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Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.
Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

Article Direct Reprogramming of Hepatic Myo?broblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis

Graphical Abstract

Highlights

d Transcription factor induction converts hepatic

myo?broblasts to iHeps in vitro

d Lineag

e tracing documents in vivo reprogramming of

myo?broblasts into iHeps

d iHeps induced in vivo closely resembl

e hepatocytes

d In vivo induction of iHeps ameliorates chemically induced

liver?brosis Authors

Guangqi Song,Martin Pacher,

Asha Balakrishnan,...,Tobias Cantz, Michael Ott,Amar Deep Sharma

Correspondence

ott.michael@mh-hannover.de(M.O.), sharma.amar@mh-hannover.de(A.D.S.) In Brief

Sharma,Ott,and colleagues show that expression of a key set of four transcription factors can reprogram hepatic myo?broblasts to induced hepatocyte-like cells in vivo and reduce liver?brosis,suggesting that direct

in vivo reprogramming may be an effective treatment approach for chronic liver disease.

Accession Numbers

GSE76843 Song et al.,2016,Cell Stem Cell18,1–12

June2,2016a2016Elsevier Inc.

https://www.wendangku.net/doc/0515207837.html,/10.1016/j.stem.2016.01.010

Direct Reprogramming of Hepatic Myo?broblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis

Guangqi Song,1,2,3,13Martin Pacher,1,4,13Asha Balakrishnan,1,4Qinggong Yuan,1,4Hsin-Chieh Tsay,1,2,4Dakai Yang,1,2 Julia Reetz,5Sabine Brandes,1,4Zhen Dai,1,2Brigitte M.Pu¨tzer,5Marcos J.Arau′zo-Bravo,6,7Doris Steinemann,8

Tom Luedde,9Robert F.Schwabe,10Michael P.Manns,1Hans R.Scho¨ler,11Axel Schambach,12Tobias Cantz,1,3 Michael Ott,1,4,14,*and Amar Deep Sharma1,2,14,*

1Department of Gastroenterology,Hepatology,and Endocrinology,Hannover Medical School,Hannover30625,Germany

2Junior Research Group MicroRNA in Liver Regeneration,Cluster of Excellence REBIRTH,Hannover Medical School,Hannover30625, Germany

3Translational Hepatology and Stem Cell Biology,Cluster of Excellence REBIRTH,Hannover Medical School,Hannover30625,Germany 4Twincore Centre for Experimental and Clinical Infection Research,Hannover30625,Germany

5Institute for Experimental Gene Therapy and Cancer Research,Rostock University Medical Center,Rostock18057,Germany

6Group of Computational Biology and Systems Biomedicine,Biodonostia Health Research Institute,San Sebastia′n20014,Spain

7IKERBASQUE,Basque Foundation for Science,Bilbao48013,Spain

8Institute of Human Genetics,Hannover Medical School,Hannover30625,Germany

9Division of Hepatobiliary Oncology,Department of Medicine III,University Hospital RWTH,Aachen52074,Germany

10Department of Medicine,Columbia University,New York,NY10032,USA

11Department of Cell and Developmental Biology,Max Planck Institute for Molecular Biomedicine,Mu¨nster48149,Germany

12Institute for Experimental Hematology,Hannover Medical School,Hannover30625,Germany

13Co-?rst author

14Co-senior author

*Correspondence:ott.michael@mh-hannover.de(M.O.),sharma.amar@mh-hannover.de(A.D.S.)

https://www.wendangku.net/doc/0515207837.html,/10.1016/j.stem.2016.01.010

SUMMARY

Direct induction of induced hepatocytes(iHeps)from ?broblasts holds potential as a strategy for regenera-tive medicine but until now has only been shown in culture settings.Here,we describe in vivo iHep forma-tion using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3,GATA4,HNF1A,and HNF4A from a polycistronic lentiviral vector converts mouse myo?-broblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription fac-tors from a p75neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the genera-tion of hepatocyte-like cells from myo?broblasts in ?brotic mouse livers and reduced liver?brosis.We have therefore been able to convert pro-?brogenic myo?broblasts in the liver into hepatocyte-like cells with positive functional bene?ts.This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease. INTRODUCTION

The utility of transcription factors(TFs)for the acquisition of novel cell fates has been unlocked through landmark studies of induced pluripotent stem cell(iPSC)reprogramming(Takahashi et al., 2007;Takahashi and Yamanaka,2006).Recently,by circumvent-ing the pluripotent cell state,direct reprogramming of?broblasts into neurons(Grande et al.,2013;Guo et al.,2014;Han et al., 2012;Kim et al.,2011;Lujan et al.,2012;Ring et al.,2012;Son et al.,2011;Su et al.,2014;Thier et al.,2012;Torper et al.,2013; Vierbuchen et al.,2010),cardiomyocytes(Ieda et al.,2010;Qian et al.,2012),and hepatocytes(Du et al.,2014;Huang et al., 2011,2014;Morris et al.,2014;Sekiya and Suzuki,2011)through overexpression of speci?c TFs has been demonstrated.For example,functional neurons were generated from mouse?bro-blasts by ectopic expression of Ascl1,Brn2,and Myt1l(Vierbu-chen et al.,2010).Similarly,a combination of three cardiac-speci?c TFs,Gata4,Mef2c,and Tbx5,directly reprogrammed mouse cardiac?broblasts into cardiomyocyte-like cells in vitro and in vivo in a heart infarction model(Ieda et al.,2010;Song et al.,2012).Lineage reprogramming of mouse?broblasts into hepatocytes has been achieved in vitro by ectopic expression of HNF4A plus Foxa1,Foxa2or Foxa3or by the combination of Gata4,Hnf1a,and Foxa3and inactivation of p19(Arf)in culture (Huang et al.,2011;Sekiya and Suzuki,2011).Recently,two other groups independently reprogrammed human?broblasts into he-patocytes by forced ectopic expression of FOXA3,HNF1A,and HNF4A or the combination of HNF1A,HNF4A,and HNF6together with the maturation factors ATF5,PROX1,and CEBPA(Du et al., 2014;Huang et al.,2014).The direct conversion of?broblasts into hepatocyte-like cells in vivo remains to be investigated,and the question needs to be addressed whether such an approach could also ameliorate the degree of?brosis in damaged livers. RESULTS

FOXA3,GATA4,HNF1A,and HNF4A Convert Hepatic Myo?broblasts into iHeps In Vitro

Based on previously reported results(Huang et al.,2011;Iacob et al.,2011;Sekiya and Suzuki,2011),we screened TFs Cell Stem Cell18,1–12,June2,2016a2016Elsevier Inc.1

(FOXA1,FOXA2,FOXA 3,GATA 4,HNF1A ,HNF4A ,and CEBP A)for direct reprogramming of myo?broblasts derived from pri-mary hepatic stellate cells into hepatocytes.FOXA 3,GATA 4,HNF1A ,and HNF4A transcription factors (4TFs)were selected for further experiments,since the absence of each of these TFs substantially reduced albumin secretion and CYP3A activity in our screening experiments (Figure 1A).To overexpress the selected 4TFs simultaneously,we cloned the cDNAs into a poly-cistronic lentiviral vector (henceforth referred to as LV.4TF)(Figure 1B).

In response to persistent in?ammatory injuries large numbers of stellate cells,which have undergone ‘‘activation’’to pro-?bro-genic myo?broblasts,accumulate in the liver.To harness the po-tential therapeutic effects of direct reprogramming in chronic liver disease,we ?rst tested whether forced expression of 4TFs would induce a hepatocyte phenotype in cultured myo?bro-blasts (Figure 1C).The expression of 4TFs in transduced myo?-broblasts was con?rmed on day 4by qRT-PCR (Figure S1A).Within 14days after transduction,we observed profound changes in morphology of the transduced cells now

resembling

Figure 1.FOXA3,GATA4,HNF1A ,and HNF4A Directly Reprogram Mouse Myo?broblasts into iHeps

(A)Screening of transcription factors in myo?broblasts.Albumin secretion and CYP3A activity were measured as read out during screening of seven transcription factors.The data represent values from three independent experiments.

(B)Lentiviral vector maps,the LV.4TF used to generate iHeps,and a reporter lentiviral vector,used to detect albumin-positive cells.

(C)Schematic of iHep generation from myo?broblasts.To prepare myo?broblasts,primary HSCs were isolated from BALB/c mice and cultured in the presence of platelet-derived growth factor (PDGF)before transduction with LV.4TF.

(D)Phase-contrast microscopy (3100)of myo?broblasts and iHeps at day 14after lentiviral transduction with LV.4TF.Scale bars,200m M.

(E)iHeps express dTomato while myo?broblasts,cultured in HCM for 14days and transduced with reporter lentiviral vector,show absence of dTomato.Scale bars,200m M.

(F)A representative FACS plot (n =3independent experiments)showing the percentage of albumin promoter-driven dTomato-positive cells.

(G)Representative RT-PCR (n =3independent experiments)showing the expression of hepatic genes in iHeps,whereas ?broblast genes such as Acta1,Col1a1,and Col2a1were downregulated.Murine primary hepatocytes (Pr.Hc)cultured for 24hr on a collagen matrix were used as positive control.

(H)2D principal component analyses indicate that the global expression pro?les of iHeps (n =3)are distinct from myo?broblasts and more similar to primary mouse hepatocytes cultured for 24hr.

(I)Representative pictures from three independent experiments showing PAS staining and LDL uptake in iHeps.Scale bars,200m M.

(J)Albumin secretion in the supernatant was measured in the absence of serum components.CYP1A2and 3A activities in 24hr cultured primary hepatocytes,iHeps,and myo?broblasts.The values shown are mean of three independent experiments.

(K)aCGH-based karyotype analysis of myo?broblasts derived iHeps.The data shown in (G)–(K)are obtained from FACS-puri?ed iHeps.See also Figure S1.

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an epithelial phenotype(Figure1D).To con?rm the generation of hepatocytes from myo?broblasts,we transduced cells at day10 with a reporter lentiviral vector,which expressed dTomato under transcriptional control of the albumin promoter(Figure1B). Approximately12%of the cells expressed dTomato,indicating the generation of iHeps(Figures1E and1F).We enriched dTomato protein expressing iHeps by?uorescence-activated cell sorting(FACS)for further characterization(Figures1G–1K). The iHeps showed expression,albeit at lower levels,of typical primary hepatocyte(Pr.Hc,cultured for24hr)markers and the downregulation of?broblast genes(Figures1G,1H,and S1B). Our principal-component analysis(PCA)analyses suggested that although iHeps acquired a hepatic gene expression pro?le, they remained a distinct cell type when compared to primary he-patocytes cultured for24hr.Importantly,iHeps exhibited func-tional characteristics of hepatocytes as they stored glycogen, showed uptake of low-density lipoprotein(LDL),secreted albu-min,and acquired cytochrome P450(CYP1A2and3A)activities (Figures1I and1J).Genomic integrity was con?rmed by array-based comparative genomic hybridization(aCGH)analysis(Fig-ure1K).Thus,ectopic expression of FOXA3,GATA4,HNF1A, and HNF4A converts myo?broblasts into iHeps in vitro. Establishment of a Lineage-Tracing Model to Detect

In Vivo Reprogramming

We next examined whether4TFs expression would facilitate iHep formation in vivo.To investigate4TFs-mediated lineage reprogramming in vivo,we developed a mouse model to detect iHeps derived from non-parenchymal cells(Figure2A). We used(Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)mice (henceforth referred to as mT/mG).All cells including hepato-cytes and non-parenchymal liver cells from these mT/mG mice express membrane-targeted tdTomato(mT)before Cre-medi-ated recombination(Muzumdar et al.,2007)(Figure2B).To label endogenous hepatocytes,adult mT/mG mice were injected intrasplenically with431011adeno-associated virus(AAV)sero-type8particles expressing Cre recombinase under transcrip-tional control of the liver-speci?c transthyretin(Ttr)promoter (Malato et al.,2011;Sharma et al.,2011).As a result,membra-nous EGFP?uorescence in hepatocytes and tdTomato membra-nous?uorescence in non-parenchymal cells of the liver were expressed four weeks after AAV-Ttr-Cre injection(Figure2C). Then,we induced myo?broblasts in the liver of AAV-Ttr-Cre-in-jected mT/mG mice by intraperitoneal injections of carbon tetra-chloride(CCl4)twice per week for a total of8weeks.These mice developed extensive?brosis(Figure2D)and accumulated myo?broblasts in the tissue.Notably,in these?brotic livers,all non-parenchymal cells including myo?broblasts expressed tdTomato,whereas hepatocytes expressed EGFP(Figure2E). To overexpress4TFs in myo?broblasts,we designed a p75NTRp-tagged recombinant adenoviral vector(serotype5), which expressed all4TFs from a polycistronic transgene cassette.The adenoviral vector was modi?ed to target mouse myo?broblasts through coupling of adenoviral?ber knobs with a peptide(single-chain antibody fragment)of the nerve growth factor(NGFp),which was selected for speci?c and high-af?nity binding to the p75neurotrophin receptor(p75NTR)present on hepatic stellate cells and myo?broblasts(Reetz et al.,2013).Ef-?ciency of vector targeting was tested in cell culture,as previ-ously published(Reetz et al.,2013)(Figure S2A).In vivo targeting of myo?broblasts by Ad.GFP-S11-NGFp was tested by injecting 53109adenoviral particles via the portal vein of?brotic wild-type BALB/c mice as shown previously(Reetz et al.,2013). The amount of injected virus particles was suf?cient to transduce 30%of stellate cells in normal and 20%of myo?broblasts in CCl4-induced?brotic livers of BALB/c mice.Predominantly,my-o?broblasts were found to express EGFP in the liver of mice in-jected with Ad.GFP-S11-NGFp(Figure2F),whereas control adenovirus vector injected mice showed transduction of hepato-cytes(Figure2G).Co-staining of EGFP with albumin(hepatocyte marker),desmin(myo?broblast marker),cytokeratin(CK)19(bile duct and liver stem/progenitor cell marker),F4/80(Kupffer cell marker),CD31(endothelial cell marker),and CD45(hematopoi-etic cell marker)revealed preferential transduction of myo?bro-blasts,but not of hepatocytes(except<0.05%rare double-pos-itive cells),Kupffer cells,endothelial cells,bile duct cells,or liver/ stem progenitor cells(undetected)(Figure S2B).Therefore,injec-tion of Ad.GFP-S11-NGFp allows preferential targeting of myo?-broblasts.Accordingly,one week after cessation of the CCl4 treatment,we injected53109p75NTRp-tagged Ad5.FOXA3. GATA4.HNF1A.HNF4A(henceforth referred to as Ad.4TF)via the portal vein of AAV-Ttr-Cre injected?brotic wild-type BALB/c mice.This led to successful overexpression of4TFs in sorted myo?broblasts from these mice within4days after injection(Fig-ure2H).Importantly,Ad.4TF administration in normal BALB/c mice neither affected liver function tests nor histology(Figure2I). In Vivo iHep Formation via Direct Reprogramming

After establishing a model that faithfully identi?es iHep forma-tion,we examined iHep formation in mT/mG mice injected with AAV-Ttr-Cre followed by CCl4injections and subsequent administration of Ad.4TF.The control animals were treated exactly in the same manner,except for injection with empty adenoviral vector instead of Ad.4TF.In our lineage-tracing model,the endogenous hepatocytes would express membra-nous EGFP,while the iHeps could be detected by tdTomato positive membrane?uorescence(Figure2J).To identify iHeps and distinguish them from endogenous hepatocytes,we stained liver tissues30days after Ad.4TF injection for albumin,major urinary protein(MUP),fumarylacetoacetate hydrolase(FAH), alpha-1antitrypsin(AAT),and HNF4A,all characteristic hepato-cyte markers in the liver.Immuno?uorescence analyses of the liver harvested at30days after Ad.4TF injection showed albu-min,MUP,FAH,AAT,and HNF4A expression mainly in endog-enous hepatocytes with EGFP-positive membranes(Figures 2K and S2C).However,few cells with tdTomato-positive mem-branes also stained positive for albumin,MUP,FAH,AAT,and HNF4A in the liver of Ad.4TF-injected mice.These tdTomato-positive cells that also stained positive for hepatocyte markers, either in clusters or as single cells,indicate the presence of iHeps.The control animals(n=10),injected with empty adeno-viral vector,did not express albumin,MUP,FAH,and AAT in any of the tdTomato-positive cells.The ef?ciency of direct reprogramming in vivo was calculated from the average of tdTomato-expressing cells,which stained positive for albumin, MUP,FAH,AAT,and HNF4A relative to the total hepatocyte population(n=10mice).The percentage of in-vivo-generated iHeps among the total hepatocyte population ranged from Cell Stem Cell18,1–12,June2,2016a2016Elsevier Inc.

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0.2%to 1.2%in Ad.4TF-injected mice,whereas control mice did not show any reprogrammed cells (Figure 2L).Of note,we did not detect iHeps when Ad.4TF was administered in unin-jured mice (data not shown).Since the number of myo?bro-blasts in ?brotic livers at 1week after cessation of 8weeks of

CCL 4treatment is between 15.4%and 21.7%of the total num-ber of liver cells (Figure S2D),it can be estimated that 0.2%to 1.2%of iHeps detected in AD.4TF-injected mice is equivalent to a reprogramming ef?ciency of less than 4%.It is noteworthy to mention that reprogramming ef?ciency may be even lower

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Figure 2.Establishment of a Lineage-Tracing Model to Detect In Vivo iHep Formation

(A)Schematic of the model.

(B)Representative pictures showing native tdTomato membranous ?uorescence (red)is present in all liver cells including hepatocytes of adult mT/mG mice (n =4).Nuclei (blue)are stained with DAPI.Scale bar,100m M.

(C)All hepatocytes express membranous EGFP in liver of 431011AAV-Ttr -Cre injected mT/mG mice (n =4),while non-parenchymal cells retain tdTomato expression.Nuclei (blue)are stained with DAPI.Arrowheads indicate non-parenchymal cells that retained tdTomato.Scale bar,100m M.

(D)Representative picture of Sirius red staining of liver (n =5)after 16intraperitoneal CCl 4injections (twice per week for 8weeks).Scale bar,200m M.

(E)Cells in the vicinity of emerging ?brous septa express native tdTomato ?uorescence in CCl 4-treated AAV-Ttr -Cre -injected mT/mG mice (n =5).Scale bar,200m M.

(F)Preferential in vivo transduction of HSC and myo?broblasts in liver of mice (n =4)by Ad.GFP-S11-NGFp adenoviral vector.Scale bar,200m M.

(G)In contrast,injection of control adenoviral vector Ad.GFP leads to the homogenous expression of GFP in hepatocytes (n =4mice).Scale bar,200m M.(H)In vivo overexpression of 4TFs in myo?broblasts of CCl 4-treated BALB/c mice that were injected with Ad.4TF was con?rmed by qRT-PCR.Four days after injection,myo?broblasts were sorted by staining with P75NTR antibody followed by FACS.Primary human hepatocytes were used as positive control.The data represent values from three independent experiments.

(I)Similar levels of serum transaminases,H&E staining,and desmin staining suggest normal liver functions and absence of any histological abnormality in uninjured BALB/c mice (n =

4)injected with Ad.4TF.

(J–L)p75NTR tagged Ad5.FOXA3.GATA4.HNF1A.HNF4A induces hepatocyte-speci?c gene expression in ?brotic mice.(J)Schematic illustration shows that endogenous hepatocytes can be identi?ed by EGFP expression,whereas reprogrammed hepatocytes are identi?ed by tdTomato expression.(K)Immuno?u-orescence staining with antibodies (blue)for hepatocyte markers such as albumin,MUP,FAH,and AAT on cryosections obtained from mT/mG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP))mice injected with 431011AAV-Ttr -Cre .These confocal ?gures are representative of livers obtained from ten mice in each group.Scale bars,100m M.(L)Table shows percentage of average tdTomato-expressing cells that stained positive for ALB,MUP,FAH,and AAT in the total hepatocyte population.Ten random sections were stained for each antigen per mouse (n =10mice).See also Figure S2.

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than 4%if iHeps proliferate between the time when they appear and 30days after Ad.4TF administration.Thus,our data indicate that expression of 4TFs in myo?broblasts facilitates the forma-tion of iHeps in chronic liver disease.

Functional Analyses of In-Vivo-Generated iHeps

One of the prerequisites for in-vivo-generated iHeps to be considered as hepatocytes is to possess the functions of mature hepatocytes.To characterize iHeps at the functional level,we isolated iHeps by Percoll density-gradient centrifugation fol-lowed by FACS from the liver of Ad.4TF-injected mice (Figures 3A and 3B).We did not ?nd iHeps in control mice by FACS as well (Figure S2E).We speci?cally sorted for single-tdTomato-positive cells,because double-positive cells may represent endogenous cells that have not silenced tdTomato completely.The iHeps that were tdTomato positive,cultured for 24hr,showed albumin secretion and urea synthesis similar to EGFP-positive endogenous hepatocytes (eHeps)(Figures 3C and 3D).The iHeps showed the ability to uptake indocyanine green (ICG);stained positive for oil red O,indicating the presence of tri-glycerides and lipids;stained positive for PAS,thus demon-strating the ability to store glycogen (Figure 3E);and

exhibited

Figure 3.Functional Characterization of In-Vivo-Reprogrammed iHeps and Evidence for Amelioration of Liver Fibrosis in Ad.4TF-Injected Mice

(A)Schematic of the FACS sorting and analyses of ?brosis.

(B)Isolation of in-vivo-generated iHeps (tdTomato positive)and eHeps (EGFP positive)by FACS sorting.Representative pictures and FACS sorting (n =6mice)are shown.

(C–G)The tdTomato-positive cells were pooled together and data are shown from technical triplicates.Scale bars,200m M.(C)Albumin ELISA revealed comparable levels of secreted albumin in iHeps and eHeps.(D)Urea synthesis was also found to be similar in iHeps and eHeps.(E)The iHeps show ICG uptake,oil red O staining,and PAS staining.Scale bars,200m M.(F)The iHeps and eHeps showed activities for CYP3A,CYP1A1,CYP2C9,and CYP1A2.(G)Evidence for drug response in iHeps was demonstrated by elevated levels of Cyp1a1,Abcc2,Ugt1a1,and Oatp .

(H–K)Amelioration of liver ?brosis in Ad.4TF-injected mice.(H)Reduced levels of Col1a1mRNA in Ad.4TF-injected mice (n =9)compared to control mice (n =9).(I)Hydroxyproline assay showed decreased levels of entire collagen content,measured in whole liver.(J)H&E,Sirius red,and immunohistochemical staining for desmin and p75NTR showed less ?brosis in Ad.4TF injected mice (n =9)than respective controls (n =9).Scale bars,100m M for H&E and desmin and 100m M for Sirius red and p75NTR staining.(K)Quanti?cation of Sirius red,desmin,and p75NTR stainings shown in (J).

(L)Reduced levels of serum transaminases suggest improved liver functions in Ad.4TF-administered,CCl 4-treated,AAV-Ttr -Cre -injected mT/mG mice (n =3).(M)A representative photograph of H&E staining after an 8-month follow-up study of Ad.4TF-administered and CCl 4-treated AAV-Ttr -Cre -injected mT/mG mice (n =3)showing normal histology and no tumor formation.

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cytochrome activity (CYP3A,1A1,2C9,and 1A2)similar to eHeps (Figure 3f).Furthermore,we tested whether isolated iHeps are drug inducible.The treatment of iHeps with phenobar-bital and rifampicin led to induction of Cyp1a1(phase 1),Ugt1a1(phase 2),Abcc2,and Oatp (Figure 3G).Importantly,the in-vivo-generated iHeps showed chromosomes with no obvious numer-ical or structural aberrations (Figure S2F)and the absence of exogenous 4TFs (Figure S2G),thus indicating the stable reprog-ramming of myo?broblasts into iHeps.In addition,Ki67staining indicated that iHeps have the ability to proliferate in response to two-thirds partial hepatectomy in vivo (Figure S2H).Therefore,our thorough characterization suggests that in-vivo-generated iHeps possess functional properties of hepatocytes.

Overexpression of 4TFs Ameliorates Chemical-Induced Liver Fibrosis

To examine whether in vivo reprogramming ameliorates chronic liver disease,we determined the extent of liver ?brosis in CCl 4-in-jected mice.First,qPCR showed lower levels of Col1a1in the liver of Ad.4TF-injected mice,suggesting less liver ?brosis compared to respective controls (Figure 3H).Second,we performed hydroxyproline assay,which measures the entire collagen con-tent of a liver.The Ad.4TF-injected mice showed signi?cantly reduced levels of hydroxyproline,indicating decreased liver ?brosis (Figure 3I).In addition,histological grading of ?brosis,Sirius red staining,immunohistochemical staining of desmin and p75NTR,as well as serum levels of aminotransferases con?rmed decreased liver ?brosis in Ad.4TF-injected mice compared to control mice (Figures 3J–3L).In addition,an 8-month follow-up study of mice injected with Ad.4TF showed normal histology and no signs of liver tumors (Figure 3M).

Next,we tested whether injection of Ad.4TF during ongoing liver injury can lead to in vivo iHep formation and a reduction of liver ?brosis (Figure 4A).Indeed,we detected the presence of iHeps and less liver ?brosis in mice injected with Ad.4TF during an ongoing injury (Figures 4B–4E).Hence,our results

indicate

Figure 4.Administration of Ad.4TF during Ongoing Injury in AAV-Ttr -Cre -Injected mT/mG Mice Also Generates iHeps and Leads to a Reduc-tion in Fibrotic Markers

(A)Schematic of the experimental https://www.wendangku.net/doc/0515207837.html,l 4was injected twice weekly for 4weeks before administration of Ad.4TF.Mice were injected again for 4more weeks.(B)Immuno?uorescence staining with antibodies (blue color)for hepatocyte markers,albumin,and MUP.The ?gures are representative of livers obtained from four mice in each group.Scale bars,100m M.

(C)Reduced levels of Col1a1mRNA in Ad.4TF-injected mice (n =4)compared to control mice (n =4).(D)Hydroxyproline assay showed decreased levels of entire collagen content,measured in whole liver.

(E)Sirius red and immunohistochemical staining for desmin showed less ?brosis in Ad.4TF-injected mice (n =4)compared to respective controls (n =4).Scale bars,200m M for Sirius red and 100m M for desmin staining.Right,quanti?cations of Sirius red and desmin stainings are shown.See also Figure S3.

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that 4TFs-induced in vivo reprogramming ameliorates liver ?brosis in mice injected with CCl 4for 8weeks.

CCl 4administration for up to 8weeks induces a signi?cant amount of liver ?brosis;however,it is still reversible.We there-fore examined whether the forced expression of 4TF is capable of ameliorating liver injury in mice injected for 12weeks with CCl 4resembling an irreversible cirrhosis model (Figure S3A).Although,we detected the presence of iHeps,a bene?cial effect on liver ?brosis was not observed in mice injected with Ad.4TF (Figures S3B–S3F).This might be due to the fact that the number of Ad.4TF reprogrammed iHeps in mice injected for 12weeks with CCl 4remained similar to those in mice injected for 8weeks,whereas total myo?broblast numbers further increased after pro-longed CCl 4administration.This phenomenon can be explained,at least in part,by a modest but similar number of myo?broblasts transduced by Ad.4TF vector in both 8-week and 12-week CCl 4models,but these were most likely outnumbered by the massive increase of ?brotic cells in the 12-week CCl 4model.

Characterization of In-Vivo-Generated iHeps

To further prove the hepatic lineage identity of in vivo iHeps,we performed mRNA microarrays after pooling several livers isolated from different animals and compared them with myo?broblasts,myo?broblast-derived iHeps in vitro,and endogenous hepato-cytes (eHeps,serving as positive control)in vivo.In silico ana-lyses,such as hierarchical clustering and PCA,demonstrated that in vivo iHeps are more similar to eHeps than to in vitro iHeps (Figures 5A and 5B).This suggests that the in vivo hepatic envi-ronment further contributes to the maturation of in vivo iHeps.The identity of reprogrammed cells can be faithfully deter-mined by the CellNet platform with very high accuracy (Cahan et al.,2014;Morris et al.,2014).We therefore extracted a list of genes comprising either the liver-typical or ?broblast-typical gene regulatory networks (GRNs)from CellNet and compared our in vivo or in vitro myo?broblast-derived iHeps (Figure 5C).In iHeps derived from myo?broblasts in vitro,1,281genes were upregulated by more than 4-fold and 1,576genes were downregulated by more than 4-fold compared to 24-hr-cultured primary mouse hepatocytes.22(of 1,281)upregulated genes and 128(of 1,576)downregulated genes were in common with the set of genes comprising the CellNet liver GRN.To assess ge-netic memory of myo?broblasts,we compared in vitro myo?bro-blast-derived iHeps with ?broblast GRN.Deregulated genes common with the ?broblast GRN included 184(156upregulated and 28downregulated)genes.When in vivo iHeps were compared to in vivo eHeps (endogenous hepatocytes),a total of 426genes were upregulated by more than 4-fold and 43were downregulated by more than 4-fold.Only 8(of 426)upregu-lated genes and 5(of 43)downregulated genes were in common with the set of genes comprising the CellNet liver GRN.Deregu-lated genes common with the ?broblast GRN include 94(94up-regulated and 0downregulated)genes.

Deregulated genes (>4-fold up or down)in the in vitro iHeps derived from myo?broblasts thus represent 35%of the CellNet

A B

Myofibroblasts

In vivo eHEP

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Myofibroblasts-derived

iHep

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= 28= 5 Foxc1, Hoxa5, Oxct1, Pdk3, Podxl.

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Figure 5.Microarray Analyses of Myo?broblast-Derived iHeps

(A)Whole-transcriptome heatmap demonstrates clustering of in vivo iHeps (n =

3),eHeps,myo?broblasts,and in vitro iHeps.

(B)2D principal component analyses indicate that the global expression pro?les of in vivo iHeps (n =3)resembles freshly isolated eHeps compared to in vitro iHeps.(C)Comparison of our in vitro and in vivo iHeps with liver GRN or ?broblasts GRN obtained from CellNet.

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listed genes comprising the liver GRN.In contrast to the in-vitro-generated iHeps,the in vivo iHeps show only 3.3%deregulated genes among the genes comprising the liver GRN.From these data,together with our thorough analyses of various liver meta-bolic functions,we conclude that the in vivo iHeps provide a similar liver GRN with only minor differences in 13of 421genes.In addition,we also provide evidence for (limited)genetic mem-ory in a number of (myo)?broblast GRNs.

Con?rmation of iHep Formation in a Direct Lineage-Tracing Model

Although,the AAV-Ttr -Cre -injected mT/mG model in combina-tion with Ad.4TF is an appropriate model to detect iHeps in vivo,we examined the generation of iHeps in a second independent

mouse model.To speci?cally detect myo?broblast-derived iHeps,we used mice expressing Cre under the transcriptional control of the lecithin-retinol acyltransferase (Lrat)promoter,which also contain the mT/mG transgenes (Figure 6A).LratCre-based lineage tracing has been shown to effectively trace myo-?broblasts derived from the hepatic stellate cell (HSC)lineage (Mederacke et al.,2013).We ?rst con?rmed the labeling of HSCs and myo?broblasts expressing EGFP in LratCre-mT/mG mice,consistent with a previous report (Mederacke et al.,2013)(Figures S4A and S4B).We then examined the generation of iHeps after administration of Ad.4TF in mice injected for 8weeks with CCl 4.Again,we conclusively observed iHeps (either in clusters or as single cells)expressing hepatocyte markers in mice injected with Ad.4TF with a percentage of

total

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% of iHep / total hepatocytes / mouse

MUP

R L U /10,000c e l l s

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10,000c e l l s

ICG FAH

AAT

Figure 6.Demonstration of iHep Formation and Their Functional Characterization in LratCre-mT/mG Mice

(A)Schematic of the model.

(B)In LratCre-mT/mG model,eHeps are tdTomato positive whereas iHeps are EGFP positive.

(C)Immuno?uorescence staining with albumin,MUP,FAH,and AAT antibody (blue)on cryosections obtained from LratCre-mT/mG mice showed the presence of iHeps (green)only in mice injected with Ad.4TF.The ?gures are representative of livers obtained from four mice in each group.Scale bars,100m M.(D)Percentage of iHeps in the total hepatocyte population (n =4mice).Ten sections were stained for each antigen per mouse (n =4mice).

(E)Isolation of in-vivo-generated iHeps (EGFP positive)and eHeps (tdTomato positive)by FACS sorting.Representative pictures and FACS (n =4mice)are shown.Scale bars,200m M.

(F–J)The iHeps were pooled and data are shown from triplicates.(F)Albumin secretion and (G)urea synthesis were similar in iHeps and eHeps.(H)The iHeps show ICG uptake,oil red O staining,and PAS staining.Scale bars,100m M for ICG uptake,50m M for oil red O,and 200m M for PAS staining.(I)iHeps and eHeps showed activity for CYP3A,CYP1A1,CYP2C9,and CYP1A2.(J)Evidence for drug response in iHeps was demonstrated by elevated levels of Cyp1a1,Abcc2,Ugt1a1,and Oatp .

See also Figures S4–S7.

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hepatocytes ranging from 0.9%to 2.5%(Figures 6B–6D and S4C).Notably,we did not ?nd iHeps in control LratCre-mT/mG mice,as all of the isolated hepatocytes expressed tdTomato only (Figure S4D).All EGFP-positive cells were myo?broblasts (Figure S4E).Furthermore,sorted in vivo iHeps exhibited func-tional characteristics similar to endogenous hepatocytes (Fig-ures 6E–6J).In addition,isolated iHeps expressed hepatocyte markers (Fah ,Ck18,Hnf4a ,Hnf1a ,Gjb1,Ck8,and Apoa1),but not the myo?broblast markers (Acta1,Col1a1,and Col2a1)or an intestinal marker (Cdx2)(Figure S4F).Importantly,in vivo iHeps possess the ability to proliferate in response to stimuli such as epidermal growth factor,in vitro (Figure S4G),or after two-thirds partial hepatectomy in vivo (Figure S4H).These iHeps regulated expression of genes involved in glucose metabolism such as G6pc and Pck1(Figures S4I and S4J),and more impor-

tantly,glucose levels upon treatment with glucagon and insulin (Figures S4K and S4L).Thus,our ?ndings in the LratCre-mT/mG model con?rm that forced expression of 4TFs in myo?-broblasts generates functional iHeps in vivo.

Demonstration of iHep Formation in a Cholestasis-Induced Liver Fibrosis Model

To rule out whether formation of in vivo iHeps is restricted to only CCl 4-induced liver ?brosis,we investigated this phenome-non in a well-established cholestasis-induced liver ?brosis model.To address this,we analyzed iHep formation in Lrat-Cre-mT/mG mice fed an 3.5-diethoxycarbonyl-1,4-dihydrocolli-dine (DDC)diet (Figure 7A).Similar to the CCl 4model,we de-tected iHeps that express EGFP and hepatocyte markers in mice injected with Ad.4TF (Figures 7B and 7C).We

then

Figure 7.Demonstration of iHep Formation in DDC-Induced Liver Fibrosis

(A)Schematic of the experimental design.Mice were kept on a DDC diet for a total of 4weeks.Ad.4TF was administered 2weeks after beginning the DDC diet.(B)Immuno?uorescence staining with antibodies (blue)for hepatocyte markers,albumin,MUP,FAH,and AAT.The ?gures are representative of livers obtained from three mice in each group.Scale bars,100m M.

(C)Table shows percentage of average EGFP-positive iHeps among the total hepatocyte population.(D)Reduced levels of Col1a1mRNA in Ad.4TF injected mice (n =4)compared to control mice (n =4).(E)Hydroxyproline assay showed decreased levels of entire collagen content,measured in whole liver.

(F)Sirius red and immunohistochemical staining for desmin showed less ?brosis in Ad.4TF-injected mice (n =4)compared to respective controls (n =4).Scale bars,200m M for Sirius red and 100m M for desmin staining.Right,quanti?cation of Sirius red and desmin stainings are shown.

(G–I)The iHeps were pooled,and data are shown from triplicates.(G)The iHeps show ICG uptake,oil red O staining,and PAS staining.Scale bars,100m M for ICG uptake,50m M for oil red O,and 200m M for PAS staining.(H)The iHeps and eHeps showed activity for CYP3A,CYP1A1,CYP2C9,and CYP1A2.(I)Evidence for a drug response in iHeps was demonstrated by elevated levels of Cyp1a1,Abcc2,Ugt1a1,and Oatp .

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evaluated the effect of overexpression of 4TFs on DDC-induced ?brosis and characterized those iHeps similar to CCl 4-induced liver ?brosis.Col1a1expression analyses,hydroxyproline con-tent,and Sirius red and desmin staining revealed the reduced ?brosis in 4TFs overexpressing mice (Figures 7D–7F).Further-more,iHeps isolated from the DDC model also showed the func-tional characteristics of hepatocytes (Figures 7G–7I).Therefore,in vivo iHep formation can be induced by overexpression of 4TFs in the DDC model,and 4TFs overexpression ameliorates DDC-induced ?brosis.

In-Vivo-Generated iHeps Lack Any Fibrolytic Activity

In order to determine whether in-vivo-generated iHeps exert any direct ?brolytic effect,we co-cultured sorted eHeps or iHeps from LratCre-mT/mG mice with the Col-GFP hepatic stellate cell line.Unchanged mRNA expression of myo?bro-blast markers,such as Acta1,Col1a1,and Col2a1,suggests that in-vivo-generated iHeps were unable to suppress activa-tion of stellate cells (Figure S4M).It further indicates that the observed reduction in ?brosis was due to decreased number of myo?broblasts rather than a ?brolytic effect of in-vivo-gener-ated iHeps.

Ad.4TF-Based Reprogramming Does Not Give Rise to Cells Other Than iHeps

To assess whether other types of liver cells are produced via in vivo reprogramming,we stained livers for SOX9(a liver stem/progenitor cell marker),CK19(a bile duct and liver stem/progenitor cell marker),and MIC1(an oval cell marker).Immuno?uorescence analyses showed the absence of positive staining for any of the above-mentioned markers in cells with EGFP-positive membranes in the Ad.4TF-administered,CCl 4-treated,LratCre-mT/mG model (Figure S5).Thus,we con?rmed that Ad.4TF injection in ?brotic livers leads to formation of iHeps in vivo without forming cholangiocytes or liver stem/pro-genitor cells.

Since p75NTR is also expressed in tissues other than ?brotic livers,we also considered the possibility whether Ad.4TF admin-istration converts cells of other organs into iHeps.However,our immuno?uorescence staining for albumin and HNF4A revealed absence of positive cells in the brain,heart,lung,and kidney (Fig-ure S6).Thus,our results indicate the absence of iHeps forma-tion in organs other than the liver.

In-Vivo-Generated iHeps Are Not the Result of Cell Fusion

Next,we sought to investigate whether in-vivo-generated iHeps could be a result of cell fusion.To address this,we stained un-sorted iHeps and eHeps,isolated from LratCre-mT/mG mice,for p75NTR,a myo?broblast-speci?c marker.By analyzing immuno?uorescence stainings on more than 1,000iHeps,we could not detect any iHeps,which were also positive for p75NTR (Figure S7).Furthermore,our aCGH analyses of in-vivo-generated iHeps did not show any increase in ploidy compared to equal DNA amounts of eHeps used as the refer-ence sample (Figure S2F),thus suggesting absence of cell fusion.Together,these data suggest that in-vivo-generated iHeps are the result of direct reprogramming rather than cell fusion events.

DISCUSSION

Our ?ndings indicate that simultaneous expression of FOXA3,GATA4,HNF1A ,and HNF4A can reprogram mouse myo?bro-blasts into cells with a hepatocyte-like phenotype.Targeted,cell-type-speci?c expression of the TFs in mice results in a phenotype change of liver myo?broblasts into hepatic cells,which triggers tissue remodeling in chronic liver failure.Degrada-tion of extracellular matrix,a decrease in myo?broblast numbers,and hepatocyte proliferation are considered necessary steps for effective resolution of ?brosis and recovery of the liver (Friedman et al.,2013;Kendall et al.,2009).Hence,the conversion of pro?-brogenic myo?broblasts into hepatocyte-like cells would not only attenuate the development of ?brosis but also improve liver function in chronic liver failure.Indeed,we provide evidence for amelioration of liver ?brosis following up to 8weeks of CCl 4treat-ment in mice with iHeps.The observed amelioration of liver ?brosis by reprogramming of myo?broblasts into iHeps in vivo can be explained by two synergistic effects.On one hand,conversion of certain numbers of myo?broblasts,the major contributor of liver ?brosis,into iHeps,reduces the number of pro-?brogenic myo?broblasts.In fact,reduced numbers of my-o?broblasts can indeed ameliorate liver ?brosis (Puche et al.,2013).Notably,in both lineage-tracing mouse models,iHeps often appeared near the portal vein or central vein regions.Therefore,it is possible that a few iHeps may be generated from perivascular mesenchymal cells,including portal ?bro-blasts,smooth muscle cells around the portal vein,and ?bro-blasts around the central vein.On the other hand,generation of numerous new iHeps,which possess functional characteris-tics of primary hepatocytes,restores the deteriorating liver func-tion that is frequently observed during liver ?brosis.

There are a few unanswered questions that would require future studies to address them.First,it remains unclear why iHeps were detected only in ?brotic livers and not in normal mice.A plausible explanation is that quiescent HSCs present in normal liver express p75NTR at very low levels compared to its very high expression in myo?broblasts seen in ?brotic livers.This may,in turn,lead to suboptimal expression of 4TF in quiescent HSCs compared to their optimal expression in myo?-broblasts.Additionally,the ?brotic milieu may also facilitate gen-eration of iHeps from myo?broblasts.

Second,it remains a possibility that iHeps are formed via an intermediate stage that resembles liver progenitor cells.A comprehensive characterization of iHeps between the time when they ?rst appear and 30days after Ad.4TF administration would not only answer whether iHeps are generated via a pro-genitor stage but also uncover key regulatory pathways involved in iHep formation.

Third,we cannot rule out the possibility that overexpression of 4TFs in endogenous hepatocytes may contribute to reduction in ?brosis,since overexpression of HNF4A in hepatocytes has recently been suggested to revert long-term CCl 4induced dam-age in a rat model (Nishikawa et al.,2015).Our data,however,indicate that the direct conversion of myo?broblasts into hepato-cyte-like cells is the more prominent factor in our experimental setup,since the delivery of 4TFs was preferentially targeted to myo?broblasts.The modest transduction ef?ciency of myo?bro-blasts in our in vivo experiments might explain the observation

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that amelioration of liver?brosis(8-week CCl4),but not of cirrhosis(12-week CCl4),was detectable.This latter?nding can be attributed to a higher total number of myo?broblasts in cirrhotic livers,from which only a limited number(similar to that in?brotic livers)was ef?ciently transduced.

In summary,our study demonstrates the direct conversion of pro-?brogenic myo?broblasts in vivo into hepatocyte-like cells in the liver and shows that this can indeed ameliorate?brosis in damaged livers.This approach provides a promising therapeutic potential for the treatment of chronic liver disease.Therefore, future studies should be aimed to enhance the ef?ciency of iHep generation through improvement in targeted transduction in order to provide bene?cial therapeutic effects through iHep formation in more advanced liver?brosis.

EXPERIMENTAL PROCEDURES

Animals

12-week-old BALB/c and mT/mG(Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP) mice were obtained from Charles River Laboratories and Jackson Laboratory, respectively.Lrat-Cre transgenic mice were kindly provided by Robert Schwabe (Columbia University,New York,NY).To induce liver?brosis,mice were injected with4m l/g10%CCl4dissolved in corn oil.To induce cholestasis-induced liver ?brosis,LratCre-mT/mG mice were fed a DDC diet.The use of animals for this study was approved by the Institutional Animal Care and Use Committee of the Hannover Medical School.

Lentivirus Expression Plasmids

Third-generation self-inactivating VSV-G lentiviral vectors were derived from the vector backbone pRRL.PPT.SF.pre*(Maetzig et al.,2011;Schambach et al.,2006).The human cDNAs of the TFs FOXA3,GATA4,HNF1A,and HNF4A were cloned together into the pRRL.PPT.SF.pre*plasmid.The cDNAs were separated by proteolytic cleavage sites.All2A sites and the HNF4A cDNA were codon optimized according to favored human tRNA codons(Gen-script).This process improves translatability and mRNA half-life and removes cryptic splice and poly(A)sites.For the reporter construct,a dTomato reporter gene was cloned under the transcriptional control of minimal albumin pro-moter/enhancer sequence.

Lineage Reprogramming

13105primary mouse myo?broblasts cells were transduced with lentiviral vector encoding for4TFs at MOI5.One day after transduction,complete DMEM medium was replaced by HCM medium.On day10,cells were trans-duced with reporter lentiviral vector before trypsinization and reseeding them on collagen-coated dishes the next day.Afterward,cells were main-tained in HCM medium.Medium was changed every2days.14days after?rst transduction,the cells were tested for protein secretion and CYP activity or harvested for mRNA isolation.

Generation of Recombinant Adeno-Associated Virus and Adenovirus Vectors

AAV8.Ttr.Cre vector was prepared as described previously(Sharma et al., 2011).Adenovirus serotype-5-derived wild-type vector(Ad.GFP)expressing GFP and Ad5.FOXA3.GATA4.HNF1A.HNF4A expressing the4TFs were generated by homologous recombination following cotransfection with pAdEasy1in E.coli BJ5183.Ad vectors were propagated in HEK293cells, puri?ed by CsCl buoyant density centrifugation,and measured at an optical density of260.Peptide coupling and titration of the vectors were performed as previously described(Reetz et al.,2013).

Histology,Immunohistochemistry,and Immuno?uorescence

Liver tissues were?xed with4%formalin,embedded in paraf?n,and cut into 5-m m-thick sections for histological and immunohistochemical analysis.For Sirius red staining,following deparaf?nization,the sections were stained with Picro-Sirius red solution(0.1%direct red80plus0.1%fast green dis-solved in1.2%saturated aqueous picric acid solution,all from Sigma-Aldrich) and incubated for60min.Sections were rinsed with water,dehydrated,and mounted in xylene.Immuno?uorescence stainings for albumin(Abcam, 19196),MUP(Santa Cruz,21856),FAH(Abcam,81087),AAT(Abcam, 117307),p75-NTR(Abcam,8874),desmin(Thermo Scienti?c,RB-9140), CD45(BioLegend,103106),CD31(Abcam,56299),F4/80(Abcam,6640), SOX9(Millipore,AB5535),HNF4A(Santa Cruz,6556),CK19(Abcam-15463),MIC1(Thermo Scienti?c,MA5-16136),and Acta1(Abcam,5694) were performed on frozen sections following a standardized protocol.Quanti-?cation of immuno?uorescence or immunohistochemical staining was per-formed using ImageJ software in a blinded manner.

Global Gene Expression Analysis

Whole Mouse Genome Oligo Microarray v2(4x44K)(Agilent Technologies)was used to characterize global gene expression pro?les of iHeps compared to myo?broblasts and primary mouse hepatocytes.All microarrays were per-formed at the Research Core Unit Transcriptomics of the Hanover Medical School.Brie?y,total RNA was used to prepare the aminoallyl-UTP-modi?ed (aaUTP)cRNAs(Amino Allyl MessageAmp II Kit,#AM1753;Life Technologies) as directed by the company.The aaUTP-cRNAs were labeled with Alexa Fluor 555Reactive Dye(#A32756;Life Technologies).Prior to the reverse transcrip-tion reaction,1m l of a1:5,000dilution of Agilent’s One-Color spike-in Kit stock solution(#5188-5282,Agilent Technologies)was added to100ng total RNA of each analyzed sample.

The cRNA fragmentation,hybridization,and washing steps were carried out according to Agilent’s One-Color Microarray-Based Gene Expression Analysis Protocol V5.7,except that500ng of each labeled cRNA sample was used for hybridization.Slides were scanned on the Agilent Micro Array Scanner G2565 CA(pixel resolution5m m,bit depth20).Data extraction was performed with the Feature Extraction Software V10.7.3.1.

Statistical Analyses

Signi?cance was determined with two-tailed,two-sample equal variance Student’s t test.A p value of<0.05was considered as signi?cant.Error bars represent±SEM.*p<0.05,**p<0.005,and***p<0.0005.

ACCESSION NUMBERS

The accession number for the gene expression data reported in this paper is GEO:GSE76843.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and seven?gures and can be found with this article online at http://dx.doi. org/10.1016/j.stem.2016.01.010.

AUTHOR CONTRIBUTIONS

M.O.and A.D.S.conceived the idea,designed experiments,provided the con-ceptual framework for the study,and wrote the manuscript.G.S.,A.B.,and T.C.further contributed to manuscript preparation.G.S.,M.P.,A.B.,Q.Y., H.-C.T.,Z.D.,and D.S.performed the experiments.G.S.,M.P.,and Q.Y. analyzed the data. D.Y.and S.B.prepared AAV-Ttr-Cre virus.J.R.and B.M.P.provided S11-NGF p peptide and Ad5.FOXA3.GATA4.HNF1A.HNF4A for targeting of myo?broblasts in vivo.A.B.and M.A.-B.analyzed microarray data.A.S.provided lentiviral vector for overexpression of4TFs.R.F.S.pro-vided LratCre mice.T.L.,R.F.S.,M.P.M.,H.R.S.,and T.C.contributed to con-ceptual evaluation of the project.

ACKNOWLEDGMENTS

The study was?nanced by the Deutsche Forschungsgemeinschaft(DFG SH640/1-2,DFG EXC62/2,DFG188/9-1,and SFB-738),Gilead Sciences In-ternational Research Scholars Program in Liver Diseases,and the Bundesmi-nisterium fu¨r Bildung und Forschung(Biodisc6,START-MSC II).G.S.and Z.D.

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1、 项目提出的背景和依据 信息系统项目可行性研究报告(建议书) 编制要求 (带*号的内容建议书不作要求) 第一章 项目概述 1 、 项目名称 2 、 项目建设单位及负责人、项目负责人 3 、 编制单位 4 、 编制依据 5 、 项目建设目标、规模、内容、建设期 6 、 项目总投资及资金来源 7 、 经济与社会效益* 8 、 相对项目建议书批复的调整情况* 9 、 主要结论与建议 第二章 项目建设单位概况 1、项目建设单位与职能 业务功能、业务流程、业务量、信息量等分析与预测 * 2、 项目实施机构与职责 第三章 项目建设的必要性 2、

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六、地块SWOT分析 七、项目评价 第六部分项目定位 一、项目目标设置 二、项目整体定位策略 三、项目定位建议 第七部分项目整体规划分析 一、项目规划设计可行性分析 二、项目规划设计的主题及概念 第八部分项目开发建设进度安排与销售节点 一、项目分期开发设置 二、工程计划 三、销售节点 第九部分投资估算与资金筹措 一、成本预测 二、税务分析 三、资金筹措 四、资金投放使用计划 第十部分销售收入测定 一、销售收入测算 二、销售利润测算 第十一部分财务与敏感性分析 一、项目盈利能力分析 二、项目盈亏平衡分析 三、项目敏感性分析 第十二部分综合评价 一、经济评价(定性) 二、社会评价(定性) 三、环境评价 四、市场预测

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时,以防止再次出血。对于上述病例,通常的治疗时间是120小时。对胰瘘、胆瘘、肠瘘的辅助治疗:应采用每小时250微克的速度静脉连续点滴给药,直到瘘管闭合(2至20天),这种治疗可作为全胃肠外营养的辅助措施。当瘘管闭合后,思他宁静脉点滴应继续进行1至3天,而后逐渐停药,以防反跳作用。对胰腺外科手术后并发症的预防和治疗:手术开始时,作为辅助治疗,以每小时250微克速度点滴思他宁;手术后,持续点滴给药5天。对糖尿病酮症酸中毒的辅助治疗:对酮症酸中毒的患者,以每小时100至500微克的速度静脉点滴思他宁同时配合胰岛素治疗,3小时内可缓解酮症酸中毒,4小时内可使血糖恢复正常。 【不良反应】 少数病例用药后产生恶心、眩晕、脸红等反应。当滴注思他宁的速度高于每分钟50微克时,病人会发生恶心和呕吐现象。 【禁忌】 已证实对于思他宁药物过敏的病人,不得使用此药。避免孕妇使用本品,除非无其它安全替代措施。 【注意事项】 1 由于本品抑制胰岛素及胰高血糖素的分泌,在治疗初期会引起短暂的血糖水平下降。特别是胰岛素依赖型糖尿病患者,使用本品后每隔3-4小时应测试一次血糖浓度。同时,给药期间应避免给予胰岛素所要求的葡萄糖。在必要情况下,应同时使用胰岛素; 2 本品必须在医生指导下使用。 【特殊人群用药】 妊娠与哺乳期注意事项: 避免孕妇使用本品,除非无其它安全替代措施。

项目情况简介

项目情况简介: 一:《织金县江西煤矿》采矿权项目: (一):情况简介: 织金县化起镇江西煤矿位于织金县城东50km,隶属贵州省织金县化起镇管辖,面积2.4993Km2,平面范围由9个直角坐标拐点圈定,准采标高+1490~+1100m,矿山北西部有织金至化起公路由通过,交通方便。矿区范围地质勘探达到勘探程度,地质总储量1544.48万吨(其中准采标高范围内地质储量1239.92万吨,+1100m标高以下地质储量304.56万吨),全区可采煤层4层。总厚度平均5.6m,设计生产规模30万吨/年, 矿区为典型的低中山构造溶蚀—剥蚀地貌。区内最高点为北西部山顶(海拔标高为+1553.6m),最低点位于勘探区南东部小河洞落水洞(海拔标高+1365m),区内相对高差50~188m。区内地表水系属乌江流域鸭池河水系,区内主要发育季节性河流及冲沟。 《织金县江西煤矿》采矿权项目为资源整合矿井项目,企业性质为合伙制企业。现有一设计能力3万吨/年的矿井正在生产,江西煤矿新井设计规模30万吨/年,该矿井预计2010年6月份建成投产。 在矿区北部原永胜煤矿为上世纪90年开采的小煤窑(年产量在3万吨以下),在2005年以前已停采。2006年下半年,国家对小煤矿进行整顿,将江西煤矿和永胜煤矿进行整合,整合后的矿区范围为上述拐点坐标范围,开采量提升为30万吨/年。在建设新的矿井前,现有矿井继续生产,目前正在生产中。

(二):矿区地质构造情况: 矿区大地构造位属于扬子准地台贵阳复杂构造变形区西段。区内位于牛场向斜北西翼,以发育北东向的断裂构造和南北向断裂构造为特征,断裂以发育北东向正断层和逆断层为主,其次为南北向正断层。断层对区内煤层均有不同程度有切错。根据区内地质构造特征和遵照《煤、泥炭地质勘探规范》要求,确定区内总体构造复杂程度为中等复杂类型 (一)褶皱 矿区位于牛场向斜北西翼。地层总体倾向130~210o,倾角8~31o。 (二)断层 矿区断裂构造有3条北东向断层和一条南北向断层。除南北向断层规模较大外,其它断层规模较小。断层具体情况如表: (三):煤层情况及特性: 根据钻孔控制情况,区内发育不稳定或较稳定的可采煤层有M6、M15、M16、M18、M20、M21、M27、M29、M32九层。其中M6、M15、M20、M27、M32五层煤局部可采;M18、M21、M16、M29大部可采,为区内主要可采煤层。各可采煤层瓦斯平均含量10.31~

专利项目可行性研究报告(提纲)(1)

项目可行性研究报告 (提纲) 一、概述 1.申请项目的概述。应包括项目中专利的基本情况、项目的主要内容、技术水平,主要用途及 应用范围(限400字以内。)。 2.简述项目的社会经济意义、目前的进展情况、申请专利实施资金的必要性。 3.简述本企业实施项目的优势和风险。 4.项目计划目标 二、申报企业情况 包括企业基本情况、项目负责人及实施人员情况、企业转化能力、企业财务经济状况、企业管理情况、企业发展思路等。 三、技术可行性分析 1.详细说明本项目的基本原理及关键技术内容及项目涉及专利的情况。 2.国内外同类产品的专利检索情况(产品核心技术的专利情况),本项目产品技术性能水平与其的比较。 3.本企业及技术依托单位或合作单位的研究开发实力。 四、项目成熟程度 1.产品的专利侵权分析。 2.成果的技术鉴定文件或产品性能检测报告、产品鉴定证书。 3.产品质量稳定性和成品率情况等。 五、市场需求情况 1.国内市场状况及产品的发展前景,在国内市场的竞争能力和市场占有率。 2.国际市场状况及产品的发展前景,在国际市场的竞争能力,产品替代进口或出口的可能性。 六、投资估算及资金筹措 1.项目投资估算 2.资金筹措方案 3.投资使用计划

七、项目实施进度计划 八、经济和社会效益分析 1.生产成本估算、销售收入估算。 2.财务分析,以动态分析为主,提供财务内部收益率、贷款偿还期、投资回收期、投资利润率和利税率、财务净现值等指标。 3.不确定性分析,主要进行盈亏平衡分析和敏感性分析,对项目的抗风险能力作出判断。 4.财务分析结论。 5.社会效益分析。 九、结论 十、其它 1.项目实施所需的基础设施及原辅材料(包括燃料)的来源、供应渠道等情况。 2.环境保护措施。 3.劳动保护和安全。 4.必要的证明材料: (1)特殊行业许可证(如食品、医药、农药、化肥产品生产许可证及批文);通信产品入网许可证;公共安全产品生产许可证;压力容器生产许可证等。 (2)可提供项目立项证明、高新技术企业证书、产品质量认证、环保证明;产品订货意向、合同等补充材料。

生长抑素

注射用生长抑素 【适应症】 1.严重急性食道静脉曲张出血; 2.严重急性胃或十二指肠溃疡出血,或并发急性糜烂性胃炎或出血性胃炎;3.胰腺外科手术后并发症的预防和治疗; 4.胰、胆和肠瘘的辅助治疗; 5.糖尿病酮症酸中毒的辅助治疗。 【用法与用量】 静脉给药。 通过慢速冲击注射(3~5分钟)0.25mg或以每小时0.25mg的速度连续滴注给药(一般每小时每公斤体重用药量为0.0035mg)。 临使用前,每支冻干剂用1ml生理盐水溶解。 对于连续滴注给药,须用本品3mg配备够使用12小时的药液(溶剂可为生理盐水或5%的葡萄糖注射液),输液量调节在每小时0.25mg。 1.严重急性上消化道出血包括食道静脉曲张出血的治疗:首先缓慢静脉推注0.25mg(用1ml生理盐水配制)作为负荷量,而后立即进行以每小时0.25mg的速度持续静脉滴注给药。当两次输液给药间隔大于3-5分钟的情况下,应重新静脉注射本品0.25mg,以确保给药的连续性。当出血停止后(一般在12-24小时内),继续用药48-72小时,以防再次出血。通常常的治疗时间是120小时。 2.胰瘘、胆瘘和肠瘘的辅助治疗:以每小时0.25mg的速度静脉连续滴注,直到瘘管闭合(2~20天),这种治疗可以用作全胃肠外营养的辅助措施。当瘘管闭合后,应继续用药1~3天,而后逐渐停药,以防反跳作用。 3.胰腺外科手术后并发症的治疗:在手术开始时,以每小时0.25mg的速度静脉滴注,术后持续静滴5天。 4.糖尿病酮症酸中毒的辅助治疗:以每小时0.1~0.5mg的速度静脉滴注,作为胰岛素治疗(10单位冲击后每小时1~4.8单位静滴)的辅助措施,在4小时内可以使血糖恢复正常,在3小时之内缓解酮症酸中毒。 【不良反应】 少数病例用药后出现恶心、眩晕、面部潮红。当注射速度超过每分钟0.05mg时,病人会发生恶心和呕吐现象。 【禁忌症】对本品过敏者禁用。 【注意事项】 (1)由于本品抑制胰岛素及胰高血糖素的分泌,在治疗初期会导致血糖水平短暂的下降;

项目概述及现状分析

第一章项目概述及现状分析 蟠桃居住区地处徐州市经济开发区中部,北临开发区主干道杨山路,东靠经六路、西至经五路。总规划用地面积约270亩(18ha)。该项目是开发区管委会贯彻中央建设社会主义新农村第一批试点项目,是开发区重点工程,安民工程。 居住区的服务定位是集中安置拆迁村民及企业产业工人,二者在工作,生活习惯上并不相同,设计中既要保证二者有一定联系,又要区别对待,因此蟠桃居住区不同与以往的居住区规划。其规划设计应充分利用现有的有利条件,通过合理的设想,完善的规划理念进行统一规划、实施。如何能适应当代的农民生活需要,体现其地方特点,是设计中首要考虑的。本设计以现代农村居住水准为目标,积极采用新方法和新观念,在兼顾居住环境质量和综合经济效益的同时,以“地方性居住环境”为主题力求创造舒适优美、方便的居住环境,促进该地区住宅建设和新型住宅产业的形成和发展。 第二章设计依据 一、徐州市经济开发区管委会发出的设计邀标文件。 二、徐州市经济开发区管委会提供的“蟠桃居住区地形图”及居住区相关资料; 三、国家有关城市规划、建筑设计法规、标准、规范等。 第三章设计理念 如何体现出社会主义新农村的特点,使之既具有现代化的特点又有其自身的底蕴是设计中面临的最大矛盾。蟠桃居住区整个用地达18公顷,势必要有一套完善的系统。 人作为自然的产物,处于天地之间,社会之中,对于自然具有依赖性和亲和

力,随着人们对自然的渴望,都希望营造一个幽美典雅的环境。因此,设计中以生态环境优先为原则,充分体现对人的关怀,坚持以人为本,大处着眼,整体设计。在规划的同时,辅以景观设计,最大限度的体现居住区本身的底蕴,设计中尽量保留居住区原有的积极元素,如居住区主要干道及商业街道路均由原有主干道发展而来,既节约了建设投资又有利于分期建设。 在设计中,规划布局不拘泥于传统模式,以现代的手法体现传统民居的内涵,力求神似。通过用现代建筑及空间形式,巧于因借的设计手法,很好地诠释了一个有着自己文化韵味居住区。 第四章总体规划 一、总平面布局: 如何合理利用原有条件:社会主义新农村不是彻底抛弃原有的,而是在其基础上发展创新。从原有主道路出发设计既可以保留居住区积极元素,又对居住区分期建设有利。 由于生活工作习惯的不同,为了避免造成不必要的干扰,设计中把企业产业工人和拆迁安置居民分开安置,并在各自内部以组团形式存在,形成居住区--居住组团的结构.采取这种结构形式的优点是:最大限度的延续了原有村子中邻里之间的关系,而不同性质的企业产业工人也可以相对集中安置.有利于各自管理。各组团空间的开敞性和通透性方面体现着传统韵味,最大限度与自然亲和。组团间通过步行景观通道串联各个内庭,形成景观轴线和广场空间。组团封闭式管理,大区开放。 将原有部分居住区内部干道演变成商业步行街,并通过一条东西向绿化步行带连通了企业产业工人公寓和拆迁安置居民小区,使之即分离又有着一定的联系。公建则安置在满足其服务半径的位置。公建适当集中安置,形成商业步行街。辐射至绿化景观带上,为居住区中心聚集了足够的人气。

项目可行性研究报告范本

项目可行性研究报告范本 第一章项目总论 第二章项目背景和发展概况 第三章市场分析与建设规模 第四章建设条件与厂址选择 第五章工厂技术方案 第六章环境保护与劳动安全 第七章企业组织和劳动定员 第八章项目实施进度安排 第九章投资估算与资金筹措 第十章财务效益、经济与社会效益评价 第十一章可行性研究结论与建议 第一章项目总论 总论作为可行性研究报告的首章,要综合叙述研究报告中各章节的主要问题和研究结论,并对项目的可行与否提出最终建议,为可行性研究的审批提供方便。总论章可根据项目的具体条件,参照下列内容编写。 §1.1 项目背景

§ 1.1.1 项目名称 企业或工程的全称,应和项目建议书所列的名称一致。 § 1.1.2 项目承办单位 承办单位系指负责项目筹建工作的单位(或称建设单位),应注明单位的全称和总负责 人。 §1.1.3 项目主管部门 注明项目所属的主管部门。或所属集团、公司的名称。中外合资 项目应注明投资各方所属部门。集团或公司的名称、地址及法人代表的姓名、国籍。 § 1.1.4 项目拟建地区、地点 § 1.1.5 承担可行性研究工作的单位和法人代表 如由若干单位协作承担项目可行性研究工作,应注明各单位的名称及其负责的工程名称、总负责单位和负责人。如与国外咨询机构合作进行可行性研究的项目,则应将承担研究工作的中外各方

的单位名称、法人代表以及所承担的工程、分工和协作关系等,分别说明。 §1.1.6 研究工作依据 在可行性研究中作为依据的法规、文件、资料、要列出名称、来源、发布日期。并将其中必要的部分全文附后,作为可行性研究报告的附件,这些法规、文件、资料大致可分为四个部分: (1)项目主管部门对项目的建设要求所下达的指令性文件;对项目承办单位或可行性研究单位的请示报告的批复文件。 (2)可行性研究开始前已经形成的工作成果及文件。 (3)国家和拟建地区的工业建设政策、法令和法规。 (4)根据项目需要进行调查和收集的设计基础资料。 §1.1.7 研究工作概况

项目概述

目录 第一章.项目概述 (2) 第二章.项目整体性规划 (4) 第三章.需求分析及项目建设方案 (6) 第四章.市政基础设施保障 (8) 第五章.资源利用和能源消耗 (10) 第六章.土地性质及出让方式 (11) 第七章.环境和生态影响分析 (14) 第八章.经济影响分析 (15) 第九章.结论 (16)

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