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Report CopyRight/DMCA Form For : Quantitative Evaluation Of Human Bone Mesenchymal Stem
Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, these cells application as a rst line therapy 11 Therefore funda University and all animals received humane care according to. mental understanding of stem cell recipient interactions is the criteria of the Guide for the Care and Use of Laboratory. required for the successful development and optimisation of Animals 17 The experimental methods were carried out in. stem cell based therapies 12 accordance with the approved guidelines Brie y male Chinese. Stem cell application in FHF treatment is attractive but many experimental miniature pigs Taihe Biotechnology Jiangsu. questions remain unanswered 13 As we and others have China weighing 8 10 kg and aged 2 5 months underwent FHF. reported liver repair through stem cell proliferation and trans induction with D Gal at a dose of 1 5 g kg body weight via. differentiation into hepatocytes has been postulated as the major jugular vein catheterisation Additionally male Sprague Dawley. mechanism of this therapy s action 5 14 Recent studies also indi rats Zhejiang Academy of Medical Sciences Hangzhou China. cated a substantial role for paracrine effects in delivering overall weighing 200 250 g and aged 2 months underwent FHF induc. bene ts although no speci c signalling molecule has yet been tion with D Gal at a dose of 3 0 g kg body weight via intraperi. identi ed to mediate these paracrine effects 15 16 The relative toneal injection 14. importance of these two mechanisms is also a topic of contro. versy 12 Furthermore we still do not know the timeframe in. hBMSC transplantation in pigs with FHF, which stem cells act to stop disease progression Answers to. Two groups n 15 group randomised assignment of pigs with. these key questions will be the foundation of designing further. FHF were studied an intraportal hBMSC transplantation T. translational studies on the development and optimisation of. group in which the animals received a transfusion with 3 106. stem cell based FHF therapy, hBMSCs kg suspended in 10 mL of normal saline NS via the. To overcome these barriers to clinical translation in the. intrahepatic portal vein under B ultrasound guidance immedi. present study we used our own in house translational model. ately after D Gal injection and a control C group in which. namely human bone marrow mesenchymal stem cell, the animals underwent a sham procedure with an equal volume. hBMSC mediated rescue of D galactosamine D Gal induced. of NS without cells No animal received any medical support. FHF in pigs to delineate stem cells activities after transplant. eg infusions drugs during the entire experimental period. ation The stem cell recipient interactions were quantitatively. evaluated by biochemical function cytokine array metabolite. pro le messenger RNA sequencing mRNA seq and immuno Delta like ligand 4 treatment in both pigs and rats with FHF. histochemistry IHC gure 1 Two groups n 15 group randomised assignment of pigs with. FHF were studied a Delta like ligand 4 DLL4 treatment. group in which the animals received multiple injections of. MATERIALS AND METHODS, DLL4 at a dose of 0 03 mg kg 2 mL through the jugular vein. Isolation culture and identi cation of hBMSCs, at 12 36 60 and 84 h after D Gal induction and a control. hBMSCs were isolated by bone marrow aspiration from the iliac. CTL group in which the animals underwent a sham procedure. crest of healthy male volunteers More details on the methods. with an equal volume of NS without DLL4 The bene cial role. of hBMSC isolation culture phenotypic identi cation and mul. of DLL4 was also validated in the male SD rat model 200. tilineage differentiation are available in online supplementary. 250 g with FHF Two groups n 50 group randomised assign. materials and methods Cells were used for experiments during. ment of rats with FHF were also studied a DLL4 group in. passages 3 7, which the animals received multiple injections of DLL4 at a. dose of 0 2 mg kg 1 mL through the intraperitoneal route at 8. FHF models 14 20 26 32 38 and 44 h after D Gal induction and a CTL. All experimental protocols were approved by the Animal Care group in which the animals underwent a sham procedure with. Ethics Committee of the First Af liated Hospital Zhejiang an equal volume of NS without DLL4 No animal received any. Figure 1 Scheme of human bone,marrow mesenchymal stem cell. hBMSC transplantation for rescuing,fulminant hepatic failure FHF in pigs. To understand the fundamental,interactions between implanted stem. cells and recipient hosts with FHF we,quantitatively pro led stem cells. activities after transplantation using an,automated biochemistry analyser. cytokine arrays ultra performance,liquid chromatography tandem mass. spectrometry UPLC MS messenger,RNA sequencing mRNA seq and. immunohistochemistry IHC, 2 Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, medical support eg infusions drugs during the entire experi osteocytes adipocytes and hepatocyte like cells These ndings. mental period indicated that the cells used in this study possessed typical. hBMSC phenotypes and multipotential stem cell characteristics. Survival curve analyses see online supplementary gure S1 All pigs in the control. Animal survival was monitored for up to 2 weeks after hBMSC group died within an average of 3 22 days whereas 13 15 pigs. transplantation and DLL4 treatment in pigs and rats with FHF in the transplantation group lived beyond day 7 gure 2A and. Survival curves were compared using the non parametric all of these 13 pigs survived for up to 6 months The other two. Mantel Cox test Animal survival was observed by a blinded pigs died within 4 days and subsequent autopsy indicated that. expert with 10 years of experience in animal studies FHF pathology resulted in rapid death. Biochemical assay analysis showed that 21 biomarkers in the. Materials reagents antibodies and PCR primers control group and 22 biomarkers in the transplantation group. The materials reagents and antibodies and quantitative reverse exhibited signi cant changes at day 3 relative to day 0. transcription PCR qRT PCR and primers for RT PCR used in gure 2B Among these biomarkers 20 were shared between. the study are provided in online supplementary tables S1 S3 the two groups Transplantation attenuated the 18 of the 20 bio. respectively marker changes see online supplementary gure S2 Cytokine. array analysis indicated that hBMSC transplantation altered liver. Sample collection responses to damage conferring a protective effect at day 3. Serum peripheral blood mononuclear cells PBMCs and liver detailed later in a dedicated section UPLC MS pro ling of. tissues were collected from the experimental animals according the serum metabolites indicated a signi cant difference in. to the experimental design for a period of 14 days after FHF hepatic metabolic functions between the two groups which. induction The serum samples were used in biochemical tests was observed as early as at day 1 p 0 012 gure 2C. ultra performance liquid chromatography tandem mass spec These results indicated that implanted hBMSCs produced imme. trometry UPLC MS analysis cytokine array detection and diate protective effects within the rst three days after. ELISA The PBMCs were pro led via mRNA seq Finally the transplantation. liver tissues were pro led analysed using UPLC MS H E stain At day 7 most 24 35 of the biochemical markers were. ing IHC mRNA seq and qRT PCR Sequencing reads from our restored to their baseline levels in the transplantation group see. mRNA seq experiments are available in the Sequence Read gure 2D for six representative markers except two signi. Archive database accession number SRP063325 Detailed cantly downregulated biomarkers and nine signi cantly upregu. methods are available in online supplementary materials and lated ones gure 2B Similarly in the transplantation group. methods no monitored cytokine exhibited a signi cant difference. between days 0 and 7 gure 2E Consistent with these results. Statistical analysis UPLC MS analysis gure 2C of 2768 distinguishable com. The results of the measurements are presented as the mean pounds in the serum revealed no signi cant differences between. SEM unless otherwise noted No statistical method was used the metabolite pro les at days 0 and 7 in the transplantation. to predetermine sample sizes Comparisons between two groups group p 0 813 In contrast these compounds shifted to right. with a sample size 3 except the comparisons of the most sig on day 3 in the control group p 0 001 H E staining. ni cant pig derived cytokine expression were performed using gure 2F further con rmed that transplantation was notably. the non parametric Mann Whitney U test assuming two tailed coupled with repair of the damaged liver structure at day 7. distributions Comparisons between two groups with a sample whereas the deceased control animals showed a typical FHF. size of 3 and the comparisons of the most signi cant pig derived histology with extensive hepatic necrosis and haemorrhage. cytokine expression n 5 per group were performed with the These results indicated that hBMSC transplantation stabilised. heteroscedastic Welch s t test to ensure sensitivity assuming two FHF within the rst seven days after D Gal administration. tailed distributions The normality assumption of Welch s t test Furthermore as shown in the online supplementary gure S2. was validated using the Shapiro Wilk test The sample data in the levels of low very low and high density lipoprotein and. each compared group showed p 0 05 Comparison of metabol total cholesterol were signi cantly decreased in both groups. ite pro les represented by the rst two principal components after D Gal administration and they restored to baseline on day. was performed using the nonparametric MANOVA permutation 5 7 after hBMSC transplantation These ndings also indicated. test the R vegan Package False discovery rate FDR calcu that the implanted hBMSCs restored hepatic synthetic functions. lated with the Benjamini Hochberg procedure was used to and stabilised FHF within the rst seven days. control 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 Human derived hepatocytes replaced approximately 4 5. p 0 05 FDR 0 05 in detecting transcript changes of pig hepatocytes. Hierarchical clustering was performed using the R stats To understand the interactions between stem cells and recipient. Package involving the Euclidean distance matrix and the pig hosts we quanti ed the relative contribution of cell replace. Pearson product moment correlation coef cient ment to the course of hBMSC assisted liver restoration IHC. using human speci c antibodies showed that both undifferenti. RESULTS ated hBMSCs CD90 and CD29 and human derived hepato. Human BMSC transplantation stabilised FHF in pigs within cytes albumin ALB and hepatocyte speci c antibody. 7 days HSA were found in the pigs liver tissue at day 3 but only. Phenotypic analysis by standard ow cytometry showed that human derived hepatocytes were found after day 7 gure 3A. seventh passage hBMSCs were positive for CD29 and CD90 and see positive and negative controls in online supplementary. but negative for CD34 and CD45 The results of multipotential gure S3 These data suggested that transdifferentiation of the. differentiation showed that the hBMSCs could differentiate into implanted hBMSCs was close to completion at day 7. Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146 3. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, 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 pro les plotted with their rst two principal. components PC1 PC2 D Temporal changes in six typical biochemical markers of liver function E Differentially regulated cytokines F H E. staining of liver tissue hBMSC transplantation led to notable improvements in the liver tissue structure at D7 C control group T transplantation. group 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 m A n 15. group B D n 5 15 group C n 10 15 group E n 3 group F n 5 group. The percentage of human derived hepatocytes in the liver the ALB in the liver tissue was human derived gure 3F G. tissue was estimated by counting HSA cells gure 3B Therefore proliferation and transdifferentiation of the. Human derived hepatocytes constituted approximately 4 5 implanted hBMSCs replaced 4 5 of liver hepatocytes when. and 4 7 of the liver hepatocytes at days 7 and 14 respectively FHF was stabilised However the percentage of liver function. see online supplementary gures S4 S7 Consistent with these compensated for by transplantation derived cells could be. results mRNA seq analysis showed that 1 3 1 8 of the total lower as evidenced by the lower levels of human derived gene. transcripts in the liver were human derived gure 3C D expression and ALB production in the liver tissue. UPLC MS analysis of peptides in the liver tissue and of secreted. peptides in the serum indicated that 3 5 5 1 and 1 2 1 7 hBMSCs altered liver responses to damage through. respectively were human derived gure 3E ALB secretion is a paracrine effects. characteristic function of hepatocytes and UPLC MS analysis of To explore the paracrine interactions between hBMSCs and. three pairs of peptides that distinguish between human recipient pig hosts we measured the changes in the serum levels. originated and pig originated ALB indicated that only 0 4 of of 215 cytokines using cytokine arrays see online. 4 Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, Figure 3 Proliferation and transdifferentiation of transplanted human bone marrow mesenchymal stem cells A immunohistochemistry of liver. tissue 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 of. human 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 m 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. supplementary table S4 and randomly selected six of signi induced between the two groups indicating a fundamental dif. cantly changed cytokines for validation using ELISA see online ference in the regulation of cell behaviours see online. supplementary gure S8 In the control group at day 3 relative supplementary gure S9C D Interestingly the altered cytokines. to day 0 and in the transplantation group at days 3 7 and 14 in the control group were more frequently involved in the regu. relative to day 0 cytokine changes were clustered gure 4A lation of apoptosis and cell death 5 27 changes in the control. The D Gal induced life threatening cytokine storms observed group vs 1 38 changes in the transplantation group By con. in both groups were suppressed in the transplantation group at trast the altered cytokines in the transplantation group were. day 7 In the transplantation group in particular 46 cytokines more enriched with functions in immunoregulation 1 27. showed signi cant changes at one or more time points see changes in the control group vs 12 38 changes in the transplant. online supplementary table S5 Hierarchical clustering analysis ation group and developmental control 2 27 changes in the. revealed four co regulated cytokines clusters see online control group vs 5 38 changes in the transplantation group. supplementary gure S9 Most 36 46 of the cytokines exhib gure 4C and annotation enrichment analysis results in online. ited a change pattern with a peak at day 3 that returned to a supplementary table S6 Comparison of liver gene expression. near baseline level at day 7 and remained stable at day 14 see between pigs with transplantation at day 7 and pigs without. online supplementary gure S9A cluster 1 This pattern at day 3 con rmed this trend of response divergence see. aligned with the progression of FHF and its restoration after online supplementary table S7. hBMSCs transplantation Other 10 46 cytokines showed alter The qRT PCR using human speci c mRNA further con rmed. native temporal change patterns see online supplementary that the pigs cells produced the three most signi cant changes. gure S9A clusters 2 4 Functionally these cytokines were in the serum cytokine levels that were speci cally observed in. related to in ammation regeneration apoptosis immunoregula the transplantation group changes in EpCAM VEGF R2 and. tion or metabolism see online supplementary gure S9B endoglin levels gure 4D These results indicated that the. In the cytokine storms at day 3 57 cytokines showed signi implanted hBMSCs remodelled the pigs liver responses to. cant changes in either one or both groups gure 4B damage by releasing small amounts of regulatory cytokines. Surprisingly speci c changes observed in only one group 49. changes were more frequent than common changes observed in Pro ling analysis indicated DLL4 Notch activation. both groups 8 changes suggesting a disparity in the liver To identify the important signalling processes through which. responses to damage between the two groups From a functional the implanted hBMSCs exerted their paracrine effects we ana. perspective most 48 57 of the altered cytokines had functions lysed the gene expression changes in PBMCs PBMCs respond. related to signal transduction metabolism apoptosis cell death ing to soluble factors without the complication of the tissue. immunoregulation or development see online supplementary microenvironment may exhibit signalling changes that are more. table S5 Cytokines from many pleiotropic signal transduction directly related to the paracrine effects of implanted hBMSCs. pathways and many metabolic processes were differentially We also identi ed the differentially expressed genes in both the. Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146 5. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, Figure 4 Paracrine effects of transplanted human bone marrow mesenchymal stem cells A Heat map of 215 protein level changes relative to day. 0 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 Heat. maps of differentially regulated cytokines involved in the most signi cantly altered functional themes immunoregulation top development left. bottom 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 factor. receptor 2 VEGF R2 and ENG endoglin in the liver tissues Only pig derived mRNAs were detected at D3 in T group Glyceraldehyde phosphate. dehydrogenase GAPDH was used as an internal reference The results are representative of ve 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. control and transplantation groups and differentially Of the identi ed 23 signalling processes the Notch signalling. co expressed genes between the two groups see online pathway is of particularly interest The differentially expressed. supplementary table S8 Differential co expression is de ned as genes in the control and the transplantation groups and the dif. a cluster of genes that show highly correlated expression in one ferentially co expressed genes between the two groups all have. group evidence of physiologically relevant activation of a bio frequent functional interactions with the Notch signalling. logical process but not in another group Functional synergy pathway gure 5A Notch receptors have ve known. analysis of expression changes showed 23 altered signalling pro ligands 22 we compared the expression of these ligands in the. cesses table 1 including several well known signalling processes liver tissue the transplantation group at day 7 vs the control. TLR 18 tumour necrosis factor alpha interleukin IL 1 and group at day 3 and found that in the transplantation group the. IL 619 that respond to liver damage Two cytokines regulating expression of DLL1 was suppressed p 0 026 and that the. these signalling processes stem cell factor and IL 6 have been expression of DLL4 was induced p 0 004 gure 5B. reported to exert prophylactic effects against FHF 20 21 The Validation using qRT PCR and ELISA con rmed that DLL4 was. rediscovery of these signalling processes suggested the potential speci cally induced in the transplantation group at both the. to exploit other identi ed processes in FHF treatment mRNA and the protein levels p 0 05 gure 5C D. 6 Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, Table 1 Twenty three generalised biological process annotations analysed by gene set linkage analysis in peripheral blood mononuclear cells. Generalised biological process gene 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 TLR4. T 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. C DcoE D0 3 D0 7 N A GO 0034130 GO 0008063 1e 5 MAP3K1. TGF beta receptor signalling C DE D3 D0 Down PID 200228 1e 5 TGFBR2. Regulation of SMAD protein and SMAD2 3 signalling T DcoE D0 7 D0 3 N A GO 0010862 1e 5 CEBPB VDR CNNM371 JUN. ALK1 signalling C DE D3 D0 Down PID 200149 1e 5 TGFBR2. IFN gamma mediated signalling and IFN gamma C DE D3 D0 Down GO 0060333 GO 0032729 1e 5. pathway C DcoE D0 3 D0 7 N A PID 200130 1e 5 STAT1. Notch 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 NOTCH3. Regulation 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 LCN2. Regulation of IL 2 biosynthetic process and C DE D3 D0 Down PID 200185 GO 0045086 1e 5 LCK. IL 2 signalling, 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 LCK. IL 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 LCK. SHP2 signalling C DE D3 D0 Down PID 200083 1e 5 LCK. c 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 TNFAIP3. Cellular response to TNF T DE D7 D0 Up GO 0071356 1e 5 LCN2. HIV 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 CASP2, Alpha 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 ITGAV. ErbB signalling T DcoE D0 7 D0 3 N A PID 200134 1e 5 ATF1 JUN BAD MAP2K1. C 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 the. transplantation 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 not. applicable, 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. Survival bene t of DLL4 in pig and rat FHF models regulation may rapidly die within several weeks 2 4 Stem cell. DLL4 treatment notably improved the survival of both pigs and transplantation holds substantial promise for the clinical treat. rats with FHF p 0 05 gure 5E In the validation using the ment of FHF 6 To overcome the barriers to clinical application. pig model the pigs in the control group had a mean survival many questions such as questions about the optimal timing. time of 2 6 days whereas the pigs in the DLL4 treatment group transdifferentiation and paracrine effects of stem cell therapies. survived for 4 6 days on average two pigs survived for up to need to be answered 13 23 In the present study we quantitatively. 6 months Similarly in the validation using the rat model the delineated stem cell recipient interactions via multifaceted pro. rats survived for 5 3 days on average in the control group and ling and demonstrated that implanted stem cells rescue. for 7 1 days in the DLL4 treatment group No animal received D Gal induced FHF by suppressing cytokine storms and alter. additional medical support eg nutrient infusions drugs during the recipient s here the pig s response to liver damage. the entire experiment H E staining indicated that the liver Importantly we discovered that DLL4 as a potential therapeutic. structure was notably repaired after DLL4 treatment gure 5F molecule may participate in the rescue of FHF via hBMSC. These results demonstrated that DLL4 treatment alone improves transplantation gure 6. animal survival Understanding the action time of implanted stem cells is. useful guidance to ensure optimal timing of treatment in future. DISCUSSION clinical applications 24 Certain evidence in cardiomyocyte trans. Patients with FHF with life threatening cytokine storms and plantation research has suggested the existence of a temporal. de ciencies in liver detoxi cation and metabolic and immune window of opportunity bound by the acute in ammatory. Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146 7. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, 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 but. which showed no correlation in the control C group between D0 and D3 B Differences in the expression levels of Notch related ligands in the. liver tissue between the T group at day 7 D7 after D galactosamine administration and the C group at D3 C Quantitative reverse transcription. PCR 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 staining. of liver tissue DLL4 treatment led to notable improvements in the liver tissue structure at D7 Scale bar 50 m 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. response on the one hand and by scar formation on the other 25 seems too low to explain the signi cantly improved animal sur. However the experimental and clinical data on the action timing vival which suggests the importance of paracrine effects in. after stem cell transplantation are limited In the current study hBMSC assisted liver restoration. our in house large animal model of FHF rescue demonstrated The pivotal role of paracrine effects in stem cell therapies has. that immediate transplantation of hBMSCs after D Gal ad been recognised to contribute to many biological processes such. ministration prevented death from FHF in the initial three days as by preventing in ammation inhibiting apoptosis improving. Serum metabolite pro les and biochemical changes indicated an metabolism and promoting regeneration 15 12 Certain positive. immediate protective effect of the implanted hBMSCs by sup results based on paracrine mechanisms have been reported in. pressing D Gal induced life threatening cytokine storms This ischaemic stroke myocardial infarction and kidney injury 7 12 29. timeframe provides evidence supporting direct investigation of In the present study the importance of paracrine effects was. the timing of stem cell based therapy in a clinical setting further supported by the observation of immediate protective. How stem cells act and speci cally whether cell replacement effects within the rst three days after transplantation when. mechanisms or the cells paracrine effects contribute most to hBMSC differentiation was far from complete Furthermore the. stem cell assisted organ repair remains a topic of controversy 12 paracrine effects of the implanted hBMSCs altered the recipient. Liver repair through stem cell proliferation and transdifferentia response to damage from producing more apoptosis cell death. tion has been postulated as the major mechanism of stem cell related cytokines to producing more cytokines related to immu. action in treating FHF 5 14 Several studies showed that only 1 noregulation and developmental control but the most altered. 12 26 28 of hepatocyte mass was replaced in the recipient liver cytokines were not produced by transplantation derived cells. such estimates were typically generated by counting immunohis These results indicated a high level regulatory role for the stem. tochemically stained cells or by ow cytometry To obtain a cells paracrine effects and suggested potential for the discovery. more reliable estimate here we used IHC mRNA seq and of stem cell inspired molecular therapies via understanding the. UPLC MS to quantitatively evaluate the number of human components of stem cells secretome and their functions. derived hepatocytes at two time points Our results show that at However many metabolic processes speci cally induced by the. day 7 when FHF was stabilised and differentiation completed implanted hBMSC need to be further clari ed. implanted hBMSCs replaced 4 5 of the pig hepatocytes Although recent studies indicated that systemic infusion of. However the liver function compensated for by these cells BMSC conditioned medium improved survival in a. could be even lower 0 4 1 8 as indicated by the lower small animal rat model of FHF 16 30 certain components may. levels of human derived gene expression and ALB production be potentially harmful 31 32 Searching for key therapeutic. The fraction of liver cells replaced by differentiated hBMSCs molecules that may be useful for developing a balanced. 8 Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, 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 to. damage signals and proliferate and transdifferentiate to repair the liver tissue Through paracrine effects the hBMSCs also induce the expression of. cytokines in the host these cytokines are involved in immunoregulation and development which protect liver cells and programme the liver. responses to damage We found evidence of an important role for Delta like ligand 4 DLL4 Notch activation in the hBMSC directed liver restoration. cocktail instead of whole cells is emerging as an exciting potential therapeutic molecule might be useful for developing a. concept in regenerative medicine However no single novel therapy that simulates stem cell actions for the treatment of. molecule based treatment for disease had been developed FHF However the precise mechanisms of DLL4 expression and. based on stem cell actions prior to the current study 15 Because how it works to improve liver function need to be further clari ed. the bene cial factors are mostly high level regulatory factors and for future therapy to be clinically effective ne tuning of the. present in small quantities in pro ling analysis changes in intervention with regard to timing dosing the route of adminis. these factors are easily masked by changes in downstream tration and long term follow up also needs to be performed. factors and effector proteins that exist in large quantities In. this study we used a novel procedure that cross references the Author af liations. functional changes in biological processes against the expres State Key Laboratory for Diagnosis and Treatment of Infectious Diseases. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases. sion changes in their known regulators to discover small bene The First Af liated Hospital Zhejiang University School of Medicine Hangzhou. cial regulatory changes and discovered that differential China. expression of DLL4 in the transplantation group was bene cial 2. Institute of Biochemistry College of Pharmaceutical Sciences Zhejiang University. for liver restoration We observed that a characteristic feature Hangzhou China. Department of Pathology The First Af liated Hospital Zhejiang University School of. of the transplantation effects was the induction of cytokines. Medicine Hangzhou China, that direct development DLL4 Notch activation might be one 4. Joint Institute for Genetics and Genome Medicine between Zhejiang University and. important step underlying the hBMSC directed liver restor University of Toronto Zhejiang University Hangzhou China. ation programme Recent studies have indicated that DLL4. along with other Notch ligands contributes to biliary injury Acknowledgements The authors thank Dr Guoqiang Mo for giving helpful. repair 33 Moreover our validation results demonstrated that suggestions in animal experiments and Dr Yu Chen for providing animal. DLL4 treatment at 8 12 h post D Gal administration signi administration. cantly improved survival in both pig and rat models of FHF Contributors DS JZ QZ JX and JJ contributed equally The study was designed by. These results are useful for further designing a novel JL and supervised by JL XC and LL The manuscript was written by JL XC ML JZ. and DS The experiment and data analysis were performed by DS JZ QZ JX JJ LJ. single molecule based therapy by simulating stem cell actions TW JL WD JL SS JL NZ LZ LJ SH PC HC ML LL XC and JL All authors were. for future clinical applications involved in critical revision of manuscript. In summary our quantitative evaluations delineated an inte Funding This work was supported by the National Basic Research Program of. grated model of the multifaceted interactions between stem cells China 2012CB944900 the Zhejiang Provincial and National Natural Science. and their recipients Furthermore we discovered that DLL4 as a Foundation of China LR13H030001 LR13C020001 81271708 81571818 and. Shi D et al Gut 2016 0 1 10 doi 10 1136 gutjnl 2015 311146 9. Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Hepatology, 31571356 the Chinese High Tech Research and Development 863 Program 17 Alleva E Santucci D Guide for the care and use of laboratory animals Ethology. 2013AA020102 the National S T Major Project 2012ZX10004503 006 and 1997 103 1072 3. 2012ZX10002004 001 and the Research Fund for the Doctoral Program of Higher 18 Kar P Singla A Polipalli S et al Expression pro les and polymorphism of toll like. Education 20130101110008 receptors 2 and 4 in peripheral blood mononuclear cells of fulminant hepatic failure. patients Am J Gastroenterol 2009 104 S123,Competing interests None declared. 19 Sekiyama KD Yoshiba M Thomson AW Circulating proin ammatory cytokines IL 1. Patient consent Obtained beta TNF alpha and IL 6 and IL 1 receptor antagonist IL 1Ra in fulminant. Ethics approval The Clinical Research Ethics Committee of the First Af liated hepatic failure and acute hepatitis Clin Exp Immunol 1994 98 71 7. Hospital Zhejiang University 20 Hu B Colletti LM Stem cell factor and c kit are involved in hepatic recovery after. acetaminophen induced liver injury in mice Am J Physiol Gastrointest Liver Physiol. Provenance and peer review Not commissioned externally peer reviewed 2008 295 G45 53. 21 Hecht N Pappo O Shouval D et al Hyper IL 6 gene therapy reverses fulminant. 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Downloaded from http gut bmj com on November 21 2016 Published by group bmj com. Quantitative evaluation of human bone,mesenchymal stem cells rescuing fulminant. hepatic failure in pigs, Dongyan Shi Jianing Zhang Qian Zhou Jiaojiao Xin Jing Jiang. Longyan Jiang Tianzhou Wu Jiang Li Wenchao Ding Jun Li Suwan. Sun Jianzhou Li Ning Zhou Liyuan Zhang Linfeng Jin Shaorui Hao. Pengcheng Chen Hongcui Cao Mingding Li Lanjuan Li Xin Chen and. Gut published online February 16 2016,Updated information and services can be found at. http gut bmj com content early 2016 02 14 gutjnl 2015 311146. These include, References This article cites 33 articles 3 of which you can access for free at. http gut bmj com content early 2016 02 14 gutjnl 2015 311146 BIBL. Email alerting Receive free email alerts when new articles cite this article Sign up in the. service box at the top right corner of the online article. To request permissions go to, http group bmj com group rights licensing permissions. 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Princeton University Press,  1974), 9. Nietzsche's influence on modern thought and his role within the various academic disciplines will be discussed only inasmuch as they affected his broader impact upon German politics, culture, and identity.  For a statement on the general nature of reception processes see Hans Robert Jauss, Toward an