Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology

Abstract

Although organ fibrosis causes significant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specific cellular contributors mediating fibrogenesis hampers the design of effective antifibrotic therapies. Different cellular sources, including tissue-resident and bone marrow-derived fibroblasts, pericytes and epithelial cells, have been suggested to give rise to myofibroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specific pericyte population, to demonstrate that HSCs give rise to 82-96% of myofibroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis these findings, HSCs should be considered the primary cellular target for antifibrotic therapies across all types of liver disease.

ARTICLE

Received 16 Oct 2013 | Accepted 25 Oct 2013 | Published 22 Nov 2013

Ingmar Mederacke1, Christine C. Hsu1,*, Juliane S. Troeger1,*, Peter Huebener1, Xueru Mu1, Dianne H. Dapito2, Jean-Philippe Pradere1 & Robert F. Schwabe1,2

Although organ brosis causes signicant morbidity and mortality in chronic diseases, the lack of detailed knowledge about specic cellular contributors mediating brogenesis hampers the design of effective antibrotic therapies. Different cellular sources, including tissue-resident and bone marrow-derived broblasts, pericytes and epithelial cells, have been suggested to give rise to myobroblasts, but their relative contributions remain controversial, with profound differences between organs and different diseases. Here we employ a novel Cre-transgenic mouse that marks 99% of hepatic stellate cells (HSCs), a liver-specic pericyte population, to demonstrate that HSCs give rise to 8296% of myobroblasts in models of toxic, cholestatic and fatty liver disease. Moreover, we exclude that HSCs function as facultative epithelial progenitor cells in the injured liver. On the basis these ndings, HSCs should be considered the primary cellular target for antibrotic therapies across all types of liver disease.

DOI: 10.1038/ncomms3823

Fate tracing reveals hepatic stellate cells as dominant contributors to liver brosis independent of its aetiology

1 Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA. 2 Institute of Human Nutrition, Columbia University, New York, New York 10032, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.F.S. (email: mailto:[email protected]

Web End [email protected] ).

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Fibrotic diseases account for up to 45% of deaths in developed countries, but we still lack effective antibrotic therapies1. Knowledge about cells contributing to the myobroblast

pool and extracellular matrix (ECM) production in organ brosis is important for designing targeted therapies2,3. Several cell populations, including tissue-resident or bone marrow (BM)-derived broblasts, pericytes and epithelial cells, have been suggested as relevant contributors to the myobroblast pool2,3, but their contribution varies between organs and different diseases, and remains a matter of debate.

Liver brosis occurs as a result of chronic liver disease and is associated with severe morbidity and mortality4. Hepatic stellate cells (HSCs), a pericyte-like cell population of the liver, and portal broblasts are widely considered the most relevant sources of hepatic myobroblasts, but their precise contribution remains unknown. It is thought that the underlying liver disease determines the cell types that contribute to the myobroblast pool, with profound differences between toxic and cholestatic liver diseases, and portal broblast exerting a key role in the latter47. Other cell types such as BM-derived brocytes may also contribute to the myobroblast pool, but to a lesser degree8,9. However, most evidence about relevant myobroblast sources and functions derives from cell isolates and in vitro studies1012, whereas in vivo studies quantifying the relative contribution of different cell populations in the brotic liver are missing. In this regard, fate-tracing studies have excluded a contribution of hepatocytes and cholangiocytes to the myobroblast pool1315, but positive identication of cell types contributing to the myobroblast pool by fate tracing is lacking. As such, previous studies employing collagen-driven Cre or Wt1-Cre have tracked either all collagen-producing myobroblasts, but not specic cellular sources of myobroblasts12, or mesothelial cells, which give rise to subcapsular broblasts within 150 mm of the liver surface but not to intrahepatic myobroblasts, that is, those that cause organ brosis and its deleterious complications16. Attempts at fate-tracing HSCs, one of the prime candidates4,11,17, using Cre driven by the human glial brillary acidic protein promoter (hGFAPCre) are hampered by the fact that hGFAPCre marks cholangiocytes18,19 (https://ndp.jax.org/NDPServe.dll?ViewItem?ItemID=1741&XPos=11536496&YPos=5169511&ZPos=0&Lens=1.25&SignIn=Sign%20in%20as%20Guest

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Web End =SignIn=Sign%20in%20as%20Guest ), thereby limiting interpretation.

Here we employ a novel fate tracing method for HSCs to demonstrate that HSCs are the dominant contributors to collagen-producing myobroblasts in models of toxic, biliary and fatty liver brosis. Moreover, we exclude that HSCs serve as facultative epithelial progenitors in the injured liver.

ResultsLratCre but not hGFAPCre labels HSCs. Using three different Cre reporters, we conrmed that hGFAPCre mice labelled bile ducts and cytokeratin 19-expressing cholangiocytes (Fig. 1a, eh). Of note, hGFAPCre mice did not efciently mark HSCs or collagen-producing myobroblasts (Fig. 1bg). Likewise, mice expressing Cre under the murine Gfap promoter did not label HSCs (Fig. 1i). To achieve faithful labelling of HSCs, we generated a bacterial artical chromosome (BAC) transgenic mouse in which Cre expression is driven by lecithin-retinol acyltransferase (Lrat; Supplementary Fig. S1B). Lrat was selected in screens among several candidates because of high expression in HSCs, virtually undetectable expression in hepatocytes, Kupffer cells and cholangiocytes (Fig. 2a), and its key role in the formation of retinyl ester-containing lipid droplets, one of the dening features of HSCs20. In contrast to a previous study21, we also did not detect Lrat protein or messenger RNA expression

in endothelial cells (Fig. 2a; Supplementary Fig. S1A). LratCre transgenic mice marked 99% of HSCs as demonstrated by ow cytometry and immunohistochemistry in two different Cre reporter mice, showing nearly complete overlap of HSC markers desmin and Pdgfrb with LratCre-induced ZsGreen Cre reporter by confocal microscopy, no signicant overlap with hepatocyte, macrophage and cholangiocyte markers, and only few spots of overlap with endothelial cell markers, most likely to be due to their very close proximity to HSCs (Fig. 2be and Supplementary Fig. S1CG).

HSCs are the main contributors to toxic liver brosis. To determine the contribution of HSCs to the myobroblast pool, we rst subjected LratCre mice to the well-established model of carbon tetrachloride (CCl4)-induced liver brosis. In this model, LratCre-induced ZsGreen expressionmarking HSCs and a-smooth muscle actin (aSMA) expressionmarking myobroblastscompletely overlapped, and displayed micro-and macroscopic ZsGreen uorescence with the characteristic septal pattern of liver brosis (Fig. 3a,b). To determine the relative contribution of HSCs to the myobroblast pool, we quantied the overlap between LratCre-induced Cre reporter and aSMA. In CCl4-induced liver brosis, the overlap between

LratCre-induced ZsGreen and aSMA was 93.6% (2.3% s.d., n 4, Fig. 3b), providing evidence that HSCs are the almost

exclusive source of myobroblasts in toxic liver brosis. To conrm this data by a second approach, we generated LratCre mice co-expressing red-uorescent Cre reporter tdTomato and a green-uorescent Col-GFP reporter, which faithfully marks collagen-producing cells22. LratCre-induced tdTomato almost completely overlapped with Col-GFP uorescence in the CCl4 model (96.0%2.4% s.d., n 4, Fig. 3b), thus matching the

degree of overlap that we found between aSMA and LratCre-induced ZsGreen. Accordingly, nearly all aSMA-positive cells were also Col-GFP-positive (Fig. 3c). To exclude that our data may be specic to the CCl4 model, we conrmed our ndings in a second well-established model of toxic liver brosis, induced by thioacetamide (TAA) treatment. Livers from TAA-induced treated mice also displayed a characteristic macro- and microscopic septal pattern of ZsGreen uorescence, and the same range of co-localization between LratCre-induced TdTomato and Col-GFP uorescence (94.80.3% s.d., n 3)

as in the CCl4 model (Supplementary Fig. S2AC). To further substantiate the functional contribution of HSCs to brogenesis, we ablated HSCs via LratCre-induced diphtheria toxin receptor. This strategy strongly reduced HSC numbers in untreated and CCl4-treated livers, albeit not completely (Fig. 3d,e and

Supplementary Fig. S3A). However, it should be noted that this approach typically does not result in complete ablation unless a strongly intensied regimen of diptheria toxin injection is employed23. The reduction of aSMA and probrogenic gene expression by at least the same degree as ablation of HSCs, assessed by desmin mRNA and immunohistochemisty (Fig. 3d,e), conrms the relevant contribution of HSCs to brogenesis. In contrast, hepatic inammation, injury and expression of endothelial marker vWf were not affected by HSC ablation (Supplementary Fig. S3BD).

HSCs are the main contributors to biliary liver brosis. After having established HSCs as the main contributors to toxic liver brosis, we next determined the contribution of HSCs to cholestatis-induced liver brosis. In this setting, portal broblasts are believed to be an essential contributor to brogenesis4,5,7. In three well-established models of cholestasis-induced liver brosis, bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydro-

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Figure 1 | GFAPCre marks extra- and intrahepatic bile ducts but not HSCs. (a) Macroscopic images demonstrate hGFAPCre-induced ZsGreen Cre reporter uorescence in extrahepatic bile ducts, the brain and brain sections (n 1). (b,c) HSCs were isolated from hGFAPCre mice expressing ZsGreen (n 2) and

were either plated (b) or analysed by ow cytometry (c). LratCre mice served as positive control (n 3). (d) HSCs were isolated from hGFAPCre mice

expressing mTom/mGFP Cre reporter and plated (n 1). (e) Sections from untreated (n 1), bile duct-ligated (n 1) and CCl4-treated livers (n 2) were

stained for desmin, showing no co-localization of ZsGreen and HSC marker desmin by confocal microscopy. (f) Representative western blotting (out of n 3)

showing cytokeratin 19 expression but no desmin expression in uorescence-activated cell sorting-sorted hGFAPCre-labelled ZsGreen-positive cells.(g) hGFAPCre mice undergoing either BDL (n 1) or CCl4 treatment (n 1) show no overlap between tdTomato Cre reporter and Col-GFP reporter, thus

excluding a contribution of hGFAPCre-labelled cells to ECM-producing myobroblasts. (h) Cytokeratin staining of bile duct-ligated hGFAPCre mice shows almost complete overlap of cytokeratin, marking the bile duct proliferates, and hGFAPCre-induced ZsGreen expression (n 1). (i) Sections from untreated

mice expressing Cre under the murine Gfap promoter and mTom/mGFP Cre reporter show no GFP expression in the liver. The brain served as the positive control (Inlet). Scale bars, 4 mm (a, left panel), 2 mm (a, upper middle and right panel), 200 mm (a, lower middle and right panel) and 50 mm (b,d,e,gi).

collidine (DDC)-containing diet and Mdr2ko mice, tdTomato expression almost completely overlapped with Col-GFP reporter expression, reaching X89% co-localization and thus almost the same degree as in toxic liver brosis (Fig. 4ac,e). This data was conrmed by ow cytometric analysis of the entire nonparenchymal cell pool, in which 8285% of collagen-producing cells were tdTomato-positive HSCs (Fig. 4d,e). A high contribution of HSCs to collagen-producing cells was even noted during early cholestatic liver brosis (8790% co-localization, Supplementary Fig. S4AD, G) and in the methioninecholine-decient diet model of non-alcoholic steatohepatitis (Supplementary Fig. S4EG). Similar to CCl4-induced liver brosis, LratCre not only marked collagen-producing cells but also aSMA-expressing cells, with 80 and 88% overlap between aSMA and LratCre-induced tdTomato after 7 and 14 days BDL, respectively, and a strong overlap between aSMA-positive and Col-GFP-positive cells (Supplementary Fig. S5A,B).

Lrat-negative portal broblasts-like cells are distinct from HSCs. In addition to LratCre-labelled, Col-GFP-positive HSCs, we also found a small population of Col-GFP-positive but LratCre-unlabelled broblasts, both in the liver and in cell isolates of cholestatic brosis models. These cells were predominantly located around the portal tracts and did not contain retinoid lipid droplets, consistent with characteristics of portal broblasts (Fig. 4f,h). This LratCre-negative, Col-GFP-positive population of portal broblast-like cells (PFLCs) showed low expression of genes characteristic for HSCs, such as Lrat, Lhx2 and HGF, and was morphologically distinct from HSCs (Fig. 4g,h). PFLCs were aSMA- and Col-GFP-positive, and abundantly expressed Acta2,

Col1a1, Lox, Timp1 and Vim in comparison with whole liver (Supplementary Fig. S6A,B), conrming them as myobroblasts. However, they expressed signicantly lower levels of brogenic genes than HSCs in our isolates (Fig. 4g), thus excluding that

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Figure 2 | LratCre efciently labels HSCs. (a) Lrat expression was determined by western blotting in pure and never-plated primary murine HSCs (n 8),

hepatocytes (n 2), Kupffer cells (n 2), endothelial cells (n 2) and cholangiocytes (n 2). (b,c) HSCs were isolated from LratCre-negative (n 2)

and LratCre-positive mice (n 3) expressing ZsGreen Cre reporter and were either plated for 24 h (b) or analysed by ow cytometry (c), using vitamin A

(VitA, determined in the violet uorescence-activated cell sorting channel) uorescence as HSC marker in both approaches. (d) Co-localization of LratCre-induced ZsGreen expression and desmin was determined in untreated livers using confocal microscopy (n 4). (e) Co-localization of LratCre-

induced ZsGreen expression and CD31 (marking endothelial cells), F4/80 (marking macrophages), HNF4a (marking hepatocytes) and cytokeratin (marking cholangiocytes) was determined by confocal microscopy in untreated and CCl4-treated mice. Scale bars, 100 mm (b,d,e).

PFLC might represent a less abundant but more brogenic population than HSCs. Together, these data indicate that portal broblast constitute a myobroblast population that is not HSC-derived and signicantly less abundant than HSCs, and probably fulls specialized functions in cholestatic liver disease.

Myobroblasts derive from LratCre-labelled retinoid-positive HSCs. aSMA staining in normal liver (where HSCs are quiescent and do not express aSMA) revealed that LratCre also labelled some vascular smooth muscle cells (VSMCs, Supplementary Fig. S7A). This may be explained by the fact that VSMCs and pericytes/HSCs are considered to be of the same lineage24, sharing a common precursor in the liver25. Although LratCre-labelled HSCs outnumbered LratCre-labelled VSMCs by a factor of 4200 (Supplementary Fig. S7B), we wanted to further exclude a major

contribution of LratCre-labelled VSMCs, or any other non-HSC population to the hepatic collagen-producing broblast pool. As specic labelling of hepatic VSMCs is not feasible, we employed retinoid uorescence to identify LratCre-labelled myobroblasts of HSC origin. Retinoid uorescence was observed in 490% of tdTomato- and Col-GFP-positive cells in the CCl4, BDL and Mdr2ko brosis models (Supplementary Fig. S7C,D), albeit at a lower level than in quiescent HSCs, thus conrming that these myobroblasts are of HSC and not of VSMC origin. Collectively, our data establish HSCs as principal contributors to the collagen-producing myobroblast pool across all types of liver brosis.

HSCs are not epithelial progenitors. Next, we determined whether HSCs may exert functions besides brogenesis in liver injury. Previous studies, employing hGFAPCre and aSMA-

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Un CCI4 100

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Figure 3 | HSCs are the principal source of myobroblasts in CCl4-induced liver brosis. (a) Representative uorescent images of whole livers from untreated and CCl4-treated mice (n 3) show LratCre-labelled ZsGreen-positive macroscopic brotic septa in CCl4-treated liver. (b). Frozen liver sections

from CCl4-treated LratCre-positive mice were stained with desmin (upper panel) or aSMA (middle panel) to demonstrate co-localization of HSC marker desmin or aSMA and LratCre-induced ZsGreen by confocal microscopy. Confocal microscopy was employed to show co-localization of Col-GFP reporter, marking activated myobroblasts and LratCre-induced tdTomato expression (lower panel). Quantication of aSMA-expressing cells that are derived from LratCre-labelled ZsGreen-positive HSCs in brosis induced by 9 CCl4 (n 4, upper graph) or Col-GFP-expressing cells that are derived from

LratCre-labelled tdTomato-positive HSCs in brosis induced by 9 CCl4 treatment (n 4, lower graph). (c) Co-localization of aSMA with tdTomato

and Col-GFP in 9 CCl4-induced liver brosis was determined by confocal microscopy employing far-red secondary antibody for aSMA detection.

(d,e) LratCre mice, expressing Cre-inducible diphteria toxin receptor (iDTR) received either vehicle (n 4) or dipheria toxin (DT, n 4) during

CCl4-induced liver brosis induction. Expression of aSMA and desmin was determined by immunohistochemstry and quantied (d), expression of brogenic genes was determined by qPCR (e). Scale bars, 1 mm (a), 100 mm (b,c) and 200 mm (d). Data are shown as meanss.d. *Po0.05, **Po0.01 (determined by Students t-test).

CreERT2 mice for fate tracing, suggested that up to 24% of hepatocytes were derived from HSCs in the methioninecholinedecient ethionine-supplemented (MCDE) diet and BDL models18,19. By employing LratCre mice, we tested whether short- and long-term injury induced by BDL, Mdr2ko, MCDE diet, DDC diet, CCl4, TAA or 70% partial hepatectomy resulted in the generation of HNF4a-positive hepatocytes expressing ZsGreen Cre reporter, thus identifying them as progeny of

LratCre-expressing HSCs. Although we found rare HNF4a-positive hepatocytes (at a frequency of E0.2 per 1,000 cells) expressing Cre reporter ZsGreen, none of the injury models increased this rate in the liver (Fig. 5ag and Supplementary Fig. S8AC). We also did not observe increased ZsGreen-positive cells with hepatocyte morphology (positive control shown in the insert of Fig.5A) in liver sections (Fig. 5af and Supplementary Fig. S8BC) or in primary hepatocyte isolates (Fig. 5h,i and

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LratCre x ZsGreen

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Figure 4 | HSCs are the principal source of myobroblasts in cholestatic liver brosis. (ac) Liver sections of 14 day bile duct-ligated (BDL) mice (a, ZsGreen n 3, tdTom/Col-GFP n 4), 9-week-old Mdr2ko mice (b, ZsGreen n 4, tdTom/Col-GFP n 5), or 0.1% DDC-diet-treated mice

(c, ZsGreen n 4, tdTom/Col-GFP n 3) were stained with desmin to demonstrate co-localization of HSC marker desmin and LratCre-induced ZsGreen by

confocal microscopy (upper panel). Confocal microscopy was employed to show co-localization of Col-GFP reporter, marking activated myobroblasts and LratCre-induced tdTomato expression (lower panel). (d) Flow cytometric images from 2-week-old BDL mice (upper panel) and 9-week-old Mdr2ko

(lower panel) mice co-expressing LratCre, tdTomato and Col-GFP. Images next to uorescence-activated cell sorting (FACS) plots show sorted cells freshly after plating. (e) Quantication of Col-GFP-expressing cells, derived from LratCre-labelled tdTomato-positive HSCs, was performed in liver sections(14 days BDL: n 4; Mdr2ko n 5; 0.1% DDC diet: n 3) or in non-parenchymal cell fractions using ow cytometry (14 days BDL: n 4, Mdr2ko n 6).

(f) Images demonstrating small and large bile ducts surrounded by Col-GFP-positive and LratCre-negative portal broblasts. (g) Quantitative PCR analysis of FACS-sorted unplated LratCre-labelled tdTom-positive and Col-GFP-positive cells (HSC, n 5 isolates), and tdTom-negative and Col-GFP-

positive cells (PFLCs, n 5 isolates). (h) Representative images of HSCs and PFLCs show morphologically distinct cell populations. Scale bars, 100 mm

(ac), 10 mm (d), 100 mm (f) and 50 mm (h). Data are shown as meanss.d. *Po0.05; **Po0.01; ***Po0.001 (determined by Students t-test).

Supplementary Fig. S8D). Furthermore, LratCre-marked cells did not give rise to cytokeratin-positive liver progenitor cells or cholangiocytes in any of the chronic injury models (Fig. 2e and Supplementary Fig. S9AE). Collectively, these data exclude that HSCs function as epithelial progenitors.

LratCre-positive HSCs are not BM derived. Finally, we employed LratCre mice to assess whether HSCs constitute a

liver-resident or BM-derived cell population, a point of substantial controversy2628. We did not detect LratCre-labelled HSCs in normal liver or brotic livers after BDL or long-term injury induced by 20 CCl4 injections despite successful BM transplantation as evidenced by mTom-positive (that is, non-recombined) BM-derived cells in the spleen and mTom- and F4/80-double-positive BM-derived macrophages in the liver (Fig. 6ac). These data conrm HSCs as liver-resident cell population.

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+ctrl Overlay +ctrl

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Figure 5 | HSCs do not contribute to the generation of newly formed hepatocytes. (ag) Determination of possible co-localization of hepatocyte marker HNF4a and LratCre-induced ZsGreen was performed by confocal microscopy in untreated mice (n 4; a), mice receiving MCDE diet for 3 weeks

followed by 3 weeks recovery on regular chow (n 4; b), mice receiving DDC diet for 4 weeks followed by 3 weeks recovery (n 3; c), mice undergoing

BDL for 2 weeks (n 3; d), 9- to 15-week-old Mdr2ko mice (n 3; e), or 2 weeks after partial hepatectomy (n 3; f). The number of HNF4a-expressing

hepatocytes, positive for ZsGreen, was quantied from confocal microscope images (g). (h,i) Representative images of primary hepatocytes isolated from control mice (n 3) and mice that received MCDE diet for 3 weeks (n 4), followed by 3 weeks recovery (h). Positive control ( ctrl) from AAV8-

TBG-Cre-injected mice showing ZsGreen-positive hepatocytes (n 1; h, upper right insert). The rare ZsGreen-positive small-size cells were identied

as HSCs by their characteristic uorescent retinoid-containing lipid droplets (h, lower right inserts). ZsGreen-positive hepatocytes were quantied (i). Scale bar, 100 mm. Data are shown as meanss.d.; NS, non-signicant (one-way analysis of variance).

DiscussionDespite the lack of solid in vivo evidence that HSCs are the primary drivers of liver brosis, much of the current research and drug discovery work focus on this cell type. After the discovery that HSCs produce signicantly larger amounts of collagen than hepatocytes and endothelial cells in vitro and thus constitute a prime candidate for hepatic myobroblast precursors11, little progress has been made to further establish and precisely quantify the relative contribution of HSCs to the hepatic myobroblast

pool and liver brosis in vivo. Most subsequent studies have investigated HSC activation in cell cultures, a process that differs substantially from in vivo activation10,29, or studied HSC isolates from normal and brotic livers10,12. Because of the lack of fate tracing techniques that specically label precursor populations such as HSCs, sources of hepatic myobroblasts have not yet been established or precisely quantied in vivo. Fate tracing has excluded a contribution for epithelial cells to the hepatic myobroblast pool1315 and characterized the

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LratCre mTom/mGFP BM transplant into wt mice

LratCre mTom/mGFP (positive control)

Confirmation of LratCre mTom/mGFP BMT

mGFP mTom Hoechst

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Figure 6 | LratCre-labelled HSCs are not BM-derived. (ac) BM from mice expressing LratCre and mTom/mGFP Cre reporter was transplanted into lethally irradiated wild-type recipients. Untreated (n 2), 20 CCl4 (n 1) and 3 weeks BDL (n 1) treated mice were killed 56 months after BM

transplantation and showed no LratCre-induced mGFP expression, thus excluding a contribution of the BM to generation of LratCre-expressing HSCs. In contrast, LratCre-positive mice expressing mTom/mGFP, serving as positive control, showed abundant mGFP signal (a). Successful BMT was conrmed by the presence mTom-positive cells in the spleen (see inserts) and demonstration of F4/80-positive mGFP-expressing liver macrophages (b). HSCs isolated from BM-transplanted mice (n 1) showed no mGFP signal or by ow cytometric analysis, whereas controls showed abundant mGFP signal

(n 4; c). Scale bar, 100 mm.

embryonic origin of HSCs16. However, positive identication of myobroblast precursors has not been achieved, as fate-tracing approaches have labelled all myobroblast populations but not specic precursors13 or subpopulations, such as subcapsular broblasts, which contribute little to organ brosis and the deleterious complications of brosis due to their sparsity and anatomic location16.

The most important nding of our study is the in vivo identication of HSCs as a universal and liver-resident source for myobroblasts, exerting a dominant role across toxic, biliary and fatty liver diseases. These data suggest that HSCs should be considered the primary target for the development of new antibrotic therapies, which has become an important focus of the eld3032. Revealing HSCs as the by-far dominant source in cholestatic brosis is unexpected, as portal broblasts have been considered a key contributor in cholestatic liver brosis4,5,7. The consistently high contribution of HSCs was observed in three different models of cholestasis, including the Mdr2ko model, which mimicks defects in cholestatic patients33. Thus, our data refute the hypothesis that the underlying disease dictates the cell type that contributes to the myobroblast pool in liver brosis, and instead establish HSCs as universal responders that trigger wound repair across different types of liver injury. Despite the dominant role of HSCs, there was a higher contribution of non-HSC myobroblast sources in biliary than in toxic liver brosis. Accordingly, we identied a population of LratCre-negative broblasts around portal tracts that were distinct from HSCs in terms of gene expression and anatomic localization. Despite being less abundant than HSCs, these cells most likely exert important functions in cholestatic liver disease. Although portal broblasts probably do not make a major contribution to organ brosis and associated complications such as portal hypertension owing to their low abundance, their anatomic localization suggests that they may exert specialized functions related to bile ducts. This

might be the mechanical stabilization of bile ducts as an adaptive response to increased pressure, or a contribution to biliary stenosis as a maladaptive response. It is also conceivable that portal broblasts represent a cell population that has a role in early biliary brosis serving as rapid responders7, and that it is essential in recruiting HSCs, which then in turn contribute to the majority of ECM production. Further studies should also determine whether LratCre-negative portal broblasts may produce different type of ECM than HSCs.

The second relevant nding of our study is that HSCs are not progenitor cells and do not contribute to the generation of hepatocytes in the injured liver. Previous studies had suggested that HSCs contribute up to 24% of newly formed hepatocytes18,19. As we labelled 499% of HSCs by LratCre and investigated their contribution to hepatocyte generation in liver sections from seven different injury models and in primary hepatocyte isolates from two injury models, in the liver and in hepatocyte isolates without nding increased number of HNF4aand ZsGreen-double positive cells, or ZsGreen-positive cells with hepatocyte shape, we can exclude HSCs as a source for epithelial regeneration in the liver. Our ndings are congruent with studies showing that hepatocytes and, to a lesser degree, bipotential progenitors function as efcient cellular sources for epithelial regeneration3436.

Finally, our data, showing that GFAPCre transgenic mice efciently label cholangiocytes but not a signicant amount of HSCs, suggest that previous studies employing GFAPCre13,18,19,37,38 are likely to be not HSC-specic. We cannot completely rule out that there may be differences in the recombination in different oxed alleles or Cre reporters, but have tested three Cre reporters, conrmed GFAPCre by sequencing, tested functional Cre expression in brain sections and have used mice expressing Cre under the human GFAP as well as the murine Gfap promoter. Furthermore, previous publications18,19 and Jackson

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Laboratories (https://ndp.jax.org/NDPServe.dll?ViewItem?ItemID=1741&XPos=11536496&YPos=5169511&ZPos=0&Lens=1.25&SignIn=Sign%20in%20as%20Guest

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Web End =ID=1741&XPos=11536496&YPos=5169511&ZPos=0& https://ndp.jax.org/NDPServe.dll?ViewItem?ItemID=1741&XPos=11536496&YPos=5169511&ZPos=0&Lens=1.25&SignIn=Sign%20in%20as%20Guest

Web End =Lens=1.25 &https://ndp.jax.org/NDPServe.dll?ViewItem?ItemID=1741&XPos=11536496&YPos=5169511&ZPos=0&Lens=1.25&SignIn=Sign%20in%20as%20Guest

Web End =SignIn=Sign%20in%20as%20Guest) have reported Cre activity in cholangiocytes.

Our study has several limitations. First of all, murine brogenesis may not fully reect human brogenesis due to its shorter duration, different biology and difculties to model all relevant diseases adequately in mice. We have assessed the HSC contribution in a large number of murine models, but human conrmation is warranted. This may be achieved by developing markers that clearly distinguish between HSC and non-HSC myobroblast populations such as portal broblasts. Second of all, LratCre also labelled some VSMCs in our study. This is likely to be because of the fact that pericytes and VSMCs share a common precursor in many organs, including the liver24,25. However, we found that 490% of LratCre-labelled myobroblasts contained retinoid lipid droplets characteristic for HSCs, thus providing strong evidence that LratCre-labelled myobroblasts are indeed derived from HSCs. Third, it is conceivable that LratCre activity is turned on in non-HSC populations during brogenesis, and that we overestimate the contribution of HSCs. However, we found that non-HSC myoboblast populations such as PFLCs were LratCre-negative and distinct from HSC. We also did not nd a signicant amount of LratCre-labelled hepatocytes, cholangiocytes, endothelial cells or Kupffer cells in CCl4-induced liver injury, providing further evidence that LratCre activity is not signicantly induced in non-HSC populations in the injured liver. Moreover, the above discussed nding that 490% of LratCre-positive myobroblasts also contained retinoids characteristic for HSCs also excludes a signicant contribution of non-HSC populations. As such, retinoid lipid droplets are a dening feature of HSCs and the only parameter by which they could be clearly dened to date. Tamoxifen-inducible LratCre would be helpful to further conrm these data and further exclude LratCre activity in other cell types during disease, but is likely to achieve a lower labelling efcacy than constitutive LratCre and a less precise quantication of HSC contribution to the hepatic myobroblast pool. Fourth, our isolation of PFLCs may contain cells other than portal boblasts as the employed isolation method relied on exclusion of HSCs and not on positive identication. Further establishment of high purity portal broblast isolates will allow more powerful qualitative comparison between HSCs and portal broblasts.

In contrast to the dominant role of HSCs in liver brosis, the contribution of pericytes in brosis of other organs remains controversial with some studies in the kidney, lung and spinal cord3941 showing a key contribution but other studies reporting opposite ndings42,43. Conrming a key role for pericytes in brogenesis in additional organs may allow to determine common targets for antibrotic therapies across different organs.

Methods

Generation of LratCre mice. A Cre-containing cassette was PCR-amplied with 60-bp overhangs homologous to the upstream and downstream sequence surrounding the ATG site of the mouse Lrat gene. The PCR product was inserted into a BAC containing the mouse Lrat gene by recombineering. After removal of the Neo cassette by arabinose-induced ippase, BAC DNA was microinjected into the pronucleus of fertilized CBAxC57BL/6J oocytes. Out of six positive founders, the one showing the strongest LratCre-induced Cre reporter expression in the liver and HSCs was used for further studies. Pups were born in a male/female ratio of 1:1(50.3% versus 49.7%, n 179).

Mice and genotyping. Mice in which Cre expression is driven by the human GFAP promoter (hGFAPCre)44, the murine Gfap promoter (mGfapCre)45 and mice expressing Cre reporters ZsGreen, TdTomato46 or mTom/mGFP47 were obtained from Jackson. Mdr2ko mice48 in FVB/N background have been described. For all experiments, LratCre mice were maintained in a mixed background after breeding one to two times into the ZsGreen, tdTomato reporter and mTom/mGFP

strains of the C57Bl/6 background, or Mdr2ko mice. Mice for HSC isolations were used at ages 1218 weeks. Mice used for brosis or injury models, or for nonparenchymal cell isolation were used at ages 812 weeks unless otherwise indicated. Mice of both genders were used with the exception of the BDL and DDC models for which only male mice were employed. Genotyping for LratCre was done using forward primer 50-CCTTTCTTTGACCCCCTGCAC-30 and reverse primer 50-GACCGGCAAACGGACAGAAG-30. Genotyping for hGFAPCre mice was done using forward primer 50-ACTCCTTCATAAAGCCCT-30 and reverse primer 50-CGCCGCATAACCAGTGAAAC-30. The PCR product for hGFAPCre genotyping was sequenced and presence of human GFAP promoter sequence was conrmed.

Liver brosis and injury models. Toxic liver brosis was induced by intraperitoneal injections of either CCl4 (0.5 ml g 1, dissolved in corn oil at a ratio of 1:3) for various intervals, or of TAA (dissolved in NaCl 0.9%) for 6 weeks (three injections per week) at increasing concentrations (rst dose: 50 mg kg 1, second dose: 100 mg kg 1, third to sixth dose: 200 mg kg 1, all following doses:300 mg kg 1) as previously described49. For the induction of cholestatic liver brosis, mice underwent ligation of the common bile duct27. Briey, after abdominal incision, the common bile duct was ligated distally. For additional models of cholestatic liver brosis, mice were either fed a 0.1% DDC-containing diet for 4 weeks or LratCre mice were crossed with Mdr2ko mice48 in FVB/N background. ZsGreen, tdTomato and mTom/mGFP Cre reporter mice46,47, as well as Col-GFP reporter22 mice, have been described elsewhere. As a model of fatty liver disease, liver brosis was induced by feeding mice a methionecholinedecient diet for 9 weeks. Seventy per cent partial hepatectomy was performed as described50. Briey, after midline abdominal incision, the left lateral and the median liver lobes were mobilized, ligated and cut off50. As models of liver injury with progenitor expansion, we employed the above described DDC diet and methioninecholine-decient diet combined with 0.15% ethionine supplementation in drinking water (MCDE diet)18,19. All animal procedures were in accordance with guidelines by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Columbia University.

Primary cell isolations. HSCs were isolated from mice as described previously49,51. Briey, after cannulation of the inferior vena cava, the portal vein was cut, allowing retrograde stepwise perfusion with pronase (Sigma-Aldrich, St Louis, MO, USA)- and collagenase (Roche, Germany)-containing solutions, and subsequent 9.7% Nycodenz gradient centrifugation. Purity was assessed by vitamin A autouorescence under a uorescent microscope (Olympus 71IX). The entire population of non-parenchymal cells was isolated using pronase/collagenase perfusion and a 16.95% Nycodenz gradient. Murine hepatocytes were isolated by collagenase perfusion and low-density centrifugation with Percoll52. F4/80-positive hepatic macrophages were isolated after collagenase/pronase perfusion, followed by a 16.95% Nycodenz gradient and subsequent positive selection of F4/80-positive cells by magnetic-activated cell sorting (MACS) using biotinylated F4/80 antibody (clone: BM8; eBioscience, San Diego, CA, USA) and anti-biotin MACS beads (Miltenyi Biotec, Auburn, CA, USA)53. On the basis of the specic marking of bile ducts by hGFAPCre, cholangiocytes were isolated by ow cytometry from hGFAPCre mice expressing ZsGreen Cre reporter after pronase/collagenase perfusion of mouse livers. Cholangiocyte identity was conrmed by western blotting for cytokeratin 19. Liver sinusoidal endothelial cells were isolated by collagenase perfusion followed by MACS using anti-CD146 MACS beads (Miltenyi Biotec) and subsequent FACS analysis to exclude HSCs in the isolate54.

Analysis of vitamin A content and Col-GFP expression. Vitamin A uorescence was analysed by ow cytometry in isolated HSCs or non-parenchymal liver cells using 405407 nm lasers for excitation and a 450/50-nm bandpass lter for detection. To determine vitamin A expression of activated HSCs, Col-GFP- and tdTomato-double-positive cells were gated. Vitamin A-negative cells expressing Col-GFP were used as negative controls to set the threshold for positive signals for Vitamin A. GFP expression was analysed using 488 nm lasers and 530/30 nm bandpass lter for detection. Non-parenchymal liver cell isolations from untreated Col-GFP-positive, LratCre-positive and tdTomoto-positive mice were used to set the threshold for positive Col-GFP signals. TdTomato expression was analysed using 488 nm lasers and 582/15 nm bandpass lter for detection. Four to six independent cell isolates were analysed for each model, using a minimum for 100,000 events for data evaluation. Flow cytometric data were analysed by FlowJo software.

BM transplantation. BM transplantation was performed as described previously27. Briey, wild-type mice were macrophage depleted by injection of liposomal clodronate, followed by lethal irradiation with 2 6 Gy and intravenous

injection of 10 106 BM cells from LratCre-positive, mTom/mGFP mice.

Successful BM transplantation was conrmed by the presence of mTom-positive cells in the spleen and mTom-positive macrophages in the liver.

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Immunohistochemical staining and microscopy. Immunohistochemistry was performed on frozen liver sections. Mouse livers were perfused with 4% paraformaldehyde and embedded. Frozen tissue blocks were cut to yield 5 mm sections for immunohistochemical staining49 using primary antibodies against desmin (1:300, rabbit, Lab Vision catalogue number RB-9014-P; Thermo Fisher Scientic, Fremont, CA, USA), aSMA (1:500, mouse, uorescein isothiocyanate-conjugated,

Sigma-Aldrich F3777), cytokeratin (1:250, rabbit, DAKO Z0622), F4/80 (1:250, rat, AbD serotec MCA497A64), CD31 (1:500, rat, Pharmingen 553369), PDGFRb (1:50, rabbit, Cell Signaling 3169) and HNF4a (1:100, goat, Santa Cruz Biotechnology SC-6556), and matching secondary anti-rabbit (1:500, donkey, A21207), anti-rat (1:500, chicken, A21472), anti-uorescein isothiocyanate (1:1,000, rabbit, A11090) and anti-goat (1.500, chicken, A21468) with various uorescent conjugates (all from Invitrogen). Confocal microscopy was performed on a Nikon A1 confocal laser microscope (Nikon Instruments, Melville, NY, USA) using a 20 lens or 40 and 60 oil-immersion lenses. For some pictures and

for quantication, four to six sections were merged. For macroscopic imaging, livers were visualized under a Leica MZ 16F uorescent dissecting microscope.

Immunoblotting. Immunoblotting for Lrat, desmin or cytokeratin was performed on isolated primary hepatic cell populations using a mouse anti-Lrat antibody (dilution 1:5,000)55, desmin (Lab Vision, 1:2,000) and cytokeratin 19 (rat, Troma-III, 1:1,000, Developmental Studies Hybridoma Bank, University of Iowa). Briey, cell or liver lysates were electrophoresed on 10% acrylamide SDS gels and transferred to nitrocellulose membrane. Loading was conrmed by Ponceau S staining. Blots were blocked in 5% non-fat dry milk, followed by overnight incubation at 4 C with primary antibody, 1 h incubation with horseradish peroxidase-conjugated secondary antibody and detection by luminescence (SuperSignal, Thermoscientc). Specicity of the Lrat antibody was determined using liver extracts from wild-type and Lrat knockout mice (SupplementaryFig. S10C). Blots were reprobed with actin antibody (1:5,000; MP Biomedicals) to conrm equal loading. Full-length images of all immunoblots are shown in Supplementary Fig. S10.

Quantication of positively stained cells. HNF4a-positive hepatocytes were quantied in merged 20 pictures representing 70100 random 20 elds per

mouse. Three to four mice per treatment group were analysed. To determine Col-GFP-positive cells or aSMA-positive cells originating from HSCs, at least 50 random 40 pictures were analysed per mouse. Experiments were performed in

three to ve animals per treatment group. Quantication of HNF4a-positive, aSMA-positive cells and Col-GFP-positive cells, and determination of co-localization with uorescent Cre reporters were performed using ImageJ software.

Statistical evaluation. Statistical analysis was performed using Prism (GraphPad, San Diego, CA). Differences between two groups were calculated by Students t-test. Signicance of differences between multiple groups was determined by oneway analysis of variance, followed by Dunnetts post-hoc test. All data are expressed as meanss.d.

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Acknowledgements

This study was supported by NIH grants U54CA163111 (Sub 5298) and 5R01DK76920

and 5R01DK075830 (to R.F.S.). I.M. was supported by a postdoctoral fellowship from the

American Liver Foundation and a fellowship from the German Research Foundation

(ME 3723/1-1). P.H. was supported by a fellowship from the German Research Foun

dation (Hu 1953/1-1). D.H.D. was supported by 1F31DK091980. We thank Timothy

Wang and Daniel Worthley, Columbia University, for help with HSC ablation experi

ments. We thank David A. Brenner (University of California, San Diego) for kindly

providing the transgenic Col1a1-GFP mouse line and Krzysztof Palczewski (Case

Western Reserve University) for providing Lrat antibody.

Author contributions

I.M. performed in vivo injury models, primary cell isolations, generation of double, triple

and quadruple transgenic mice, data acquisition, data analysis and drafted the manu

script. C.C.H. performed immunohistochemistry and data analysis. J.S.T. generated

LratCre transgenic mice. P.H. isolated primary hepatocytes. X.M. performed immuno

histochemistry. D.H.D. performed immunohistochemistry and cell isolations. J.-P.P.

performed BM transplantations and macrophage isolations. R.F.S. designed and oversaw

the study, performed data analysis and drafted the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Mederacke, I. et al. Fate tracing reveals hepatic stellate cells as

dominant contributors to liver brosis independent of its aetiology. Nat. Commun.

4:2823 doi: 10.1038/ncomms3823 (2013).

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