expressed genes in both the

Figure 3 Proliferation and transdifferentiation of transplanted human bone marrow mesenchymal stem cells. (A) immunohistochemistry of livertissue from pigs in the T group. (B) Percentage (4.5%) of human-derived hepatocytes in the livers of T-group pigs at day 7 (D7). (C) Separation ofhuman-originated and pig-originated mRNA sequencing reads. (D) Percentage of human-derived gene expression in liver tissues from two pigs (T1,T2). (E) Percentage of human-derived peptides from liver tissue and serum. (F) Typical MS/MS spectra of the human-derived albumin (ALB) peptides.(G) Percentage of human-derived ALB from liver tissue. P1, P2, P3: three pairs of peptides that distinguish between human- and pig-derived ALB. C,control group; T, transplantation group. Scale bar, 100 μm. Error bars in (D) and (E), SEM. (A, B) n=5; (D) n=2; (E) n=3, 5 at D7 and D14 for serum;n=3 both at D7 and D14 for liver tissue; (G) six samples harvested from three pigs were pooled together for analysis.

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control and transplantation groups, and differentiallyco-expressed genes between the two groups (see onlinesupplementary table S8). Differential co-expression is defined asa cluster of genes that show highly correlated expression in onegroup (evidence of physiologically relevant activation of a bio-logical process), but not in another group. Functional synergyanalysis of expression changes showed 23 altered signalling pro-cesses (table 1) including several well-known signalling processes(TLR,18 tumour necrosis factor-alpha, interleukin (IL)-1 andIL-619) that respond to liver damage. Two cytokines regulatingthese signalling processes (stem cell factor and IL-6) have beenreported to exert prophylactic effects against FHF.20 21 Therediscovery of these signalling processes suggested the potentialto exploit other identified processes in FHF treatment.

Of the identified 23 signalling processes, the Notch signallingpathway is of particularly interest. The differentially expressedgenes in the control and the transplantation groups and the dif-ferentially co-expressed genes between the two groups all havefrequent functional interactions with the Notch signallingpathway (figure 5A). Notch receptors have five knownligands;22 we compared the expression of these ligands in theliver tissue (the transplantation group at day 7 vs the controlgroup at day 3) and found that in the transplantation group, theexpression of DLL1 was suppressed (p=0.026) and that theexpression of DLL4 was induced (p=0.004, figure 5B).Validation using qRT-PCR and ELISA confirmed that DLL4 wasspecifically induced in the transplantation group at both themRNA and the protein levels (p<0.05, figure 5C,D).

Figure 4 Paracrine effects of transplanted human bone marrow mesenchymal stem cells. (A) Heat map of 215 protein-level changes relative to day0 (D0). (B) Venn diagram of the differentially regulated cytokines from D0 to D3 in the control (C) and transplantation (T) groups (p<0.05). (C) Heatmaps of differentially regulated cytokines involved in the most significantly altered functional themes: immunoregulation (top), development (leftbottom) and apoptosis (right bottom). See online supplementary table S4 for full cytokine names. (D) Quantitative reverse transcription PCR(qRT-PCR) measurement of human- and pig-derived EPCAM (EpCAM, Epithelial cell adhesion molecule), KDR (vascular endothelial growth factorreceptor 2 (VEGF-R2)) and ENG (endoglin) in the liver tissues. Only pig-derived mRNAs were detected at D3 in T group. Glyceraldehyde phosphatedehydrogenase (GAPDH) was used as an internal reference. The results are representative of five independent experiments. H, human; P, pig; HN,human normal liver tissue; PN, pig normal liver tissue; T-D3, liver tissue harvested from the T group at D3. Error bars, SEM. Student’s t test,*p<0.05. D3:D0, D7:D0, D14:D0, day 3 or day 7 or day 14 vs day 0. (A–C) n=3/group; (D) n=5/group.

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Survival benefit of DLL4 in pig and rat FHF modelsDLL4 treatment notably improved the survival of both pigs andrats with FHF (p<0.05, figure 5E). In the validation using thepig model, the pigs in the control group had a mean survivaltime of 2.6 days, whereas the pigs in the DLL4 treatment groupsurvived for 4.6 days on average (two pigs survived for up to6 months). Similarly, in the validation using the rat model, therats survived for 5.3 days on average in the control group andfor 7.1 days in the DLL4 treatment group. No animal receivedadditional medical support (eg, nutrient infusions, drugs) duringthe entire experiment. H&E staining indicated that the liverstructure was notably repaired after DLL4 treatment (figure 5F).These results demonstrated that DLL4 treatment alone improvesanimal survival.

DISCUSSIONPatients with FHF with life-threatening cytokine storms anddeficiencies in liver detoxification and metabolic and immune

regulation may rapidly die within several weeks.2 4 Stem celltransplantation holds substantial promise for the clinical treat-ment of FHF.6 To overcome the barriers to clinical application,many questions (such as questions about the optimal timing,transdifferentiation and paracrine effects of stem cell therapies)need to be answered.13 23 In the present study, we quantitativelydelineated stem cell-recipient interactions via multifaceted pro-filing and demonstrated that implanted stem cells rescueD-Gal-induced FHF by suppressing cytokine storms and alterthe recipient’s (here, the pig’s) response to liver damage.Importantly, we discovered that DLL4, as a potential therapeuticmolecule, may participate in the rescue of FHF via hBMSCtransplantation (figure 6).

Understanding the action time of implanted stem cells isuseful guidance to ensure optimal timing of treatment in futureclinical applications.24 Certain evidence in cardiomyocyte trans-plantation research has suggested the existence of a temporalwindow of opportunity bound by the acute inflammatory

Table 1 Twenty-three generalised biological process annotations analysed by gene set linkage analysis in peripheral blood mononuclear cells

Generalised biological processType ofgene set* Up/down GO/PID term p Value† Overlapping gene(s)‡

TLR signalling and TLR-like receptor signalling C-DE, D3:D0 Up PID:200209 ≤1e-5 S100A8, S100A9, TLR4T-DcoE, D0-7:D0-3 N/A GO:0034138, GO:0002224, GO:0034134, GO:0002756,

GO:0034130, GO:0034142,GO:0002755, GO:0008063

≤1e-5 MAP2K1,ATF1, JUN

C-DcoE, D0-3:D0-7 N/A GO:0034130, GO:0008063 ≤1e-5 MAP3K1TGF-beta receptor signalling C-DE, D3:D0 Down PID:200228 ≤1e-5 TGFBR2Regulation of SMAD protein and SMAD2/3 signalling T-DcoE, D0-7:D0-3 N/A GO:0010862 ≤1e-5 CEBPB, VDR, CNNM371, JUNALK1 signalling C-DE, D3:D0 Down PID:200149 ≤1e-5 TGFBR2IFN-gamma mediated signalling and IFN-gammapathway

C-DE, D3:D0 Down GO:0060333, GO:0032729 ≤1e-5 –

C-DcoE, D0-3:D0-7 N/A PID:200130 ≤1e-5 STAT1Notch receptor and Notch signalling C-DE, D3:D0 Up GO:0007219 ≤1e-5 NRG1

T-DE, D7:D0 Down GO:0007220 ≤1e-5 –

T-DcoE, D0-7:D0-3 N/A PID:200015 ≤1e-5 NOTCH3Regulation of IL-6 biosynthetic process C-DE, D3:D0 Up GO:0045410 ≤1e-5 –

Response to IL-1 C-DE, D3:D0 Up GO:0071347 ≤1e-5 LCN2Regulation of IL-2 biosynthetic process andIL-2 signalling

C-DE, D3:D0 Down PID:200185, GO:0045086 ≤1e-5 LCK

IL-4-mediated signalling events C-DE, D3:D0 Down PID:200022 ≤1e-5 –

IL-5-mediated signalling events C-DE, D3:D0 Down PID:200107 ≤1e-5 –

IL-12 signalling and IL-12-mediated signalling events C-DE, D3:D0 Down PID:200045, PID:200230 ≤1e-5 GZMA, LCKIL-23-mediated signalling events C-DE, D3:D0 Down PID:200155 ≤1e-5 –

CXCR4-mediated signalling events C-DE, D3:D0 Down PID:200099 ≤1e-5 LCKSHP2 signalling C-DE, D3:D0 Down PID:200083 ≤1e-5 LCKc-Kit-mediated signalling events T-DcoE, D0-7:D0-3 N/A PID:200182 ≤1e-5 SH2B3

C-DE, D3:D0 Down PID:200182 ≤1e-5 –

TNF receptor signalling T-DE, D7:D0 Down PID:200102 ≤1e-5 TNFAIP3Cellular response to TNF T-DE, D7:D0 Up GO:0071356 ≤1e-5 LCN2HIV-1 Nef: negative effector of Fas and TNF-alpha C-DcoE, D0-3:D0-7 N/A PID:200156 ≤1e-5 –

C-DE, D3:D0 Down PID:200156 ≤1e-5 FAS, CASP2Alpha-synuclein signalling C-DE, D3:D0 Down PID:200220 ≤1e-5 –

Syndecan-1-mediated signalling events C-DE, D3:D0 Down PID:200157 ≤1e-5 –

Plexin-D1 signalling C-DE, D3:D0 Up PID:200122 ≤1e-5 ITGB1, NRP1, ITGA1, ITGAVErbB signalling T-DcoE, D0-7:D0-3 N/A PID:200134 ≤1e-5 ATF1, JUN, BAD, MAP2K1,

RALGDSC-DE, D3:D0 Down PID:200205, PID:200011 ≤1e-5 LCK

*Type of gene set. C-DE, D3:D0, genes differentially expressed between days 0 and 3 in the control (C) group; T-DE, D7:D0, genes differentially expressed between days 0 and 7 in thetransplantation (T) group; T-DcoE, D0-7:D0-3, differentially co-expressed gene clusters, with co-expression in the transplantation (T) group (days 0 and 7) but not in the control (C)group (days 0 and 3); C-DcoE, D0-3:D0-7, differentially co-expressed gene clusters, with co-expression in the C group (days 0 and 3) but not in the T group (days 0 and 7). N/A, notapplicable.†The p value reflects the reliability/robustness/biological significance of the identified functional interaction.‡Overlapping genes are those that have been annotated to the biological process.GO/PID, gene ontology/pathway interaction database; IFN, interferon; IL, interleukin; TGF-beta, transforming growth factor-beta; TLR, toll-like receptor; TNF, tumour necrosis factor.

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response, on the one hand, and by scar formation, on the other.25

However, the experimental and clinical data on the action timingafter stem cell transplantation are limited. In the current study,our in-house large-animal model of FHF rescue demonstratedthat immediate transplantation of hBMSCs after D-Gal ad-ministration prevented death from FHF in the initial three days.Serum metabolite profiles and biochemical changes indicated animmediate protective effect of the implanted hBMSCs by sup-pressing D-Gal-induced life-threatening cytokine storms. Thistimeframe provides evidence supporting direct investigation ofthe timing of stem cell-based therapy in a clinical setting.

How stem cells act, and specifically whether cell replacementmechanisms or the cells’ paracrine effects contribute most tostem cell-assisted organ repair, remains a topic of controversy.12

Liver repair through stem cell proliferation and transdifferentia-tion has been postulated as the major mechanism of stem cellaction in treating FHF.5 14 Several studies showed that only 1–12%26–28 of hepatocyte mass was replaced in the recipient liver;such estimates were typically generated by counting immunohis-tochemically stained cells or by flow cytometry. To obtain amore reliable estimate, here, we used IHC, mRNA-seq andUPLC-MS to quantitatively evaluate the number of human-derived hepatocytes at two time points. Our results show that atday 7, when FHF was stabilised and differentiation completed,implanted hBMSCs replaced ∼4.5% of the pig hepatocytes.However, the liver function compensated for by these cellscould be even lower (0.4%–1.8%), as indicated by the lowerlevels of human-derived gene expression and ALB production.The fraction of liver cells replaced by differentiated hBMSCs

seems too low to explain the significantly improved animal sur-vival, which suggests the importance of paracrine effects inhBMSC-assisted liver restoration.

The pivotal role of paracrine effects in stem cell therapies hasbeen recognised to contribute to many biological processes, suchas by preventing inflammation, inhibiting apoptosis, improvingmetabolism and promoting regeneration.15 12 Certain positiveresults based on paracrine mechanisms have been reported inischaemic stroke, myocardial infarction and kidney injury. 7 12 29

In the present study, the importance of paracrine effects wasfurther supported by the observation of immediate protectiveeffects within the first three days after transplantation, whenhBMSC differentiation was far from complete. Furthermore, theparacrine effects of the implanted hBMSCs altered the recipientresponse to damage, from producing more apoptosis/cell death-related cytokines to producing more cytokines related to immu-noregulation and developmental control, but the most alteredcytokines were not produced by transplantation-derived cells.These results indicated a high-level regulatory role for the stemcells’ paracrine effects and suggested potential for the discoveryof stem cell-inspired molecular therapies via understanding thecomponents of stem cells’ secretome and their functions.However, many metabolic processes specifically induced by theimplanted hBMSC need to be further clarified.

Although recent studies indicated that systemic infusion ofBMSC-conditioned medium improved survival in asmall-animal (rat) model of FHF,16 30 certain components maybe potentially harmful.31 32 Searching for key therapeuticmolecules that may be useful for developing a balanced

Figure 5 Delta-like ligand 4 (DLL4)-Notch activation in human bone marrow mesenchymal stem cell (hBMSC)-directed liver restoration. (A)Summary of evidence linking DLL4-Notch signalling to liver restoration. DE, differentially expressed genes; D3:D0, D7:D0, day 3 or day 7 vs day 0;T-DcoE, D0-7:D0-3, differentially co-expressed gene clusters, which were co-expressed in the transplantation (T) group between D0 and D7 butwhich showed no correlation in the control (C) group between D0 and D3. (B) Differences in the expression levels of Notch-related ligands in theliver tissue between the T group at day 7 (D7) after D-galactosamine administration and the C group at D3. (C) Quantitative reverse transcriptionPCR (qRT-PCR) measurement of DLL4 expression in liver tissue. *p<0.05, Mann–Whitney U test. (D) ELISA measurement of serum DLL4 protein.*p<0.05, Student’s t test. (E) Survival analysis of the DLL4-treated pigs/rats (p=0.033/0.045, Mantel–Cox test). CTL, control group. (F) H&E stainingof liver tissue. DLL4 treatment led to notable improvements in the liver tissue structure at D7. Scale bar, 50 μm. Error bars in (C), SEM. (B) n=2/group; (C) n=5/group; (D) n=3/group; (E) n=15/group (pigs), n=50/group (rats); (F) n=5/group.

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cocktail, instead of whole cells, is emerging as an excitingconcept in regenerative medicine. However, no singlemolecule-based treatment for disease had been developedbased on stem cell actions prior to the current study.15 Becausethe beneficial factors are mostly high-level regulatory factorspresent in small quantities, in profiling analysis, changes inthese factors are easily masked by changes in downstreamfactors and effector proteins that exist in large quantities. Inthis study, we used a novel procedure that cross-references thefunctional changes in biological processes against the expres-sion changes in their known regulators to discover small bene-ficial regulatory changes and discovered that differentialexpression of DLL4 in the transplantation group was beneficialfor liver restoration. We observed that a characteristic featureof the transplantation effects was the induction of cytokinesthat direct development. DLL4-Notch activation might be oneimportant step underlying the hBMSC-directed liver restor-ation programme. Recent studies have indicated that DLL4,along with other Notch ligands, contributes to biliary injuryrepair.33 Moreover, our validation results demonstrated thatDLL4 treatment at 8–12 h post-D-Gal administration signifi-cantly improved survival in both pig and rat models of FHF.These results are useful for further designing a novelsingle-molecule-based therapy by simulating stem cell actionsfor future clinical applications.

In summary, our quantitative evaluations delineated an inte-grated model of the multifaceted interactions between stem cellsand their recipients. Furthermore, we discovered that DLL4, as a

potential therapeutic molecule, might be useful for developing anovel therapy that simulates stem cell actions for the treatment ofFHF. However, the precise mechanisms of DLL4 expression andhow it works to improve liver function need to be further clarified,and for future therapy to be clinically effective, fine-tuning of theintervention with regard to timing, dosing, the route of adminis-tration and long-term follow-up also needs to be performed.

Author affiliations1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou,China2Institute of Biochemistry, College of Pharmaceutical Sciences, Zhejiang University,Hangzhou, China3Department of Pathology, The First Affiliated Hospital, Zhejiang University School ofMedicine, Hangzhou, China4Joint Institute for Genetics and Genome Medicine between Zhejiang University andUniversity of Toronto, Zhejiang University, Hangzhou, China

Acknowledgements The authors thank Dr Guoqiang Mo for giving helpfulsuggestions in animal experiments and Dr Yu Chen for providing animaladministration.

Contributors DS, JZ, QZ, JX and JJ contributed equally. The study was designed byJL and supervised by JL, XC and LL. The manuscript was written by JL, XC, ML, JZand DS. The experiment and data analysis were performed by DS, JZ, QZ, JX, JJ, LJ,TW, JL, WD, JL, SS, JL, NZ, LZ, LJ, SH, PC, HC, ML, LL, XC, and JL. All authors wereinvolved in critical revision of manuscript.

Funding This work was supported by the National Basic Research Program ofChina (2012CB944900), the Zhejiang Provincial and National Natural ScienceFoundation of China (LR13H030001, LR13C020001, 81271708, 81571818 and

Figure 6 Proposed mechanisms of human bone marrow mesenchymal stem cell (hBMSC) action in liver restoration. D-galactosamine (D-Gal)injection destroys hepatocytes, inducing the release of detrimental cytokines, which further damage the liver tissue. Implanted hBMSCs respond todamage signals and proliferate and transdifferentiate to repair the liver tissue. Through paracrine effects, the hBMSCs also induce the expression ofcytokines in the host; these cytokines are involved in immunoregulation and development, which protect liver cells and programme the liverresponses to damage. We found evidence of an important role for Delta-like ligand 4 (DLL4)-Notch activation in the hBMSC-directed liver restorationprogramme.

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31571356), the Chinese High Tech Research and Development (863) Program(2013AA020102), the National S&T Major Project (2012ZX10004503-006 and2012ZX10002004-001), and the Research Fund for the Doctoral Program of HigherEducation (20130101110008).

Competing interests None declared.

Patient consent Obtained.

Ethics approval The Clinical Research Ethics Committee of the First AffiliatedHospital, Zhejiang University.

Provenance and peer review Not commissioned; externally peer reviewed.

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hepatic failure in pigsmesenchymal stem cells rescuing fulminant Quantitative evaluation of human bone

Jun LiPengcheng Chen, Hongcui Cao, Mingding Li, Lanjuan Li, Xin Chen and Sun, Jianzhou Li, Ning Zhou, Liyuan Zhang, Linfeng Jin, Shaorui Hao,Longyan Jiang, Tianzhou Wu, Jiang Li, Wenchao Ding, Jun Li, Suwan Dongyan Shi, Jianing Zhang, Qian Zhou, Jiaojiao Xin, Jing Jiang,

published online February 16, 2016Gut 

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