WO2023180122A1 - Utilisation de cellules progénitrices allogéniques humaines dérivées du foie pour traiter et/ou prévenir la sénescence cellulaire - Google Patents

Utilisation de cellules progénitrices allogéniques humaines dérivées du foie pour traiter et/ou prévenir la sénescence cellulaire Download PDF

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WO2023180122A1
WO2023180122A1 PCT/EP2023/056454 EP2023056454W WO2023180122A1 WO 2023180122 A1 WO2023180122 A1 WO 2023180122A1 EP 2023056454 W EP2023056454 W EP 2023056454W WO 2023180122 A1 WO2023180122 A1 WO 2023180122A1
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cells
liver
halpc
population
senescence
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PCT/EP2023/056454
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English (en)
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Giulia JANNONE
Mustapha Najimi
Etienne Sokal
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Université Catholique de Louvain
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    • 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
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/30—Animals modified by surgical methods
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases

Definitions

  • the present invention relates to human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC, and their use for treating and/or preventing cellular senescence and diseases associated therewith, in a subject in need thereof.
  • HALPC liver-derived progenitor cells
  • Senescence is a process of cellular aging, leading to an irreversible inhibition of cell proliferation and to metabolic modifications. Accelerated senescence occurs in numerous chronic hepatobiliary diseases and has been associated to disease severity and cirrhosis. Senolytic therapies applied to genetically modified preclinical models allowed to further emphasize the connection between senescence and hepatobiliary diseases. Clearance of senescent cells by suicide gene-mediated ablation or reintroduction of telomerase activity improved liver function, histological fibrosis and steatosis in mouse models of hepatobiliary diseases.
  • WO2020/193714 discloses adult HALPC for use in the treatment of Acute- on-Chronic Liver Failure.
  • WO2020/221842 describes a process for the manufacture of HALPC, the process comprising the use of microcarriers and a bioreactor.
  • WO2020/221843 describes a process for the manufacture of HALPC, process comprising the use of a xeno- and serum-free culture medium comprising purified native or recombinant human serum albumin.
  • EP3881853 discloses HALPC for use in the treatment of inflammatory lung diseases, infectious lung diseases and/or systemic inflammation.
  • Wiemann Stefanie et al., 2002, doi: 10.1096/fj.01- 0977com discloses that telomeres shortening and senescence are makers in liver cirrhosis.
  • MSCs Mesenchymal stem cells transplantation in chronic liver disease has been repeatedly shown to improve liver function and histology. MSCs also have senolytic properties as they reduced heart and skin senescence in aging rodents. Recently, MSCs-derived exosomes improved senescence in cholangioids treated with H2O2 and in the CCI4 mouse model of liver injury (Chen W et al., Stem Cell Res Then, 2021). However, the effect of cell therapy on liver senescence has never been investigated.
  • liver-derived progenitor cells are a population of cells obtained from healthy adult human liver and approved for clinical applications. Those cells display a hepatocytic differentiation ability and secrete important bioactive factors with paracrine activity, including anti-inflammatory, anti-fibrotic and regenerative effects.
  • the present invention is directed towards the treatment and/or prevention of senescence using HALPC, and thus towards the improvement of the treatment of diseases involving or related to senescence.
  • This invention thus relates to a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or a conditioned medium obtainable by culturing said HALPC, for use in the prevention and/or treatment of cellular senescence.
  • HALPC liver-derived progenitor cells
  • the cellular senescence is induced by a stress signal such as telomeric shortening, DNA damage, oxidative stress, oncogenic activation or metabolic dysfunction, or combinations thereof.
  • a stress signal such as telomeric shortening, DNA damage, oxidative stress, oncogenic activation or metabolic dysfunction, or combinations thereof.
  • the cellular senescence is induced by a disease or damage, an oncogene, a therapy, a diet, and the like.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease.
  • the at least one disease is selected from the group comprising or consisting of metabolic, genetic, infectious, toxic and autoimmune liver diseases, chronic biliary diseases, cholestatic diseases, age-related diseases, bone and cartilage disorders, pancreatic diseases, kidney diseases, pulmonary diseases, cardiovascular diseases, metabolic diseases, eye diseases, neurodegenerative diseases, skin diseases, inflammatory diseases and cancer.
  • the at least one disease is selected from the group comprising or consisting of hepatic fibrosis, pre-cirrhotic conditions, cirrhosis including liver and biliary cirrhosis, biliary atresia, Alagille syndrome, progressive familial intrahepatic cholestasis, primary biliary cholangitis, primary sclerosing cholangitis, chronic hepatitis, chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection, cholestasis, osteoporosis, osteoarthritis, atherosclerosis, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, thrombosis, cataracts, glaucoma, macular degeneration, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, renal dysfunction, pancreatic fibrosis, type 2 diabetes, Alzheimer disease, Huntington's disease, Parkinson's disease,
  • COPD chronic ob
  • the at least one disease is cirrhosis, preferably liver or biliary cirrhosis.
  • the cellular senescence is determined by measuring expression of the markers selected from the group comprising or consisting of p21, pl6 INK4A , pi4 ARF z 19 ARF , pRB, p53, high-mobility group box 1, lamin Bl, SA-p-Gal, lipofuscin, DNA damage (eg yH2AX), preferably p21, and senescence-associated secretory phenotype.
  • the markers selected from the group comprising or consisting of p21, pl6 INK4A , pi4 ARF z 19 ARF , pRB, p53, high-mobility group box 1, lamin Bl, SA-p-Gal, lipofuscin, DNA damage (eg yH2AX), preferably p21, and senescence-associated secretory phenotype.
  • the population, lysates thereof and/or conditioned medium is to be administered in a therapeutic effective amount to a subject in need thereof.
  • the population comprises a dose of 0.25 to 20 million of said human allogenic liver-derived progenitor cells per kg of body weight.
  • the population, lysates thereof and/or conditioned medium is to be administered in combination with another therapeutic agent.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or a conditioned medium obtainable by culturing said HALPC, according to the invention, and a pharmaceutically acceptable vehicle, for use in the prevention and/or the treatment of cellular senescence.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to a combination kit comprising (i) a population of HALPC, lysates thereof and/or a conditioned medium obtainable by culturing said HALPC, or a pharmaceutical composition comprising the same and (ii) another therapeutic agent, for use for the prevention and/or the treatment of cellular senescence.
  • the present invention further relates to a method for the prevention and/or the treatment of cellular senescence in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or a conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to a method for reducing cellular senescence in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or a conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • liver progenitor cell refers to an unspecialized and proliferation-competent cell which is produced by culturing cells that are isolated from liver or part thereof and which or the progeny of which can give rise to at least one relatively more specialized cell type.
  • a liver progenitor cell give rise to descendants that can differentiate along one or more lineages to produce increasingly more specialized cells (but preferably hepatocytes or hepato-active cells), wherein such descendants may themselves be progenitor cells, or even to produce terminally differentiated liver cells (e.g. fully specialized cells, in particular cells presenting morphological and functional features similar to those of primary human hepatocytes).
  • stem cell refers to a progenitor cell capable of self-renewal, i.e., can proliferate without differentiation, whereby the progeny of a stem cell or at least part thereof substantially retains the unspecialized or relatively less specialized phenotype, the differentiation potential, and the proliferation competence of the mother stem cell.
  • the term encompasses stem cells capable of substantially unlimited self-renewal, i.e., wherein the capacity of the progeny or part thereof for further proliferation is not substantially reduced compared to the mother cell, as well as stem cells which display limited self-renewal, e., wherein the capacity of the progeny or part thereof for further proliferation is demonstrably reduced compared to the mother cell.
  • liver progenitor or stem cells refers to adult-derived human liver stem/progenitor cells (ADHLSC or ADHLSCs) and to human allogenic liver- derived progenitor cells or human allogenic liver progenitor cells (HALPC or HALPCs) produced at higher scale for pursuing clinical studies, and is used synonymously with "human adult liver-derived progenitor cells”, “heterologous human adult liver- derived progenitor cells”, abbreviated as “HHALPC” or “HHALPCs”. These cells represent a specific type of human liver-derived progenitor cells, obtainable as described herein.
  • a progenitor or stem cell may be usually described as totipotent, pluripotent, multipotent or unipotent.
  • a single “totipotent” cell is defined as being capable of growing, i.e. developing, into an entire organism.
  • a "pluripotent” cell is not able of growing into an entire organism, but is capable of giving rise to cell types originating from all three germ layers, i.e., mesoderm, endoderm, and ectoderm, and may be capable of giving rise to all cell types of an organism.
  • a “multipotent” cell is capable of giving rise to at least one cell type from each of two or more different organs or tissues of an organism, wherein the said cell types may originate from the same or from different germ layers, but is not capable of giving rise to all cell types of an organism.
  • a “unipotent” cell is capable of differentiating to cells of only one cell lineage.
  • pharmaceutically acceptable excipient refers to an excipient 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.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • therapeutically effective amount refers to an amount sufficient to effect beneficial or desired results including clinical results.
  • a therapeutically effective amount can be administered in one or more administrations. In one embodiment, the therapeutically effective amount may depend on the individual to be treated.
  • treating or “treatment” or “alleviation” or “prevention” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) cellular senescence.
  • Those in need of treatment include those already affected with cellular senescence, as well as those prone to have the disease or condition, preferably cellular senescence, or those in whom cellular senescence is to be prevented.
  • a subject or mammal is successfully "treated" for cellular senescence if, after receiving a therapeutic amount of a population of cells according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, one or more of the symptoms associated with cellular senescence; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in cellular senescence are readily measurable by routine procedures familiar to a physician.
  • the term "individual” or “subject” refers to an animal individual, preferably a mammalian individual, more preferably a human individual. In some embodiments, an individual may be a mammalian individual. Mammalians include, but are not limited to, all primates (human and non-human), cattle (including cows), horses, pigs, sheep, goats, dogs, cats, and any other mammal 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 cellular senescence.
  • an individual 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 cellular senescence.
  • the individual is an adult (e.g., an individual above the age of 18).
  • the individual is a child (e.g., an individual below the age of 18).
  • the individual is a male.
  • the individual is a female.
  • extract means a lysed cell content.
  • such extract or lysate has not been further purified and thus contains the whole cell lysate content.
  • such extract refers to a protein extract, an RIMA extract, a lipid, or a membrane vesicle.
  • extracts include cellular and extracellular extracts.
  • extracts according to the present invention include metabolites from the cells.
  • lysate may include soluble fractions and/or insoluble fractions.
  • cell medium or "culture medium” or “medium” refers to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells.
  • Cell culture medium can contain serum or can be serum-free.
  • conditioned medium refers to a medium that has been exposed to (/.e., contacted with, cultured with) cells grown in culture for a time sufficient to include at least one additional component in the medium, said component produced by the cells, that was not present in the medium before exposing the same to the cells.
  • a "conditioned medium” may be deemed as a composition comprising cell secretion products, such as inter alia cell secretion proteins and cellular metabolites, which has previously supported the maintenance and/or the proliferation of cells.
  • a "conditioned medium” may include soluble fractions and/or insoluble fractions.
  • fraction refers to a part, composition or derivative obtainable from the conditioned medium of the invention.
  • the present invention relates to a population of human allogenic liver- derived progenitor cells (HALPC) for use in the prevention and/or treatment of cellular senescence.
  • HLPC liver- derived progenitor cells
  • the cells according to the invention are preferably generated from cells that have been isolated from mammalian liver or part of a liver, where the term "mammalian” refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs and primates, e.g., monkeys and apes.
  • mammalian refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs and primates, e.g., monkeys and apes.
  • the liver progenitor cell or stem cell is generated from cells that have been isolated from human liver or a part thereof, preferably human adult liver or a part thereof.
  • the term "adult liver” refers to liver of subjects that are postnatal, i.e., any time after birth, preferably full term, and may be, e.g., at least 1 day, 1 week, 1 month or more than 1 month of age after birth, or at least 1, 5, 10 years or more.
  • an "adult liver”, or mature liver may be found in human subjects who would otherwise be described in the conventional terms of "infant”, “child”, “adolescent”, or “adult”.
  • infant infant
  • child infant
  • adult adult liver
  • adult liver may attain substantial developmental maturity in different time postnatal intervals in different animal species, and can properly construe the term "adult liver” with reference to each species.
  • the adult liver or part thereof may be from a non-human animal subject, preferably a non-human mammal subject.
  • Progenitor or stem cells or cell lines, or progeny thereof, derived as described herein from livers of non-human animal or non-human mammal subjects can be advantageously used.
  • particularly suitable non-human mammal cells for use in human therapy may originate from pigs.
  • a donor subject may be living or dead, as determined by art-accepted criteria, such as, for example, the "heart-lung” criteria (usually involving an irreversible cessation of circulatory and respiratory functions) or the "brain death” criteria (usually involving an irreversible cessation of all functions of the entire brain, including the brainstem).
  • Harvesting may involve procedures known in the art, such as, for example, biopsy, resection or excision.
  • liver or part thereof from donor subjects may be subject to respective legal and ethical norms.
  • harvesting of liver tissue from a living human donor may need to be compatible with sustenance of further life of the donor.
  • liver may typically be removed from a living human donor, e.g., using biopsy or resection, such that an adequate level of physiological liver functions is maintained in the donor.
  • harvesting of liver or part thereof from a non-human animal may, but need not be compatible with further survival of the non-human animal.
  • the non- human animal may be humanely culled after harvesting of the tissue.
  • Liver or part thereof may be obtained from a donor, preferably a human donor, who has sustained circulation, e.g., a beating heart, and sustained respiratory functions, e.g., breathing lungs or artificial ventilation.
  • a donor preferably a human donor
  • sustained circulation e.g., a beating heart
  • sustained respiratory functions e.g., breathing lungs or artificial ventilation.
  • the donor may need to be or need not be brain dead (e.g., removal of entire liver or portion thereof, which would not be compatible with further survival of a human donor, may be allowed in brain dead human beings).
  • Harvesting of liver or part thereof from such donors is advantageous, since the tissue does not suffer substantial anoxia (lack of oxygenation), which usually results from ischemia (cessation of circulation).
  • liver or part thereof may be obtained from a donor, preferably a human donor, who at the time of harvesting the tissue has ceased circulation, e.g., has a non-beating heart, and/or has ceased respiratory functions, e.g., has non-breathing lungs and no artificial ventilation. While liver or part thereof from these donors may have suffered at least some degree of anoxia, viable progenitor or stem cells can also be isolated from such tissues.
  • Liver or part thereof may be harvested within about 24h after the donor's circulation (e.g., heart-beat) ceased, e.g., within about 20h, e.g., within about 16h, more preferably within about 12h, e.g., within about 8h, even more preferably within about 6h, e.g., within about 5h, within about 4h or within about 3h, yet more preferably within about 2h, and most preferably within about I h, such as, within about 45, 30, or 15 minutes after the donor's circulation (e.g., heart-beat) ceased.
  • the donor's circulation e.g., heart-beat
  • the harvested tissues may be cooled to about room temperature, or to a temperature lower than room temperature, but usually freezing of the tissue or parts thereof is avoided, especially where such freezing would result in nucleation or ice crystal growth.
  • the tissue may be kept at any temperature between about 1°C and room temperature, between about 2°C and room temperature, between about 3°C and room temperature or between about 4°C and room temperature, and may advantageously be kept at about 4°C.
  • the tissue may also be kept "on ice” as known in the art.
  • the tissue may be cooled for all or part of the ischemic time, i.e., the time after cessation of circulation in the donor.
  • the tissue can be subjected to warm ischemia, cold ischemia, or a combination of warm and cold ischemia.
  • the harvested tissue may be so kept for, e.g., up to 48h before processing, preferably for less than 24h, e.g., less than 16h, more preferably for less than 12h, e.g., less than lOh, less than 6h, less than 3h, less than 2h or less than Ih.
  • the harvested tissue may advantageously be kept in, e.g., completely or at least partly submerged, in a suitable medium and/or may be perfused with the suitable medium, before further processing of the tissue.
  • a suitable medium which can support the survival of the cells of the tissue during the period before processing.
  • Isolation of primary liver cells from a liver or part of a liver may be performed according to methods known in the art, for example as described in EP1969118, EP3039123, EP3140393, EP3423566.
  • the cells according to the invention and methods for isolating the latter are known in the art, evidenced for instance by EP1969118, EP3039123, EP3140393, EP3423566, EP3947644, EP3947645, WO2020221842 or WO2020221843, which are incorporated as references herein.
  • Cell-free compositions obtained by culturing human allogenic liver-derived progenitor cells in cell culture medium are in detail reported EP3016665 and WO2021069553, which are incorporated as references herein.
  • the cells according to the invention may be obtained by any suited method known in the art, for instance as described in EP1969118, EP3039123, EP3140393, EP3423566, EP3947644, EP3947645, WO2020221842 or WO2020221843 (see Example 2). Briefly, a population of liver primary cells is first obtained from disassociating of liver or part thereof, to form a population of liver primary cells from said liver or part thereof. In other words, in one embodiment, a population of liver primary cells is obtained from a liver or a part thereof, which is disassociated in order to separate liver primary cells and generate a preparation of primary liver cells.
  • cells comprised in this preparation are cultured under adherent conditions, preferably as to allow adherence and growth of cells onto a support.
  • these cells are passaged at least once, preferably at 70 %, 80 % or 90 % confluence.
  • cells are isolated and are positive for at least one hepatic marker and at least one mesenchymal marker and that have at least one liver-specific activity.
  • such method comprises the steps of:
  • a suitable method for disassociating liver or part thereof to obtain a population (suspension) of primary cells therefrom may be any method well known in the art, including but not limited to, enzymatic digestion, mechanical separation, filtration, centrifugation and combinations thereof.
  • the dissociation step involves obtaining a liver or a part thereof that comprises, together with fully differentiated hepatocytes, an amount of primary cells that can be used for producing liver progenitor or stem cells.
  • the liver or part thereof is obtained from a "subject", “donor subject” or “donor”, interchangeably referring to a vertebrate animal, preferably a mammal, more preferably a human.
  • a part of a liver can be a tissue sample derived from any part of the liver and may comprise different cell types present in the liver.
  • liver primary cells are first obtained from disassociating of liver or part thereof, to form a population of primary cells from said liver or part thereof.
  • cells comprised in this preparation are cultured under adherent conditions, preferably as to allow adherence and growth of cells onto a support.
  • these cells are passaged at least once, preferably at 70% confluence.
  • cells, which are positive for at least one hepatic marker and at least one mesenchymal marker and that have at least one liver-specific activity are isolated.
  • the population of primary cells as defined and obtained herein by disassociating liver or part thereof may typically be heterogeneous, i.e., it may comprise cells belonging to one or more cell types belonging to any liver-constituting cell type, including progenitor or stem cells, that may have been present in liver parenchyma and/or in the liver non-parenchymal fraction.
  • Exemplary liver-constituting cell types include but are not limited to hepatocytes, cholangiocytes (bile duct cells), Kupffer cells, hepatic stellate cells (Ito cells), oval cells and liver endothelial cells.
  • the above terms have art-established meanings and are construed broadly herein as encompassing any cell type classified as such.
  • a primary cell population may comprise hepatocytes in different proportions (0.1 %, 1 %, 10 %, or more of total cells), according to the method of disassociating liver and/or any methods for fractioning or enriching the initial preparation for hepatocytes and/or other cell types on the basis of physical properties (dimension, morphology), viability, cell culture conditions, or cell surface marker expression by applying any suitable techniques.
  • the population of primary cells as defined and obtained herein by disassociating liver can be used immediately for establishing cell cultures as fresh primary liver cells or, preferably, stored as cryopreserved preparations of primary liver cells using common technologies for their long-term preservation.
  • liver primary cells obtained in step (b) is then cultured directly onto a fully synthetic support (e.g., plastic or any polymeric substance) or a synthetic support pre-coated with feeder cells, protein extracts, or any other material of biological origin that allow the adherence and the proliferation of similar primary cells and the emergence of a population of adult liver progenitor or stem cells having the desired markers, such markers being identified preferably at the level of protein, by means of immunocytochemistry or immunohistochemistry, flow cytometry, or other antibody based technique.
  • Primary cells are cultured in a cell culture medium sustaining their adherence and the proliferation of and the emergence of a homogenous cell population.
  • This step of culturing of primary liver cells as defined above leads to emergence and proliferation of liver progenitor or stem cells in the culture and can be continued until liver progenitor or stem cells have proliferated sufficiently. For example, culturing can be continued until the cell population has achieved a certain degree of confluence (e.g., at least 50 %, 70 %, 80 % or at least 90 % or more confluent).
  • a certain degree of confluence e.g., at least 50 %, 70 %, 80 % or at least 90 % or more confluent.
  • Liver progenitor or stem cells obtained at step (c) can be further characterized by technologies that allow detecting relevant markers already at this stage (that is, before passaging cells as indicated in step (d)), as described in EP3140393, EP3423566, EP3947644, EP3947645, WO2020221842 or
  • the technologies used for identifying such markers and measuring them as being positive or negative western blot, flow cytometry, immunocytochemistry, and ELISA are preferred since these allow marker detection at the protein level even with the low amount of liver progenitor or stem cells that are available at this step.
  • liver progenitor cells can then be made based on the confirmation of the cells' identity, i.e., the marker profile, morphology and/or activity.
  • the liver progenitor cells are positive for at least one mesenchymal marker.
  • Mesenchymal markers include but are not limited to Vimentin, CD13, CD90, CD73, CD44, CD29, o-smooth muscle actin (ASMA) and CD140b.
  • the liver progenitor cells may secrete HGF and/or PGE2.
  • they can optionally be positive for at least one hepatic marker and/or exhibit at least one liver-specific activity.
  • the cells are positive for at least one hepatic marker and/or exhibit at least one liver-specific activity.
  • hepatic markers include but are not limited to HNF-3B, HNF-4, CYP1A2, CYP2C9, CYP2E1, CYP3A4 and alpha-1 antitrypsin and may also include albumin (ALB).
  • Liver-specific activities may include but are not limited to urea secretion, bilirubin conjugation, alpha-l-antitrypsin secretion and CYP3A4 activity.
  • the liver progenitor cells are heterologous human adult liver-derived progenitor cells (HALPC) that express at least one mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o- smooth muscle actin (ASMA), and which also secrete HGF.
  • the liver progenitor cells are heterologous human adult liver-derived progenitor cells (HALPC) that express at least one mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o-smooth muscle actin (ASMA), and which also secrete HGF and PGE2.
  • liver progenitor or stem cells are cultured in a cell culture medium sustaining their adherence and the proliferation of and the emergence of a homogenous cell population that, following at least one passage, is progressively enriched for liver progenitor or stem cells.
  • These liver progenitor or stem cells can be rapidly expanded for generating sufficient cells for obtaining progeny having the desired properties (as described in EP3140393, EP3423566, EP3947644, EP3947645, WO2020221842 or WO2020221843), with cell doubling that can be obtained within 48-72 hours and maintenance of liver progenitor or stem cells having the desired properties for at least for 2, 3, 4, 5 or more passages.
  • passage or “passaging” is common in the art and refers to detaching and dissociating the cultured cells from the culture substrate and from each other.
  • first passage or passage 1, Pl
  • the cells may be passaged at least one time and preferably two or more times.
  • Each passage subsequent to passage 1 is referred to herein with a number increasing by 1, e.g., passage 2, 3, 4, 5, or Pl, P2, P3, P4, P5, etc.
  • the isolated liver progenitor or stem cells are plated onto a substrate which allows adherence of cells thereto, and cultured in a medium sustaining their further proliferation, generally a liquid culture medium, which may contain serum or may be serum-free.
  • a substrate which allows adherence of cells thereto may be any substantially hydrophilic substrate.
  • Current standard practice for growing adherent cells may involve the use of defined chemical media with or without addition of bovine, human or other animal serum. These media, that can be supplemented with appropriate mixture of organic or inorganic compounds may, besides providing nutrients and/or growth promoters, also promote the growth/adherence or the elimination/detachment of specific cell types.
  • the added serum may also promote cell adhesion by coating the treated plastic surfaces with a layer of matrix to which cells can better adhere.
  • the cells may be counted in order to facilitate subsequent plating of the cells at a desired density.
  • the term "serum”, as conventionally defined is obtained from a sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation.
  • An inert catalyst e.g., glass beads or powder, can facilitate clotting.
  • serum can be prepared using serum-separating vessels (SST), which contain the inert catalyst to mammals.
  • the environment in which the cells are plated may comprise at least a cell medium, in the methods of the invention typically a liquid medium, which supports the survival and/or growth of the isolated liver progenitor cells.
  • the liquid culture medium may be added to the system before, together with or after the introduction of the cells thereto.
  • the term "cell medium” or “cell culture medium” or “medium” refers to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells.
  • Cell culture media can contain serum or be serum-free.
  • the medium will comprise a basal medium formulation as known in the art.
  • basal media formulations can be used to culture the primary cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM/F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's MB 752/1 or Williams Medium E, and modifications and/or combinations thereof.
  • MEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • alpha-MEM alpha modified Minimum Essential Medium
  • BME Basal Medium Essential
  • BGJb medium F-12
  • compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured.
  • a preferred basal medium formulation may be one of those available commercially such as Williams Medium E, IMDM or DMEM, which are reported to sustain in vitro culture of adult liver cells, and including a mixture of growth factors for their appropriate growth, proliferation, maintenance of desired markers and/or biological activity, or long-term storage, or DMEM with 10 % FBS (Gibco).
  • Another preferred medium is commercially available serum-free medium that supports the growth of liver progenitor or stem cells, such as e.g., StemMacsTM from Miltenyi, Prime-XV from FUJIFILM Irvine Scientific.
  • Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se.
  • these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g., glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), etc.
  • physiological buffers e.g., HEPES, bicarbonate
  • nucleotides e.g., nucleosides and/or nucleic acid bases
  • ribose deoxyribose
  • amino acids e.g., vitamins, antioxidants (e.g
  • basal media can be supplied with one or more further components.
  • additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion.
  • Such supplements include insulin, transferrin, selenium salts, and combinations thereof.
  • These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution.
  • Further antioxidant supplements may be added, e.g., [3-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution.
  • a medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin.
  • antibiotic and/or antimycotic compounds such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neo
  • Hormones can also be advantageously used in cell culture and include, but are not limited to D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin/human growth hormone (HGH), thyrotropin, thyroxine, L-thyronine, epidermal growth factor (EGF) and hepatocyte growth factor (HGF). Liver cells can also benefit from culturing with triiodothyronine, o-tocopherol acetate, and glucagon.
  • DES diethylstilbestrol
  • dexamethasone estradiol
  • hydrocortisone insulin
  • prolactin progesterone
  • HGH somatostatin/human growth hormone
  • thyrotropin thyroxine
  • L-thyronine L-thyronine
  • EGF epidermal growth
  • Lipids and lipid carriers can also be used to supplement cell culture media.
  • Such lipids and carriers can include, but are not limited to cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others.
  • Albumin can similarly be used in fatty-acid free formulations.
  • Suitable plasma or serum for use in the media as described herein may include human plasma or serum; or plasma or serum derived from non-human animals, preferably non-human mammals, such as, e.g., non-human primates (e.g., lemurs, monkeys, apes), fetal or adult bovine, horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc.
  • non-human primates e.g., lemurs, monkeys, apes
  • fetal or adult bovine horse, porcine, lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc.
  • the any combination of the above plasma and/or serum may be used in the cell medium.
  • the cultured cells are detached and dissociated from the culture substrate and from each other.
  • Detachment and dissociation of the cells can be carried out as generally known in the art, e.g., by enzymatic treatment with proteolytic enzymes (e.g., chosen from trypsin, collagenase, e.g., type I, II, III or IV, dispase, pronase, papain, etc.), treatment with bivalent ion chelators (e.g., EDTA or EGTA) or mechanical treatment (e.g., repeated pipetting through a small-bore pipette or pipette tip), or any combination of these treatments.
  • proteolytic enzymes e.g., chosen from trypsin, collagenase, e.g., type I, II, III or IV, dispase, pronase, papain, etc.
  • bivalent ion chelators e.g., EDTA or EGTA
  • mechanical treatment
  • a suitable method of cell detachment and dispersion should ensure a desired degree of cell detachment and dispersion, while preserving a majority of cells in the culture.
  • the detachment and dissociation of the cultured cells would yield a substantial proportion of cells as single, viable cells (e.g., at least 50 %, 70 %, 80 %, 90 % of the cells or more).
  • the remaining cells may be present in cell clusters, each containing a relatively small number of cells (e.g., on average, between 1 and 100 cells).
  • the so detached and dissociated cells may be re-plated onto a substrate which allows the adherence of cells thereto, and are subsequently cultured in a medium as described above sustaining the further proliferation of HALPC and HALPC progeny.
  • These cells may be then cultured by re-plating them at a density of between 10 and 10 5 cells/cm 2 , and at a splitting ratio between about 1/16 and 1/2, preferably between about 1/8 and 1/2, more preferably between about 1/4 and 1/2.
  • the splitting ratio denotes the fraction of the passaged cells that is seeded into an empty (typically a new) culture vessel of the same surface area as the vessel from which the cells were obtained.
  • the type of culture vessel, as well as of surface allowing cell adherence into the culture vessel and the cell culture media, can be the same as initially used and as described above, or may be different.
  • cells are maintained onto CellBind or any other appropriate support that is coated with extracellular matrix proteins (such as collagens, and preferably collagen type I) or synthetic peptides that are acceptable in GMP conditions.
  • extracellular matrix proteins such as collagens, and preferably collagen type I
  • synthetic peptides that are acceptable in GMP conditions.
  • the isolation of HALPC can then be made based on the confirmation of the cells' identity, i.e., the marker profile, morphology and/or activity.
  • the HALPC are positive for at least one mesenchymal marker.
  • Mesenchymal markers include but are not limited to Vimentin, CD13, CD90, CD73, CD44, CD29, o-smooth muscle actin (ASMA) and CD140b.
  • the HALPC may secrete HGF and/or PGE2.
  • they can optionally be positive for at least one hepatic marker and/or exhibit at least one liver-specific activity.
  • the cells are positive for at least one hepatic marker and/or exhibit at least one liver-specific activity.
  • hepatic markers include but are not limited to HNF-3B, HNF- 4, CYP1A2, CYP2C9, CYP2E1, CYP3A4 and alpha-1 antitrypsin and may also include albumin (ALB).
  • Liver-specific activities may include but are not limited to urea secretion, bilirubin conjugation, alpha-l-antitrypsin secretion and CYP3A4 activity.
  • the HALPC express at least one mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o-smooth muscle actin (ASMA), and also secrete HGF.
  • the HALPC express at least one mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o-smooth muscle actin (ASMA), and also secrete HGF and PGE2.
  • human liver progenitor or stem cells express at least one mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o-smooth muscle actin (ASMA), and they optionally also express at least one hepatic marker and/or exhibit a liver-specific activity.
  • mesenchymal marker selected from CD90, CD44, CD73, CD13, CD140b, CD29, vimentin and o-smooth muscle actin (ASMA)
  • ASMA o-smooth muscle actin
  • human liver progenitor or stem cells may be characterized in that they co-express (i.e., are positive for) at least one mesenchymal marker including but not limited to CD90, CD44, CD73, CD13, CD140b, Vimentin, CD29, and o-smooth muscle actin (ASMA), with at least one hepatic or hepatocyte marker including but not limited to alpha-fetoprotein (AFP), alpha-1 antitrypsin, HNF-4 and/or MRP2 transporter, optionally with the hepatocyte marker albumin (ALB).
  • mesenchymal marker including but not limited to CD90, CD44, CD73, CD13, CD140b, Vimentin, CD29, and o-smooth muscle actin (ASMA)
  • ASMA o-smooth muscle actin
  • AFP alpha-fetoprotein
  • HNF-4 alpha-1 antitrypsin
  • MRP2 transporter optionally with the hepatocyte marker albumin (ALB
  • HALPC preferably express and/or secrete HGF and PGE-2.
  • said cells are preferably human liver progenitor or stem cells positive for at least one hepatic marker and at least one mesenchymal marker and that display at least one liver-specific activity.
  • hepatic markers include but are not limited to HNF-3B, HNF-4, CYP1A2, CYP2C9, CYP2E1, CYP3A4 and alpha-1 antitrypsin and may also include albumin (ALB).
  • Mesenchymal markers include but are not limited to Vimentin, CD13, CD90, CD73, CD44, CD29, o-smooth muscle actin (ASMA) and CD140b.
  • Liver-specific activities include but are not limited to urea secretion, bilirubin conjugation, alpha-1 antitrypsin secretion and CYP3A4 activity.
  • Said liver progenitor or stem cell, or a cell population comprising such cells will be able to give rise to at least hepatocyte like cells.
  • said cells do not differentiate into osteocytes or adipocytes.
  • the used human liver progenitor or stem cells will be positive for at least one of the markers chosen from the group of o-smooth muscle actin (ASMA), albumin (ALB), CD140b, and MMP1; and negative for at least one of the markers chosen from the group sushi domain containing protein 2 (SUSD2) and cytokeratin-19 (CK-19).
  • ASMA o-smooth muscle actin
  • ALB albumin
  • CD140b CD140b
  • MMP1 MMP1
  • SUSD2 group sushi domain containing protein 2
  • CK-19 cytokeratin-19
  • the HALPC are measured (i) positive for a-smooth muscle actin (ASMA), CD140b and optionally albumin (ALB); (ii) negative for cytokeratin-19 (CK-19); (iii) and optionally negative for Sushi domain containing protein 2 (SUSD2).
  • ASMA smooth muscle actin
  • ALB optionally albumin
  • CK-19 negative for cytokeratin-19
  • SUSD2 Sushi domain containing protein 2
  • the HALPC are measured: (i) positive for a-smooth muscle actin (ASMA), CD140b and optionally albumin (ALB); (ii) negative for Sushi domain containing protein 2 (SUSD2) and cytokeratin- 19 (CK-19).
  • ASMA smooth muscle actin
  • ALB optionally albumin
  • SUSD2 Sushi domain containing protein 2
  • CK-19 cytokeratin- 19
  • the human liver progenitor or stem cells are measured positive for CD90, CD73, vimentin and ASMA.
  • the human liver progenitor or stem cells are positive for CD90, CD73, vimentin and ASMA, and exhibit in average less than about 2.5% clonal aberrations per metaphase and/or less than about 15% non-clonal aberrations per metaphase.
  • the human liver progenitor or stem cells are measured positive for CD90, CD73, vimentin and ASMA, and negative for CK-19.
  • said human liver progenitor or stem cells are measured positive for one or more markers chosen from the group of: (i) o-smooth muscle actin (ASMA), albumin (ALB), CD140b, MMP1; (ii) at least one hepatic marker selected from HNF-3B, HNF-4, CYP1A2, CYP2C9, CYP2E1, CYP3A4; (iii) at least one mesenchymal marker selected from vimentin, CD90, CD73, CD44, CD29; (iv) at least one liver-specific activity selected from urea secretion, bilirubin conjugation, alpha-1 antitrypsin secretion and CYP3A4 activity; (v) at least one marker selected from ATP2B4, ITGA3, TFRC, SLC3A2, CD59, ITGB5, CD151, ICAM1, ANPEP, CD46, CD81; and (vi) at least one marker selected from ITGA11
  • the HALPC are further measured positive for: (i) at least one hepatic marker selected from HNF-3B, HNF-4, CYP1A2, CYP2C9, CYP2E1 and CYP3A4 and optionally albumin; (ii) at least one mesenchymal marker selected from Vimentin, CD90, CD73, CD 44, and CD29; (iii) at least one liver-specific activity selected from urea secretion, bilirubin conjugation, alpha- 1-antitiypsin secretion, and CYP3A4 activity; (iv) at least one marker selected from ATP2B4, ITGA3, TFRC, SLC3A2, CD59, ITGB5, CD151, ICAM1, ANPEP, CD46, and CD81; and (v) at least one marker selected from MMP1, ITGA11, FMOD, KCND2, CCL11, ASPN, KCNK2, and HMCNl.
  • the cells can be negative for any combination of markers as given above. In a particularly preferred embodiment, said cells are negative for all markers above.
  • the HALPC are negative for HLA-DR.
  • the HALPC are negative for certain markers, such as CD133, CD45, CK19 and/or CD31.
  • the HALPC may also be measured positive for one or more of the enzymatic activities listed in W02016/030525, Table 6.
  • this type of adult liver progenitor cell can be further characterized by a series of negative markers, in particular for one or more selected from the group consisting of ITGAM, ITGAX, IL1R2, CDH5, and NCAM 1.
  • HALPC may also be measured negative for one or more selected from the group consisting of HP, CP, RBP4, APOB, LBP, ORM 1, CD24, CPM, and APOCI.
  • markers can be present in HALPC in different combinations of markers, such as: (i) positive for a-smooth muscle actin, vimentin, CD90, CD73, CD44, CD29, CD 140b, and CYP3A4 activity and optionally albumin; and (ii) negative for Sushi domain containing protein 2, Cytokeratin-19, and CD271.
  • HALPC HALPC of the above embodiment in any functional and technical combination, for instance by measuring positivity for at least one further marker selected from ATP2B4, ITGA3, TFRC, SLC3A2, CD59, ITGB5, CD151, ICAM1, ANPEP, CD46, and CD81.
  • HALPC can be measured negative for at least one further marker selected from the group consisting of ITGAM, ITGAX, IL1R2, CDH5, and NCAM1.
  • HALPC can be measured negative for at least one of HP, CP, RBP4, APOB, LBP, ORM1, CD24, CPM, and APOCI.
  • the population of HALPC is substantially pure.
  • substantially pure means that cells other than HALPC represent less than 25, 24, 23, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 0.00001, 0.000001% or less compared to the total population of cells.
  • the population of HALPC is pure, meaning devoid of any other cell types.
  • Examples of useful adult liver derived progenitor or stem cells, cell lines thereof or cell populations for the current purpose are disclosed in EP1969118, EP3039123, EP3140393 and EP3423566, and are incorporated as a reference herein.
  • the current invention equally relates to a lysate, a cell lysate or an extract of the population of HALPC of the invention, for uses as described hereinabove and hereinbelow.
  • the term "lysate” encompasses any components of the cells of the invention, in particular it encompasses both cellular and extracellular extracts which retain the capability of preventing and/or treating cellular senescence.
  • the lysate is a whole cell lysate. In some other embodiments, the lysate is an extract of the whole cell lysate. In one embodiment, the lysate is a cellular extract. In another embodiment, the lysate is an extracellular extract.
  • cellular extracts include cytoplasmic extracts, membrane extracts, and combination thereof, in particular, extracts obtained from fractionation methods.
  • Cellular extracts may be obtained by any standard chemical (implementing SDS, proteinase K, lysozyme, combinations thereof, and the like) and/or mechanical (sonication, pressure) fractionation approaches, or approaches adapted therefrom.
  • extracellular extracts may include the secreted fraction, in particular soluble compounds, or extracellular vesicles (EV).
  • extracellular vesicles encompasses exosomes, exosome-like vesicles, microvesicles (or ectosomes) and apoptotic bodies.
  • the extracellular extracts are extracellular vesicles.
  • the extracellular extracts are the secreted fraction.
  • the extracellular extracts include secreted molecules.
  • the secreted fraction may be isolated and/or purified from the culture medium, according to any suitable method known in the state of the art, or a method adapted therefrom.
  • the extracellular extracts may be isolated by differential centrifugation from culture medium; by polymer precipitation; by high-performance liquid chromatography (HPLC), combination thereof, and the like.
  • the lysate is a protein extract, a RIMA extract, a lipid, or a membrane vesicle.
  • the current invention equally relates to a conditioned medium obtainable or obtained by culturing the population of HALPC of the invention, for uses as described hereinabove and hereinbelow.
  • the conditioned medium of the invention is cell-free.
  • the cell-free nature can be obtained by conventional methods in the art such as filtration, enzymatic digestion, centrifugation, absorption, and/or separation by chromatography, or repetitions and/or combinations of such methods.
  • Said conditioned medium further comprises soluble proteins, microvesicles and exosomes.
  • the conditioned medium obtainable by culturing the liver progenitor or stem cells described above was found to comprise soluble proteins, among others, growth factors, chemokines, matrix metalloproteases, and pro- and anti-inflammatory cytokines whose presence can provide useful biological activities. These components are presumed to be secreted by the cells in the medium.
  • HALPC are plated onto a substrate which allows adherence of cells thereto, and cultured in a medium sustaining their further proliferation, generally a liquid culture medium, which may contain serum or may be serum-free.
  • a substrate which allows adherence of cells thereto may be any substantially hydrophilic substrate.
  • Current standard practice for growing adherent cells may involve the use of defined chemical media with or without addition of bovine, human or other animal serum.
  • such culturing medium comprises serum.
  • These media which can be supplemented with appropriate mixture of organic or inorganic compounds may, besides providing nutrients and/or growth promoters, also promote the growth/adherence or the elimination/detachment of specific cell types.
  • the added serum may also promote cell adhesion by coating the treated plastic surfaces with a layer of matrix to which cells can better adhere.
  • the cells may be counted in order to facilitate subsequent plating of the cells at a desired density.
  • such culturing medium is serum-free.
  • a method for producing a cell-free conditioned medium as taught herein may comprise the step of obtaining human liver progenitor or stem cells by any of the above methods, culturing human liver progenitor or stem cells in a cell culture medium, and separating the cell culture medium from human liver progenitor or stem cells.
  • the environment in which the cells are plated may comprise at least a cell medium, in the methods of the invention typically a liquid medium, which supports the survival and/or growth of the isolated liver progenitor or stem cells.
  • the liquid culture medium may be added to the system before, together with or after the introduction of the cells thereto. Preferred media formulations have been described above.
  • Said method can be performed by using the cell culture medium that is a serum-free medium, by modifying specific conditions of cell culture, and/or by separating the cell culture medium from said human liver progenitor or stem cells after culturing human liver progenitor or stem cells at given time points (e.g., at least 2, 4, 6, 8, 12, 24 hours).
  • Obtained conditioned medium has a composition enriched (or depleted) in soluble proteins, RNAs, exosomes and/or microvesicles that are either degraded (or unstable) within the conditioned media or secreted by human liver progenitor or stem cells not in regular manner but only before or after a certain number of hours (and thus not progressively accumulated in the conditioned medium).
  • time points can be shorter (e.g., 2 hours or less) or longer such as at 24 hours, at 36 hours or more hours.
  • said conditioned medium is a product derived from cell- free conditioned medium obtainable by culturing liver progenitor or stem cells.
  • Said product is a fraction obtained by fractioning said conditioned medium.
  • Such fractioning may comprise applying one or more technologies known in the art, such as for example filtering, enzymatically digesting, centrifuging, adsorbing, and/or separating by chromatography.
  • the conditioned medium of the invention and/or the fraction thereof typically contain soluble proteins, RNAs, exosomes and/or microvesicles.
  • the conditioned medium comprises one or more components chosen from the group of hepatocyte growth factor (HGF), interleukin 6 (IL-6), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF).
  • HGF hepatocyte growth factor
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • VEGF vascular endothelial growth factor
  • the conditioned medium and/or the fraction thereof comprises at least hepatocyte growth factor (HGF), interleukin 6 (IL-6), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF).
  • HGF hepatocyte growth factor
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • VEGF vascular endothelial growth factor
  • the conditioned medium and/or the fraction thereof comprise:
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • CCL11 interleukin-6
  • IL-8 interleukin-8
  • soluble proteins selected from the group consisting of matrix metalloproteinases, growth factors, chemokines, and cytokines.
  • Such soluble proteins may be preferably present in the conditioned medium and/or in a fraction thereof, at a concentration of at least 1 ng/ml.
  • one or more of HGF, VEGF, CCL11, IL-6, or IL-8 may be present at a concentration of at least 1 ng/ml.
  • one or more soluble proteins selected from the group consisting of HGF, VEGF, CCL11, IL-6, and IL-8 are present in the conditioned medium and/or fraction thereof, at a concentration of at least 1 ng/ml/million cells.
  • the conditioned medium and/or the fraction thereof contain microvesicles that are characterized and, when appropriate, selected according to their size (in certain embodiments, size smaller than 0.40 pm), molecular weight, and/or composition.
  • the conditioned medium and/or the fraction thereof contain exosomes that are characterized (in certain embodiments, size smaller than 80 nm) and, when appropriate, selected according to their size, molecular weight, and/or composition.
  • the conditioned medium and the fraction thereof comprise RNAs, for example miRNAs.
  • compositions of such soluble proteins, RNAs, exosomes and/or microvesicles in conditioned medium and/or in a fraction thereof can be obtained for example by appropriately concentrating (or diluting) the respective preparation at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold.
  • certain embodiments provide so-concentrated or so-diluted conditioned medium and/or in a fraction thereof.
  • the present invention provides a fraction obtainable from said conditioned medium, said fraction comprising hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), eotaxin (CCL11), interleukin-6 (IL-6), and interleukin-8 (IL-8), each one being present at a concentration of at least 1 ng/ml.
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • eotaxin CCL11
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • the fraction obtained from said conditioned medium comprises one or more soluble proteins selected from the group consisting of HGF, VEGF, CCL11, IL-6, and IL-8, each one, at a concentration of at least 1 ng/ml/million cells.
  • the conditioned medium of current invention comprises at least one component from the group of FGF family, angiopoietin-1, sphingosine- l-phosphate, TGF-p, HGF, TIMP1, and TIMP2.
  • the conditioned medium of current invention comprises at least one component selected from sphingosine-l-phosphate, TGF-pi, HGF and TIMP2.
  • the conditioned medium of the current invention comprises at least sphingosine-l-phosphate (SIP).
  • SIP sphingosine-l-phosphate
  • the conditioned medium of the current invention comprises at least 30 ng/million total cells of sphingosine-l-phosphate, preferably at least 50 ng/million total cells, at least 100 ng/million total cells, at least 150 ng/million total cells, at least 200 ng/million total cells, at least 250 ng/million total cells or at least 300 ng/million total cells.
  • the conditioned medium and/or fraction thereof is suitable for the use as described above.
  • Such medical, e.g., prophylactic or therapeutic, use may involve using conditioned medium and/or fraction thereof, alone or in combination with one or more exogenous active ingredients, which may be suitably added.
  • exogenous active ingredients include cells (e.g., liver progenitor or stem cells or other cells suitable for ex vivo or in vivo applications), proteins (e.g., matrix metalloproteases, growth factors, chemokines, cytokines, hormones, antigens, or antibodies), nucleic acids (e.g., miRNAs), lipids, nutrients (e.g., sugars or vitamins) 1 and/or chemicals (e.g., drugs with antimicrobial, anti-inflammatory, or antiviral properties) that were not initially present in conditioned medium and/or fraction thereof, and that are known to be effective as medicaments for the desired indication.
  • cells e.g., liver progenitor or stem cells or other cells suitable for ex vivo or in vivo applications
  • proteins e.g., matrix metalloproteases, growth factors, chemokines, cytokines, hormones, antigens, or antibodies
  • nucleic acids e.g., miRNAs
  • lipids e.
  • cellular senescence can be used interchangeably with the terms “cell senescence”, “induced cellular senescence”, “induced cell senescence”, and “cell aging”.
  • Cellular senescence refers to an irreversible cell cycle arrest. Mechanisms of senescence and characteristics of senescent cells are well known in the art (see e.g., Kumari et al., Front Cell Dev Biol, 2021).
  • the cellular senescence is induced.
  • the cellular senescence is induced by a stress signal such as telomeric shortening, DNA damage, oxidative stress, oncogenic activation or metabolic dysfunction, disease or damage, oncogene, therapy, diet, or combinations thereof.
  • the cellular senescence is induced by a stress signal such as telomeric shortening, DNA damage, oxidative stress, oncogenic activation or metabolic dysfunction, or combinations thereof.
  • a stress signal such as telomeric shortening, DNA damage, oxidative stress, oncogenic activation or metabolic dysfunction, or combinations thereof.
  • the cellular senescence is induced by telomeric shortening.
  • telomeric shortening refers to the erosion of the extremities of chromosomes (/.e., telomeres) occurring after repeated cell divisions. Advanced telomeric erosion is notably found in cells of aged individuals.
  • the cellular senescence is induced by DNA damage.
  • the DNA damage is selected from the group comprising or consisting of double strand break, single strand break, cyclization, chemical alteration, and mutation including deletion, addition, substitution and frameshift.
  • the DNA damage is endogenous or exogeneous.
  • the DNA damage occurs at a locus corresponding to at least one gene, wherein said at least one gene encodes at least one protein having antitumoral, oncogenic or antioxidant properties.
  • the DNA damage occurs on at least one gene encoding a telomerase.
  • the DNA damage is induced by oxidative stress, irradiation, chemical toxicity (e.g., DNA intercalants or mutagens) or biological toxicity (e.g., nucleases or integrases), or mechanical damage.
  • chemical toxicity e.g., DNA intercalants or mutagens
  • biological toxicity e.g., nucleases or integrases
  • the cellular senescence is induced by oxidative stress.
  • oxidative stress is defined as an imbalance between the levels of reactive oxygen species (ROS) and antioxidant defenses within a cell, notably the inability of the antioxidant defenses of the cell to fully contravene, alleviate or suppress the deleterious effects of ROS.
  • ROS reactive oxygen species
  • Non-limitative examples of ROS include hydrogen peroxide, hydroxyl radical, peroxyl radical, peroxinitrite, nitrous oxide, superoxide and singlet oxygen (dioxidene).
  • Non- limitative examples of antioxidant defenses include antioxidant enzymes, e.g., superoxide dismutase, catalase and glutathione peroxidase, and antioxidant molecules, e.g., glutathione, ascorbate and o-tocopherol.
  • the oxidative stress damages soluble proteins, transmembranous proteins, organelles, RNA and/or DNA.
  • the oxidative stress results in an oxidation reaction that is biologically irreversible, e.g., sulfonylation reaction.
  • high oxidative stress is associated with faster aging.
  • the cellular senescence is induced by oncogenic activation.
  • oncogenic activation refers to the increased expression of at least one gene that has an oncogenic or protooncogenic activity.
  • oncogenes or protooncogenes include vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), Raf, Ras, c-Myc, c-Kit or cyclin DI (CCND1).
  • VEGFR vascular endothelial growth factor receptor
  • EGFR epidermal growth factor receptor
  • Raf Raf
  • Ras Ras
  • c-Myc c-Myc
  • c-Kit cyclin DI
  • CCND1 cyclin DI
  • oncogenic activation is associated with increased risk of cancer and/or metabolic reprogramming.
  • oncogenic activation is associated with higher cell division rates.
  • the cellular senescence is induced by metabolic dysfunction.
  • metabolic dysfunction refers to an imbalance in at least one pathway, cycle or process of the metabolism of a cell, tissue, organ or organism.
  • Non limitative examples of metabolic dysfunctions include diabetes, hypercholesterolemia or Gaucher disease.
  • the cellular senescence is induced by a disease or damage, an oncogene, a therapy, a diet, and the like.
  • the cellular senescence is induced by a disease.
  • the disease is selected from the group comprising genetic, infectious, parasitic, metabolic, degenerative, physiological and idiopathic diseases.
  • the disease is chronic.
  • the disease is acute.
  • diseases inducing senescence include cirrhosis, hepatitis, cancer, diabetes, Alzheimer's disease, cardiovascular conditions such as thrombosis, and the like.
  • the cellular senescence is induced by a damage to the organism.
  • damages include oxidative stress, irradiation, chemical toxicity (e.g., DNA intercalants or mutagens) or biological toxicity (e.g., nucleases or integrases), mechanical damage, or combinations thereof.
  • the damage directly promotes or induces senescence.
  • the damage induces a disease or condition that promotes senescence.
  • the cellular senescence is induced by an oncogene.
  • oncogenes include vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), Raf, Ras, c-Myc, c-Kit or cyclin DI (CCND1).
  • the cellular senescence is induced by therapy.
  • the therapy involves deleterious or toxic side effects.
  • the therapy is radiotherapy, chemotherapy, pharmacotherapy, invasive surgery, antibiotic, antifungal and the likes.
  • the cellular senescence is induced by a medicament with some degree of toxicity for a tissue, organ or organism.
  • the cellular senescence is induced by an overdose of a medicament, i.e., a biologically active molecule and/or excipient comprised in the medicament, that is not toxic when taken within prescribed quantities, e.g., paracetamol.
  • the cellular senescence is induced by a medicament used as prescribed, e.g., chemotherapy for cancer like etoposide or doxorubicin.
  • the cellular senescence is induced by diet.
  • the diet is imbalanced.
  • imbalanced it is meant that at least one category of nutrient is overrepresented in the individual's regimen (e.g., carbohydrates or fats) and/or underrepresented (e.g., vitamins or proteins).
  • the cellular senescence is induced by overeating, i.e., an excessive intake of caloric nutrient.
  • the cellular senescence is induced by undereating, i.e., an insufficient intake of caloric nutrient. It is to be understood that the terms "excessive intake” and “insufficient intake” refer to quantities recommended by health authorities of a country or a region, and vary depending on the age, gender, health condition, physiology and culture of each individual.
  • the cellular senescence is associated with the development of another disease.
  • Cellular senescence and the disease may evolve independently or interdependently.
  • the cellular senescence induces partially or completely another disease. In some embodiments, the cellular senescence induces, increases and/or potentiates the formation, expression and/or development of at least one risk factor for the development of at least one disease. In some embodiments, the cellular senescence inhibits and/or decreases the formation, expression and/or development of factors preventing the development of at least one disease, such as e.g., tumor suppressors such as the protein p53. In some embodiments, the cellular senescence increases the risks of developing at least one disease.
  • the cellular senescence affects at least one cell type in at least one tissue or organ selected from the group comprising or consisting of liver, bones, cartilage, pancreas, kidney, lungs, heart, blood vessels, blood, eyes, central nervous system including the brain, nerves, and skin.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease or condition.
  • treating and/or preventing cellular senescence with the population of HAPLC of the invention thereby treats and/or prevents another disease or condition.
  • the treatment and/or prevention of cellular senescence prevents, slows or blocks the progression or development of at least one disease or condition.
  • the treatment and/or prevention of cellular senescence alleviates the symptoms of at least one disease or condition.
  • the treatment and/or prevention of cellular senescence cures at least one disease or condition.
  • the positive effect may be a decrease of liver dysfunction, a decrease of liver inflammation or a decrease of fibrosis, or combinations thereof.
  • the treatment and/or prevention of cellular senescence decreases liver dysfunction, liver inflammation and/or fibrosis.
  • HALPC but not a combination of the pro-apoptotic drugs dasatinib (D) and quercetin (Q) reduced the early marker of senescence p21 in BDL rats and improved biliary injury (serum yGT and Sox9 gene expression) and hepatocytes mass loss (Hnf4a).
  • HALPC reduced early senescence and improved liver disease in a preclinical model of BA, providing encouraging preliminary results regarding the use of senotherapies in pediatric biliary cirrhosis.
  • HALPC are used for the treatment of preclinical model of BA.
  • the positive effect may be a decrease of ductular reaction.
  • the treatment and/or prevention of cellular senescence decreases ductular reaction.
  • ductular reaction refers to an increased number of ductules (the finest ramifications of the biliary tree), accompanied by polymorphonuclear leukocytes and an increase in matrix, leading to periportal fibrosis and eventually biliary cirrhosis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of metabolic, genetic, infectious, toxic and autoimmune liver diseases, chronic biliary diseases, cholestatic diseases, age-related diseases, bone and cartilage disorders, pancreatic diseases, kidney diseases, pulmonary diseases, cardiovascular diseases, metabolic diseases, eye diseases, neurodegenerative diseases, skin diseases, inflammatory diseases and cancer.
  • at least one disease selected from the group comprising or consisting of metabolic, genetic, infectious, toxic and autoimmune liver diseases, chronic biliary diseases, cholestatic diseases, age-related diseases, bone and cartilage disorders, pancreatic diseases, kidney diseases, pulmonary diseases, cardiovascular diseases, metabolic diseases, eye diseases, neurodegenerative diseases, skin diseases, inflammatory diseases and cancer.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of hepatic fibrosis, precirrhotic conditions, cirrhosis including liver and biliary cirrhosis, biliary atresia, Alagille syndrome, progressive familial intrahepatic cholestasis, primary biliary cholangitis, primary sclerosing cholangitis, chronic hepatitis, chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection, cholestasis, osteoporosis, osteoarthritis, atherosclerosis, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, thrombosis, cataracts, glaucoma, macular degeneration, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, renal dysfunction, pancreatic fibro
  • at least one disease selected from
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of liver diseases, biliary diseases and/or cholestatic diseases.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of hepatic fibrosis, precirrhotic conditions, cirrhosis including liver and biliary cirrhosis, biliary atresia, Alagille syndrome, progressive familial intrahepatic cholestasis, primary biliary cholangitis, primary sclerosing cholangitis, chronic hepatitis, chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection, cholestasis.
  • hepatic fibrosis precirrhotic conditions
  • cirrhosis including liver and biliary cirrhosis
  • biliary atresia biliary atresia
  • Alagille syndrome progressive familial intrahepatic cholestasis
  • primary biliary cholangitis
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of biliary diseases.
  • the biliary disease is chronic or acute.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cholestatic diseases.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of cirrhosis preferably biliary cirrhosis, biliary atresia, primary biliary cholangitis, primary sclerosing cholangitis, cholestasis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of liver diseases.
  • the liver disease is a metabolic, genetic, infectious, toxic and/or autoimmune disease.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of hepatic fibrosis, precirrhotic conditions, cirrhosis preferably liver cirrhosis, chronic hepatitis, chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection.
  • at least one disease selected from the group comprising or consisting of hepatic fibrosis, precirrhotic conditions, cirrhosis preferably liver cirrhosis, chronic hepatitis, chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of hepatic fibrosis, precirrhotic conditions, cirrhosis preferably liver cirrhosis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of hepatic fibrosis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cirrhosis, preferably liver or biliary cirrhosis. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of liver cirrhosis. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of biliary cirrhosis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of pre-cirrhotic conditions. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of biliary atresia. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of primary biliary cholangitis. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of primary sclerosing cholangitis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of chronic hepatitis, chronic hepatitis B virus (HBV) infection and/or chronic hepatitis C virus (HCV) infection. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cholestasis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of age-related diseases.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease selected from the group comprising or consisting of osteoporosis, osteoarthritis, cardiomyopathy, cataracts, glaucoma, macular degeneration, renal dysfunction, type 2 diabetes, neurodegenerative disorders, dementia, lipodystrophy, sarcopenia, age-related cachexia, skin aging and cancer.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of lipodystrophy, sarcopenia and/or age- related cachexia.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of bone and cartilage disorders. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on osteoporosis and/or osteoarthritis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of pancreatic diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on pancreatic fibrosis and/or type 2 diabetes.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of kidney diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on renal dysfunction. [0168] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of pulmonary diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on chronic obstructive pulmonary disease (COPD) and/or pulmonary fibrosis.
  • COPD chronic obstructive pulmonary disease
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cardiovascular diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, atherosclerosis and/or thrombosis.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of metabolic diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on diabetes, preferably type 2 diabetes.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of eye diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on cataracts, glaucoma and/or macular degeneration.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of neurodegenerative diseases or disorders.
  • the treatment and/or prevention of cellular senescence has a positive effect on dementia.
  • dementia include Lewy Body dementia, vascular dementia, frontotemporal dementia and the like.
  • neurodegenerative diseases include Alzheimer's disease and other memory disorders, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy and the like.
  • the neurodegenerative disease is selected from the group comprising or consisting of Lewy Body dementia, vascular dementia, frontotemporal dementia, Alzheimer's disease, memory diseases, ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, multiple system atrophy and progressive supranuclear palsy.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of skin diseases. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on skin aging and/or skin cancer.
  • the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of inflammatory diseases.
  • cancer is selected from the group comprising or consisting of adenoid cystic carcinoma, adrenal gland cancer, anal cancer, ataxia-telangiectasia, atypical mole syndrome, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, carcinoid tumor, cervical cancer, colorectal cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, liver cancer, lobular carcinoma, hepatocellular carcinoma, lung cancer, glioma, melanoma, meningioma, nasopharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, peritoneal cancer, pituitary gland tumor, polycythemia vera, prostate cancer
  • the cellular senescence is determined by measuring expression of the markers selected from the group comprising or consisting of p21, pl6 INK4A , pi4 ARF z 19 ARF , pRB, p53, high-mobility group box 1, lamin Bl, SA-p-Gal, lipofuscin, preferably p21, and senescence-associated secretory phenotype.
  • the cellular senescence is determined by measuring expression of one of these markers. In another embodiment, the cellular senescence is determined by measuring expression of a combination of these markers. In some embodiments, the cellular senescence is determined by measuring expression of at least one, at least two, at least three, at least four, at least five or more of these markers, preferably at least one.
  • the markers are measured at protein and/or RIMA level. [0179] In some embodiments, the markers are measured at protein level.
  • Methods to measure protein expression include, inter alia, enzyme- linked immunosorbent assay (ELISA), Western blot, Dot blot, immunofluorescence, immunochemistry, immunoprecipitation, fluorescent activated cell sorting (FACS), high-performance liquid chromatography (HPLC), and liquid chromatography - mass spectrometry (LC-MS).
  • the markers are measured at RIMA level.
  • Methods to measure RNA expression include, inter alia, RT-PCR, RT- qPCR, Northern Blot and hybridization techniques.
  • At least two markers are measured at different levels (e.g., one marker measured at protein level, and one marker measured at RNA level).
  • a marker is measured by measuring several proteins or RNA molecules, e.g., the measurement of senescence-associated secretory phenotype involves measuring more than one of e.g., cytokine, growth factor and protease.
  • the cellular senescence is determined by measuring expression of p21. In some embodiments, the cellular senescence is determined by measuring expression of pl6 INK4A . In some embodiments, the cellular senescence is determined by measuring expression of pl4 ARF . In some embodiments, the cellular senescence is determined by measuring expression of 19 ARF . In some embodiments, the cellular senescence is determined by measuring expression of pRB. In some embodiments, the cellular senescence is determined by measuring expression of p53. In some embodiments, the cellular senescence is determined by measuring expression of high-mobility group box 1. In some embodiments, the cellular senescence is determined by measuring expression of lamin Bl.
  • the cellular senescence is determined by measuring expression of SA-p-Gal. In some embodiments, the cellular senescence is determined by measuring expression of lipofuscin. In some embodiments, the cellular senescence is determined by measuring expression of senescence-associated secretory phenotype, e.g., TGFP, IL-6, IL-8, IL-lo, IL-1 [3 and/or CXCL1. [0184] In some embodiments, the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, is, or is to be, administered in a therapeutic effective amount to a subject in need thereof.
  • therapeutic effective amount it is meant a level or amount that is necessary and sufficient for slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of senescence, or disease related thereof; or alleviating the symptoms of senescence, or disease related thereof; or curing senescence, or disease related thereof, without causing significant negative or adverse side effects to the individual.
  • the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC is to be administered locally. In some embodiments, the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, is to be administered systemically.
  • the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, to be administered is contained in a suitable solution, gel, biogel, biomaterial, biocompatible synthetic material (e.g., biocompatible polymer), medical device and/or combinations thereof.
  • a suitable solution gel, biogel, biomaterial, biocompatible synthetic material (e.g., biocompatible polymer), medical device and/or combinations thereof.
  • the administration of the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC involves invasive surgery, noninvasive surgery, injection, targeted delivery and/or combination thereof.
  • multiple administrations of the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, are performed on the same subject.
  • the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC is administered in vitro or ex vivo, for example to another population of cells or to a tissue, e.g., in culture.
  • the treatment of senescence is performed ex vivo, then the treated tissue, organ or parts thereof is grafted in a subject in need thereof.
  • the population of HALPC is administered at a dose of 0.25 to 20 million of cells per kg of body weight.
  • 0.25 to 15 million of cells per kg of body weight include from 0.25 million, 0.30 million, 0.35 million, 0.40 million, 0.45 million, 0.50 million, 0.55 million, 0.60 million, 0.65 million, 0.70 million, 0.75 million, 0.80 million, 0.90 million, 0.95 million, 1.00 million, 1.10 million, 1.20 million, 1.30 million, 1.40 million, 1.50 million, 1.60 million, 1.70 million, 1.80 million, 1.90 million, 2.00 million, 2.10 million, 2.20 million, 2.30 million, 2.40 million, 2.50 million, 2.60 million, 2.70 million, 2.80 million, 2.90 million, 3.00 million, 3.10 million, 3.20 million, 3.30 million, 3.40 million, 3.50 million, 3.60 million, 3.70 million, 3.80 million, 3.90 million, 4.00 million, 4.10 million, 4.20 million, 4.30 million, 4.40 million
  • the population of HALPC is administered at a dose of about 0.25 to about 20 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.25 to about 15 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.25 to about 10 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.25 to about 5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.25 to about 2.5 million of cells per kg of body weight.
  • the population of HALPC is administered at a dose of about 0.25 to about 1.5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.5 to about 20 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.5 to about 15 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.5 to about 10 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.5 to about 5 million of cells per kg of body weight.
  • the population of HALPC is administered at a dose of about 0.5 to about 2.5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 0.5 to about 1.5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1 to about 20 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1 to about 15 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1 to about 10 million of cells per kg of body weight.
  • the population of HALPC is administered at a dose of about 1 to about 5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1 to about 2.5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1 to about 1.5 million of cells per kg of body weight. In some embodiments, the population of HALPC is administered at a dose of about 1.25 million of cells per kg of body weight.
  • the effective amount of the lysates of population of HALPC corresponds to lysates of a population of HALPC at a dose as described hereinabove.
  • the effective amount of a conditioned medium obtainable by culturing HALPC corresponds to the conditioned medium of the culture of a population of HALPC at a dose as described hereinabove.
  • the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC is administered in combination with another therapeutic agent.
  • the another therapeutic agent is to be administered in combination with, concomitantly or sequentially, the HALPC population, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, according to the invention.
  • the another therapeutic agent is administered at the same time as the HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC (/.e., simultaneous administration optionally in a coformulation).
  • the another therapeutic agent is administered at a different time than the HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC (/.e., sequential administration, where the another therapeutic agent is administered before or after the HALPC are administered).
  • the another therapeutic agent may be administered in the same way as the HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, or by using the usual administrative routes for that another therapeutic agent.
  • the another therapeutic agent is a pharmacologically active molecule, e.g., a small molecule, or a biologically active peptide/protein.
  • the another therapeutic agent is comprised in pharmacological composition approved (e.g., FDA or EMA approved) for the treatment of at least one disease.
  • the another therapeutic agent is undergoing at least one preclinical or clinical trial.
  • the another therapeutic agent is for treating and/or preventing senescence.
  • the another therapeutic agent is a senolytic agent.
  • a senolytic agent is a molecule used to treat and/or prevent senescence.
  • the another therapeutic agent is for treating and/or preventing at least one disease or condition as disclosed hereinabove.
  • the another therapeutic agent is for treating and/or preventing hepatic, biliary or cholestatic diseases, in particular hepatic fibrosis and cirrhosis.
  • the another therapeutic agent is selected from the group comprising or consisting of ursodeoxycholic acid, rifampicin, obeticholic acid, selonsertib, elifibranor, cenicriviroc, liraglutide, semaglutide, statins, losartan, telmisartan, candesartan, emricasan, pioglitazone, saroglitazar, solithromycin, cholestyramine and ASBT inhibitors (e.g., maralixibat).
  • the another therapeutic agent is ursodeoxycholic acid or rifampicin.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC, and a pharmaceutically acceptable vehicle, for use in the prevention and/or the treatment of cellular senescence.
  • HALPC liver-derived progenitor cells
  • the pharmaceutically acceptable vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, and any combinations thereof.
  • the carrier, diluent, solvent or excipient must be "acceptable" in the sense of being compatible with the HALPC, and not be deleterious upon being administered to an individual.
  • the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.
  • the pharmaceutical compositions should meet general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).
  • FDA Food and Drugs Administration
  • EMA European Medicines Agency
  • the pharmaceutical composition comprises HALPC in a sterile liquid at a concentration of 0.25 to 20 million cells per mL, preferably at a concentration of 0.5 to 5 million cells per mL.
  • Said sterile liquid may be prepared from a reconstituted suspension of HALPC, prepared for example by the dilution of a thawed concentrated HALPC suspension with a sterile diluent, such as a sterile aqueous solution, optionally comprising excipients such as pH-modifiers and/or human serum albumin, which is physiologically compatible with the patient and adapted for intravenous infusion.
  • a sterile diluent such as a sterile aqueous solution, optionally comprising excipients such as pH-modifiers and/or human serum albumin, which is physiologically compatible with the patient and adapted for intravenous infusion.
  • the composition is administered via intravenous infusion, optionally using a peripheral catheter.
  • the composition may be administered to the patient through a central line.
  • the volume and concentration of the composition in the form of a sterile liquid comprising the HALPC is preferably adapted for intravenous infusion.
  • the composition may be administered to the patient in the form of a sterile liquid comprising, after final adjustment, the pharmaceutical composition comprises HALPC in a sterile liquid at a concentration of 0.25 to about 20 million of cells per mL.
  • the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 15 million of cells per mL.
  • the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 10 million of cells per mL.
  • the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 2.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 1.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 20 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 15 million of cells per mL.
  • the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 10 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 2.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.5 to about 1.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 20 million of cells per mL.
  • the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 15 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 10 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 2.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1 to about 1.5 million of cells per mL. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 1.25 million of cells per mL. These final cell concentrations may be obtained by appropriately diluting a more concentrated HALPC composition.
  • the present invention further relates to a combination kit comprising (i) a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC, or a pharmaceutical composition comprising said population, lysates thereof and/or conditioned medium obtainable by culturing said population, and (ii) another therapeutic agent, for use for the prevention and/or the treatment of cellular senescence.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to a method for the prevention and/or the treatment of cellular senescence in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to the use of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC, for the manufacture of a medicament for treating and/or preventing cellular senescence.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to the treatment of senescence using a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • the present invention further relates to a method for reducing cellular senescence in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • the method is for reducing cellular senescence in cells or tissues cultured in vitro or ex vivo.
  • Another object of the present invention is a method for reducing liver dysfunction, liver inflammation and/or fibrosis in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC.
  • HALPC human allogenic liver-derived progenitor cells
  • Still another object of the present invention is a method for reducing ductular reaction in a subject in need thereof comprising a step of administrating a therapeutically effective amount of a population of human allogenic liver-derived progenitor cells (HALPC), lysates thereof and/or conditioned medium obtainable by culturing said HALPC.
  • HALPC liver-derived progenitor cells
  • Figures 1 A-F are a combination of scatter plots and a graph showing that senescence increases over time in BDL rats.
  • A SA-p-gal activity and
  • B [3- galactosidase gene expression (Glbl) in BDL livers versus controls at different timepoints after surgery.
  • C p21 protein and gene expression levels in BDL livers at different timepoints after surgery.
  • D p21 expression in cholangiocytes and hepatocytes of BDL rats at different timepoints after surgery.
  • E p21 expression in cholangiocytes and hepatocytes of BDL rats at different timepoints is compared to the correspondent cellular type of control rats.
  • BDL bile duct ligation
  • CPA collagen proportionate area
  • SA-p-gal senescence-associated p-galactosidase
  • Mann-Whitney U tests. Data is presented as mean ⁇ SEM; *p ⁇ 0.05; **p ⁇ 0.01. Scale bars 100pm.
  • Figures 2 A-G are a combination of scatter plots showing that senescence progression correlates to liver disease in BDL. Intrahepatic collagen deposition evidenced by Sirius Red staining (A), CollAl gene expression (B) and senescence evolution (SA-p-gal activity) (C) at different timepoints after surgery. Biliary damage (serum yGT) (D), bile ducts proliferation (Sox9 gene expression) (E), senescence progression (Cdknla expression) (F) and Serum ALT (G) at different timepoints after surgery.
  • Sirius Red staining A
  • CollAl gene expression B
  • SA-p-gal activity senescence evolution
  • C senescence evolution
  • Biliary damage sex9 gene expression
  • E senescence progression
  • Cdknla expression Serum ALT
  • FIGS. 3 A-F are a set of histology images showing that both fibrosis and senescence develop from the periportal area in bile duct ligation (BDL).
  • A-C Serial staining of o-SMA (top panels) and SA-p-gal (bottom panels) activity shows that both fibrosis and senescence at different timepoints following surgery.
  • SA-p- gal senescence-associated p-galactosidase.
  • P periportal area.
  • C centro-lobular area.
  • Figures 4 A-G are a combination of scatter plots showing the effect of HALPC injection on senescence and liver disease evolution in BDL rats.
  • Serum yGT A
  • bile duct proliferation Sox9 gene expression
  • B serum ALT
  • C CollAl gene expression
  • D Cdknla gene expression
  • E Cdkn2a gene expression
  • G SA-p-gal activity
  • BDL bile duct ligation
  • SA-p-gal senescence-associated p-galactosidase
  • ULN upper limit of normal
  • yGT gamma-glutamyl transferase. Mann-Whitney U tests. Data is presented as mean ⁇ SEM; *p ⁇ 0.05; **p ⁇ 0.01.
  • FIGS 5 A-I are an illustration of HALPC administration method and regimen used in BDL rats and a combination of scatter plots showing the effect of HALPC injection on early senescence and liver disease in the BDL model.
  • HALPC or vehicule are injected throught peripheral vein 48h after BLD.
  • SA-p-gal activity B
  • p21 gene expression C
  • pl6 gene expression D
  • Serum total bilirubin E
  • Serum AST F
  • Hnfa gene expression G
  • Histological fibrosis H
  • Gpxl gene expression I
  • BDL bile duct ligation
  • HALPC human allogenic liver- derived progenitor cells.
  • SA-p-gal senescence-associated p-galactosidase
  • ULN upper limit of normal. Data is presented as mean ⁇ SEM; **p ⁇ 0.01. Non-parametric Kruskal-Wallis One-Way ANOVA with Dunn's post-hoc tests were performed to compare continuous variables between subgroups.
  • Figure 6 A-C are combination of a set of histology images and scatter plots showing fibrosis and bile ducts proliferation in BA livers versus controls.
  • A Histological fibrosis increases in late BA patients compared to controls and to early stage BA;
  • B Myofibroblasts activation increases in BA patients compared to controls and weakly correlates with senescence development;
  • C Bile ducts proliferation increases in BA patients compared to controls and correlates with senescence development.
  • Figure 7 A-E are a combination of a set of histology images and scatter plots showing increase of senescence in BA livers and predominates in cholangiocytes and perinodular hepatocytes.
  • A SA-p-gal activity increases in cholangiocytes (arrowheads) and surrounding perinodular hepatocytes (arrows) in BA livers.
  • B pl6 protein expression also increases in cholangiocytes (arrowheads) and surrounding perinodular hepatocytes (arrows) in BA livers.
  • C Gene expression of pl6 progresses until liver transplantation in BA.
  • E DNA damage yH2AX-positive foci increase in both hepatocytes and cholangiocytes in BA livers and progress until liver transplantation in cholangiocytes.
  • Figure 8 A-C are a combination of histology images, microscopy images and scatter plots showing increase of DNA and telomere damages in BA.
  • A BA livers do not display an increased p21 protein expression.
  • B H2AX protein expression increases in BA and predominates in cholangiocytes (arrowheads) and perinodular hepatocytes (arrows).
  • telomere damage (co-localization of H2AX and TRF2 reported to total DNA damage) do not increase in late BA livers.
  • Figure 9 A-B are set of histology images showing cholangiocytes and perinodular hepatocytes display cellular senescence in BA livers.
  • A Co-staining of SA-p-gal activity and CK-19 IHC on BA livers: some bile ductules cholangiocytes show staining co-localization (arrowheads) while perinodular hepatocytes are only positive for SA-p-gal (arrows);
  • B Serial staining of SA-p-gal activity and CK-19 IHC on BA livers cryosections.
  • bile ductules (arrowheads) and perinodular hepatocytes (arrows) are positive for SA-p-gal in both early and late stage BA.
  • Right images remaining large septal bile duct display SA-p-gal activity.
  • FIG. 10 A-D presenting visualization of results of digital spatial whole transcriptomic analysis in BA livers.
  • A Design of the analysis.
  • B PCA of the dataset.
  • C Enrichment analysis of published senescence gene lists between cholangiocytes subgroups.
  • D Heatmaps of SASP genes obtained from two different published datasets in cholangiocytes and hepatocytes subgroups.
  • BA biliary atresia
  • Ctrl control
  • FC fold change
  • NES normalized enrichment score
  • p-adj adjusted p-value
  • PCA principal component analysis
  • ROI region of interest
  • SASP senescence-associated secretory phenotype.
  • Figure 11 A-C are a combination of histology images and scatter plots showing correlation of liver disease to senescence progression in BDL.
  • Liver fibrosis increases post-BDL and correlates with senescence progression.
  • B Biliary damage (serum GT) and proliferation (Sox9) increase in BDL rats and biliary proliferation correlates with senescence progression.
  • C Serum AST maximal increase occurs 48 hours post-BDL and is followed by a loss of hepatocytes mass (Hnf4a).
  • BDL bile duct ligation
  • CPA collagen proportionate area
  • Figure 12 A-D are a combination of histology images and scatter plots showing senescence progressively developing in BDL rats and appears in cholangiocytes before hepatocytes.
  • A SA-p-gal activity increases after the surgery.
  • B p21 gene expression progressively increases in BDL livers as compared to controls. Senescence (p21-positive cell percentage) is maximal in cholangiocytes 48 hours post-BDL, while hepatocytes senescence increases progressively to become significant only two weeks after the surgery. The percentages of p21-positive cells at different timepoints are compared to the correspondent cellular type of control rats.
  • C pl6 gene expression increases in diseased rats two weeks post-surgery.
  • BDL bile duct ligation
  • SA-p-gal senescence-associated p- galactosidase
  • SASP senescence-associated secretory phenotype
  • Figure 13 A-D are a combination of scatter plots showing the results of D+Q administration in BDL rats.
  • D+Q had no effect on liver senescence.
  • C D+Q had no effect on biliary injury (serum GT) and proliferation (Sox9) nor on biochemical cholestasis (serum total bilirubin).
  • liver models are depicted in specific examples as possible senescence models that HALPC treatment can be applied on, it should be clear to a skilled person that invention is also suitable for any other senescence model.
  • the non-limiting examples of such models can be for instance, a mouse model of idiopathic pulmonary fibrosis, card io myocytes of aging rats, an ischemiareperfusion model of kidney injury, aging-related skin senescence models or ultraviolet B-induced skin senescence models.
  • BDL Bile Duct Ligation
  • Bile duct ligation animal model Liver tissue was obtained from wild-type male Wistar rats. Biliary cirrhosis was induced in 2-month-old rats by performing the extrahepatic BDL surgical procedure as previously described (Sokal EM et al., Hepathology, 1992). Animals were sacrificed at various timepoints after the surgical procedure. Controls underwent sham procedure (bile duct dissection without resection) at the same age and were sacrificed at the same time points. All animals were fed with branched-chain amino acid enriched diet (Ssniff-Spezialdiaten GmbH, Soest, Germany) after the surgery and were supplemented with daily oral vitamin E and weekly intraperitoneal vitamin K.
  • branched-chain amino acid enriched diet Ssniff-Spezialdiaten GmbH, Soest, Germany
  • Senescence-associated p-galactosidase activity assay Senescence-associated p-galactosidase (SA-p-gal) activity assay was performed on cryopreserved liver tissue as previously described (Jannone G et al., Tissue. J Histochem Cytochem, 2020). The reaction was performed at pH 4 and the staining solution was removed after two hours of incubation at 37°C. Stained sections were washed with PBS before subsequent immunohistochemical staining when indicated.
  • RT-qPCR Reverse transcription quantitative polymerase chain reaction
  • RT-qPCR was carried out in duplicate using TaqMan universal MasterMix (Applied Biosystems) and pre-designed TaqMan probes obtained from Integrated DNA Technologies (Coralville, IA, USA) on a StepOnePlus real-time PCR machine (Applied Biosystems). Relative gene expression was determined with the AACt method using Gapdh and B2m as housekeeping genes (Livak KJ et al., Methods, 2001).
  • Senescence increases over time in the BDL model BDL rats displayed a higher SA-p-gal activity compared to controls from one week after the surgery (p ⁇ 0.05) (Fig. 1A).
  • [3-galactosidase (Glbl) gene expression increased as well in diseased animals two weeks after the surgery (p ⁇ 0.01) (Fig. IB).
  • the protein expression of senescence marker p21 progressively increased in both cholangiocytes and hepatocytes of operated rats as soon as 48 hours after the surgery (p ⁇ 0.05) (Fig. 1C). Cholangiocytes senescence was maximal 48 hours postsurgery and subsequently diminished, while hepatocytes senescence progressively increased (Fig. ID).
  • Liver fibrosis and ductular reaction progression correlate to senescence in the BDL model: Histological fibrosis increased in BDL rats from one-week postsurgery as compared to controls and was strongly correlated to senescence progression in the animals (r 0.96; p ⁇ 0.0001) (Fig. 2A and 2C). Gene expression confirmed that fibrosis developed over time in BDL rats and increased significantly from 48 hours after the surgery (CollAl; p ⁇ 0.05) (Fig. 2B). Biliary injury was evidenced through serum y-glutamyl transferase (yGT) elevation (Fig.
  • yGT serum y-glutamyl transferase
  • DR ductular reaction
  • Fig. 2F Hepatocytes injury also occurred and serum alanine aminotransferase (ALT) elevation was maximal 48 hours post-BDL.
  • Example 2 Human allogenic liver-derived progenitor cells (HALPC) injections improve senescence and biliary impairment in BDL
  • HHLPC Human allogenic liver-derived progenitor cells
  • PBS containing heparin 300UI/5xl0 6 cells.
  • Cells were subsequently injected to the animals at high dose (12.5xl0 6 cells/kg) versus low dose (1.25xl0 6 cells/kg) through the penile vein 48 hours after BDL procedure. Controls underwent BDL as well and were injected with the vehicle only at the same timepoint. All animals were sacrificed three days after the injection ( Figure 5A).
  • Intravenous HALPC injection reduced the gene expression of the early marker of senescence p21 in both the low dose and the high dose HALPC injected animal groups compared to control group (Fig. 5C).
  • No difference in pl6 gene expression Fig. 4F, Fig. 5D
  • SA-p-gal activity Fig. 4G, Fig 5B
  • No significant amount of HALPC was identified in the livers of injected rats through human SRY gene expression when animals were sacrificed.
  • Serum total bilirubin is slightly decreased in the presence of HALPC (Fig. 5E).
  • Serum AST is decreased in the presence of HALPC (Fig. 5F), suggesting a potential improvement of the hepatocytes injury.
  • the cells slightly improved the hepatocytes mass loss (hepatocyte nuclear factor 4-alpha - Hnf4a - expression) (Fig. 5G).
  • Liver histological fibrosis (Fig. 5H) showed a tendency of decrease in the presence of both low and high dose of HALPC.
  • HALPC antisenescence properties might be related to anti-oxidative effects and suggest their use for treating or preventing senescence in plurality of organs including lungs, heart and kidneys. after BDL suraerv in rats
  • BDL was performed in two-months-old rats to generate a model of biliary senescence in which we could test senotherapeutics.
  • the operated rats developed progressive cholestasis, biliary proliferation, loss of hepatocytes mass and liver fibrosis as expected (Fig. 11).
  • Senescence progression was confirmed in the animal model as demonstrated by an increased SA-p-gal activity, p21 protein and gene expression, pl6 gene expression and SASP-related gene expression (Fig. 12).
  • Biliary proliferation and liver fibrosis significantly correlated with senescence development (Fig. 11A-B).
  • the earliest marker of senescence was p21 as it was significantly increased in whole liver homogenates from 1 week after the surgery (Fig. 12B).
  • the percentage of senescent p21-positive cholangiocytes was already maximal after 48 hours and subsequently decreased, while senescence progressively increased in hepatocytes of the parenchyma (Fig. 12B).
  • Example 4 HALPC but not D+Q decrease early senescence and liver disease in the BDL model
  • Example 4 refers to example 2 for Material and Methods section. Briefly, as the treatment regime, human allogenic liver-derived progenitor cells (HALPC) or a combination of dasatinib (5 mg/kg) and quercetin (50 mg/kg) (D+Q) were administrated to two-month-old Wistar rats 48 hours after bile duct ligation (BDL). Dasatinib and quercetin are two senotherapeutic options suitable for clinical applications.
  • HLPC human allogenic liver-derived progenitor cells
  • dasatinib and quercetin 50 mg/kg
  • Example 5 Senescence and senotherapies in biliary atresia and biliary cirrhosis, human samples
  • Senescence is a process of cellular ageing, resulting in metabolic modifications and in an irreversible cell cycle arrest.
  • Various cellular changes reflect this complex phenomenon, including upregulation of cell cycle inhibitors (e.g. p21 and pl6) and anti-apoptotic pathways, increased lysosomal protein content and senescence-associated beta-galactosidase (SA-p-gal) activity, accumulation of DNA damage foci (e.g. y-H2AX and/or 53BPl-positive foci), and a senescence-associated secretory phenotype (SASP). Because there are no completely specific senescence features, the gold standard is to assess cellular senescence through the evaluation of multiple markers.
  • Accelerated senescence occurs in numerous chronic adult hepatobiliary diseases and has been associated to disease severity and cirrhosis. Senescence progression worsens the prognosis as it participates in tissue remodeling and liver dysfunction. Furthermore, clearance of senescent cells or inhibition of the paracrine transmission of senescence improves liver function, histological fibrosis or steatosis in mouse models of hepatobiliary diseases. Existing data demonstrate that liver disease improves when senescence decreases, thus supporting the development of senotherapeutics that could be translated to clinical applications.
  • Biliary atresia is a severe pediatric disease caused by the progressive fibro-inflammatory obliteration of extrahepatic bile ducts, leading to biliary cirrhosis and end-stage liver disease.
  • BA is the first cause of liver transplantation in children, its underlying mechanisms are still not completely elucidated.
  • a hepatoportoenterostomy procedure can be attempted in case of early diagnosis, but liver transplantation remains the only curative treatment in about two thirds of the cases.
  • a few studies described premature senescence in BA, but the possibility of using anti-senescence therapies was never explored in pediatric biliary cirrhosis.
  • Table 2 Primary antibodies. Ag: antigen; C: citrate; H: human; IF: immunofluorescence; IHC: immunohistochemistry; M: Mouse; mAb: monoclonal antibody; pAb: polyclonal antibody; R: rat; Rb: rabbit; T: Tris-EDTA.
  • Results were expressed as (stained area/total tissue area) x 100 to obtain a percentage of stained area or as (stained cells/total number of cells) x 100 to obtain a percentage of stained cells.
  • the parameters of the designed Visiopharm APPs were kept constant for all sections.
  • RT-qPCR performed as described in example 1. Relative gene expression was determined with the AACt method using TBP and PPIA as housekeeping genes for human experiments and Gapdh and B2m for rat experiments. Used TaqMan universal MasterMix (Applied Biosystems) and pre-designed TaqMan probes are listed in Table 3.
  • Table 3 Pre-designed TaqMan probes used for reverse transcription quantitative polymerase chain reaction. Digital spatial whole transcriptome of BA livers:
  • DSP Digital spatial profiling
  • RNA targets were exposed through incubation with Proteinase K at 37C for 15min and a post-fixation step was performed to preserve the samples.
  • the slides were hybridized overnight with GeoMx Human Whole Transcriptome Atlas Human RIMA probe mix for Illumina Systems (NanoString Technologies). Stringent washes were performed to remove off-target probes and samples were blocked with Buffer W (NanoString Technologies). Prepared slides were then stained for one hour with immunofluorescent antibodies to allow the identification of tissue morphology and the detection of specific cell types during the regions of interest (ROI) selection step.
  • ROI regions of interest
  • the morphological markers used were pan-cytokeratin (2 pg/ml; NBP2- 33200AF532, Novus Biologicals, Centennial, CO, USA), alpha smooth muscle actin (1.25 pg/ml; ab202368, Abeam, Cambridge, UK) and Sytol3 (S7575, Thermo Fisher Scientific). Slides were then washed and loaded into the GeoMx DSP machine for scanning (x20 magnification) and ROI selection. Selection of 4-5 ROI for hepatocytes and 4-5 ROI for cholangiocytes was manually performed in each sample.
  • GeoMx Human Whole Transcriptome Atlas RNA assay contains in situ hybridization probes conjugated to unique DNA oligonucleotides (DSP barcodes) via a UV-photocleavable linker. After ROI selection, the DSP barcodes were tagged according to their ROI location and then UV-cleaved and collected to be sequenced on an Illumina sequencer (San Diego, CA, USA). Sequenced oligonucleotides were processed and then imported back into the GeoMx DSP analysis software for integration with the ROI selection information and generation of spatially- and cell type-resolved transcriptomic data.
  • DSP barcodes unique DNA oligonucleotides
  • liver tissue samples were obtained from thirty BA patients undergoing liver transplantation (BA late group) and five patients during hepatoportoenterostomy procedure (BA early group), the latter procedure being performed very shortly after the diagnosis. All BA patients were pediatric, while the ten control livers were obtained from pediatric and adult individuals (four adults aged 28 - 41 years old). General characteristics of the study population are summarized in Table 4. As expected, fibrosis and biliary proliferation increased in BA livers ( Figure 6).
  • Table 4 Description of the study population. In the first column, standard values are indicated for all biochemical parameters. Continuous data is presented as median (range) or mean ⁇ SEM. D: days; m: months; NA: not available; y: years.
  • Fig. 7C & 7E there was still a clear progression of senescence between the time of diagnosis and liver transplantation.
  • cholangiocytes and hepatocytes ROI were selected in BA early, BA late and control livers (Fig. 10A).
  • Principal component analysis (PCA) revealed that the transcriptomes differed according to cell type (cholangiocytes versus hepatocytes) and disease stage (Fig. 10B).
  • Early and late stage BA transcriptomes were clustered together and differed from controls for each cell type. Differential expression analysis was performed between disease stages subgroups for each cell type.
  • a geneset corresponding to genes that were upregulated in senescent HepG2 cells as well as in both senescent HepG2 and diseased human adult livers was significantly enriched in diseased cholangiocytes (BA early and late stages) as compared to controls, supporting the presence of senescence in BA cholangiocytes.
  • the same gene lists as well as two other datasets obtained from senescent HepG2 cells and from an organ-independent literaturebased senescence signature database were significantly enriched in BA late stage cholangiocytes as compared to BA early stage, underlying the progression of senescence until liver transplantation in BA cholangiocytes.
  • a core transcriptome database obtained from senescent human fibroblasts, melanocytes, keratinocytes and astrocytes was not significantly enriched in our diseased cholangiocytes. None of those publically available senescence datasets were enriched in our diseased hepatocytes. SASP genes obtained from two published datasets were overexpressed in both diseased cholangiocytes and hepatocytes in our cohort (Fig. 10D).
  • a publically available SASP gene list obtained from senescent HepG2 cells was significantly enriched in BA late stage cholangiocytes as compared to BA early stage (NES 1.6; p-adj 0.007), and in diseased cholangiocytes as compared to hepatocytes (NES 1.47; p-adj 0.04).
  • those results confirm the progressive development of senescence and SASP in cholangiocytes until liver transplantation and highlight the predominance of senescence in BA cholangiocytes as compared to hepatocytes.

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Abstract

La présente invention concerne une population de cellules progénitrices allogènes humaines dérivées du foie (HALPC), leurs lysats et/ou le milieu conditionné obtenu en cultivant lesdites HALPC, pour traiter et/ou prévenir la sénescence cellulaire chez un sujet en ayant besoin. Le traitement et/ou la prévention de la sénescence cellulaire à l'aide de la population cellulaire de l'invention, d'un lysat de celle-ci et/ou d'un milieu conditionné obtenu par sa culture, a un effet positif sur d'autres maladies, en particulier les maladies associées à la sénescence cellulaire.
PCT/EP2023/056454 2022-03-24 2023-03-14 Utilisation de cellules progénitrices allogéniques humaines dérivées du foie pour traiter et/ou prévenir la sénescence cellulaire WO2023180122A1 (fr)

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