WO2017005886A1 - Reverted stellate cells and uses thereof - Google Patents

Reverted stellate cells and uses thereof Download PDF

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Publication number
WO2017005886A1
WO2017005886A1 PCT/EP2016/066213 EP2016066213W WO2017005886A1 WO 2017005886 A1 WO2017005886 A1 WO 2017005886A1 EP 2016066213 W EP2016066213 W EP 2016066213W WO 2017005886 A1 WO2017005886 A1 WO 2017005886A1
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stellate cells
acid
fibrosis
composition
fgf2
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Mustapha Najimi
Adil EL TAGHDOUINI
Leonardus VAN GRUNSVEN
Étienne SOKAL
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Universite Catholique de Louvain UCL
Vrije Universiteit Brussel VUB
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Universite Catholique de Louvain UCL
Vrije Universiteit Brussel VUB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells

Definitions

  • the present invention relates to compositions and methods for treating fibrosis.
  • the present invention relates to a composition comprising EGF and/or FGF2, and to the use thereof for reverting stellate cells or for treating fibrosis.
  • US 2013/0101553 discloses reversal of hepatic stellate cells in mouse models and identified markers of activated HSCs, such as Hspala/b, PPARa and PPARy. Moreover, US 2013/0101553 suggests that compounds upregulating the expression of these markers may reduce symptoms of fibrosis. However, US 2013/0101553 does not exemplify the effect of any of the claimed compounds in the treatment of fibrosis.
  • the present invention relates to a composition comprising EGF and/or FGF2 and to the use thereof for reverting stellate cells or for treating fibrosis.
  • Another object of the present invention is a composition comprising EGF, FGF2, oleic acid, palmitic acid and retinol, wherein the concentration of EGF ranges from 0.1 to 100 ng/mL, the concentration of FGF2 ranges from 0.05 to 80 ng/mL, the concentration of oleic acid ranges from 1 to 1000 nmol/mL, the concentration of palmitic acid ranges from 1 to 1000 nmol/mL and the concentration of retinol ranges from 0.01 to 100 nmol/mL.
  • Another object of the present invention is a culture medium comprising the composition as described hereinabove.
  • the present invention further relates to an in vitro method for determining the pro-fibrotic activity of a tested agent comprising the steps of in vitro incubating stellate cells in a culture medium as described hereinabove; adding the tested agent in the culture medium; and determining the phenotype of said stellate cells, preferably the activated or quiescent phenotype of said stellate cells.
  • Subject refers to a mammal, preferably a human.
  • a subject may be a "patient”, i.e. a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of fibrosis.
  • Treating refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted fibrosis.
  • Those in need of treatment include those already with the disease as well as those prone to have the disease or those in whom the disease is to be prevented.
  • dietary fatty acids such as oleic, palmitic acids and retinol (also named retinoic acid) leads to the reversion of activated stellate cells to a quiescent-like state (see Examples).
  • the concentration of retinol in the composition of the invention ranges from 0.01 to 100 nmol/mL, preferably from 0.1 to 50 nmol/mL, more preferably from 0.5 to 20 nmol/mL, more preferably the concentration of retinol in the composition of the invention is of about 5 nmol/mL.
  • oleic acid preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably about 100 nmol/mL, from 1 to 1000 nmol/mL of palmitic acid, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably about 100 nmol/mL, and
  • the composition of the invention has a pH ranging from 6.8 to 8.0, preferably from 7.0 to 7.8, more preferably from 7.2 to 7.6.
  • gene markers of activated stellate cells include, but are not limited to, ACTA2, COL1A1, LOX, LOXL1, LOXL2, LOXL3, PDGFRB, COL4A1, COL4A2, COL5A1, ADAM 12, ADAMTS2, ACTG2, NOTCH3 and CRYAB, preferably ACTA2, COL1A1 and LOX.
  • expression may refer alternatively to the transcription of a gene marker of activated stellate cells (i.e. expression of the RNA) or to the translation (i.e. expression of the protein) of a gene marker of activated stellate cells.
  • a tested agent has a pro-fibrotic activity if the expression of the at least one gene marker of activated stellate cells is higher than the reference expression profile.
  • the term "higher” means at least 10% higher, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher or more than the reference expression profile.
  • determining the phenotype of said stellate cells corresponds to evaluating the presence or the number of lipid containing droplets in said stellate cells.
  • a tested agent has a pro-fibrotic activity if lipid containing droplets are absent of stellate cells incubated with said tested agent.
  • a tested agent as a pro-fibrotic activity if the number of lipid containing droplets is lower in stellate cells incubated with said tested agent than in stellate cells incubated in the absence of the tested agent.
  • the term "lower” means at least 10% lower, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower or more than the number of lipid containing droplets in stellate cells incubated in the absence of the tested agent.
  • the method for determining the pro-fibrotic activity of a test agent comprising the steps of:
  • determining the phenotype of said stellate cells by: determining the expression profile of at least one gene marker of activated stellate cells in said stellate cells, and comparing this expression profile with a reference expression profile, and
  • the reference expression profile is the expression profile of the at least one gene marker of activated stellate cells in stellate cells incubated in a culture medium according to the invention without the tested agent.
  • salts with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.
  • salts with a basic amino acid include salts with arginine, lysine and ornithine.
  • salts with an acidic amino acid include salts with aspartic acid and glutamic acid.
  • a list of suitable salts is disclosed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p 1418, 1985, the entire disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition of the invention comprises EGF and at least one dietary fatty acid in combination with at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition of the invention comprises EGF and oleic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, oleic acid and palmitic acid in combination with at least one pharmaceutically acceptable excipient.
  • Another object of the invention is a medicament comprising EGF and/or FGF2 or a pharmaceutically acceptable salt or solvate thereof, as described hereinabove.
  • the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof.
  • the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof.
  • the medicament of the invention comprises EGF and FGF2 or a pharmaceutically acceptable salt or solvate thereof.
  • the medicament further comprises at least one dietary fatty acid.
  • the at least one dietary fatty acid is selected from oleic acid, palmitic acid, retinol and mixture thereof.
  • the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and oleic acid and retinol. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof, palmitic acid and retinol. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof, oleic acid, palmitic acid and retinol.
  • the subject is affected with a liver disease, preferably selected from the list comprising significant fibrosis, cirrhosis, the fibrosis being from alcoholic or nonalcoholic origin and/or the subject is a patient affected with a chronic disease, preferably said chronic disease is selected from the group comprising chronic viral hepatitis C, chronic viral hepatitis B, chronic viral hepatitis D, chronic viral hepatitis E, non-alcoholic fatty liver disease (NAFLD), alcoholic chronic liver disease, autoimmune hepatitis, primary biliary cirrhosis, hemochromatosis and Wilson disease.
  • a liver disease preferably selected from the list comprising significant fibrosis, cirrhosis, the fibrosis being from alcoholic or nonalcoholic origin and/or the subject is a patient affected with a chronic disease, preferably said chronic disease is selected from the group comprising chronic viral hepatitis C, chronic viral hepatitis B, chronic viral hepatitis D, chronic viral hepatitis
  • Nycodenz-isolated HSCs cultured for 1 day or for 3 passages were washed with PBS and fixed for 10 minutes with 4% buffered formaldehyde (Merck, Darmstadt, Germany). Following permeabilization with 0.1% Triton-X 100 (in PBS containing 1% bovine serum albumin), cells were incubated overnight with anti-aSMA (1/1000) (Sigma). Primary antibody binding was visualized using an Alexa488-labeled secondary antibody (1/200) (Invitrogen, Eurgene, OR). Images were taken with an AxioCam MRc5 digital camera (Carl Zeiss).
  • qHSCs reverted by this mix of growth factors and dietary components (further referred to as RM) present a thinner cell body and intra-cytoplasmic lipid droplets (Fig. 7).
  • An important functional hallmark of qHSCs is their ability to store vitamin A in their cytoplasmic lipid droplets.
  • Example 5 In vitro reverted human HSCs have a reduced proliferation rate
  • aHSCs functionally differ from qHSCs by their increased proliferation rate, unbalanced ECM homeostasis and higher migratory potential (Friedman, Physiological Reviews. 2008, 88: 125-172). Therefore, it was further investigated whether human in vitro rHSCs functionally differed from their activated counterparts by comparing their proliferation (EdU-incorporation), ECM degradation (in situ zymography) and PDGFbb-induced migration (transwell assay). Results show that rHSCs (obtained with RM) displayed a 75% reduction in proliferation compared to aHSC (Fig. 11A).
  • results show that the global gene expression profile of rHSCs resembles more closely to that of aHSCs than qHSCs, with 2277 (rHSC vs aHSC) against 9122 (rHSC vs qHSC) genes significantly differentially regulated (student t-test, p ⁇ 0,05).
  • Many of the top upregulated genes in rHSCs are inflammation-related, i.e. IL-8, IL-33, IL- ⁇ , CXCL1, and CXCL6 (Table 2).
  • Over 60% of the >2-fold deregulated genes in rHSCs compared to aHSCs are downregulated and include genes such as TNNT2, SULT1E1, ACTG2, and SYNP02L (Table 2).
  • Table 2 The fold-change of the top 15 upregulated and top 15 downregulated g rHSCs, compared to the aHSCs
  • mice were male BALB/c (Charles River) of 6-7 weeks. Treatment Administrations
  • CC1 4 solution a freshly-prepared mix of volume of 15 ⁇ ⁇ CC1 4 (Sigma Fluka, 87031) and 85 ⁇ ⁇ of corn oil) or same volume of corn oil via the IP routes according to QA/PROD34.
  • IP intraperitoneal
  • EGF and FGF2 pump placement EGF/FGF2 solution preparation
  • EGF/FGF2 solution containing EGF (Peprotech London, M315-09) at 20 ng/ml and FGF2 (Peprotech London, 450-33) at 10 ng/ml was prepared with PBS for all the Alzet pumps. The solution was aliquoted and stored at -20°C.
  • the ALZET pump model 2002 (volume of 200 ⁇ for 2 weeks with a 0.5 ⁇ /h rate) was used.
  • mice All mice were euthanized by cervical dislocation according to SOP QA/PROD31 and necropsied. Two control mice of GPl to GP4 were euthanized after 2 weeks of treatment at Day+14.
  • liver slices were stained using Sirius red and counterstained with fast green and light microscopic images were captured.
  • PCR amplification mixtures (25 ⁇ ) contained 25 ng template cDNA, Master Mix buffer (12.5 mL; Applied Biosystems), and the corresponding Taqman assay were run in duplicate and performed on a StepOnePlus Real-time PCR (Applied Biosystems).
  • the cycling conditions comprised 10 min polymerase activation at 95 °C and 40 cycles at 95 °C for 15 s and 60°C for 1 min. Relative quantification was normalized against the house keeping gene ⁇ -2-microglobulin.
  • the Applied Biosystems assays used for the current study are listed in Table 5.
  • the EGF/FGF2 solution of the invention is able to reduce liver fibrosis.

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Abstract

The present invention relates to a composition for treating fibrosis comprising EGF and/or FGF2, and optionally at least one dietary fatty acid. The present invention also relates to a process for reverting activated stellate cells, preferably hepatic stellate cells, and to reverted stellate cells obtained by such process. The present invention also relates to in vitro methods for determining the pro- or anti-fibrotic activity of an agent.

Description

REVERTED STELLATE CELLS AND USES THEREOF
FIELD OF INVENTION
The present invention relates to compositions and methods for treating fibrosis. In particular, the present invention relates to a composition comprising EGF and/or FGF2, and to the use thereof for reverting stellate cells or for treating fibrosis.
BACKGROUND OF INVENTION
In response to injury, tissue can heal and restore its normal function through a controlled sequence of events known as wound-healing. However, when the insult is of chronic nature it can impair the wound-healing response and subsequently develop into tissue fibrosis, a condition characterized by the excessive accumulation of matrix proteins and associated with severe morbidity and mortality. Different cellular sources, including tissue- specific fibroblasts, bone-marrow derived progenitor cells, pericytes and epithelial cells have been suggested to give rise to myofibroblasts, the major source of extracellular matrix (ECM) components in the fibrotic organ.
In the liver however, the resident hepatic stellate cells (HSCs) have unambiguously been identified as the predominant source of myofibroblasts, irrespective of the underlying disease etiology (Mederacke et al. Nat Commun. 2012, 4). In the normal liver, quiescent HSCs (qHSCs) reside in a virtual space (of Disse) between the hepatocytes and liver sinusoidal endothelial cells and are characterized by the abundance of cytoplasmic lipid droplets containing up to 80% of total vitamin A body reserve (Friedman, Physiological Reviews. 2008, 88: 125-172). Besides their well-known role in the regulation of retinoid and ECM homeostasis, there is evidence that HSCs can regulate the sinusoidal blood flow and stimulate angiogenesis (Reynaert et al. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology. 2008, 291: 693-698; Taura et al. Gastroenterology. 2008, 135: 1729-1738). Following chronic liver injury, qHSCs undergo a process of activation, during which they transdifferentiate into cells with a fibrogenic, myofibroblast-like phenotype characterized by increased ACTA2 expression which leads to an enhanced contractility, and an augmentation in ECM production and secretion (Friedman, Physiological Reviews. 2008, 88: 125-172; Taura et al. Gastroenterology. 2008, 135: 1729-1738). Initially, this activation process was considered to be unidirectional while the principal ability of the fibrotic liver to revert to normal state upon cessation of injury (Ellis et al. Journal of Hepatology. 2012, 56: 1171-1180) was mainly attributed to apoptotic clearance of activated HSCs (aHSCs) that undergo apoptosis (Iredale et al. J Clin Invest. 1998, 102: 538-549; Issa et al. Gut. 2001, 48: 548-557). However, different studies strongly imply that the activated phenotype of HSCs can be modulated and reverted to quiescent-like state in vivo in rodents (Kisseleva et al. PNAS. 2012; Troeger et al. Gastroenterology. 2012, 143: 1073-1083.el022). Although these studies describe HSCs deactivation after cessation of injections which cause liver fibrosis in mouse models, no component was identified to induce such HSCs deactivation in vivo.
As activation of hepatic stellate cells is the central event in hepatic fibrosis, reversal of HSC activation could contribute to fibrosis cure. Stellate cell-like cells are present in various tissues and organs that can be affected by fibrosis, such as for example lung, kidney, pancreas, intestine, and the like. Therefore, for treating fibrosis, it could be of interest to develop compositions and/or methods for reversing HSC-like cells to a non- activated state.
The patent application US 2013/0101553 discloses reversal of hepatic stellate cells in mouse models and identified markers of activated HSCs, such as Hspala/b, PPARa and PPARy. Moreover, US 2013/0101553 suggests that compounds upregulating the expression of these markers may reduce symptoms of fibrosis. However, US 2013/0101553 does not exemplify the effect of any of the claimed compounds in the treatment of fibrosis.
Different studies also demonstrated the reversal of HSCs in vitro. Hazra et al. discloses that depletion of PPARy is a key feature in the activation of HSCs and demonstrates that restoration of this component reverses the activated HSCs to a quiescent phenotype (Journal of Biological Chemistry. 2004, 279: 11392-11401). She et al. extends these findings and describes the use of an adipocyte differentiation mixture comprising isobutylmethylxanthine, dexamethasone, and insulin, or the ectopic expression of PPARy or SREBP-lc, to reverse activated HSCs to a quiescent phenotype (Journal of Biological Chemistry. 2005, 280: 4959-4967).
Nevertheless, to the Applicant knowledge, there is currently no molecule restoring a quiescent phenotype of stellate cells which has been validated in human by clinical trials. Therefore, there is still a need for compounds that allow reversing activated stellate cells, thereby treating fibrosis. Identification of a potential pro-fibrotic activity of agents could be useful to prevent or minimize drug-related fibrotic adverse effects. However, actual conditions for in vitro stellate cells culture are limited by cell activation as soon as they are cultured. Consequently, identification of anti- and pro-fibrotic agents is currently performed in vivo in animal models. Therefore, a compound allowing reversing activated stellate cells may also be of interest for developing culture media for maintaining HSCs in a non-activated state in culture. Such culture media could thus be used for screening agents for their pro- or anti-fibrotic activity.
The inventors herein surprisingly demonstrate that the presence of EGF and/or FGF2 in a culture medium reverses activated stellate cells to a quiescent-like state (see Examples). Therefore, the present invention relates to a composition comprising EGF and/or FGF2 and to the use thereof for reverting stellate cells or for treating fibrosis.
SUMMARY
The present invention thus relates to a composition for treating fibrosis comprising EGF and/or FGF2. In one embodiment, the composition further comprises at least one dietary fatty acid. In one embodiment, said at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, and retinol, preferably oleic acid, palmitic acid or retinol, or a mixture thereof.
In one embodiment, said fibrosis is selected from the group comprising liver fibrosis, pulmonary fibrosis, heart fibrosis, Crohn's Disease, progressive kidney disease, pancreatic fibrosis, scleroderma/systemic sclerosis, post-surgery adhesions, peritoneal adhesions, retroperitoneal fibrosis, pleural fibrosis, pericardial fibrosis, uterine fibroid, and graft fibrosis.
In a particular embodiment, said fibrosis is liver fibrosis, wherein said liver fibrosis is cirrhosis. In another particular embodiment, fibrosis is pulmonary fibrosis, wherein said pulmonary fibrosis is idiopathic pulmonary fibrosis or cystic fibrosis.
The present invention also relates to an in vitro process for reverting stellate cells comprising a step of incubating stellate cells into a composition comprising EGF and/or FGF2. In one embodiment, said composition further comprises at least one dietary fatty acid. In one embodiment, said at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, and retinol, preferably oleic acid, palmitic acid or retinol, or a mixture thereof.
In one embodiment, said stellate cells are selected from the group comprising hepatic stellate cells (HSCs), pancreatic stellate cells (PaSCs), lung stellate cells, intestinal stellate cells, kidney stellate cells, spleen stellate cells, adrenal gland stellate cells, ductus deferens stellate cells and vocal cords stellate cells, preferably HSCs, PaSCs, lung stellate cells and intestinal stellate cells, more preferably HSCs.
The present invention further relates to an isolated reversed stellate cells population obtained by the in vitro process as described hereinabove.
Another object of the present invention is a composition comprising EGF, FGF2, oleic acid, palmitic acid and retinol, wherein the concentration of EGF ranges from 0.1 to 100 ng/mL, the concentration of FGF2 ranges from 0.05 to 80 ng/mL, the concentration of oleic acid ranges from 1 to 1000 nmol/mL, the concentration of palmitic acid ranges from 1 to 1000 nmol/mL and the concentration of retinol ranges from 0.01 to 100 nmol/mL. Another object of the present invention is a culture medium comprising the composition as described hereinabove.
The present invention further relates to an in vitro method for determining the pro-fibrotic activity of a tested agent comprising the steps of in vitro incubating stellate cells in a culture medium as described hereinabove; adding the tested agent in the culture medium; and determining the phenotype of said stellate cells, preferably the activated or quiescent phenotype of said stellate cells.
The present invention also relates to a kit of part comprising two parts, wherein the first part comprises a composition as described hereinabove and the second part comprises a stellate cells population.
DEFINITIONS
In the present invention, the following terms have the following meanings: - "About" preceding a value means plus or less 10% of said value.
"Culture medium" refers to a composition comprising components for supporting the growth and maintenance of cells in culture. Usually, a culture medium comprises a carbon source, various salts and water.
"Fibrosis" refers to the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. In one embodiment, fibrosis involves excessive collagen mRNA production and deposition (mostly Type I collagen). In another embodiment, fibrosis is caused, at least in part, by injury, e.g. chronic injury (e.g. an insult, a wound, a toxin, a disease). In another embodiment, fibrosis is associated with an inflammatory, an autoimmune or a connective tissue disorder. Tissues that may be affected by fibrosis include, without limitation, liver tissue, lung tissue, heart tissue, kidney tissue, skin tissue, gut tissue, peritoneal tissue, bone marrow, and the like. "Subject" refers to a mammal, preferably a human. In one embodiment, a subject may be a "patient", i.e. a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of fibrosis.
"Treating" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted fibrosis. Those in need of treatment include those already with the disease as well as those prone to have the disease or those in whom the disease is to be prevented. A subject is successfully "treated" for fibrosis if, after receiving a therapeutic amount of the composition, pharmaceutical composition or medicament of the present invention, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction or absence of fibrotic area; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, of one or more of the symptoms associated with the specific fibrosis; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the respiratory disease are readily measurable by routine procedures familiar to a physician.
"Therapeutically effective amount" means level or amount of the composition, pharmaceutical composition or medicament of the invention that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of fibrosis; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of fibrosis; (3) bringing about ameliorations of the symptoms of fibrosis; (4) reducing the severity or incidence of fibrosis; or (5) curing fibrosis. A therapeutically effective amount may be administered prior to the onset of the fibrosis, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the fibrosis, for a therapeutic action.
"Pharmaceutically acceptable carrier or excipient" refers to an excipient or carrier that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, injected preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.
DETAILED DESCRIPTION
The inventors herein demonstrated that the presence of epithelial growth factor (EGF) and/or fibroblast growth factor 2 (FGF2) in a culture medium leads to the reversion of activated stellate cells, such as, for example, stellate cells in culture, to a quiescent-like state (see Examples). This invention thus relates to a composition comprising EGF and/or FGF2. In one embodiment, the composition comprises EGF. In another embodiment, the composition comprises FGF2. In another embodiment, the composition comprises EGF and FGF2.
In one embodiment of the present invention, the concentration of EGF in the composition of the invention ranges from 0.1 to 100 ng/mL, preferably from 1 to 75 ng/mL, more preferably from 5 to 50 ng/mL, more preferably the concentration of EGF in the composition of the invention is of about 20 ng/mL.
In one embodiment of the present invention, the concentration of FGF2 in the composition of the invention ranges from 0.05 to 80 ng/mL, preferably from 0.1 to 50 ng/mL, more preferably from 1 to 30 ng/mL, more preferably the concentration of FGF2 in the composition of the invention is of about 10 ng/mL.
The inventors herein also demonstrated that the presence of dietary fatty acids such as oleic, palmitic acids and retinol (also named retinoic acid) leads to the reversion of activated stellate cells to a quiescent-like state (see Examples).
Therefore, in one embodiment, the composition of the invention further comprises at least one dietary fatty acid.
In one embodiment of the present invention, the concentration of the at least one dietary fatty acid ranges from 1 to 1000 nmol/mL, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably the concentration of the at least one dietary fatty acid is of about 100 nmol/mL.
In one embodiment, the composition of the invention comprises EGF and at least one dietary fatty acid. In another embodiment, the composition of the invention comprises FGF2 and at least one dietary fatty acid. In another embodiment, the composition of the invention comprises EGF, FGF2 and at least one dietary fatty acid.
In one embodiment, the at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, retinol or a mixture thereof. In a particular embodiment, the at least one dietary acid is selected from oleic acid, palmitic acid and retinol or a mixture thereof. In one preferred embodiment, the composition of the invention comprises oleic acid, palmitic acid and retinol.
In one embodiment of the present invention, the concentration of oleic acid in the composition of the invention ranges from 1 to 1000 nmol/mL, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably the concentration of oleic acid in the composition of the invention is of about 100 nmol/mL.
In one embodiment of the present invention, the concentration of palmitic acid in the composition of the invention ranges from 1 to 1000 nmol/mL, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably the concentration of palmitic acid in the composition of the invention is of about 100 nmol/mL.
In one embodiment of the present invention, the concentration of retinol in the composition of the invention ranges from 0.01 to 100 nmol/mL, preferably from 0.1 to 50 nmol/mL, more preferably from 0.5 to 20 nmol/mL, more preferably the concentration of retinol in the composition of the invention is of about 5 nmol/mL.
In one embodiment of the present invention, the concentration of retinol in the composition of the invention ranges from 0.01 to 95.5 IU/mL, preferably from 0.095 to 47.7 IU/mL, more preferably from 0.48 to 19.1 IU/mL, more preferably the concentration the concentration of retinol in the composition of the invention is of about 4.8 IU/mL.
In one embodiment, the composition of the invention comprises EGF and oleic acid. In another embodiment, the composition of the invention comprises EGF and palmitic acid. In another embodiment, the composition of the invention comprises EGF and retinol. In another embodiment, the composition of the invention comprises EGF, oleic acid and palmitic acid. In another embodiment, the composition of the invention comprises EGF, oleic acid and retinol. In another embodiment, the composition of the invention comprises EGF, palmitic acid and retinol. In another embodiment, the composition of the invention comprises EGF, oleic acid, palmitic acid and retinol.
In one embodiment, the composition of the invention comprises FGF2 and oleic acid. In another embodiment, the composition of the invention comprises FGF2 and palmitic acid. In another embodiment, the composition of the invention comprises FGF2 and retinol. In another embodiment, the composition of the invention comprises FGF2, oleic acid and palmitic acid. In another embodiment, the composition of the invention comprises FGF2, oleic acid and retinol. In another embodiment, the composition of the invention comprises FGF2, palmitic acid and retinol. In another embodiment, the composition of the invention comprises FGF2, oleic acid, palmitic acid and retinol.
In one embodiment, the composition of the invention comprises EGF, FGF2 and oleic acid. In another embodiment, the composition of the invention comprises EGF, FGF2 and palmitic acid. In another embodiment, the composition of the invention comprises EGF, FGF2 and retinol. In another embodiment, the composition of the invention comprises EGF, FGF2, oleic acid and palmitic acid. In another embodiment, the composition of the invention comprises EGF, FGF2, oleic acid and retinol. In another embodiment, the composition of the invention comprises EGF, FGF2, palmitic acid and retinol. In another embodiment, the composition of the invention comprises EGF, FGF2, oleic acid, palmitic acid and retinol. In one preferred embodiment, the composition of the invention comprises:
from 0.1 to 100 ng/mL of EGF, preferably from 1 to 75 ng/mL, more preferably from 5 to 50 ng/mL, more preferably about 20 ng/mL,
from 0.05 to 80 ng/mL of FGF2, preferably from 0.1 to 50 ng/mL, more preferably from 1 to 30 ng/mL, more preferably about 10 ng/mL,
from 1 to 1000 nmol/mL of oleic acid, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably about 100 nmol/mL, from 1 to 1000 nmol/mL of palmitic acid, preferably from 10 to 500 nmol/mL, more preferably from 50 to 200 nmol/mL, more preferably about 100 nmol/mL, and
from 0.01 to 100 nmol/mL of retinol, preferably from 0.1 to 50 nmol/mL, more preferably from 0.5 to 20 nmol/mL, more preferably about 5 nmol/mL.
In one embodiment, the composition according to the invention is in form of solution. In another embodiment, the composition according to the invention is in form of powder to be dissolved or resuspended in water.
Another object of the present invention is the use of the composition as described hereinabove as a culture medium.
The invention also relates to a culture medium comprising a composition according to the invention. In one embodiment, the composition or the culture medium of the invention further comprises:
a carbon source for cell growth, which may be a sugar such as glucose, or a less energy-rich source like succinate,
various salts, which may vary among cell species and growing conditions, for example essential elements such as magnesium, nitrogen, phosphorus, or sulfur, and water.
In one embodiment, the composition or the culture medium of the invention is suitable for eukaryote cell culture, preferably for mammalian cell culture. In one embodiment, the composition for use as a culture medium or the culture medium of the invention comprises the composition according to the invention and a culture medium of the prior art such as for example MEM, DMEM, IMDM, RPMI 1640, 199/109 medium, HamF10/HamF12 or McCoy's 5 A.
In one embodiment, the composition further comprises any supplementary factors known by the person skilled in the art that may be used in cell culture. Examples of supplementary factors include, but are not limited to, FBS; glycine; amino acids, such as glutamine, asparagine, glutamic acid, aspartic acid, serine, proline or alanine, preferably the L-configuration of amino acids; and antibiotics, such as streptomycin or penicillin.
In one embodiment, the composition of the invention has a pH ranging from 6.8 to 8.0, preferably from 7.0 to 7.8, more preferably from 7.2 to 7.6.
In one embodiment, the composition of the invention is free of biological contamination such as for example bacteria, fungi (such as, for example, molds or yeasts), viruses, protozoa or mycoplasmas. In another embodiment, the composition according to the invention is sterile and stored in sterile conditions. The invention also relates to the use of the composition as described hereinabove for reverting stellate cells, preferably for in vitro reverting stellate cells.
As used herein, reverting stellate cells, or reversion of stellate cells, means allowing activated stellate cells to return to a non-activated state, preferably a quiescent-like state. In one embodiment, activated HSCs are HSCs that have transdifferentiated into cells with a fibrogenic, myofibroblast-like phenotype may be characterized by the loss of vitamin A containing lipid droplets. In another embodiment, activated HSCs may be characterized by an increased ACTA2, COL1A1 and LOX expression. In another embodiment, activated HSCs may be characterized by the loss of vitamin A containing lipid droplets and an increased ACTA2, COL1A1 and LOX expression. Alpha smooth muscle actin (ACTA1), collagen type I alpha 1 (COLlal) and lysyl oxidase (LOX) are known genes related to hepatic stellate cells activation (Mannaerts et al. PLoS ONE. 2013, 8(12):e84071). In one embodiment of the invention, a quiescent-like state or quiescent-like phenotype may be characterized by the presence of vitamin A containing lipid droplets. In another embodiment, a quiescent-like state or quiescent-like phenotype may be characterized by a low expression of ACTA2, COLlAl and LOX. In another embodiment, a quiescent- like state or quiescent-like phenotype may be characterized by the presence of vitamin A containing lipid droplets and a low expression of ACTA2, COLlAl and LOX. As used herein, the term "low level" or "low expression" refers to levels lower than level in activated stellate cells. In one embodiment, a low level is at least 10% lower than the level in activated stellate cells, preferably at least 20%, 30%, 40% or 50% lower than the level in activated stellate cells.
In one embodiment, stellate cells are selected from the group comprising hepatic stellate cells (HSCs), pancreatic stellate cells (PaSCs), lung stellate cells, intestinal stellate cells, kidney stellate cells, spleen stellate cells; preferably HSCs, PaSCs, lung stellate cells and intestinal stellate cells, and more preferably HSCs and PaSCs. In a preferred embodiment, stellate cells are HSCs.
In one embodiment, the composition according to the invention is used for reverting HSCs.
Another object of the invention is an in vitro process for reverting stellate cells, preferably HSCs. In one embodiment, the process of the invention comprises a step of culture of stellate cells within a culture medium of the invention or within a composition of the invention. In one embodiment, the composition is refreshed every day, or every two days, or every three days.
In one embodiment, the process of the invention optionally comprises a preceding step of culture of stellate cells in a composition that does not contain EGF, FGF2 and/or dietary components, which leads stellate cells to be activated.
In another embodiment, stellate cells used for the process according to the invention are freshly isolated quiescent stellate cells or not yet cultured stellate cells. This invention also relates to a reversed stellate cell population (rSC), preferably a reversed hepatic stellate cell population (rHSC).
In one embodiment, the reversed stellate cell population is an isolated reversed stellate population. In one embodiment, the reversed stellate cells population is characterized by a non- activated phenotype, preferably a quiescent-like phenotype.
In one embodiment, the reversed stellate cell population expresses a low level of ACTA2, COL1A1 and LOX. In another embodiment, the reversed stellate cell population is characterized by the presence of lipid containing droplets. In another embodiment, the reversed stellate cells population expresses a low level of ACTA2, COL1A1 and LOX, and is characterized by the presence of lipid containing droplets.
In one embodiment, the reversed stellate cell population is obtained by the in vitro process for reverting stellate cells as described hereinabove.
According to one embodiment, the reversed stellate cell population of the invention comprises reversed stellate cells and non-reversed, or activated, stellate cells. In a particular embodiment, the reversed stellate cell population at least 70%, preferably at least 75, 80, 85, 90, 95 or 100% of reversed stellate cells.
This invention also concerns a composition comprising a population of stellate cells and a composition according to the invention, wherein the stellate cells expand in said composition.
Another object of the invention is a kit of part comprising two parts, wherein the first part comprises a composition or a culture medium according to the invention and the second part comprises a stellate cells population, preferably a HSCs population.
In one embodiment, the first part of the kit of the invention comprises a composition or a culture medium according to the invention, wherein the composition or a culture medium is in the form of a solution. In another embodiment, the first part of the kit of the invention comprises a composition or a culture medium according to the invention, wherein the composition or a culture medium is in the form of a powder to be dissolved or resuspended in water.
In one embodiment, the stellate cell population of the second part of the kit of the invention is an activated stellate cell population. In another embodiment, the stellate cell population of the second part of the kit of the invention is a quiescent stellate cell population. In another embodiment, the stellate cell population of the second part of the kit of the invention is a reversed stellated cell population.
In one embodiment, the second part of the kit of the invention comprises a stellate cell population, wherein the stellate cells population is in the form of a frozen stock solution. In one embodiment, the stellate cells population is stocked in a composition according to the invention.
A further aspect of the present invention is a method for determining the pro-fibrotic activity of a tested agent, comprising the steps of:
a) in vitro incubating stellate cells in a culture medium of the invention or in a composition according to the invention,
b) adding the tested agent in the culture medium, and
c) determining the phenotype of said stellate cells, preferably the activated or quiescent phenotype of said stellate cells.
In one embodiment, "determining the phenotype of said stellate cells" corresponds to determining the expression profile of at least one gene marker of activated stellate cells in said stellate cells, and comparing this expression profile with a reference expression profile.
Examples of gene markers of activated stellate cells include, but are not limited to, ACTA2, COL1A1, LOX, LOXL1, LOXL2, LOXL3, PDGFRB, COL4A1, COL4A2, COL5A1, ADAM 12, ADAMTS2, ACTG2, NOTCH3 and CRYAB, preferably ACTA2, COL1A1 and LOX. As used herein, the term "expression" may refer alternatively to the transcription of a gene marker of activated stellate cells (i.e. expression of the RNA) or to the translation (i.e. expression of the protein) of a gene marker of activated stellate cells.
Methods for determining the expression profile are well-known from the skilled artisan, and include, without limitation, determining the transcriptome (in an embodiment wherein expression relates to transcription of a gene marker of activated stellate cells) or proteome (in an embodiment wherein expression relates to translation of a gene marker of activated stellate cells) of a stellate cell population cultured in presence of the tested agent. In one embodiment of the invention, the expression of the at least one gene marker of activated stellate cells is assessed at the RNA level. Methods for assessing the transcription level of a gene marker are well known in the prior art. Examples of such methods include, but are not limited to, RT-PCR, RT-qPCR, Northern Blot, hybridization techniques such as, for example, use of microarrays, and combination thereof including but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing such as, for example, next-generation DNA sequencing (NGS) or RNA-seq (also known as "Whole Transcriptome Shotgun Sequencing") and the like.
In one embodiment of the invention, the expression of the at least one gene marker of activated stellate cells is assessed at the protein level. Methods for determining a protein level in a sample are well-known in the art. Examples of such methods include, but are not limited to, immunohistochemistry, Multiplex methods (Luminex), western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and the like. In one embodiment of the invention, the reference expression profile is the expression profile of the at least one gene marker of activated stellate cells in stellate cells incubated in a culture medium according to the invention without the tested agent.
In one embodiment, a tested agent has a pro-fibrotic activity if the expression of the at least one gene marker of activated stellate cells is higher than the reference expression profile. In one embodiment, the term "higher" means at least 10% higher, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher or more than the reference expression profile.
In another embodiment, "determining the phenotype of said stellate cells" corresponds to evaluating the presence or the number of lipid containing droplets in said stellate cells.
In one embodiment, a tested agent has a pro-fibrotic activity if lipid containing droplets are absent of stellate cells incubated with said tested agent.
In another embodiment, a tested agent as a pro-fibrotic activity if the number of lipid containing droplets is lower in stellate cells incubated with said tested agent than in stellate cells incubated in the absence of the tested agent. In one embodiment, the term "lower" means at least 10% lower, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% lower or more than the number of lipid containing droplets in stellate cells incubated in the absence of the tested agent.
Methods for evaluating the presence of lipid containing droplets are well-known from the skilled artisan, and include, without limitation, Nile Red, BODIPY, LD450 and Oil Red O.
In another embodiment, the step c) of determining the phenotype of said stellate cells comprises the step of:
determining the expression profile of at least one gene marker of activated stellate cells in said stellate cells, and comparing this expression profile with a reference expression profile, and
evaluating the presence of lipid containing droplets in said stellate cells.
In a preferred embodiment, the method for determining the pro-fibrotic activity of a test agent, comprising the steps of:
a) in vitro incubating stellate cells in a culture medium of the invention or a composition used as a culture medium according to the invention; b) adding the tested agent in the culture medium;
c) determining the phenotype of said stellate cells by: determining the expression profile of at least one gene marker of activated stellate cells in said stellate cells, and comparing this expression profile with a reference expression profile, and
evaluating the presence of lipid containing droplets in said stellate cells.
Another aspect of the present invention is a method for determining the anti-fibrotic activity of a tested agent, comprising the steps of:
a) in vitro incubating stellate cells in a culture medium which is not a culture medium or a composition used as a culture medium according to the invention; b) adding the tested agent in the culture medium;
c) determining the expression profile of at least one gene marker of activated stellate cells in said stellate cells; and
d) comparing the expression profile obtained in step c) with a reference expression profile.
In one embodiment of the invention, the reference expression profile is the expression profile of the at least one gene marker of activated stellate cells in stellate cells incubated in a culture medium according to the invention without the tested agent.
In one embodiment, a tested agent has an anti-fibrotic activity if the expression of the at least one gene marker of activated stellate cells is similar to the reference expression level. In one embodiment, the term "similar" means that the difference between the expression levels are of less than 20%, preferably less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less.
Another object of the invention is a pharmaceutical composition comprising EGF and/or FGF2 as described hereinabove, in combination with at least one pharmaceutically acceptable excipient.
Suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol®, vegetable oils, and the like. One may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, such as, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like. In one embodiment, the pharmaceutical composition comprises EGF in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises FGF2 in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises EGF and FGF2 in combination with at least one pharmaceutically acceptable excipient.
In one embodiment, the composition may comprise a pharmaceutically acceptable salt of EGF and/or FGF2.
Examples of pharmaceutically acceptable salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like. Examples of salts with an inorganic base include alkali metal salts, such as a sodium salt and a potassium salt; an alkaline earth metal salt such as a calcium salt and a magnesium salt; an aluminum salt; and an ammonium salt. Examples of salts with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and Ν,Ν'-dibenzylethylenediamine. Examples of salts with an inorganic acid include salts with hydrochloric acid, boric acid, nitric acid, sulfuric acid and phosphoric acid. Examples of salts with an organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid. Examples of salts with a basic amino acid include salts with arginine, lysine and ornithine. Examples of salts with an acidic amino acid include salts with aspartic acid and glutamic acid. A list of suitable salts is disclosed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p 1418, 1985, the entire disclosure of which is incorporated herein by reference.
In one embodiment, the pharmaceutical composition according to the invention further comprises at least one dietary fatty acid. In one embodiment, the at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, retinoic acid (or retinol) or a mixture thereof. In a particular embodiment, the at least one dietary acid is selected from the group comprising oleic acid, palmitic acid and retinol, or a mixture thereof. In one preferred embodiment, the pharmaceutical composition of the invention further comprises oleic acid, palmitic acid and retinol.
In one embodiment, the pharmaceutical composition of the invention comprises EGF and at least one dietary fatty acid in combination with at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition of the invention comprises EGF and oleic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, oleic acid and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, oleic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, oleic acid, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient.
In one embodiment, the pharmaceutical composition of the invention comprises FGF2 and at least one dietary fatty acid in combination with at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition of the invention comprises FGF2 and oleic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2 and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2 and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2, oleic acid and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2, oleic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises FGF2, oleic acid, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient.
In one embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2 and at least one dietary fatty acid in combination with at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2 and oleic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2 and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2 and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2, oleic acid and palmitic acid in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2, oleic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition of the invention comprises EGF, FGF2, oleic acid, palmitic acid and retinol in combination with at least one pharmaceutically acceptable excipient.
Another object of the invention is a medicament comprising EGF and/or FGF2 or a pharmaceutically acceptable salt or solvate thereof, as described hereinabove. In one embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the medicament of the invention comprises EGF and FGF2 or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the medicament further comprises at least one dietary fatty acid. In one embodiment, the at least one dietary fatty acid is selected from oleic acid, palmitic acid, retinol and mixture thereof.
In one embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and at least one dietary fatty acid. In one embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and oleic acid. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and palmitic acid. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and retinol. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and oleic acid and palmitic acid. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof and oleic acid and retinol. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof, palmitic acid and retinol. In another embodiment, the medicament of the invention comprises EGF or a pharmaceutically acceptable salt or solvate thereof, oleic acid, palmitic acid and retinol.
In one embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof and at least one dietary fatty acid. In one embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof and oleic acid. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof and palmitic acid. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof and retinol. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof and oleic acid and palmitic acid. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof, oleic acid and retinol. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof, palmitic acid and retinol. In another embodiment, the medicament of the invention comprises FGF2 or a pharmaceutically acceptable salt or solvate thereof, oleic acid, palmitic acid and retinol.
In one embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, and at least one dietary fatty acid. In one embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, and oleic acid. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, and palmitic acid. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, and retinol. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, oleic acid and palmitic acid. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, oleic acid and retinol. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, palmitic acid and retinol. In another embodiment, the medicament of the invention comprises EGF, FGF2, or a pharmaceutically acceptable salt or solvate thereof, oleic acid, palmitic acid and retinol. Another object of the invention is a composition, a pharmaceutical composition or a medicament as described here above for treating fibrosis or for use in treating fibrosis.
In one embodiment, fibrosis is selected in the group comprising liver fibrosis (such as for example alcoholic hepatitis, cirrhosis, or acute-on-chronic hepatitis), pulmonary fibrosis (such as for example idiopathic pulmonary fibrosis or cystic fibrosis), heart fibrosis, Crohn's Disease, progressive kidney disease, pancreatic fibrosis, scleroderma/systemic sclerosis, post-surgery or peritoneal adhesions, retroperitoneal fibrosis, pleural fibrosis, pericardial fibrosis, uterine fibroid, and graft fibrosis.
In one embodiment, the subject is affected with a liver disease, preferably selected from the list comprising significant fibrosis, cirrhosis, the fibrosis being from alcoholic or nonalcoholic origin and/or the subject is a patient affected with a chronic disease, preferably said chronic disease is selected from the group comprising chronic viral hepatitis C, chronic viral hepatitis B, chronic viral hepatitis D, chronic viral hepatitis E, non-alcoholic fatty liver disease (NAFLD), alcoholic chronic liver disease, autoimmune hepatitis, primary biliary cirrhosis, hemochromatosis and Wilson disease.
The invention also relates to a method of treating fibrosis comprising administering to a subject in need thereof a therapeutically effective amount of a composition, pharmaceutical composition or medicament of the present invention.
In one embodiment of the invention, the subject is an animal, preferably a mammal, such as for example, a rat or a pet, such as, for example, a cat or a dog. According to a preferred embodiment, the subject is a human. In one embodiment of the invention, the human is a male, a female or a child. According to an embodiment, the subject, including a human, is at risk of presenting fibrosis in an organ or a tissue; or presents fibrosis in an organ or a tissue. In one embodiment, the subject is at risk of being affected by hepatitis. In another embodiment, the subject is an excessive alcohol drinker. In another embodiment, the subject has a history of smoking and/or breathing secondhand smoke. In another embodiment, the subject is overweight. In another embodiment, the subject is genetically predisposed to develop fibrosis. In one embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted for oral administration.
Examples of forms adapted for oral administration include, but are not limited to, tablets, orodispersing/orally disintegrating tablets, effervescent tablets, powders, granules, pills (including sugarcoated pills), dragees, capsules (including soft gelatin capsules), syrups, liquids, gels or other drinkable solutions, suspensions, slurries, liposomal forms and the like.
In one embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted for injection, such as, for example, for intramuscular, subcutaneous, intradermal, transdermal or intravenous injection or infusion and the like.
Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like. Administration of the composition, pharmaceutical composition or medicament of the invention may be accomplished by any acceptable method which allows the therapeutically components to reach its target. Any acceptable method known to one of ordinary skill in the art may be used to administer a composition, pharmaceutical composition or medicament to the subject. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being or to be treated. In one embodiment, the targeted tissue comprises stellate cells, preferably HSCs.
In one embodiment, a therapeutically effective amount of a composition, pharmaceutical composition or medicament of the present invention can be delivered via a nanoparticle- based drug delivery system. In another embodiment, the composition, pharmaceutical composition or medicament can be perfused directly through the targeted tissue, such as the liver. In another embodiment, the composition, pharmaceutical composition or medicament can be delivered using a bioerodible implant by way of diffusion or by degradation of a polymer matrix. Other suitable delivery systems include, but are not limited to, time-release, delayed release, sustained release, or controlled release delivery systems. Many types of release delivery system are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems or non-polymer systems such as for example liposome-based systems or hydrogel release systems. In one embodiment, the composition, pharmaceutical composition or medicament can be delivered by vitamin A- coupled liposomes.
It will be understood that the total daily usage of the compound of the invention, composition, pharmaceutical composition and medicament of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the composition may be varied over a wide range from about 10 to about 10000 mg of active components per adult per day, preferably 100 to about 5000, more preferably from about 200 to about 2000 mg per adult per day. Preferably, the compositions contain 10, 50, 100, 250, 500, 1000 and 2,000 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 10 to about 10000 mg of the active ingredient, preferably 100 to about 5000, more preferably from about 200 to about 2000 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.1 mg/kg to about 100 mg/kg of body weight per day, preferably from about 1 mg/kg to 40 mg/kg of body weight per day, more preferably from about 2 mg/kg to 20 mg/kg of body weight per day. In another embodiment of the invention, the composition, pharmaceutical composition or medicament of the invention may further comprise at least one anti-fibrotic agent.
In another embodiment of the invention, the composition, pharmaceutical composition or medicament of the invention may be used in combination with at least one anti-fibrotic agent.
Examples of anti-fibrotic agents include, but are not limited to, corticosteroids, mucolytics, anti-inflammatory agents, antiviral agents (such as for example an anti-HCV or anti-HBV agent), angiotensin II receptor antagonists, and immunosuppressant drugs.
The use of the anti-fibrotic agents described above is generally well characterized in the fibrosis therapy arts, and their use herein falls under the same considerations for monitoring tolerance and effectiveness and for controlling administration routes and dosages, with some adjustments. Typical dosages of an effective anti-fibrotic agent can be in the ranges recommended by the manufacturer, and where indicated by in vitro responses or responses in animal models, can be reduced by up to about one order of magnitude concentration or amount. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based on the in vitro responsiveness of cells or tissue samples, or the responses observed in the appropriate animal models.
Another object of the invention is an in vivo process for reverting stellate cells, preferably HSCs, thereby treating fibrosis. In one embodiment, the in vivo process of the invention comprises administering a composition, pharmaceutical composition or medicament according to the invention to a subject in need thereof.
Another object of the present invention is a method for inhibiting gene markers of activated stellate cells, preferably HSCs, comprising administering a composition, pharmaceutical composition or medicament according to the invention to a subject in need thereof.
Examples of gene markers of activated stellate cells include, but are not limited to, ACTA2, COL1A1, LOX, LOXL1, LOXL2, LOXL3, PDGFRB, COL4A1, COL4A2, COL5A1, ADAM 12, ADAMTS2, ACTG2, NOTCH3 and CRYAB, preferably ACTA2, COLlAl and LOX.
The invention also relates to a method for inducing inactivated stellate cells, preferably HSCs, specific markers, comprising administering a composition, pharmaceutical composition or medicament according to the invention to a subject in need thereof.
Examples of inactivated stellate cells specific markers include, but are not limited to, CXCL1, CXCL2, XCL10, CTSS, LY86, CAPN6, RND1, KRT20, CX3CR1 and GPC3.
The invention also relates to a method for inducing vitamin A storage in cytoplasmic lipid droplets of stellate cells, preferably HSCs, comprising administering a composition, pharmaceutical composition or medicament according to the invention to a subject in need thereof.
The invention also relates to a method for reducing stellate cells, preferably HSCs, proliferation rate, comprising administering a composition, pharmaceutical composition or medicament according to the invention to a subject in need thereof. This invention will be better understood from the Experimental Details that follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter, and are not to be considered in any way as limited thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows light microscopic and a-SMA immunocytochemistry images of the freshly isolated, qHSC-enriched cell population and fully culture activated HSCs (passage 4).
Figure 2 is a set of 2 graphs showing log2 mRNA expression levels of COLlAl and LOX in freshly isolated, non-plated qHSCs and aHSCs from 5 different donors. In the graphs, **p<0.01. Figure 3 is a photograph showing protein levels for PDGFRB and GAPDH in aHSCs (passage 4).
Figure 4 is a histogram showing mRNA expression levels of ACTA2, COL1 Al and LOX in human aHSCs incubated with EGF, FGF2 or a combination of both, presented as relative fold change to untreated control cells (gray line). In the graph, ns = not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001.
Figure 5 is a histogram showing mRNA expression levels of ACTA2, COL1 Al and LOX in human aHSCs incubated with a combination of oleic acid (OA), palmitic acid (PA) and 5μΜ retinol (RE), or a combination of OA+PA+R+EGF+FGF2 (further called RM), presented as relative fold change to untreated control cells (gray line). In the graph, ns = not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001.
Figure 6 is a set of 3 histograms showing mRNA expression levels of ACTA2, COLlAl and LOX in non-cultured quiescent HSCs (qHSCs), culture aHSCs and in vitro aHSCs reverted to quiescence-like by RM (rHSCs) from 3 different donors. The expression levels are presented as relative fold change to aHSCs. In the graphs, ns = not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001.
Figure 7 shows light microscopic (lOx magnification) and Oil Red O staining images (63x magnification) of aHSCs and rHSCs.
Figure 8 is two graphs showing FACS-detection and quantification of the intrinsic fluorescence (at wavelength of -328 nm) of all-trans retinyl esters in UV-excited aHSCs and rHSCs.
Figure 9 is a set of 3 histograms showing relative expression of ACTA2, COLlAl and LOX in rHSCs after a recovery of 2 days in presence or absence of recombinant TGFpi (lOng/mL) in FBS free medium, from 5 different donors. The expression levels are presented as relative fold change to rHSCs. In the graphs, ns = not significant p>0.05, *p<0.05, **p<0.01.
Figure 10 is a set of 3 histograms showing relative mRNA expression of Actal, Collal and Lox in freshly isolated, uncultured qHSCs, aHSCs (day 6 of culture) and rHSCs (day 6 of culture in RM). Figure 11 is a set of 3 histograms showing ratio of EdU-positive nuclei over DAPI- positive nuclei in aHSCs and rHSCs cultured for 6 hours in the presence of EdU and stained with DAPI (A), percentage of degraded gelatin (stained area) over the total analyzed area after aHSCs or rHSCs culture for 5 days on quenched- gelatin coated coverslips under control conditions or in RM (B) and the number of migrated aHSCs and rHSCs seeded in collagen-coated boyden-chamber and stimulated with PDGFbb or with its solvens as a control in the lower compartment, in a trans-well migration assay (C). In the graphs, ns = not significant p>0.05, *p<0.05.
Figure 12 is a set of 6 photographs showing Sirius Red staining of liver slices from Group 1 (A), Group 2 (B), Group 3 (C), and Group 4 (D) mice.
EXAMPLES
The present invention is further illustrated by the following examples. Patient samples The protocol and experiments were approved by the ethical committees of the St-Luc Hospital and faculty of Medicine of Universite Catholique de Louvain. An agreement from the Belgian Ministry of Health was obtained for the Hepatocytes and Hepatic Stem Cells Bank. Five livers were used for the following experiments, for which the clinical characteristics are summarized in Table 1. Table 1: Clinical characteristics of livers
Donor number Health status Age Gender Ischemia time
L4 Healthy 12 years Female 16h30
L8 Healthy 1 day Male 4h40
L10 Healthy 7 months Female 5h20
Lll Healthy 7 days Male 4h25
L12 FH* 13 years Male lh30
*FH=Familial Hypercholesterolemia Isolation of high purity human quiescent HSCs
The human liver parenchymal and non-parenchymal cell fractions were separated from each other by sequential perfusion of liver pieces with pre-warmed EGTA-containing EBSS medium (Lonza, Venders, Belgium) and a digestion enzyme solution (EBSS supplemented with 0.9 mg/mL collagenase P and 0.03 mg/mL soybean trypsin inhibitor (Roche)) for 9 to 12 minutes. Collagenase digestion was stopped with ice-cold M199 wash medium (Lonza) containing 0.03 mg/mL of soybean trypsin inhibitor and 100 mL/L of human plasma (Najimi et al, Cell Transplant. 2007, 16(7):717-28). After filtration, the non-parenchymal cells were separated from the parenchymal cells by subsequent low- speed centrifugation steps (50g) and submitted to an additional centrifugation step (700g for 8 minutes). Non parenchymal cell pellets were then resuspended and cryopreserved in Dulbecco's modified Eagle's medium (DMEM) (Lonza) supplemented with 20% fetal bovine serum (FBS) (Biochrom GmbH, Berlin, Germany) and 5% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO). The isolation of high purity human qHSCs was performed according to the following method (adapted from Guimaraes et al., Journal of Hepatology. 2010, 52(3):389-397): dissociated and washed single non-parenchymal cells were suspended in a 5% FBS, 2mM EDTA (Sigma) buffer (106cells/10(^L) and incubated for 30 minutes at 4°C with 500 ng/106 cells anti-CD32 (Abeam, Cambridge, United Kingdom) and 1 μg/106 cells anti-CD45 (BD Biosciences, San Jose, CA). 7- aminoactinomycin (7-AAD) (eBioscience, San Diego, CA) was used for the exclusion of non-viable cells. Pure populations of qHSCs were sorted as CD32-CD45-UV+ cells, using a FACSAria (BD Biosciences). RNA from freshly isolated qHSCs was obtained using RNEasy Micro Kit (Qiagen). RNA samples were amplified using Ovation Pico WTA system V2 (NuGEN) producing microgram quantities of cDNA. Isolation and in vitro reversion of HSCs
Human aHSCs were obtained by plating the qHSC-enriched population obtained after Nycodenz (Myegaard, Oslo, Norway) gradient centrifugation of the non-parenchymal cell fraction (Berardis et al. PLoS ONE. 2014, 9:e86137). Homogeneous populations of aHSCs were obtained after 3 passages in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% C02. Prior to each passage, cells were washed with phosphate buffered saline (PBS) and lifted using 0.05 % Trypsin (Lonza). For reversion of the activated phenotype, human aHSCs were seeded at a density of 10.000 cells/cm2 (unless stated differently) and 24 hours later the cells were washed and incubated with DMEM supplemented with 1% FBS, 20 ng/mL epidermal growth factor (EGF) (Peprotech, London, UK), 10 ng/mL fibroblast growth factor 2 (FGF2) (Peprotech), 100 μΜ oleic acid (Sigma), 100 μΜ palmitic acid (Sigma) and 5 μΜ retinol (Sigma). The medium was refreshed every two days for 5 days (day 1, 3 and 5) and the cells were harvested for further analysis on day 6. The effect of this mixture of growth factors and dietary components (further referred to as human HSC reverting medium (RM)) on the activation status of the cells was assessed by relative comparison to control cells cultured in DMEM with 1% FBS for the same period of time.
Mouse HSCs were isolated from male BalbC mice (age 20-25 weeks) (Charles River Laboratories, L'arbresle, France) and cultured in DMEM with 10% FBS, as described previously (Mannaerts et al. Hepatology. 2010, 51 : 603-614). For treatment experiments with mouse HSC reverting medium (mRM) (DMEM supplemented with 10% FBS, 40 ng/mL human EGF, 20 ng/mL human FGF2, 100 μΜ oleic acid, 100 μΜ palmitic acid and 5 μΜ retinol), freshly isolated mouse qHSCs were seeded at a density of 7.500 cells/cm2. The inhibition of activation was assessed by relative comparison to control cells cultured in DMEM supplemented with 10% FBS for the same period of time. All procedures on animals were carried out in accordance with University's guidelines for the care and use of laboratory animals in research. The performed experiments were approved by the ethical committee of the Vrije Universiteit Brussel in project 12-212-1.
Gene expression profiling and analysis
Double- stranded cDNA was synthesized from total RNA originating from cultured (activated and reverted HSCs; n=3 from corresponding donors) and uncultured (FACS- sorted quiescent HSCs; n=3 from two corresponding donors) human HSCs. cDNA was labeled and fragmented using Encore Biotin Module (NuGEN) and hybridized to the Affymetrix HG-U219 genechip (Affymetrix, Santa Clara, California, USA). Data normalization and analysis was performed using GeneSpring GX12 (Agilent, Santa Clara, CA) as described previously (Mannaerts et al. PLoS ONE. 2013, 8:e84071). Briefly, Affymetrix gene expression data were normalized using the robust multi-array algorithm (Irizarry et al. Nucleic Acids Research. 2003, 31:el5). For the detection of differentially expressed genes, a p-value cut-off of 0.05 was used in combination with a fold-change cut-off of 2.0. Functional analysis of gene expression data (gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)-pathways) was conducted using an open access, high-level cross-platform microarray dataset analysis tool (InCroMAP) (http://www.ra.cs.uni-tuebingen.de/software/InCroMAP/).
Immunocytochemistry
Nycodenz-isolated HSCs cultured for 1 day or for 3 passages were washed with PBS and fixed for 10 minutes with 4% buffered formaldehyde (Merck, Darmstadt, Germany). Following permeabilization with 0.1% Triton-X 100 (in PBS containing 1% bovine serum albumin), cells were incubated overnight with anti-aSMA (1/1000) (Sigma). Primary antibody binding was visualized using an Alexa488-labeled secondary antibody (1/200) (Invitrogen, Eurgene, OR). Images were taken with an AxioCam MRc5 digital camera (Carl Zeiss).
Lipid staining
The cells were washed with PBS and fixed in 10% formalin solution. The cells were then washed with deionized water, incubated with isopropanol for 5 minutes and stained with diluted (3/2 in water) and filtered Oil Red O (Sigma-Aldrich) at 0.3% (w/v) in 99% isopropanol for 20 minutes. The red stained lipid droplets were visualized with light microscopy (Carl Zeiss).
Western blot
Cells were washed with ice-cold PBS and scraped with ice-cold lysis buffer (170 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.4, 50 mM NaF, 0,2 mM dithiothreitol and 0.5% NP-40) supplemented with protease (Roche Diagnostics, Mannheim, Germany) and phosphatase (Roche Diagnostics) inhibitors. Protein concentrations were determined using the BCA protein assay kit (Pierce Chemical Co, Rockford, IL). Thirty microgram of protein was separated on a 8% tris-glycine SDS-polyacrylamide gel and electroblotted onto polyvinylidene difhioride membranes (Amersham Biosciences, Little Chalfront, UK) using a wet blotting apparatus (Mini Trans-Blot Cell, BioRad, Nazareth, Belgium). Blots were blocked with 5% milk powder in tris buffered saline (TBS) with 0.2% tween (Sigma) and subsequently incubated overnight with primary anti-PDGFRB (diluted 1/1000 in blocking buffer) (Abeam) or anti-GAPDH (diluted 1/30.000) (Abeam). The membranes were washed and incubated with a horseradish peroxidase conjugated secondary antibody (1/20000) (Dako, Glostrup, Denmark) for 1 hour and the antigen was visualized by enhanced chemiluminescence using ECL substrate (Pierce Chemical co.).
Proliferation assay Cell proliferation of human HSCs was assessed with the Click-iT EdU Cell Proliferation Assay Kit (Invitrogen, Eugene, OR). HSCs were cultured under control conditions or in RM for 6 days. On day 6, the cells were labeled with 10 μΜ EdU for 6 hours and subsequently formalin fixed and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). EdU incorporation was visualized according to the manufacturer's instructions. The percentage proliferation was calculated as the ratio of EdU positive cells to DAPI positive cells. The quantification was performed on at least 500 cells per donor and is presented as the mean percentage measured in 3 different donors.
In situ Zymography
The gelatinase activity of human HSCs was assessed using the highly quenched, fluorescein-labeled pig skin gelatin (DQ gelatin, Invitrogen). Upon proteolytic digestion, its green fluorescence is revealed and can be used to measure enzymatic activity. A 1 mg/mL stock solution of DQ-gelatin was prepared using deionized water and stored at 4°C. Prior to cell seeding (20.000 cells/cm2), glass coverlips (12 mm diameter) were coated with 50μg DQ-gelatin for 1 hour. 24 Hours post-seeding, the cells were washed with serum- free DMEM and cultured for 6 days under control conditions or RM. On day 6, the cells were formalin fixed and mounted with Prolong Gold antifade reagent with DAPI. The percentage green stained area was calculated using ImageJ (http://imagej.nih.gov/ij/index.html) at the adjusted threshold of 34-255. The quantification was performed on 10 images per donor and is represented as the mean percentage measured in 3 different donors.
Migration assay
Both aHSCs and rHSCs were seeded in collagen-coated Boyden chambers (Millipore, 40.000 cells/chamber) in serum-free DMEM. After 60 minutes, the chambers were transferred to wells with 20 ng/mL platelet derived growth factor (PDGFbb) (R&D systems, Minneapolis, MN) or its solvent as a control. After 16 hours, non-migrated cells were cleared and migrated cells were fixed with ice-cold 100% methanol and mounted with Prolong Gold antifade reagent with DAPI. The quantification was performed by manually counting the totality of migrated cells on images covering the entire membrane and is represented as the mean of 3 different donors.
RNA purification and RTq-PCR
RNA was extracted and purified from cultured and uncultured cells using the Reliaprep RNA cell Miniprep system (Promega, Madison, WI). Total RNA was converted to cDNA by reverse transcription using the Revert Aid Kit (ThermoFisher Scientific, St. Leon-Rot, Germany). Quantitative real-time polymerase chain reaction was performed using the GoTaq qPCR Master Mix with BRYTE green (Promega). A 7500 real time PCR system was used and data was analyzed using System SDS software v2.0.6 (Applied biosystems). Fold change differences between samples were determined using the comparative Ct method (AACt). The expression level of different target genes, relative to glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and the calibrator, was given by 2-ΔΔΟ:. Gene specific primers were produced by Integrated DNA technologies (Leuven, Belgium).
Statistical analysis
GraphPad Prism v4.0.0 (GraphPad Software, La Jolla, CA) was used for statistical analysis. Data in the figures are expressed as means + SEM. Differences among groups were tested for statistical significance by Student t-test or analysis of variance (ANOVA) followed by Tukey' s test, depending on the number of groups. ns= not significant p<0.05, * p<0.05, ** p<0.01, ***p<0.001. Example 1: EGF and FGF2 revert the culture activated phenotype of human HSCs
Human HSCs were isolated by density gradient centrifugation from the non-parenchymal liver fraction, allowed to activate by seeding on plastic culture dishes and expanded for at least 3 passages. The activated status of the cells was assessed by the typical myofibroblastic phenotype (Fig. 1), the strong increase in alpha smooth muscle actin (ACTA2) protein by immunocytochemistry and collagen type 1 alpha 1 (COL1A1) and lysyl oxidase (LOX) expression by RT-qPCR (Fig. 2), and the high protein level of platelet-derived growth factor receptor beta (PDGFRB), a membrane receptor associated with HSCs (Fig. 3). These features have been demonstrated on all the HSC isolated from different donor livers.
Thereafter, different culture conditions have been tested for their ability to revert the activated phenotype of culture expanded human primary HSCs. These conditions included pharmacological agents previously shown to inhibit rodent HSC activation, i.e. TWS119, valproic acid, Trichostatin A, valinomycin, Carbonyl cyanide-p- trifluoromethoxyphenylhydrazone, bafilomycin, hydroxychloroquine, 3-methyladenine; an adipogenic differentiation mixture and a diverse pool of growth factors and cytokines (data not shown). Among these agents, results show that FGF2 and EGF significantly down-regulate the expression of ACTA2, COL1A1 and LOX, alone or in combination (Fig.4). Example 2: Oleic acid, palmitic acid and retinol revert the culture activated phenotype of human HSCs
The same experiment has been realized with a medium containing dietary fatty acids. Results show that oleic acid (OA), palmitic acid (PA) and retinol (R) also down-regulate the expression of ACTA2, COL1A1 and LOX (Fig. 5, "OA+PA+R" columns). Moreover, aHSCs cultured in medium containing the mixture of dietary fatty acids displayed a quiescent-like phenotype, characterized by a thinner cell body and the presence of intra-cytoplasmic lipid droplets (data not shown). Example 3: EGF and FGF2 act synergistically with dietary fatty acids and retinol to revert the culture activated phenotype of human HSCs
By combining the tested agents in the culture medium, it was observed that the effect of EGF and FGF2 on the expression of ACTA2, COLlAl and LOX expression was strongly potentiated by the mixture of dietary fatty acids (OA, PA and R) to reach levels of expression that are similar to those measured in freshly isolated, non-cultured qHSCs (Fig. 5 and 6). HSCs reverted by this mix of growth factors and dietary components (further referred to as RM) present a thinner cell body and intra-cytoplasmic lipid droplets (Fig. 7). An important functional hallmark of qHSCs is their ability to store vitamin A in their cytoplasmic lipid droplets. In order to assess whether the in vitro reverted HSCs (rHSCs) had the molecular machinery both to metabolize and store vitamin A, the retinyl ester auto-fluorescence at 328 nm was measured by fluorescence-activated cell sorting (FACS). Results show that around 75% of the aHSCs cultured in RM became UV- positive. Analysis of the rHSCs under UV-light shows that the auto-fluorescent signal co- localizes with the intra-cytoplasmic lipid droplets, indicating that vitamin A is indeed metabolized and stored inside the lipid droplets (Fig. 8).
Example 4: Analysis of the reversibility of the quiescent- like state of HSCs
To investigate whether this induced quiescent-like state is reversible, the cells were allowed to recover for 2 days in the absence or presence of transforming growth factor beta (TGFP), a profibrogenic molecule, in serum-free medium. In the absence of TGFp, the cells maintained low expression levels of profibrogenic genes. However, in the presence of TGFp, the cells again upregulated the expression of ACTA2, COLlAl and LOX (Fig. 9). To investigate whether the described mix of growth factors and dietary components can prevent the activation process and maintain a quiescent phenotype, freshly isolated mouse qHSCs were seeded in normal control conditions or RM optimized for mouse HSC cultures (mRM) until day 6. It was observed that cells grown under mRM conditions maintained low expression levels of Acta2, ColAl and Lox, similar to uncultured DO mouse qHSCs (Fig. 10) and have an intermediary phenotype with a large intra-cytoplasmic lipid droplet content (data not shown).
Example 5: In vitro reverted human HSCs have a reduced proliferation rate
It is known that aHSCs functionally differ from qHSCs by their increased proliferation rate, unbalanced ECM homeostasis and higher migratory potential (Friedman, Physiological Reviews. 2008, 88: 125-172). Therefore, it was further investigated whether human in vitro rHSCs functionally differed from their activated counterparts by comparing their proliferation (EdU-incorporation), ECM degradation (in situ zymography) and PDGFbb-induced migration (transwell assay). Results show that rHSCs (obtained with RM) displayed a 75% reduction in proliferation compared to aHSC (Fig. 11A). Although rHSCs express slightly higher levels of TIMP1 and show no difference in MMP2 and MMP9 expression (data not shown), no difference in overall gelatinase activity between aHSCs and rHSCs is observed (Fig. 11B). As this assay is dependent on cell density and that we observed a difference in proliferation between both conditions, only areas with a similar number of cells were used for quantification. In concordance with the in situ zymography results, we measured no difference in PDGFbb- induced migration (Fig. 11C).
Example 6: The overall gene expression profile of in vitro rHSCs is distinct from that of aHSCs and qHSCs To gain more insight into the gene expression changes elicited by this transient reversion into a quiescent-like state, the global gene expression profile of rHSCs was assessed and compared to that of freshly isolated, non-cultured qHSCs and culture aHSCs using the human genome U219 arrays. Results show that the global gene expression profile of rHSCs resembles more closely to that of aHSCs than qHSCs, with 2277 (rHSC vs aHSC) against 9122 (rHSC vs qHSC) genes significantly differentially regulated (student t-test, p<0,05). Many of the top upregulated genes in rHSCs are inflammation-related, i.e. IL-8, IL-33, IL-Ιβ, CXCL1, and CXCL6 (Table 2). Over 60% of the >2-fold deregulated genes in rHSCs compared to aHSCs are downregulated and include genes such as TNNT2, SULT1E1, ACTG2, and SYNP02L (Table 2). Table 2: The fold-change of the top 15 upregulated and top 15 downregulated g rHSCs, compared to the aHSCs
Figure imgf000039_0001
Example 7: In vitro reverted human HSCs upregulate the expression of in vivo inactivated HSC specific markers To determine whether human HSCs reverted to quiescence-like by RM share characteristics with in vivo inactivated HSCs (iaHSCs) found in the mouse liver after recovery from experimentally induced fibrosis, the expression of a panel of genes (n=17) identified as highly specific for in vivo iaHSCs was measured and compared to aHSCs and qHSCs (Kisseleva et al. PNAS. 2012; Troeger et al. Gastroenterology. 2012, 143: 1073-1083.el022; Friedman and Arthur, Science & Medicine. 2002, 8(4): 194; Xiao Liu et al. Curr Pathobiol. 2013, Rep 1: 209-214) in aHSCs and rHSCs from 3 liver donors. Although there was some variation between HSCs from different donors, 10 genes of the 17 genes tested are consistently upregulated in rHSCs of each donor, i.e. CXCL1, CXCL2, CXCL10, CTSS, LY86, CAPN6, RND1, KRT20, CX3CR1 and GPC3 (Table 3). Table 3: Overview of the fold-difference in expression of in vivo iaHSC signature genes in rHSCs and aHSCs
Gene Accession Donor L4 Donor L8 Donor L12
CXCL1 NM_001511 52 30.7 26
CXCL2 NM_002089 39.1 7.3 223.6
CXCL10 NM_001565 3.7 32.7 nd
CTSS (tv2) NM_001199739 27.6 20.5 10.1
LY86 NM_004271 1.8 26.4 6.2
CAPN6 NM_014289 2.6 5.2 14.1
RND1 NM_014470 1.9 11.7 6.6
KRT20 NM_019010 6 nd 3.9
CX3CR1 (tvl) NM_001171174 3.2 nd 5.4
GPC3 (tv4) NM_001164619 1.5 1.8 1.2 tv: transcript variant; nd: not detected
Example 8: In vivo evaluation of EGF and FGF2 impact on liver fibrosis Material and methods
Mouse model
The proposed mouse model is well-documented and recapitulates the important cellular and molecular events characterizing the development of liver fibrosis in patients. This model is used routinely by the researchers to study the activation process of HSCs. It is known that a 4-week treatment induces remarkable fibrosis that is well-suited to study the impact of potential anti-fibrotic agents (Mannaerts et al. 2010; Mannaerts et al., 2015). Therefore, we opt for a 4- week treatment of CC14 with or without a co-treatment of the EGF/FGF2 solution in the first or last two weeks of treatment.
Mice were male BALB/c (Charles River) of 6-7 weeks. Treatment Administrations
CCh and corn oil injections
The scheme of treatment administrations is detailed in Table 4. Each animal received 100 μΐ of CC14 solution (a freshly-prepared mix of volume of 15 μΐ^ CC14 (Sigma Fluka, 87031) and 85 μΐ^ of corn oil) or same volume of corn oil via the IP routes according to QA/PROD34.
The injection was done 2 times a week: one IP on the right side of the mouse and one on the left side of the mouse. Table 4: Groups of Animals, Doses and Administration Routes
Figure imgf000041_0001
IP: intraperitoneal
EGF and FGF2 pump placement EGF/FGF2 solution preparation
The EGF/FGF2 solution containing EGF (Peprotech London, M315-09) at 20 ng/ml and FGF2 (Peprotech London, 450-33) at 10 ng/ml was prepared with PBS for all the Alzet pumps. The solution was aliquoted and stored at -20°C. The ALZET pump model 2002 (volume of 200 μΐ for 2 weeks with a 0.5 μΐ/h rate) was used.
Analgesia
Five (5) mg/kg BW of carprofen - Rimadyl® was injected subcutaneously at the base of the neck before surgery. Surgery
Surgery was done under general anaesthesia (isoflurane gaz anaesthesia, B132 equipment) according to SOP QA/PROD31. Animals were left in a recovering chamber till they recover from the anaesthesia. The mice of GP2 were subjected to anaesthesia without pump placement at Day +14. This anaesthesia was lasted the same time then the anaesthesia of the animals with pump placement. Animals were left on a heating pad till they recover from the anaesthesia.
Sampling of organs and histological analysis
All mice were euthanized by cervical dislocation according to SOP QA/PROD31 and necropsied. Two control mice of GPl to GP4 were euthanized after 2 weeks of treatment at Day+14.
One day after the last injection for GPl to GP4 (Day+26), the mice were euthanized and the organ sampling was done.
Livers were harvested under Ketamine/xylasine anesthesia. Liver tissue was fixed and processed for paraffin sections. Formalin fixed and paraffin imbedded 5 μιη liver sections were deparaffinized and rehydrated in graded alcohol bathes. To evaluate liver fibrosis, liver slices were stained using Sirius red and counterstained with fast green and light microscopic images were captured.
Sampling of blood and RT-qPCR analysis At necropsy, the blood was collected by retroorbital punction. The blood serum was aliquoted (100 μΐ/tube) and stored at -20°C until the end of the study.
Total RNA was extracted from the liver samples using Tripure isolation Reagent (Roche, Belgium) and Fastprep lysing matric (ceramic beads). DNAse I treated samples, were thereafter processed for First-strand cDNA synthesis using high capacity cDNA Reverse Transcription kit according to the manufacturer's instructions (Applied Biosystems). Samples were then and subsequently diluted with nuclease-free water (Invitrogen) to lO ng/mL cDNA.
PCR amplification mixtures (25 μί) contained 25 ng template cDNA, Master Mix buffer (12.5 mL; Applied Biosystems), and the corresponding Taqman assay were run in duplicate and performed on a StepOnePlus Real-time PCR (Applied Biosystems). The cycling conditions comprised 10 min polymerase activation at 95 °C and 40 cycles at 95 °C for 15 s and 60°C for 1 min. Relative quantification was normalized against the house keeping gene β-2-microglobulin. The Applied Biosystems assays used for the current study are listed in Table 5.
Table 5: Genes studied by RT-qPCR
Figure imgf000043_0001
Results
Histological analysis
To examine whether the EGF/FGF2 solution of the invention is able to modulate liver fibrosis, CC14 was administrated to mice for 4 weeks and the effect of these two growth factors was investigated both at early phase of liver fibrosis induction (Group 3) and late phase of liver fibrosis induction (Group 4).
Subsequently to CC14 administration to Balb/c male mice, liver fibrosis was induced in CCl4-treated mice, as demonstrated by Sirius Red staining commonly used to visualize collagen fibers (Group 2) as compared to sham mice (Group 1) (Figures 12 A and B respectively). On the contrary, Figures 12 C and D show that mice treated with EGF/FGF2 solution presented a reduction in liver fibrosis, in both groups 3 and 4 respectively.
Therefore, the EGF/FGF2 solution of the invention is able to reduce liver fibrosis.
RT-qPCR analysis Recovered livers of the studied mice were processed for mRNA extraction and analysis of genes related to fibrosis. RT-qPCR using Taqman probes and dCT analysis have revealed that CC14 treatment leads to an upregulation of the expression of alpha smooth muscle actin (ACTA1) and collagen type I alpha 1 (COLlal) mRNAs (Table 6, GP1 compared to GP2), two widely used markers reflecting an in situ activation of hepatic stellate cells.
In mice treated with EGF and FGF2, results presented in Table 6 (GP3 and GP4 compared to GP2) show a downregulation of both mRNAs expression.
Table 6: RT-qPCR values
Figure imgf000044_0001
Therefore, treatment with a composition comprising EGF and FGF2 leads to an inhibition of the CC14 induced fibrosis.

Claims

1. A composition for treating fibrosis comprising EGF and FGF2.
2. The composition for treating fibrosis according to claim 1, wherein said composition further comprises at least one dietary fatty acid.
3. The composition for treating fibrosis according to claim 2, wherein said at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, and retinol, preferably oleic acid, palmitic acid or retinol, or a mixture thereof.
4. The composition for treating fibrosis according to claims 1 to 3, wherein said fibrosis is selected from the group comprising liver fibrosis, pulmonary fibrosis, heart fibrosis, Crohn's Disease, progressive kidney disease, pancreatic fibrosis, scleroderma/systemic sclerosis, post-surgery adhesions, peritoneal adhesions, retroperitoneal fibrosis, pleural fibrosis, pericardial fibrosis, uterine fibroid, and graft fibrosis.
5. The composition for treating fibrosis according to claim 4, wherein said liver fibrosis is cirrhosis.
6. The composition for treating fibrosis according to claim 4, wherein said pulmonary fibrosis is idiopathic pulmonary fibrosis or cystic fibrosis.
7. In vitro process for reverting stellate cells comprising a step of incubating stellate cells into a composition comprising EGF and/or FGF2, and optionally at least one dietary fatty acid.
8. The in vitro process according to claim 7 wherein said at least one dietary fatty acid is selected from the group comprising oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, myristic acid, and retinol, preferably oleic acid, palmitic acid or retinol, or a mixture thereof.
9. The in vitro process according to anyone of claims 7 or 8 wherein said stellate cells are selected from the group comprising hepatic stellate cells (HSCs), pancreatic stellate cells (PaSCs), lung stellate cells, intestinal stellate cells, kidney stellate cells, spleen stellate cells, adrenal gland stellate cells, ductus deferens stellate cells and vocal cords stellate cells, preferably HSCs, PaSCs, lung stellate cells and intestinal stellate cells, more preferably HSCs.
10. An isolated reversed stellate cells population obtained by the in vitro process according to anyone of claims 7 to 9.
11. A composition comprising EGF, FGF2, oleic acid, palmitic acid and retinol, wherein
the concentration of EGF ranges from 0.1 to 100 ng/mL,
the concentration of FGF2 ranges from 0.05 to 80 ng/mL,
the concentration of oleic acid ranges from 1 to 1000 nmol/mL, the concentration of palmitic acid ranges from 1 to 1000 nmol/mL, and - the concentration of retinol ranges from 0.01 to 100 nmol/mL.
12. A culture medium comprising the composition according to claim 11.
13. An in vitro method for determining the pro-fibrotic activity of a tested agent comprising the steps of:
a) in vitro incubating stellate cells in a culture medium according to claim 12, b) adding the tested agent in the culture medium,
c) determining the phenotype of said stellate cells, preferably the activated or quiescent phenotype of said stellate cells.
14. A kit of part comprising two parts, wherein the first part comprises a composition according to claim 11 or a culture medium according to claim 12, and the second part comprises a stellate cells population.
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