research article isolation and time lapse microscopy of...

Research ArticleIsolation and Time Lapse Microscopy ofHighly Pure Hepatic Stellate Cells

Matthias Bartneck1 Klaudia Theresa Warzecha1

Carmen Gabriele Tag12 Sibille Sauer-Lehnen12 Felix Heymann1

Christian Trautwein1 Ralf Weiskirchen2 and Frank Tacke1

1Department of Medicine III RWTH University Hospital Aachen Pauwelsstrasse 30 52074 Aachen Germany2Institute of Molecular Pathobiochemistry Experimental Gene Therapy and Clinical Chemistry RWTH University Hospital AachenPauwelsstrasse 30 52074 Aachen Germany

Correspondence should be addressed to Frank Tacke franktackegmxnet

Received 17 April 2015 Revised 23 June 2015 Accepted 24 June 2015

Academic Editor Nady Braidy

Copyright copy 2015 Matthias Bartneck et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis We aimed at optimizing HSC isolation by an additional stepof fluorescence-activated cell sorting (FACS) via a UV laser HSC were isolated from livers of healthy mice and animals subjectedto experimental fibrosis HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method withsubsequent FACS-based sorting We assessed cellular purity viability morphology and functional properties like proliferationmigration activation marker and collagen expression FACS-augmented isolation resulted in a significantly increased purityof stellate cells (gt99) compared to iohexol-based density centrifugation (60ndash95) primarily by excluding doublets of HSCand Kupffer cells (KC) Importantly this method is also applicable to young animals and mice with liver fibrosis Viabilitymigratory properties andHSC transdifferentiation in vitrowere preserved upon FACS-based isolation as assessed using time lapsemicroscopy During maturation of HSC in culture we did not observe HSC cell division using time lapse microscopy StrikinglyFACS-isolated differentiated HSC showed very limited molecular and functional responses to LPS stimulation In conclusionisolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cellpopulations especially KC without affecting HSC viability migration or differentiation

1 Introduction

Hepatic stellate cells (HSC) are the main effector cells inliver fibrosis [1] In homeostatic conditions they reside inthe perisinusoidal space of Disse store vitamin A and areinvolved in maintaining tissue integrity [2] In case of liverinjury HSC can be activated by different stimuli such asmacrophages [3] or danger-associated signals [4] ActivatedHSC were found to release proinflammatory mediators andtransdifferentiate into myofibroblasts which are highly pro-liferative and produce large amounts of extracellular matrixproteins such as collagen types I and IIIThis process leads tothe excess production of hepatic connective tissue ultimatelyleading to hepatic fibrosis and reduced in liver functionality[5]

ActivatedHSCare considered one of themajor target cellsfor antifibrotic therapies because they are the main contrib-utors of hepatic extracellular matrix [6] In order to studyHSC biology and to evaluate therapeutic strategies affectingHSC activation or functionality primary HSC isolation fromhuman mouse or rat liver is an evitable tool in experi-mental fibrosis research Early attempts to isolate HSC frommouse or rat livers were based on centrifugal fractionationandor centrifugal elutriation [7 8] Subsequent methodsincorporated the simultaneous isolation of different hepaticcell populations based on density gradient centrifugationwith Stractan [9] With the rise of flow cytometry and flowcytometric cell sorting early attempts for flow cytometric cellsorting were based on the strong sideward scattering of HSCdue to the specific intracellular (retinol) droplets [10] Later

Hindawi Publishing CorporationAnalytical Cellular PathologyVolume 2015 Article ID 417023 13 pageshttpdxdoiorg1011552015417023

2 Analytical Cellular Pathology

strategies incorporated multiplex staining of surface markersand cell sorting to exclude cell types other than HSC fromcell purifications However the purity of all these strategiesfor HSC isolation remained disputed since antibody stainingmay affect cell populations [11] Moreover there is no reliablesurfacemarker known that is generally expressed onHSCandmyofibroblasts which hampers positive selection strategiesbased on antibody staining [5] Some surface markers thathad been suggested forHSC isolation include platelet-derivedgrowth factor 120573 (PDGFR-120573) or low-affinity nerve growth fac-tor receptor (p75) while other investigators tested intracellu-lar glial fibrillary acidic protein (GFAP) staining to identifyVitamin A+ HSC [1 5 12] FACS sorting of HSC has beenlately employed by several groups including ours by using aUV laser that specifically excites the stored retinol droplets inresting HSC [12ndash18] However it remained unclear whetherthis technique would alter functional properties of HSC suchas migratory properties relevant to wound healing responses

The current ldquogold standardrdquo for HSC isolation is stillbased on density centrifugation using iohexol (also knownunder their brand names Nycodenz Exypaque or Omni-paque) which separates the HSC due to its physical prop-erties from other hepatic cells and usually results in a highnumber of viable HSC applicable to cell culture experimentsWe hypothesized that via the ldquoconventionalrdquo density gradientmethod cell aggregates between HSC and other cell typesespecially Kupffer cells (KC) may occur which in turn resultin cellular impurities that could lead to contradictory resultson distinct HSC functions For example it is heavily debatedin the field whether HSC can act as antigen-presenting cellsas studies have led to conflicting results on this topic [11 19]Also the notion that HSC strongly respond to bacterial prod-ucts like lipopolysaccharides (LPS) [20 21] requires that thereis no relevant contamination with macrophages which areequipped with manifold danger-recognition receptors [22]

In this study we optimized the ldquoconventionalrdquo method-ology for the isolation of HSC based on iohexol densitycentrifugation and compared it to an additional step offluorescence-activated cell sorting (FACS) based on anti-bodies and UV-autofluorescence with respect to cellularpurity viability yield and cellular characteristics We furtheranalyzed the behavior of highly pureHSC in vitro by studyingtheir cellular morphology and maturation over five days ofculture using time lapse microscopy as well as migratoryproperties in an assay for cell migration and after stimulationwith LPS By implementing an additional step of cell sortingto the current ldquogold standardrdquo HSC isolation method ourprotocol results in significantly improved cellular puritywhich helps to clarify HSC functions

2 Materials and Methods

21 Ethics Statement All in vivo experimentswere performedfollowing approval by the State Animal Protection Board atthe Bezirksregierung Cologne Germany The investigationconforms to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health(NIH Publication Number 85-23 revised 1996)

22 Mice C57BL6J wild-type mice at 40ndash50 weeks of age ifnot stated otherwise were housed in a specific pathogen-freeenvironment To induce liver fibrosis carbon tetrachloride(CCl4 06mLkg Sigma-Aldrich Taufkirchen Germany)

was injected intraperitoneally two times per week for sixweeks control animals received the vehicle (corn oil) [13] Allanimal experiments have been approved by the InstitutionalReview Board and by the German legal authorities (LANUVRecklinghausen Germany)

23 Liver Perfusion Enzymatic Digestion and DensityGradient Centrifugation Mice were anaesthetized using7mgkg body weight xylazine and 105mgkg body weight ofketamine The liver was perfused via the Vena portae using a26G needle (BD Franklin Lakes USA) that was fixed usinga Bulldog clamp (Aesculap Tuttlingen Germany) Bufferswere prewarmed to 37∘C and pumped into the liver via theabdominalVena portae and drained via theVena cava inferiorusing a peristaltic pump at a flow rate of 65mLminute

Initially perfusion buffer 1 (8 gL NaCl 400mgL KCl78mgL NaH

2PO4times H2O 151mgL NaHPO

4times 2 H

2O

2380mgL HEPES 350mgL NaHCO3 190mgL EGTA

900mgL glucose and 6mgL phenol red adjusted to pH73ndash74 using 10N NaOH sterile filtered and kept at 4∘Cuntil use) was injected into the liver for 45 minutes Secondperfusion buffer 2 (8 gL NaCl 400mgL KCl 78mgLNaH2PO4times H2O 151mgL NaHPO

4times 2 H

2O 2380mgL

HEPES 350mgL NaHCO3 560mgL CaCl

2times 2 H

2O and

6mgL phenol red adjusted to pH 73ndash74 using 10N NaOHsterile filtered and kept at 4∘C until use) was applied for45 minutes and supplemented with 05mgmL pronaseE (Merck Darmstadt Germany) Third perfusion buffer2 which was supplemented with 075UmL collagenase P(Roche Basel Switzerland)was administered for 45minutesThe perfused liver was removed and the gall bladder andconnective tissue sticking to the liver were detached HSCwere removed by tearing the liver into bits using two tweezers

The liver cell suspension was further digested for 20minutes in a 37∘C water bath in perfusion buffer 2 sup-plemented with 04mgmL pronase E 15 UmL collagenaseP and 002mgmL DNase I (Roche Basel Switzerland)adjusted to a pH of 72ndash74 using NaOH Cells were fil-tered using a 70 120583m nylon gaze and rinsed using perfusionbuffer 2 tempered at 4∘C and centrifuged for ten min-utes at 4∘C at 600 g The pellet was then washed using4∘C Geyrsquos buffered salt solution (GBSS) (exact composi-tion 370mgL KCl 210mgL MgCl

2times 6 H

2O 70mgL

MgSO4times 7 H

2O 75mgL Na

2HPO4times 2 H

2O 30mgL

KH2PO4 991mgL glucose 227mgL NaHCO

3 225mgL

CaCl2times H2O 8000mgL NaCl and 6mgL phenol red

adjusted to pH 73ndash74 sterile filtered and kept at 4∘C untiluse) supplemented with 001mgmLDNase I Cells were thencentrifuged for ten minutes at 4∘C at 600 g and suspended inthe same buffer (without NaCl otherwise identical) that wassupplemented with iohexol (Nycodenz Axis-Shield DundeeScotland) to a final concentration of 8

The mixture was then pipetted to the ground of a falconcontaining GBSS and centrifuged at 4∘C and 1500 g for 22minutes without brake The interphase containing enriched

Analytical Cellular Pathology 3

HSC between the GBSS and iohexol layer was removed andwashed using 4∘C tempered GBSS (for cell culture) or 4∘CHankrsquos complete (Hankrsquos buffered salt solution without cal-cium ormagnesium containing 10mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 006bovine serumalbumin (BSA) and 03mM EDTA adjusted to pH 73ndash74(for FACS) The cells were then washed for ten minutes at600 g and the supernatant was removed

In case of optional F480 staining (here only done toidentify HSC-KC doublets) that is not required routinelythe cell pellet was resuspended in 4∘C FACS staining buffer(one part of phosphate-buffered saline without calcium ormagnesium and one part of Hankrsquos complete supplementedwith each 1 of mouse human and rabbit serum and 1BSA) Staining with anti-F480 antibody (diluted 1 400)conjugated with PE-Cy7 rat anti-mouse antibody was donefor 30minutes antibody and staining buffer were removed byone time ofwashing inHankrsquos complete for tenminutes at 4∘Cand 500 gwithout brake and suspending in the same solution

24 Fluorescence-Activated Cell Sorting Cell sorting wasdone using a BD FACS Aria II SORP Cell Sorter (BD Bio-sciences Franklin Lakes NJ USA) The pellet was resolvedin 4∘C Hankrsquos complete and filtered using 40 120583m nylon gazeThe sorting of the HSC required excitation via UV laser andmeasuring the emission in the Indo-1 channel based on a505 nm long pass and a 530 plusmn 30 nm band-pass filter Thesample loading port was set to 4∘C and 300 rpm We used a100 120583m nozzle and a pressure of 20 psi HBSS with calciumor magnesium was used as sheath fluid The flow rate wasset to 5000 events per second and the threshold was adjustedto 5000 The precision mode purity was set up using a yieldmask of 32 a purity mask of 32 and a phase mask of 0 Thecollection device was set to 4∘C The collection tube was a5mL glass tube that contained 1mL of Hankrsquos BSS withoutcalcium or magnesium 10mM HEPES and 20 of fetalbovine serum (FBS) After sorting cells were centrifuged for10 minutes at 4∘C and at 750 g without brake

25 Cell Culture Viability and Purity Cells were countedusing a hemocytometer (Neubauer chamber) and 04 try-pan blue (Sigma-Aldrich St Louis MO USA) The purityafter sorting was determined by a BD FACS Aria II SORPCell Sorter (BD Biosciences Franklin Lakes NJ USA) and ahemocytometerThe cell pellet was suspended in DMEM cellculture medium supplemented with 25mM HEPES 45 gLglucose 4mM glutamine 1 penicillinstreptomycin and10FBS (all Lonza Basel Switzerland) Cell culturewas donein 24-well plates (Greiner Kremsmunster Austria) using40000 cells per well in a volume of 1mL medium

26 Fluorescence Microscopy To stain cells for actin phallo-toxin staining was done Therefore cells were grown on glassslides that were precoated with poly-L-lysine as describedearlier [23] Cells were washed before and after each stepusing 4∘C Hankrsquos buffered salt solution (HBSS) Cells werefixed using 4 paraformaldehyde for 20 minutes at 4∘CPermeabilization was done using 02 triton X-100 for 4

minutes at 4∘C A solution of 3 of BSA dissolved in PBSwas used to inhibit unspecific binding Actin staining wasdone for 20 minutes at room temperature (21∘C) using rho-damine phalloidin (Invitrogen Carlsbad CA USA) whichwas diluted 1 40 in PBS Desmin staining was done by thesame methods for fixation permeabilization and blockingThe primary antibody was antidesmin (Novus BiologicalsLittleton Colorado USA) Cells were then blocked againand the secondary antibody was Cy3 donkey anti-rabbit(Jackson Immuno Research Bethesda MD USA) diluted1 100 in 3 BSA in PBS for 90 minutes at room temperatureNuclear stainingwas done using 01 120583gmL410158406-diamidino-2-phenylindole (DAPI) in HBSS for 5 minutes at room temper-ature Finally slides were mounted using VectashieldMount-ing medium (Vector laboratories Burlingame CA USA)Micrographsweremade using aDMLB (LeicaMicrosystemsVienna Austria)

27 Staining of Liver Sections Paraffin-embedded liver sec-tions were cleared from paraffin using xylene and ethanolin ascending line and distilled water for all staining pro-cedures Hematoxylin and eosin (HampE) staining was doneby 15 minutes of incubation in hematoxylin rinsing for 10minutes using warm tap water and 5 minutes of stainingwith eosin Sirius red staining was performed using 100mgSirius Red (Polysciences Inc Warrington USA) in 100mLsaturated picric acid for staining with the pH adjusted to 2using 2N NaOH To differentiate sections were incubatedfor two minutes in 001N HCl and rinsed with tap waterMorphometric quantification of collagen fibres was doneusing Image J Staining for 120572 smooth muscle actin (120572SMA)was done using the antibodyM0851 and the Animal ResearchKit according to the instructions of the manufacturer (allDako Hamburg Germany) All sections were dehydratedusing a descending order of ethanol and xylene

28 Quantitative Gene Expression Analysis To isolate RNAfrom cells peqGOLD (peqLab Erlangen Germany) wasadded to cell pellets and RNA was purified as describedbefore [24] ComplementaryDNAwas generated from 100 ngof RNA using the Transcriptor first strand cDNA synthesiskit (Roche Basel Switzerland) The quantitative real-timepolymerase chain reaction (PCR) was performed using SYBRGreen Reagent (Invitrogen Carlsbad CA USA) using a7500 PCR system (Applied Biosystems Carlsbad CA USA)Exon-spanning primers were used and 120573-actin regulationof transcripts was employed to normalize gene expressionPrimer sequences are available upon request

29 Time Lapse Microscopy and Migration Assays Micro-graphs were taken using an Axio Observer Z1 equipped withan Axio Cam MR and an XLmulti S1 DARK LS incubatorproviding identical conditions like in a normal incubator Toprocess data we used the ZEN pro 2012 software (Carl ZeissMicroImaging GmbH Gottingen Germany) Pictures weremade every 15 minutes To evaluate the effects of cell cultureon HSC proliferation we normalized the cell number data tothe starting point


To study HSCmigration into a defined area we used cul-ture inserts for self-insertion (IBIDI Martinsried Germany)that were inserted before adding the cells to 24-well plates(Greiner Kremsmunster Austria) Cells grow among theseinserts and to initiate the migration experiment the insertswere removed and cells began to move to the empty space(similar to a ldquoscratch assayrdquo but with a well-defined area forthe cells tomigrate and as a big advantage no cells are harmedin this assay)

210 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism 50 Unpaired t-tests were performedto test significance of data

3 Results

31 Effect of Additional Sorting on Stellate Cell Purity Viabilityand Differentiation We first compared the standard methodfor HSC isolation to an optimized protocol based on addi-tional sorting of HSCThis standard method includes digest-ing the liver in anaesthetized mice using pronase and colla-genase containing buffers Pronase reduces the abundance ofother liver cells especially of liver sinusoidal endothelial cellsand hepatocytes (Figure 1(a)) Liver cells are then harvestedinto a cell culture dish (Figure 1(b)) and a postdigestion stepis done (Figure 1(c))The cellular mixture is then subjected toiohexol-based density centrifugation (Figure 1(d)) Cells canbe cultured directly (Figure 1(e)) as shown by others [25] orbe subjected to an additional step of FACS (Figure 1(f)) TheFACS-based isolation of primary HSC frommouse livers hasbeen reported from several groups [12ndash18] with considerablevariations in the exact gating strategy

Our FACS gating strategy is based on selecting cells thatexhibit a high extent of sideward scattering due to theirvitamin A droplets (Figure 2(a)) The next step consists ofexcluding cell doublets (Figures 2(b) and 2(c)) The mostdecisive step in our method is the selection of HSC basedon their emission of retinol-based autofluorescence via UVexcitation (Figure 2(d)) In the protocol it is further impor-tant to exclude the larger retinol+ cells (larger cells exhibit ahigher forward scattering FSC) from the HSC gate as theseare HSC-KC doublets indicated by their expression of F480(Figure 2(d) right side) whereas the smaller retinol+ cells areF480minus HSC (Figure 2(d) left hand side)

Using the iohexol density gradient centrifugation asstandard method of HSC purification we normally observeHSC purities of 60ndash95 HSC strongly depending on thebatch number of the enzymes (pronase collagenase) usedto digest the liver (which therefore has to be pretestedextensively) the age of mice genetic background and thegender By direct comparison FACS of HSC resulted in finalpurities of up to 995 compared to 668after iohexol-baseddensity centrifugation only (Figure 3(a)) Earlier studies thatwere claiming to reach 95 or higher levels of purity inconventional HSC isolation may have reported on retinol+cells which however might contain a considerable quantityof HSC-KC doublets [25] As shown in Figure 2(d) theseretinol+ HSC-KC doublets cannot be removed completely

by density gradient but only by stringent gating in flowcytometric cell sorting (Figure 2(d))

This became more evident by statistical evaluations offour independent experiments which showed that the num-ber of retinol+ cells was much higher than the numberof pure HSC singlets (Figure 3(b)) Furthermore the HSCderived from sorting were highly viable indicated by 893living cells similar to that after the iohexol gradient asretrieved from trypan blue staining (90) (detailed datanot shown) To our experience it is not reliably possible todiscriminate between singularHSC andHSC-KCdoublets bymicroscopic analysis but it requires FACS to remove doublets(Figure 2(d))

The dramatic improvement in HSC purity was corrob-orated by quantitative real-time PCR analyses After flowcytometry-based sorting mRNA expression of the HSC-specific decorin was further increased (Figure 3(c)) com-pared to standard density centrifugation isolation On theother hand the expression of mRNA characteristic of otherhepatic cell types such as the C-type lectin domain fam-ily 4f (Clec4f) which is expressed by macrophages theplatelet endothelial cell adhesionmolecule 1 (Pecam-1 CD31)exhibited by liver sinusoidal endothelial cells and albuminreflective of hepatocytes were strongly and significantlyreduced by additional FACS (Figure 3(c))

To further investigate the functionality of HSC afteradditional cell sorting in vitro we isolated HSC via iohexolgradient and used half of these cells for an additional step ofpurification with FACS Both cell isolates were subsequentlycultured for up to four days If only the iohexol gradient wasperformed KC were found in the culture dishes after oneday of culture (Figure 4(a)) and more visibly after four daysHSC could hardly be identified due to excessive growth ofdiverse cell types other than HSC with KC being noticeable(Figure 4(b)) Performing an additional step of FACS-basedcell isolation resulted in a significantly higher purity of cellpopulations and HSC could clearly be identified due to theirretinol droplets (Figure 4(c)) After four days of culture HSCcould still be identified and exhibited a rather stretched mor-phology whereas KC or other contaminating cell types couldnot be observed (Figure 4(d)) Immunofluorescent stainingfor desmin (Figure 4(e)) and actin (Figure 4(f)) showedthat the FACS-isolated cells expressed these characteristicmarkers of activated HSC at day 4 indicating that the sortingprocedure did not negatively impact HSC differentiation invitro

32 Suitability of the Methodology for Stellate Cell Isolationfrom Experimental Liver Injury Models To outline the acti-vation of HSC in vivo chronic toxic liver injury was inducedin 8-week-old C57BL6 mice using CCl

4for six weeks

and twice weekly a standard method for fibrosis induction[13] Liver damage was associated with necrotic areas andcharacteristic fibrotic bridging (Figure 5(a)) whereas Siriusred staining which marks collagen reflective of fibrosis con-firmed fibrosis progression in the animals treated with CCl

4

(Figure 5(b)) similar to 120572SMA which stains for activatedHSC (Figure 5(c)) Morphometric quantifications of Sirius


Endothelial cells (EC)Kupffer cells (KC)

Hepatic stellate cells (HSC)

Hepatocytes (HC)

Out In

(a) Perfusion with enzymes (b) Liver cell harvest (c) After digestion

(d) Iohexol gradient

WasteHSC

(e) Direct cell culture (f) Retinol autofluorescencebased cell sorting

8 iohexol

Debris

Hepatocytes NPC other than HSCcell debris blood

Pronase

(1)

(2)

Collagenase

Buffer White HSC layerBrownish layer with HSC-KC doublets

Culture of highly pure HSC

Retinol+ Retinolminus

Figure 1 Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation andfluorescence-activated cell sorting (FACS) (schematic depiction) In both strategies for cell purification the mouse is anaesthetized beforesurgery and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of theenzymes pronase and collagenase (a) Liver cells are harvested by gently tearing the liver into bits (b) followed by a postdigestion using acombination of both enzymes (c) The liver cells are subjected to iohexol density gradient centrifugation after which HSC and Kupffer cellsare located in the interphase between iohexol and buffer (d)The enrichedHSC layer containingHSC HSC-Kupffer cell doublets and cellulardebris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS basedon the autofluorescence of retinol using the UV laser of the cell sorter resulting in highly pure HSC (f)

red revealed a significant increase in the fibrotic areas in theliver of fibrotic mice (Figure 5(d)) Real-time PCR confirmedfibrosis development on the level of collagen 1 and 120572SMAmRNA expression (Figure 5(e)) To investigate whether ourmethod is suitable to isolate HSC from the livers of youngercontrol and diseasedmiceHSCwere then isolated from thesevia iohexol density gradient centrifugation and additionalFACS We observed that the gating strategy could also beapplied to younger diseased (Figure 5(f)) and control mice(Figure 5(g)) which are known to have fewer numbers of

HSC and in which HSCmight contain fewer retinol dropletsIn case of the diseased mice the sorting step appeared to beeven more important than in case of healthy mice becauseafter CCl

4treatment the amount of debris or other cells

compared to HSC (286) was much higher than that forcontrol littermates that were treated with the vehicle cornoil (656) (Figure 5(h)) FACS helped to get rid of cellulardebris relating to the toxic effects of CCl

4andmade it possible

to increase the purity up to 991 (Figure 5(h)) therebyallowing comparable purity to that of healthy mice


FSC-A

(a) (b) (c)

(d)

SSC-

A

SSC-A

SSC-

W

FSC-H

FSC-

W

0

0F480 FSC-A

HSC-KC doubletsSelected HSC

HSC gate HSC-KC doublets

SSC gate

FSC gateScatter gate

Retin

ol+

104

104

103

103

102

102

105

0

Retin

ol+

104

103

102

105

0

Retin

ol+

104

103

102

105

105 0F480

104103102 105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

Figure 2 Gating strategy for the purification of hepatic stellate cells using fluorescence-activated cell sorting Cells are first gated based ontheir forward and sideward scattering (a) doublets are excluded from sideward (b) and forward scattering (c) and hepatic stellate cells (HSC)are selected based on the UV light excitation of retinol (vitamin A) (d) A detailed cell-type specific staining of the Kupffer cell marker F480demonstrated that the large (here FSC-A gt 100 in the plot dotted gate) retinol+ cells are Kupffer cell- (KC-) HSC doublets that stain positivefor F480 (right hand side) whereas the smaller retinol+ cells are F480minus HSC (left hand side) By placing a sorting gate as depicted in (d)(middle plot black line) selected HSC (left plot) are pure and do not contain contaminating KC

33 Functional Studies of FACS-Isolated Stellate Cells In VitroTo further characterize the HSC in vitro we performed timelapse microscopy under different experimental conditionsDuring the first five days of HSC culture retinol dropletsmoved within the HSC but we did not observe proliferationof HSC (Video 1 in Supplementary Material available onlineat httpdxdoiorg1011552015417023 and Figures 6(a) and6(b)) During days five to seven motility of intracellularretinol droplets was increased but no HSC proliferation wasnoted (Video 2 and Figures 6(a) and 6(b))

It is well established that HSC respond to bacterialproducts like lipopolysaccharide (LPS) via toll-like receptor4 signaling [4] When LPS was added to the cultures atthe fourth day of culture for 24 hours no changes in HSCmorphology or in the spontaneous migration of HSC inthe culture dishes were observed (Video 3 Figure 6(b))A longer LPS incubation period from days five to sevendid not result in any morphological differences comparedto control conditions either (Figure 6(b) and Video 4) Tofurther study HSC functionality we performed a horizontalmigration assaywith theHSC (similar to a ldquoscratch assayrdquo butusing culture inserts which results in well-defined regions)

at day five until day seven of culture without (Video 5and Figure 6(c)) and with LPS stimulation (Video 6 andFigure 6(d)) FACS-isolated HSC showed horizontal migra-tion and the migratory capacity of the HSC was slightlyenhanced by LPS (Figure 6(d))

To further study the differentiation and effector functionsof HSC in vitro we isolated mRNA from cells directly aftercell sorting after one day of culture after five days and afteran additional stimulation with LPS at day four until day fiveof culture We found that the HSC-characteristic activationmarkers collagen 1 (Col1A1) and 120572SMA were upregulatedstarting at the first day of culture and further increase at day5 but with comparatively low increase after treatment withlipopolysaccharides (LPS) The transforming growth factor120573 (TGF120573) however was only weakly affected during culture(Figure 6(e))

4 Discussion

Mechanistic studies in liver fibrosis research using highlypure HSC rely on a robust method that guarantees cellular


0

FSC-A

After additional sortingAfter iohexol gradient

995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)

Figure 3 Comparison of the purity of hepatic stellate cells isolated via iohexol gradient without or with subsequent cell sorting Purityof hepatic stellate cells (HSC) before (left top) and after fluorescence-activated cell sorting (FACS right top) (a) based on their retinolautofluorescence only (dashed line) or additional exclusion of HSC-Kupffer cell doublets (highly pure real HSC straight line) The statisticalsummary of retinol+ cells compared to HSC (with HSC-KC doublets excluded) is depicted (b) Analysis of purity using cell type-specificmarkers for major cell populations Decorin is considered as a marker for hepatic stellate cells C-type lectin domain family 4f (Clec4f) is agene expressed by Kupffer cells the plateletendothelial cell adhesionmolecule 1 (Pecam-1 CD31) is mainly expressed by endothelial cells andalbumin is a marker for hepatocytes (c) Data are given relative to the expression of 120573-actin of the cells derived from perfusion and digestionMean data plusmn SD of 119899 = 4 independent experiments

purity and functionality However especially ldquoconventionalrdquoHSC isolation suffers from high levels of variation causedby the batch-dependent quality of the enzymes used forliver digestion age gender genetic background and thetreatment of the mice [5] The FACS-based sorting of HSChas been reported from several groups including ours withconsiderable variations in the protocols and without com-prehensive analyses on the functional properties of FACS-isolated HSC [12ndash18] Many researchers in the field believethat FACS-based cell sorting is insufficient to retrieve highenough HSC numbers for functional in vitro experimentsand furthermore may affect the viability of HSC

In this study we demonstrate that FACS is actuallyrequired to retrieve highly pure and functional HSC and that

thismethodology allows functional in vitro experimentationswith sufficient cell numbers and unaffected viability over atleast one week of culture Moreover this technique is alsoapplicable to the isolation from young animals (eg 12 weeksof age) as well as to mice subjected to standard liver injurymodels like repetitive CCl

4administration over 6 weeks

Especially the isolation of HSC from fibrotic livers using thedensity gradient centrifugation method without FACS hasyielded conflicting results because gene array analyses fromthese HSC did not match with culture-activated HSC [26] Atthis time the authors found that the addition of KC or LPS toculturedHSC shifted the gene expression pattern towards thein vivo activation suggesting that macrophages and inflam-matory cytokines drive HSC activation in vivo [26] However


After iohexol gradientDay 1 HSC

(a)


(b)

Day 1 HSCAfter iohexol gradient and additional sorting

(c)


(d)

Desmin staining

Day 4 HSC

After iohexol gradient and additional sorting

(e)

Actin staining

Day 4 HSC


(f)

Figure 4 Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting Hepaticstellate cells (HSC) were isolated from 40-week-old C57BL6J mice using enzymatic digestion of the liver based on pronase and collagenasefollowed by density gradient centrifugation in 8 iohexol Cells were cultured directly after the gradient for one day (a) and four days (b)where Kupffer cells (indicated by arrows) can be found in the HSC culture Highly pure HSC after additional fluorescence-activated cellsorting (FACS) after one (c) and four days of culture are shown (d) Expression analysis of desmin (e) and phalloidin (f) of HSC after fourdays of culture indicating proper maturation of HSC


0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks

Control CCl4 6 weeks

CCl4 6 weeksControlCCl4 6 weeks


lowast lowastlowast

lowast lowastlowastlowastlowast

120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A

After sortingControl mice

Before sorting After sorting

286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k

FACS-based isolation of HSC from injured livers

Figure 5 Hepatic stellate cell activation in vivo and isolation of hepatic stellate cells from livers of injured mice Chronic toxic liver injurywas induced in 8-week-old C57BL6 mice by 6 weeks of carbon tetrachloride (CCl

4) treatment and control treatment was done using corn

oil Mice were sacrificed 48 hours after the last injection of (CCl4) and liver sections were stained for hematoxylin eosin (a) Sirius red (stains

collagen fibres a hallmark of fibrosis) (b) and 120572 smooth muscle action (120572SMA) which targets activated hepatic stellate cells mediators offibrosis Morphometric quantification of Sirius red confirms fibrosis progression (d) Quantitative real-time PCR indicates upregulation ofcollagen 1 (Col1A1) and 120572SMAmRNA in liver sections (e) Application of the gating strategy of fluorescence-activated cell sorting (FACS) toisolate hepatic stellate cells from livers of mice that underwent six weeks of repetitive CCl

4-based liver injury (f) compared to vehicle corn

oil-treated control mice (g) Flow cytometric analysis of HSC purity before and after sorting demonstrated that the purification was successful(h) Mean plusmn SD of three independent experiments 119899 = 12 for control and 16 for fibrotic mice lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 0005 and lowast119875 lt 005(unpaired Studentrsquos 119905-test)

our data show that in addition to other nontarget cells espe-cially doublets of HSC with KC can considerably ldquocontami-naterdquo primary HSC isolates frommouse liver if no additionalFACS-based sorting is performed Therefore it is importantto exclude that such doublets confounded prior conclusionson HSC gene expression profiles from fibrotic livers

Along the same line a solid body of literature existsdemonstrating that HSC isolated from mouse or rat liverscan produce large amounts of proinflammatory mediatorssynthesized by HSC [27] and respond vividly to bacterial

products like LPS via toll-like receptor recognition [4]Similarly LPS was also found to activate human HSC invitro [28] It will be important to determine whether someof the LPS responsiveness reported for HSC might be partlyrelated to contaminations with KC which are known to beboth efficient producers and responders to inflammatorymediators [22] Using highly pure HSC cultures after FACS-based isolation LPS had very limited effects onHSC behaviorsuch as spontaneous migration as well as transdifferentiationor proliferation


Day 0 Day 5 Day 7

50120583m

(a)

HSC proliferation in vitroC

ell n

umbe

r cha

nge (

)

Culture time (hours)24 48 72 86 110 122

0

20

ControlLPS

134 156146minus20

(b)

3 hours 24 hours 48 hours

100120583m

(c)

HSC migration in vitro

ControlLPS

0

50

100

150

3 12 24 36 48Culture time (hours)

Mig

rate

d ce

lls (c

ells

mm2)

(d)

HSC activation in vitroCol1A1

0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)

Figure 6 Hepatic stellate cell functionality in vitro Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL6J mice usingenzymatic digestion of the liver based on pronase and collagenase followed by density gradient centrifugation in 8 iohexol and fluorescence-activated cell sorting (FACS) The HSC were cultured in DMEM with 10 fetal calf serum and some plates were stimulated withlipopolysaccharides (LPS) at 100 ngmL (after five days of culture) for another 48 hours Changes in the cell number during culture weredetermined from time lapsemicroscopy (a) and statistical summary (b) HSCwere cultured for five days on tissue culture-treated polystyrenein DMEM with 10 fetal calf serum including culture inserts for self-insertion (ldquoscratch assayrdquo) To start horizontal migration the plasticinsertswere removed andHSCmigrated (c) andwere quantified using software (d)HSCwere cultured for designated periods and quantitativereal-time PCR was performed to study the expression of 120572 smooth muscle actin (120572SMA) collagen 1 (Col1A1) or the transforming growthfactor 120573 (TGF120573) as markers of HSC activation Gene expression was normalized to 120573-actin expression of cells that were lysed directly afterisolation at day zero (e) Mean plusmn SD of three independent experiments


Nevertheless one should keep inmind that upon contactwith the tissue culture material HSC begin to mature and donot reflect quiescent HSC but ldquoculture-activatedrdquo HSC [29]This is well reflected by the fact that HSC isolated either byconventional methodology or with additional FACS signifi-cantly upregulate activation andmyofibroblast differentiationmarkers upon culture

Another controversial aspect of HSC biology relates tothe question whether HSC derived from noninjured livermicroenvironment proliferate in vitro It is known thatLPS-stimulated peripheral blood mononuclear cells such asmonocytes can stimulate HSC proliferation [30] Moreoverdifferent profibrogenic stimuli such as TGF-120573 can stimulateHSC proliferation in vitro [31] However it was not clearwhether highly pure FACS-isolated HSC alone would pro-liferate In our hands highly pure HSC do not proliferatespontaneously suggesting that they rely on external stimulisuch as cytokines from inflamed liver or cell-cell contactswith macrophages [3] Furthermore one has to considerthat also HSC exhibit heterogeneity and it was reported thatretinolminus HSC proliferate whereas retinol+ cells do not [32]

In conclusion we developed and validated an optimizedisolation procedure for primary HSC from mouse liverswhich results in a highly pure viable and functionallyactive population of HSC Upcoming studies should validatecontroversial basic studies to unravel to which extent con-taminations especially with KCmay have confounded earlierconflicting data onHSCbiology as earlier studies suggest thatKC release molecules which induce HSC proliferation [33]

Disclaimer

The funding agencies had no role in study design collectionanalysis or interpretation of data

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Matthias Bartneck and Klaudia Theresa Warzecha con-tributed equally to this work

Acknowledgments

The authors thank Aline Roggenkamp for excellent technicalassistanceThis work was supported by the German ResearchFoundation (DFGTRR57 cell isolation platform Q3) and aSTART grant of the medical faculty (to Matthias Bartneck)

References

[1] I Mederacke C C Hsu J S Troeger et al ldquoFate tracing revealshepatic stellate cells as dominant contributors to liver fibrosisindependent of its aetiologyrdquo Nature Communications vol 4article 2823 2013

[2] A Geerts ldquoHistory heterogeneity developmental biology andfunctions of quiescent hepatic stellate cellsrdquo Seminars in LiverDisease vol 21 no 3 pp 311ndash335 2001

[3] J-P Pradere J Kluwe S De Minicis et al ldquoHepaticmacrophages but not dendritic cells contribute to liverfibrosis by promoting the survival of activated hepatic stellatecells in micerdquo Hepatology vol 58 no 4 pp 1461ndash1473 2013

[4] E Seki S DeMinicis C H Osterreicher et al ldquoTLR4 enhancesTGF-120573 signaling and hepatic fibrosisrdquo Nature Medicine vol 13no 11 pp 1324ndash1332 2007

[5] F Tacke and R Weiskirchen ldquoUpdate on hepatic stellate cellspathogenic role in liver fibrosis and novel isolation techniquesrdquoExpert Review of Gastroenterology amp Hepatology vol 6 no 1pp 67ndash80 2012

[6] D Schuppan and Y O Kim ldquoEvolving therapies for liverfibrosisrdquo Journal of Clinical Investigation vol 123 no 5 pp1887ndash1901 2013

[7] D L Knook A M Seffelaar and A M de Leeuw ldquoFat-storing cells of the rat liver their isolation and purificationrdquoExperimental Cell Research vol 139 no 2 pp 468ndash471 1982

[8] A Margreet de Leeuw S P McCarthy A Geerts and D LKnook ldquoPurified rat liver fat-storing cells in culture divide andcontain collagenrdquo Hepatology vol 4 no 3 pp 392ndash403 1984

[9] R Weiskirchen and A M Gressner ldquoIsolation and culture ofhepatic stellate cellsrdquo Methods in Molecular Medicine vol 117pp 99ndash113 2005

[10] A Geerts T Niki K Hellemans et al ldquoPurification of rathepatic stellate cells by side scatter-activated cell sortingrdquoHepatology vol 27 no 2 pp 590ndash598 1998

[11] S Ichikawa D Mucida A J Tyznik M Kronenberg andH Cheroutre ldquoHepatic stellate cells function as regulatorybystandersrdquo Journal of Immunology vol 186 no 10 pp 5549ndash5555 2011

[12] K Iwaisako C Jiang M Zhang et al ldquoOrigin of myofibroblastsin the fibrotic liver in micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no32 pp E3297ndashE3305 2014

[13] C Baeck A Wehr K R Karlmark et al ldquoPharmacologicalinhibition of the chemokine CCL2 (MCP-1) diminishes livermacrophage infiltration and steatohepatitis in chronic hepaticinjuryrdquo Gut vol 61 no 3 pp 416ndash426 2012

[14] L Hammerich J M Bangen O Govaere et al ldquoChemokinereceptor CCR6-dependent accumulation of 120574120575T cells in injuredliver restricts hepatic inflammation and fibrosisrdquo Hepatologyvol 59 no 2 pp 630ndash642 2014

[15] T Kisseleva M Cong Y Paik et al ldquoMyofibroblasts revertto an inactive phenotype during regression of liver fibrosisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 24 pp 9448ndash9453 2012

[16] I Mederacke D H Dapito S Affo H Uchinami and RF Schwabe ldquoHigh-yield and high-purity isolation of hepaticstellate cells from normal and fibrotic mouse liversrdquo NatureProtocols vol 10 no 2 pp 305ndash315 2015

[17] C Roderburg M Luedde D Vargas Cardenas et al ldquoMiR-133amediates TGF-120573-dependent derepression of collagen synthesisin hepatic stellate cells during liver fibrosisrdquo Journal of Hepatol-ogy vol 58 no 4 pp 736ndash742 2013

[18] J S Troeger I Mederacke G-Y Gwak et al ldquoDeactivation ofhepatic stellate cells during liver fibrosis resolution in micerdquoGastroenterology vol 143 no 4 pp 1073e22ndash1083e22 2012


[19] F Winau G Hegasy R Weiskirchen et al ldquoIto cells are liver-resident antigen-presenting cells for activating T cell responsesrdquoImmunity vol 26 no 1 pp 117ndash129 2007

[20] B Wang M Trippler R Pei et al ldquoToll-like receptor activatedhuman andmurine hepatic stellate cells are potent regulators ofhepatitis C virus replicationrdquo Journal of Hepatology vol 51 no6 pp 1037ndash1045 2009

[21] Q Zhu L Zou K Jagavelu et al ldquoIntestinal decontamina-tion inhibits TLR4 dependent fibronectin-mediated cross-talkbetween stellate cells and endothelial cells in liver fibrosis inmicerdquo Journal of Hepatology vol 56 no 4 pp 893ndash899 2012

[22] F Tacke and H W Zimmermann ldquoMacrophage heterogeneityin liver injury and fibrosisrdquo Journal of Hepatology vol 60 no 5pp 1090ndash1096 2014

[23] M Bartneck H A Keul G Zwadlo-Klarwasser and J GrollldquoPhagocytosis independent extracellular nanoparticle clearanceby human immune cellsrdquo Nano Letters vol 10 no 1 pp 59ndash632010

[24] M Bartneck T Ritz H A Keul et al ldquoPeptide-functionalizedgold nanorods increase liver injury in hepatitisrdquoACS Nano vol6 no 10 pp 8767ndash8777 2012

[25] P Maschmeyer M Flach and F Winau ldquoSeven steps to stellatecellsrdquo Journal of Visualized Experiments no 51 article e27102011

[26] S de Minicis E Seki H Uchinami et al ldquoGene expressionprofiles during hepatic stellate cell activation in culture and invivordquo Gastroenterology vol 132 no 5 pp 1937ndash1946 2007

[27] A Dangi T L Sumpter S Kimura et al ldquoSelective expansionof allogeneic regulatory T cells by hepatic stellate cells role ofendotoxin and implications for allograft tolerancerdquo Journal ofImmunology vol 188 no 8 pp 3667ndash3677 2012

[28] M Muhlbauer T S Weiss W E Thasler et al ldquoLPS-mediatedNF120581B activation varies between activated human hepatic stel-late cells from different donorsrdquo Biochemical and BiophysicalResearch Communications vol 325 no 1 pp 191ndash197 2004

[29] S WWoo J-X Nan S H Lee E-J Park Y Z Zhao and D HSohn ldquoAloe emodin suppresses myofibroblastic differentiationof rat hepatic stellate cells in primary culturerdquo Pharmacologyand Toxicology vol 90 no 4 pp 193ndash198 2002

[30] K TodaNKumagai K Tsuchimoto et al ldquoInduction of hepaticstellate cell proliferation by LPS-stimulated peripheral bloodmononuclear cells from patients with liver cirrhosisrdquo Journal ofGastroenterology vol 35 no 3 pp 214ndash220 2000

[31] Y He C Huang X Sun X-R Long X-W Lv and J LildquoMicroRNA-146a modulates TGF-beta1-induced hepatic stel-late cell proliferation by targeting SMAD4rdquo Cellular Signallingvol 24 no 10 pp 1923ndash1930 2012

[32] T Ogawa C Tateno K Asahina et al ldquoIdentification of vitaminA-free cells in a stellate cell-enriched fraction of normal rat liveras myofibroblastsrdquoHistochemistry and Cell Biology vol 127 no2 pp 161ndash174 2007

[33] X Zhang W-P Yu L Gao K-B Wei J-L Ju and J-ZXu ldquoEffects of lipopolysaccharides stimulated Kupffer cellson activation of rat hepatic stellate cellsrdquo World Journal ofGastroenterology vol 10 no 4 pp 610ndash613 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


strategies incorporated multiplex staining of surface markersand cell sorting to exclude cell types other than HSC fromcell purifications However the purity of all these strategiesfor HSC isolation remained disputed since antibody stainingmay affect cell populations [11] Moreover there is no reliablesurfacemarker known that is generally expressed onHSCandmyofibroblasts which hampers positive selection strategiesbased on antibody staining [5] Some surface markers thathad been suggested forHSC isolation include platelet-derivedgrowth factor 120573 (PDGFR-120573) or low-affinity nerve growth fac-tor receptor (p75) while other investigators tested intracellu-lar glial fibrillary acidic protein (GFAP) staining to identifyVitamin A+ HSC [1 5 12] FACS sorting of HSC has beenlately employed by several groups including ours by using aUV laser that specifically excites the stored retinol droplets inresting HSC [12ndash18] However it remained unclear whetherthis technique would alter functional properties of HSC suchas migratory properties relevant to wound healing responses

The current ldquogold standardrdquo for HSC isolation is stillbased on density centrifugation using iohexol (also knownunder their brand names Nycodenz Exypaque or Omni-paque) which separates the HSC due to its physical prop-erties from other hepatic cells and usually results in a highnumber of viable HSC applicable to cell culture experimentsWe hypothesized that via the ldquoconventionalrdquo density gradientmethod cell aggregates between HSC and other cell typesespecially Kupffer cells (KC) may occur which in turn resultin cellular impurities that could lead to contradictory resultson distinct HSC functions For example it is heavily debatedin the field whether HSC can act as antigen-presenting cellsas studies have led to conflicting results on this topic [11 19]Also the notion that HSC strongly respond to bacterial prod-ucts like lipopolysaccharides (LPS) [20 21] requires that thereis no relevant contamination with macrophages which areequipped with manifold danger-recognition receptors [22]

In this study we optimized the ldquoconventionalrdquo method-ology for the isolation of HSC based on iohexol densitycentrifugation and compared it to an additional step offluorescence-activated cell sorting (FACS) based on anti-bodies and UV-autofluorescence with respect to cellularpurity viability yield and cellular characteristics We furtheranalyzed the behavior of highly pureHSC in vitro by studyingtheir cellular morphology and maturation over five days ofculture using time lapse microscopy as well as migratoryproperties in an assay for cell migration and after stimulationwith LPS By implementing an additional step of cell sortingto the current ldquogold standardrdquo HSC isolation method ourprotocol results in significantly improved cellular puritywhich helps to clarify HSC functions

2 Materials and Methods

21 Ethics Statement All in vivo experimentswere performedfollowing approval by the State Animal Protection Board atthe Bezirksregierung Cologne Germany The investigationconforms to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health(NIH Publication Number 85-23 revised 1996)

22 Mice C57BL6J wild-type mice at 40ndash50 weeks of age ifnot stated otherwise were housed in a specific pathogen-freeenvironment To induce liver fibrosis carbon tetrachloride(CCl4 06mLkg Sigma-Aldrich Taufkirchen Germany)

was injected intraperitoneally two times per week for sixweeks control animals received the vehicle (corn oil) [13] Allanimal experiments have been approved by the InstitutionalReview Board and by the German legal authorities (LANUVRecklinghausen Germany)

23 Liver Perfusion Enzymatic Digestion and DensityGradient Centrifugation Mice were anaesthetized using7mgkg body weight xylazine and 105mgkg body weight ofketamine The liver was perfused via the Vena portae using a26G needle (BD Franklin Lakes USA) that was fixed usinga Bulldog clamp (Aesculap Tuttlingen Germany) Bufferswere prewarmed to 37∘C and pumped into the liver via theabdominalVena portae and drained via theVena cava inferiorusing a peristaltic pump at a flow rate of 65mLminute

Initially perfusion buffer 1 (8 gL NaCl 400mgL KCl78mgL NaH

2PO4times H2O 151mgL NaHPO

4times 2 H

2O

2380mgL HEPES 350mgL NaHCO3 190mgL EGTA

900mgL glucose and 6mgL phenol red adjusted to pH73ndash74 using 10N NaOH sterile filtered and kept at 4∘Cuntil use) was injected into the liver for 45 minutes Secondperfusion buffer 2 (8 gL NaCl 400mgL KCl 78mgLNaH2PO4times H2O 151mgL NaHPO

4times 2 H

2O 2380mgL

HEPES 350mgL NaHCO3 560mgL CaCl

2times 2 H

2O and

6mgL phenol red adjusted to pH 73ndash74 using 10N NaOHsterile filtered and kept at 4∘C until use) was applied for45 minutes and supplemented with 05mgmL pronaseE (Merck Darmstadt Germany) Third perfusion buffer2 which was supplemented with 075UmL collagenase P(Roche Basel Switzerland)was administered for 45minutesThe perfused liver was removed and the gall bladder andconnective tissue sticking to the liver were detached HSCwere removed by tearing the liver into bits using two tweezers

The liver cell suspension was further digested for 20minutes in a 37∘C water bath in perfusion buffer 2 sup-plemented with 04mgmL pronase E 15 UmL collagenaseP and 002mgmL DNase I (Roche Basel Switzerland)adjusted to a pH of 72ndash74 using NaOH Cells were fil-tered using a 70 120583m nylon gaze and rinsed using perfusionbuffer 2 tempered at 4∘C and centrifuged for ten min-utes at 4∘C at 600 g The pellet was then washed using4∘C Geyrsquos buffered salt solution (GBSS) (exact composi-tion 370mgL KCl 210mgL MgCl

2times 6 H

2O 70mgL

MgSO4times 7 H

2O 75mgL Na

2HPO4times 2 H

2O 30mgL

KH2PO4 991mgL glucose 227mgL NaHCO

3 225mgL

CaCl2times H2O 8000mgL NaCl and 6mgL phenol red

adjusted to pH 73ndash74 sterile filtered and kept at 4∘C untiluse) supplemented with 001mgmLDNase I Cells were thencentrifuged for ten minutes at 4∘C at 600 g and suspended inthe same buffer (without NaCl otherwise identical) that wassupplemented with iohexol (Nycodenz Axis-Shield DundeeScotland) to a final concentration of 8

The mixture was then pipetted to the ground of a falconcontaining GBSS and centrifuged at 4∘C and 1500 g for 22minutes without brake The interphase containing enriched














3 Results









4for six weeks


4





Hepatocytes (HC)

Out In



WasteHSC


8 iohexol

Debris


Pronase

(1)

(2)

Collagenase












FSC-A

(a) (b) (c)

(d)

SSC-

A

SSC-A

SSC-

W

FSC-H

FSC-

W

0

0F480 FSC-A



SSC gate


Retin

ol+

104

104

103

103

102

102

105

0

Retin

ol+

104

103

102

105

0

Retin

ol+

104

103

102

105

105 0F480

104103102 105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k






4 Discussion



0

FSC-A


995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)









(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























3 Results









4for six weeks


4





Hepatocytes (HC)

Out In



WasteHSC


8 iohexol

Debris


Pronase

(1)

(2)

Collagenase












FSC-A

(a) (b) (c)

(d)

SSC-

A

SSC-A

SSC-

W

FSC-H

FSC-

W

0

0F480 FSC-A



SSC gate


Retin

ol+

104

104

103

103

102

102

105

0

Retin

ol+

104

103

102

105

0

Retin

ol+

104

103

102

105

105 0F480

104103102 105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k






4 Discussion



0

FSC-A


995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)









(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Hepatocytes (HC)

Out In



WasteHSC


8 iohexol

Debris


Pronase

(1)

(2)

Collagenase












FSC-A

(a) (b) (c)

(d)

SSC-

A

SSC-A

SSC-

W

FSC-H

FSC-

W

0

0F480 FSC-A



SSC gate


Retin

ol+

104

104

103

103

102

102

105

0

Retin

ol+

104

103

102

105

0

Retin

ol+

104

103

102

105

105 0F480

104103102 105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k






4 Discussion



0

FSC-A


995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)









(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FSC-A

(a) (b) (c)

(d)

SSC-

A

SSC-A

SSC-

W

FSC-H

FSC-

W

0

0F480 FSC-A



SSC gate


Retin

ol+

104

104

103

103

102

102

105

0

Retin

ol+

104

103

102

105

0

Retin

ol+

104

103

102

105

105 0F480

104103102 105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k

0 50k 100 k 150 k 200 k 250 k






4 Discussion



0

FSC-A


995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)









(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

FSC-A


995 HSC

999

668 HSC

813

102

103

104

105

retinol+ retinol+

Retin

ol+

0102

103

104

105

Retin

ol+

FSC-A0 50k 100 k 150 k 200 k 250 k 0 50k 100 k 150 k 200 k 250 k

(a)

Popu

latio

n pu

rity

()

0

50

100

25

75


Retinol+ versus HSC

HSC

Retin

ol+

cells

HSC

Retin

ol+

cells

(b)

Decorin

0

2

4

6

8

mRN

A ex

pres

sion

[fold

chan

ge]

Clec4f

00

05

10

15Pecam-1

00

05

10

15Albumin

000

001

002

003

004

lowastlowastlowast

lowast lowast

(c)









(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)


(c)


(d)

Desmin staining

Day 4 HSC


(e)

Actin staining

Day 4 HSC


(f)



0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

mRN

A ex

pres

sion

[fold

chan

ge]

0

20

40

60

80

100

0

1

2

3

4 Sirius red

Are

a fra

ctio

n (

)

Col1A1

Control

Control(a)

(b)

(c)

(d) (e)

Hamp

ESi

rius r

ed

CCl4 6 weeks




lowast lowastlowast


120572SM

A

120572SMA

Figure 5 Continued


(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(f)

(g)

(h)

Before sorting

Scatter gate

Scatter gate

Control mice

SSC gate

SSC gate

FSC gate

FSC gate

HSC gate

HSC gate

FSC-A

FSC-AFSC-A

SSC-

A

SSC-A

SSC-

W

SSC-A

SSC-

W

FSC-H

FSC-

W

FSC-H

FSC-

W

FSC-A



286 991 656 988

CCl4-treated mice

CCl4-treated mice

SSC-

A

FSC-A

Retin

ol+

Retin

ol+

Retin

ol+

0

104

103

102

105

0

104

103

102

105

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

Retin

ol+

0

104

103

102

105

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0

50k

100 k

150 k

200 k

250 k

0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 k 0 100 k 200 k 0 100 k 200 k

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 kFSC-A

0 100 k 200 k












Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 0 Day 5 Day 7

50120583m

(a)


ell n

umbe

r cha

nge (

)


0

20

ControlLPS

134 156146minus20

(b)


100120583m

(c)


ControlLPS

0

50

100

150


Mig

rate

d ce

lls (c

ells

mm2)

(d)


0

10

20

30

40

50

mRN

A ex

pres

sion

[fold

chan

ge]

D0

HSC

D1

HSC

D5

HSC

0

200

400

600

800

0

1

2

3

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

D0

HSC

D1

HSC

D5

HSC

D5

HSC

+LP

S24

h

120572SMA TGF120573

(e)






Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Disclaimer






Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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