USE OF HUMAN ALLOGENIC LIVER-DERIVED PROGENITOR CELLS FOR TREATING AND/OR PREVENTING CELLULAR SENESCENCE

FIELD OF INVENTION

[0001] 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.

BACKGROUND OF INVENTION

[0002] 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.

[0003] Nevens Frederik et al.,2021, doi: 10.1016/j.jhepr.2021.100291 reports the results of a phase II clinical study of HALPC therapy in cirrhotic patients with ACLF or with AD at risk of developing ACLF.

[0004] 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.

[0005] Existing data demonstrate that liver disease improves when senescence decreases. Thus, there is a need for senolytic therapies that can be translated to clinical applications. Mesenchymal stem cells (MSCs) 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.

[0006] Human allogenic liver-derived progenitor cells (HALPC) 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.

[0007] 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.

SUMMARY

[0008] 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.

[0009] In some embodiments, 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.

[0010] In some embodiments, the cellular senescence is induced by a disease or damage, an oncogene, a therapy, a diet, and the like.

[0011] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease.

[0012] In some embodiments, 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.

[0013] In some embodiments, 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, dementia, lipodystrophy, sarcopenia, age-related cachexia, skin aging, hepatocellular carcinoma, liver cancer, lobular carcinoma, bile duct cancer, melanoma, lung cancer, and islet cell tumor.

[0014] In some embodiments, the at least one disease is cirrhosis, preferably liver or biliary cirrhosis.

[0015] In some embodiments, 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.

[0016] In some embodiments, the population, lysates thereof and/or conditioned medium, is to be administered in a therapeutic effective amount to a subject in need thereof.

[0017] In some embodiments, the population comprises a dose of 0.25 to 20 million of said human allogenic liver-derived progenitor cells per kg of body weight.

[0018] In some embodiments, the population, lysates thereof and/or conditioned medium, is to be administered in combination with another therapeutic agent.

[0019] The present invention further relates to 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.

[0020] 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.

[0021] 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.

[0022] 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.

DEFINITIONS

[0023] In the present invention, the following terms have the following meanings:

[0024] The term "about", when preceding a figure, means plus or less 10% of the value of said figure.

[0025] The term "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). [0026] The term "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.

[0027] The term "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. Based on the ability to give rise to diverse cell types, 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.

[0028] The term "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. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0029] The term "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.

[0030] The term "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.

[0031] 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. In some embodiments, 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. In some embodiments, the individual is an adult (e.g., an individual above the age of 18). In some embodiments, the individual is a child (e.g., an individual below the age of 18). In some embodiments, the individual is a male. In some embodiments, the individual is a female.

[0032] The term "extract" or "lysate", as used herein, means a lysed cell content. In one embodiment, such extract or lysate has not been further purified and thus contains the whole cell lysate content. In another embodiment, such extract refers to a protein extract, an RIMA extract, a lipid, or a membrane vesicle. In practice, extracts include cellular and extracellular extracts. In one embodiment, extracts according to the present invention include metabolites from the cells. According to the present invention, lysate may include soluble fractions and/or insoluble fractions.

[0033] The term "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.

[0034] The term "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. In other words, 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. According to the present invention, a "conditioned medium" may include soluble fractions and/or insoluble fractions.

[0035] The term "fraction" as used herein, refers to a part, composition or derivative obtainable from the conditioned medium of the invention.

DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] More preferably, 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.

[0039] Hence, 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". A skilled person will appreciate that the 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.

[0040] In an alternative embodiment of the invention, the adult liver or part thereof may be from a non-human animal subject, preferably a non-human mammal subject.

[0041] 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. By means of example and not limitation, particularly suitable non-human mammal cells for use in human therapy may originate from pigs.

[0042] 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.

[0043] A skilled person will appreciate that at least some aspects of harvesting liver or part thereof from donor subjects may be subject to respective legal and ethical norms. By means of example and not limitation, harvesting of liver tissue from a living human donor may need to be compatible with sustenance of further life of the donor.

[0044] Accordingly, only a part of 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. On the other hand, 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. For example, the non- human animal may be humanely culled after harvesting of the tissue. These and analogous considerations will be apparent to a skilled person and reflect legal and ethical standards.

[0045] 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. Subject to ethical and legal norms, 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).

[0046] Alternatively, 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.

[0047] 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. For example, 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. That is, 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.

[0048] 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 skilled person is able to select a suitable medium which can support the survival of the cells of the tissue during the period before processing.

[0049] 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.

[0050] 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. Subsequently, cells comprised in this preparation are cultured under adherent conditions, preferably as to allow adherence and growth of cells onto a support. Next, these cells are passaged at least once, preferably at 70 %, 80 % or 90 % confluence. Finally, 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.

[0051] In a preferred embodiment, such method comprises the steps of:

(a) Disassociating adult liver or a part thereof to form a population of liver primary cells;

(b) Generating preparations of the liver primary cells of (a);

(c) Culturing the cells comprised in the preparations of (b) onto a support which allows adherence and growth of cells thereto and the emergence of a population of cells;

(d) Passaging the cells of (c) at least once;

(e) Isolating the cell population that is obtained after passaging of (d) and positive for the markers identified in the description of the Invention.

[0052] 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.

[0053] Concerning step (a) of the method, 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.

[0054] Briefly, a population of liver primary cells is first obtained from disassociating of liver or part thereof, to form a population of primary cells from said liver or part thereof. Subsequently, cells comprised in this preparation are cultured under adherent conditions, preferably as to allow adherence and growth of cells onto a support. Next, these cells are passaged at least once, preferably at 70% confluence. Finally, 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.

[0055] Concerning step (b) of the method, 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.

[0056] 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.

[0057] The population of primary cells as defined and obtained herein by disassociating liver (or part of it) 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.

[0058] Concerning step (c), the preparation of 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).

[0059] 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

WO2020221843, or as described below at step (e). Among 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.

[0060] The isolation or harvesting of liver progenitor cells can then be made based on the confirmation of the cells' identity, i.e., the marker profile, morphology and/or activity. For example, 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. In addition, the liver progenitor cells may secrete HGF and/or PGE2. Moreover, they can optionally be positive for at least one hepatic marker and/or exhibit at least one liver-specific activity. In one embodiment, the cells are positive for at least one hepatic marker and/or exhibit at least one liver-specific activity. For example, 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.

[0061] In one embodiment, 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. In a further embodiment, 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. [0062] Concerning step (d) of the method, primary 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. The term "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. For sake of simplicity, the passage performed after the first time of growing the cells under adherent culture conditions is herein referred to as "first passage" (or passage 1, Pl) within the method of the invention. 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.

[0063] 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. In general, 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, besides providing nutrients and/or growth promoters, may also promote cell adhesion by coating the treated plastic surfaces with a layer of matrix to which cells can better adhere. As appreciated by those skilled in the art, 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. Advantageously, serum can be prepared using serum-separating vessels (SST), which contain the inert catalyst to mammals.

[0064] 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.

[0065] Typically, the medium will comprise a basal medium formulation as known in the art. Many 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. 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., StemMacs™ from Miltenyi, Prime-XV from FUJIFILM Irvine Scientific.

[0066] Such basal media formulations contain ingredients necessary for mammal cell development, which are known per se. By means of illustration and not limitation, 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. It will also be apparent that many media are available as low- glucose formulations with or without sodium pyruvate.

[0067] For use in culture, basal media can be supplied with one or more further components. For example, 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.

[0068] 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.

[0069] 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.

[0070] Also contemplated is supplementation of cell culture medium with mammalian plasma or serum. Plasma or serum often contains cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated. 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. In another embodiment, the any combination of the above plasma and/or serum may be used in the cell medium.

[0071] When passaged, 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.

[0072] 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. Preferably, 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).

[0073] Next, the so detached and dissociated cells (typically as a cell suspension in an isotonic buffer or a medium) 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. Preferably, 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. [0074] Concerning step (e) above, the isolation of population of HALPC applies to cells that are positive for the listed markers, further validating the criteria for initially identifying HALPC at step (c) above but that can be more easily established given the higher amount of cells that are available after passaging.

[0075] 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. For example, 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. In addition, the HALPC may secrete HGF and/or PGE2. Moreover, they can optionally be positive for at least one hepatic marker and/or exhibit at least one liver-specific activity. In one embodiment, the cells are positive for at least one hepatic marker and/or exhibit at least one liver-specific activity. For example, 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.

[0076] In one embodiment, 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. In a further embodiment, 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.

[0077] In another embodiment of the present invention, 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.

[0078] In one embodiment, 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). They optionally also exhibit a liver-specific activity which may be selected from urea secretion, bilirubin conjugation, alpha-l-antitrypsin secretion and CYP3A4 activity. Moreover, the HALPC preferably express and/or secrete HGF and PGE-2.

[0079] In one embodiment, 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. For example, 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.

[0080] 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. By preference, said cells do not differentiate into osteocytes or adipocytes.

[0081] In a preferred embodiment of the current invention, 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).

[0082] In another or further embodiment of the present invention, 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).

[0083] In another embodiment of the present invention, 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).

[0084] In another embodiment of the invention, the human liver progenitor or stem cells are measured positive for CD90, CD73, vimentin and ASMA.

[0085] In a further embodiment of the invention, 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.

[0086] In another embodiment, the human liver progenitor or stem cells are measured positive for CD90, CD73, vimentin and ASMA, and negative for CK-19.

[0087] In another or further preferred embodiment, 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, FMOD, KCND2, CCL11, ASPN, KCNK2, and HMCN1.

[0088] In still another or further embodiment of the present invention, 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.In another or further embodiment, said cells are measured negative for one or more markers chosen from the group of: (i) sushi domain containing protein 2 (SUSD2) and cytokeratin-19 (CK-19); (ii) CD271; (iii) at least one marker selected from ITGAM, ITGAX, IL1R2, CDH5, and NCAM1; and (iv) at least one marker selected from HP, CP, RBP4, APOB, LBP, ORM1, CD24, CPM, and APOCI.

[0089] It will be understood that 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.

[0090] In a further embodiment the HALPC are negative for HLA-DR. [0091] In a further embodiment, the HALPC are negative for certain markers, such as CD133, CD45, CK19 and/or CD31.

[0092] In a further embodiment, the HALPC may also be measured positive for one or more of the enzymatic activities listed in W02016/030525, Table 6. In some embodiments, 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. Additionally, 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.

[0093] The biological activities, the markers, and the morphological/functional features listed above 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.

[0094] Further features can be also determined for 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. In some of such embodiments, HALPC can be measured negative for at least one further marker selected from the group consisting of ITGAM, ITGAX, IL1R2, CDH5, and NCAM1. In some of such embodiments, HALPC can be measured negative for at least one of HP, CP, RBP4, APOB, LBP, ORM1, CD24, CPM, and APOCI.

[0095] In some embodiments, the population of HALPC is substantially pure. As used herein, 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. In some embodiments, the population of HALPC is pure, meaning devoid of any other cell types.

[0096] 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. [0097] 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.

[0098] As used herein, 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.

[0099] In some embodiments, 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.

[0100] In practice, 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.

[0101] In practice, extracellular extracts may include the secreted fraction, in particular soluble compounds, or extracellular vesicles (EV). As used herein, the term "extracellular vesicles" encompasses exosomes, exosome-like vesicles, microvesicles (or ectosomes) and apoptotic bodies. In some embodiments, the extracellular extracts are extracellular vesicles. In some embodiments, the extracellular extracts are the secreted fraction. In some embodiments, the extracellular extracts include secreted molecules. In practice, 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. Illustratively, 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.

[0102] In one embodiment, the lysate is a protein extract, a RIMA extract, a lipid, or a membrane vesicle.

[0103] 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. [0104] In one embodiment, 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.

[0105] 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.

[0106] In one embodiment, 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. In general, 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. In a particularly preferred embodiment, 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, besides providing nutrients and/or growth promoters, may also promote cell adhesion by coating the treated plastic surfaces with a layer of matrix to which cells can better adhere. As appreciated by those skilled in the art, the cells may be counted in order to facilitate subsequent plating of the cells at a desired density. In another particularly preferred embodiment, such culturing medium is serum-free.

[0107] 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.

[0108] 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.

[0109] 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). Relevant time points can be shorter (e.g., 2 hours or less) or longer such as at 24 hours, at 36 hours or more hours. By obtaining samples of said conditioned medium at these time points or at intermediate ones (such as 1, 2, 4, 6, 8, 12, or 18 hours) and testing such samples for their composition and/or activities, the optimal timing for obtaining the desired conditioned medium derived from human liver progenitor or stem cells can be determined.

[0110] In one embodiment, 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.

[0111] The conditioned medium of the invention and/or the fraction thereof, typically contain soluble proteins, RNAs, exosomes and/or microvesicles.

[0112] In a preferred embodiment, 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).

[0113] In a further preferred embodiment, 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). [0114] In certain embodiments the conditioned medium and/or the fraction thereof comprise:

(a) at least one of soluble proteins selected from the group consisting of: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), eotaxin (CCL11), interleukin-6 (IL-6), and interleukin-8 (IL-8); and, optionally

(b) at least one of soluble proteins selected from the group consisting of matrix metalloproteinases, growth factors, chemokines, and cytokines.

[0115] 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. In particular, one or more of HGF, VEGF, CCL11, IL-6, or IL-8 (preferably all of them) may be present at a concentration of at least 1 ng/ml.

[0116] In an alternative embodiment 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.

[0117] In another embodiment, 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.

[0118] In another and further embodiment 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.

[0119] In another preferred embodiment, the conditioned medium and the fraction thereof comprise RNAs, for example miRNAs.

[0120] Particularly desired concentrations 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. Hence, certain embodiments provide so-concentrated or so-diluted conditioned medium and/or in a fraction thereof.

[0121] In a further preferred embodiment, 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.

[0122] In an alternative embodiment, 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.

[0123] In an embodiment, 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.

[0124] In another embodiment, the conditioned medium of current invention comprises at least one component selected from sphingosine-l-phosphate, TGF-pi, HGF and TIMP2.

[0125] In a further preferred embodiment, the conditioned medium of the current invention comprises at least sphingosine-l-phosphate (SIP).

[0126] In a further preferred embodiment, 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.

[0127] 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. Examples of such 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.

[0128] Possible useful conditioned medium or stem cells in cell culture medium are in detail reported in WO 2015/001124 and WO 2021/069553 and herein incorporated as a reference.

[0129] As used herein, the term "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).

[0130] In some embodiments, the cellular senescence is induced. In some embodiments, 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.

[0131] In some embodiments, 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.

[0132] In one embodiment, the cellular senescence is induced by telomeric shortening. As used herein, the term "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.

[0133] In one embodiment, the cellular senescence is induced by DNA damage. In certain embodiments, 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. In some embodiments, the DNA damage is endogenous or exogeneous. In some embodiments, 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. In one embodiment, the DNA damage occurs on at least one gene encoding a telomerase. In some embodiments, 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.

[0134] In one embodiment, the cellular senescence is induced by oxidative stress. Within the scope of the present invention, the term "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. 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. In some embodiment, the oxidative stress damages soluble proteins, transmembranous proteins, organelles, RNA and/or DNA. In some embodiments, the oxidative stress results in an oxidation reaction that is biologically irreversible, e.g., sulfonylation reaction. In certain embodiments, high oxidative stress is associated with faster aging.

[0135] In one embodiment, the cellular senescence is induced by oncogenic activation. As used herein, the term "oncogenic activation" refers to the increased expression of at least one gene that has an oncogenic or protooncogenic activity. Non limitative examples of 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). In some embodiments, oncogenic activation is associated with increased risk of cancer and/or metabolic reprogramming. In some embodiments, oncogenic activation is associated with higher cell division rates.

[0136] In one embodiment, the cellular senescence is induced by metabolic dysfunction. As used herein, the term "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.

[0137] In some embodiments, the cellular senescence is induced by a disease or damage, an oncogene, a therapy, a diet, and the like. [0138] In one embodiment, the cellular senescence is induced by a disease. In some embodiments, the disease is selected from the group comprising genetic, infectious, parasitic, metabolic, degenerative, physiological and idiopathic diseases. In some embodiments, the disease is chronic. In some embodiments, the disease is acute. Non limitative examples of diseases inducing senescence include cirrhosis, hepatitis, cancer, diabetes, Alzheimer's disease, cardiovascular conditions such as thrombosis, and the like.

[0139] In one embodiment, the cellular senescence is induced by a damage to the organism. Non-limitative examples of 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. In some embodiments, the damage directly promotes or induces senescence. In some embodiments, the damage induces a disease or condition that promotes senescence.

[0140] In one embodiment, the cellular senescence is induced by an oncogene. Non limitative examples of oncogenes include vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), Raf, Ras, c-Myc, c-Kit or cyclin DI (CCND1).

[0141] In one embodiment, the cellular senescence is induced by therapy. In some embodiments, the therapy involves deleterious or toxic side effects. In some embodiments, the therapy is radiotherapy, chemotherapy, pharmacotherapy, invasive surgery, antibiotic, antifungal and the likes. In some embodiments, the cellular senescence is induced by a medicament with some degree of toxicity for a tissue, organ or organism. In some embodiments, 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. In some embodiments, the cellular senescence is induced by a medicament used as prescribed, e.g., chemotherapy for cancer like etoposide or doxorubicin.

[0142] In one embodiment, the cellular senescence is induced by diet. In some embodiment, the diet is imbalanced. By 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). In some embodiment, the cellular senescence is induced by overeating, i.e., an excessive intake of caloric nutrient. In some embodiment, 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.

[0143] In some embodiments, the cellular senescence is associated with the development of another disease. Cellular senescence and the disease may evolve independently or interdependently.

[0144] In some embodiments, 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.

[0145] In some embodiments, 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.

[0146] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of at least one disease or condition. In other words, in some embodiments, treating and/or preventing cellular senescence with the population of HAPLC of the invention thereby treats and/or prevents another disease or condition.

[0147] By "positive effect", it is meant that the treatment and/or prevention of cellular senescence prevents, slows or blocks the progression or development of at least one disease or condition. In some embodiments, the treatment and/or prevention of cellular senescence alleviates the symptoms of at least one disease or condition. In some embodiments, the treatment and/or prevention of cellular senescence cures at least one disease or condition. In one embodiment, the positive effect may be a decrease of liver dysfunction, a decrease of liver inflammation or a decrease of fibrosis, or combinations thereof. In some embodiments, the treatment and/or prevention of cellular senescence decreases liver dysfunction, liver inflammation and/or fibrosis.

[0148] Premature senescence occurs in adult chronic hepatobiliary diseases and worsens the prognosis through deleterious effects on liver tissue remodeling and hepatic functions. Some evidence suggests that senescence also arises in biliary atresia (BA), the first cause of pediatric liver transplantation. Since alternatives to liver transplantation are needed, the present invention also relates to potential use of senotherapies in biliary atresia and biliary cirrhosis.

[0149] The inventors discovered that advanced premature senescence was evidenced in BA livers from early stage and continued to progress until liver transplantation. Senescence and senescence-associated secretory phenotype (SASP) were predominant in cholangiocytes, but also present in surrounding hepatocytes. 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). Inventors found that BA livers displayed advanced cellular senescence at diagnosis that continued to progress until liver transplantation. 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.

[0150] In embodiments, HALPC are used for the treatment of preclinical model of BA.

[0151] In one embodiment, the positive effect may be a decrease of ductular reaction. In some embodiments, the treatment and/or prevention of cellular senescence decreases ductular reaction. The term "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.

[0152] In some embodiments, 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.

[0153] In some embodiments, 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 fibrosis, type 2 diabetes, Alzheimer disease, Huntington's disease, Parkinson's disease, dementia, lipodystrophy, sarcopenia, age-related cachexia, skin aging, hepatocellular carcinoma, liver cancer, lobular carcinoma, bile duct cancer, melanoma, lung cancer, and islet cell tumor.

[0154] In some embodiments, 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.

[0155] In some embodiments, 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.

[0156] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of biliary diseases. In some embodiments, the biliary disease is chronic or acute. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cholestatic diseases. [0157] In some embodiments, 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.

[0158] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of liver diseases. In some embodiments, the liver disease is a metabolic, genetic, infectious, toxic and/or autoimmune disease.

[0159] In some embodiments, 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.

[0160] In some embodiments, 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.

[0161] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of hepatic fibrosis.

[0162] In some embodiments, 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.

[0163] In some embodiments, 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. In some embodiments, 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.

[0164] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of age-related diseases. In some embodiments, 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. In some embodiments, 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.

[0165] In some embodiments, 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.

[0166] In some embodiments, 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.

[0167] In some embodiments, 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.

[0169] In some embodiments, 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.

[0170] In some embodiments, 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.

[0171] In some embodiments, 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.

[0172] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of neurodegenerative diseases or disorders. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on dementia. In some embodiments, dementia include Lewy Body dementia, vascular dementia, frontotemporal dementia and the like. In some embodiments, 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. In one embodiment, 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.

[0173] In some embodiments, 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.

[0174] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of inflammatory diseases.

[0175] In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of cancer. In some embodiments, 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, renal cell carcinoma, retinoblastoma, salivary gland cancer, sarcoma, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, uterine (endometrial) cancer and vaginal cancer. In some embodiments, the treatment and/or prevention of cellular senescence has a positive effect on the progression or development of hepatocellular carcinoma.

[0176] In some embodiments, 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.

[0177] In one embodiment, 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.

[0178] In some embodiments, 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 are known in the art and 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).

[0180] In some embodiments, the markers are measured at RIMA level. Methods to measure RNA expression are known in the art and include, inter alia, RT-PCR, RT- qPCR, Northern Blot and hybridization techniques.

[0181] In some embodiments, at least two markers are measured at different levels (e.g., one marker measured at protein level, and one marker measured at RNA level).

[0182] In some embodiments, 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.

[0183] In some embodiments, 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. In some embodiments, 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.

[0185] By "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.

[0186] In some embodiments, 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.

[0187] In some embodiments, 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.

[0188] In some embodiments, 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.

[0189] In some embodiment, multiple administrations of the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, are performed on the same subject.

[0190] In another embodiment, 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. In one embodiment, the treatment of senescence is performed ex vivo, then the treated tissue, organ or parts thereof is grafted in a subject in need thereof.

[0191] In some embodiments, the population of HALPC is administered at a dose of 0.25 to 20 million of cells per kg of body weight. [0192] Within the scope of this invention, 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, 4.50 million, 4.60 million, 4.70 million, 4.80 million, 4.90 million, 5.00 million, 5.10 million, 5.20 million, 5.30 million, 5.40 million, 5.50 million, 5.60 million, 5.70 million, 5.80 million, 5.90 million, 6.00 million, 6.10 million, 6.20 million, 6.30 million, 6.40 million, 6.50 million, 6.60 million, 6.70 million, 6.80 million, 6.90 million, 7.00 million, 7.10 million, 7.20 million, 7.30 million, 7.40 million, 7.50 million, 7.60 million, 7.70 million, 7.80 million, 7.90 million, 8.00 million, 8.10 million, 8.20 million, 8.30 million, 8.40 million, 8.50 million, 8.60 million, 8.70 million, 8.80 million, 8.90 million, 9.00 million, 9.10 million, 9.20 million, 9.30 million, 9.40 million, 9.50 million, 9.60 million, 9.70 million, 9.80 million, 9.90 million, 10.00 million, 10.10 million, 10.20 million, 10.30 million, 10.40 million, 10.50 million, 10.60 million, 10.70 million,

10.80 million, 10.90 million, 11.00 million, 11.10 million, 11.20 million, 11.30 million, 11.40 million, 11.50 million, 11.60 million, 11.70 million, 11.80 million,

11.90 million, 12.00 million, 12.10 million, 12.20 million, 12.30 million, 12.40 million, 12.50 million, 12.60 million, 12.70 million, 12.80 million, 12.90 million,

13.00 million, 13.10 million, 13.20 million, 13.30 million, 13.40 million, 13.50 million, 13.60 million, 13.70 million, 13.80 million, 13.90 million, 14.00 million,

14.10 million, 14.20 million, 14.30 million, 14.40 million, 14.50 million, 14.60 million, 14.70 million, 14.80 million, 14.90 million, 15.00 million, 16.000 million,

17.000 million, 18.000 million, 19.000 million and 20.000 million of cells per kg of body weight.

[0193] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0194] In some embodiments, the effective amount of the lysates of population of HALPC corresponds to lysates of a population of HALPC at a dose as described hereinabove.

[0195] In some embodiments, 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. [0196] In some embodiments, the population of HALPC, lysates thereof and/or conditioned medium obtainable by culturing said HALPC, is administered in combination with another therapeutic agent.

[0197] In certain embodiments, 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.

[0198] In some embodiments, 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). In one embodiment, 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). In some embodiments, 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.

[0199] In some embodiments, the another therapeutic agent is a pharmacologically active molecule, e.g., a small molecule, or a biologically active peptide/protein. In some embodiments, the another therapeutic agent is comprised in pharmacological composition approved (e.g., FDA or EMA approved) for the treatment of at least one disease. In some embodiments, the another therapeutic agent is undergoing at least one preclinical or clinical trial.

[0200] In some embodiments, the another therapeutic agent is for treating and/or preventing senescence. In some embodiments, the another therapeutic agent is a senolytic agent.

[0201] As used herein, a senolytic agent is a molecule used to treat and/or prevent senescence.

[0202] In some embodiments, the another therapeutic agent is for treating and/or preventing at least one disease or condition as disclosed hereinabove. [0203] In some embodiments, the another therapeutic agent is for treating and/or preventing hepatic, biliary or cholestatic diseases, in particular hepatic fibrosis and cirrhosis. In some embodiments, 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). In some embodiments, the another therapeutic agent is ursodeoxycholic acid or rifampicin.

[0204] The present invention further relates to 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.

[0205] In some embodiments, 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. Typically, the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.

[0206] For the particular purpose of human administration, 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).

[0207] In embodiments, 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.

[0208] 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. [0209] In one embodiment, the composition is administered via intravenous infusion, optionally using a peripheral catheter. Alternatively, the composition may be administered to the patient through a central line.

[0210] The volume and concentration of the composition in the form of a sterile liquid comprising the HALPC is preferably adapted for intravenous infusion. In an embodiment, 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. In some embodiments, the composition comprises HALPC in a sterile liquid at a concentration of about 0.25 to about 15 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 10 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 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. In some embodiments, 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. In some embodiments, 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] In some embodiments, the method is for reducing cellular senescence in cells or tissues cultured in vitro or ex vivo.

[0217] 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.

[0218] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0219] 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. (F) pl6 gene expression (Cdkn2a) in diseased rats at different timepoints after surgery. BDL: bile duct ligation; CPA: collagen proportionate area; SA-p-gal: senescence-associated p-galactosidase; 48h - Iw - 2w: rats sacrificed 48 hours (n=3) - 1 week (n=3) - 2 weeks (n=4) after BDL surgery. Mann-Whitney U tests. Data is presented as mean ± SEM; *p<0.05; **p<0.01. Scale bars = 100pm.

[0220] 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. BDL: bile duct ligation; DR: ductular reaction; 48h - Iw - 2w: rats sacrificed 48 hours (n=3) - 1 week (n=3) - 2 weeks (n=4) after BDL surgery. Mann-Whitney U tests. Data is presented as mean ± SEM; *p<0.05; **p<0.01. Scale bars = 100pm. [0221] Figures 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. (D-F) Costaining of o-SMA and SA-p-gal activity. Rats were sacrificed 48 hours (n=3; A and D), 1 week (n=3; B and E) and 2 weeks (n=4; C and F) after BLD surgery. SA-p- gal: senescence-associated p-galactosidase. P: periportal area. C: centro-lobular area. Arrows: o-SMA-positive cells. Arrowheads: hepatocytes. Scale bars = 100pm.

[0222] 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 (p21) (E), Cdkn2a gene expression (pl6) (F) and SA-p-gal activity (G) compared to the vehicle group 72h after the injection of high dose (1.25 xlO ⁶ cells/kg) and low dose (12.5xl0 ⁶ cells/kg) of HALPC. 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.

[0223] Figures 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 administration method and regimen to rat model (A). 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) compared to the vehicle group 72h after the injection of high dose (1.25 xlO ⁶ cells/kg) and low dose (12.5xl0 ⁶ cells/kg) of HALPC. 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.

[0224] 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. BA: biliary atresia; CPA: collagen proportionate area. Data is presented as mean 4 SEM; **p<0.01; ***p<0.001. Scale bars = 100 um.

[0225] 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. (D): Gene expression of SASP markers IL-8 and TGF-pi increase in BA livers versus controls. (E): DNA damage yH2AX-positive foci increase in both hepatocytes and cholangiocytes in BA livers and progress until liver transplantation in cholangiocytes. BA: biliary atresia; DAB: 3,3'-diaminobenzidine; SA-p-gal: senescence-associated beta-galactosidase; Data is presented as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001. Scale bars = 100 pm.

[0226] 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). (C): Late stage BA livers display increased DNA damage foci (vH2AX staining) and telomere damage foci (co-localization H2AX- TR.F2) in the periphery of the BA nodules (n=6) as compared to the center of the nodules (n=6) and to controls (n=3). Relative telomere damage (co-localization of H2AX and TRF2 reported to total DNA damage) do not increase in late BA livers. BA: biliary atresia; DAB: 3,3-diaminobenzidine. Data is presented as mean i SEM; *ps0.05, **p<0.01, ***p<0.001. Black scale bars = 100 pm; red scale bars = 10 pm.

[0227] 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. Left and middle images: 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. BA: biliary atresia; IHC: immunohistochemistry; SA-p-gal: senescence- associated beta-galactosidase. Scale bars = 100 pm.

[0228] Figure 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.

[0229] Figure 11 A-C are a combination of histology images and scatter plots showing correlation of liver disease to senescence progression in BDL. (A) 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; 48h - Iw - 2w : rats sacrificed 48 hours (n=3) - 1 week (n=3) - 2 weeks (n=4) after BDL surgery. Data is presented as mean 4 SEM; *p<0.05; **p<0.01. Scale bars = 100pm.

[0230] 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. (D) Gene expression of SASP markers Il-1[3 and TGF-pi increase in BDL livers as compared to controls. BDL: bile duct ligation; SA-p-gal: senescence-associated p- galactosidase; SASP: senescence-associated secretory phenotype; 48h - Iw - 2w : rats sacrificed 48 hours (n=3) - 1 week (n=3) - 2 weeks (n=4) after BDL surgery.

Data is presented as mean ± SEM; *p<0.05; **p<0.01. Scale bars = 100pm.

[0231] Figure 13 A-D are a combination of scatter plots showing the results of D+Q administration in BDL rats. (A) Operated rats received D (5 mg/kg) + Q (50 mg/kg) by oral gavage (n=5) 48 hours after BDL and were compared to controls that received the vehicle (50% PEG400; n=5). (B) 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). (DE) D+Q worsened the hepatocytes mass loss (Hnf4a) and had no significant effect on hepatocytes injury (serum AST) nor on liver histological fibrosis. BDL: bile duct ligation; D: dasatinib: Q: quercetin; SA-B-gal: senescence-associated -galactosidase; ULN: upper limit of normal. Data is presented as mean 4 SEM; *p<0.05.

EXAMPLES

[0232] The present invention is further illustrated by the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures. Although 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.

Example 1: Bile Duct Ligation (BDL) rat model of hepatobiliary disease

Materials and Methods

[0233] 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. [0234] 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.

[0235] Immunohistochemistry - immunofluorescence: Liver sections of 5 pm were deparaffinized and rehydrated in xylene and graded alcohol series. Antigen retrieval was performed with Tris-EDTA buffer (Merck, Kenilworth, NJ, USA) or with citrate buffer (Dako Target Retrieval Solution, Agilent, Santa Clara, CA, USA). Non-specific immuno-staining was prevented by 1 hour incubation in PBS containing 5% Bovine Serum Albumin (Merck) ± 5% Normal Goat Serum (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, sections were incubated with primary antibody for 1 hour (Table 1). After washing, cells were incubated with species-specific secondary antibodies (Dako EnVision-i- System HRP, Agilent). Immunohistochemical detection was performed with liquid 3,3'-diaminobenzidine (DAB) chromogen and substrate buffer (Dako, Agilent), and was followed by Mayer's hematoxylin counterstaining. For serial immunofluorescence staining, tyramide-fluorophore amplification was performed with different fluorochromes (Invitrogen, Waltham, MA, USA) for each antibody.

Table 1: Primary antibodies. C: citrate; IF: immunofluorescence; IHC: immunohistochemistry; M: Mouse; mAb: monoclonal antibody; pAb: polyclonal antibody; Rb: rabbit; T: Tris-EDTA.

[0236] Reverse transcription quantitative polymerase chain reaction (RT-qPCR): Liver homogenates were obtained with the FastPrep-24 Classic Instrument (MP Biomedicals, Irvine, CA, USA) from liver biopsies cryopreserved in Lysing Matrix D tubes (MP Biomedicals, Irvine, CA, USA) filled with Tripure isolation reagent (Roche, Basel, Switzerland). Total RNA was extracted from liver homogenates using Tripure isolation reagent (Roche), according to the manufacturer's instructions. Genomic DNA was digested by DNase I (Invitrogen). RNA was retro-transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA). 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).

[0237] Quantification of staining in whole tissue sections: Quantification of staining was assessed as previously described (Jannone G et al., Tissue. J Histochem Cytochem, 2020). Briefly, stained slides were digitalized using a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany). Scanned slides were then analyzed using the image analysis tool Author version 2017.2 (Visiopharm, Horsholm, Denmark) for staining digital quantification. Results were expressed as (stained area/total tissue area) x 100 to obtain a percentage of stained area. The quantification parameters were kept constant for all sections. For specific analysis, manual quantification was performed blindly on digitalized sections at x40 magnitude. Ten randomly chosen fields were analyzed per section and the result was expressed as (stained cells/total cells) x 100 to obtain a percentage of stained cells.

[0238] Statistical analysis: Statistical analysis was conducted using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Continuous variables were presented as mean ± standard error of the mean (SEM). The Mann-Whitney U test was used to compare continuous variables between subgroups. Correlations between variables were tested using Pearson's rank-correlation coefficient (r). A two-tailed p-value < 0.05 was considered to indicate statistical significance for all analyses.

Results

[0239] 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). Gene expression analysis confirmed an increase in p21 (Cdknla) from one-week post-BDL (p<0.05) (Fig. IE). pl6 (Cdkn2a) gene expression increased in diseased animals as well, but no sooner than two weeks after the surgery (p<0.01) (Fig. IF). Taken together, these data provide evidence that BDL is a robust model of hepatobiliary disease in which senescence progressively develops starting from 48 hours after the surgery.

[0240] 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. 2D) and resulted in the onset of a ductular reaction (DR) starting from 48 hours post-BDL (Sox9; p<0.05) (Fig. 2E). DR and senescence progressions were strongly correlated in BDL rats (r=0.97; p<0.0001) (Fig. 2F). Hepatocytes injury also occurred and serum alanine aminotransferase (ALT) elevation was maximal 48 hours post-BDL. Hepatocytes mass decreased post-surgery (Hnf4a; p<0.05), but there was no significant correlation between hepatocytes loss and senescence (r=-0.47, p=0.17) (Fig. 2G).

[0241] Cell and tissue localization of senescence: Histological investigations of fibrosis and senescence progression through o-SMA and SA-p-gal activity co- and serial staining showed that both processes start in the periportal area (Fig. 3A-F). The development of fibrosis and senescence in the centro-lobular area occurs only in advanced stages following BDL surgery (Fig. 3A-C). Histological analysis of costained BDL sections evidenced senescence in different cell types, including cholangiocytes, hepatocytes and myofibroblasts (Fig. 3D-F). To further investigate the localization of senescence, multiple immunofluorescence staining was performed on BDL and control livers. Senescence was mainly visible in cholangiocytes 48 hours after the surgery and then progressively appeared in hepatocytes with disease progression (Fig. 3D-F).

Example 2: Human allogenic liver-derived progenitor cells (HALPC) injections improve senescence and biliary impairment in BDL

Materials and Methods

[0242] For BDL animal model, RT-qPCR, and statistical analyses: see Example 1. [0243] Cell transplantation: Human allogenic liver-derived progenitor cells (HALPC) were obtained from the Hepatic Biobank of Cliniques Universitaires Saint-Luc. The cells were originally obtained from the liver of a male donor aged 4 months, cultured, expanded in vitro and cryopreserved at P4 in liquid nitrogen. For the experiments, cells were thawed and re-suspended in 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).

Results

[0244] Animals who received the high dose of HALPC displayed 72h after the injection improved biliary injury (serum yGT levels, Fig. 4A) and improved biliary proliferation (decreased ductular reaction (DR); transcription factor SOX-9 - Sox9 - expression; p<0.05; Fig. 4B). No difference was observed regarding hepatocytes mass (Fig. 4C) and liver fibrosis (Fig. 4D) between subgroups. Senescence improved in BDL rats who received both low and high HALPC dose (Cdknla (p21); p<0.05) (Fig. 4E). 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) or SA-p-gal activity (Fig. 4G, Fig 5B) was observed between subgroups in the early timepoints investigated. No significant amount of HALPC was identified in the livers of injected rats through human SRY gene expression when animals were sacrificed.

[0245] . 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. Since the anti-senescence properties of MSCs in plurality of organs, including lungs, heart and kidneys, were previously attributed to antioxidant effects, the gene expression of glutathione peroxidase 1 (Gpxl) in liver homogenates was measured. A decrease of Gpxl expression was observed in the group that received the HALPC as compared to the vehicle (Fig. 51). [0246] Our results provide evidence that HALPC display anti-senescence properties and improve liver injury in the BDL model of biliary cirrhosis.

[0247] The effects of HALPC on Gpxl gene expression shows that 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

[0248] Material and methods are carried out as described in Example 1.

Results

[0249] 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). As expected, 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).

Our results confirm that BDL is a robust model of biliary senescence. Studying early post-surgical stages allowed us to demonstrate that senescence developed first in cholangiocytes and subsequently in hepatocytes during biliary injury.

Example 4: HALPC but not D+Q decrease early senescence and liver disease in the BDL model

[0250] 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.

[0251] Once we demonstrated that biliary senescence occurs in the BDL model, we evaluated the efficacy of HALPC or D+Q in the operated animals. Both senotherapeutics were administered 48 hours after the surgery and the animals were compared to vehicle-treated controls (Fig. 5A and Fig. 13A). Results of intravenous HALPC injection is discussed in example 2. Unexpectedly, the very well described combination of D+Q had no effect on senescence nor liver disease progression as compared to the vehicle, and on the opposite appeared to aggravate the hepatocytes mass loss (Fig. 13).

[0252] Our results provide evidence that HALPC but not D+Q display antisenescence properties and improve liver injury in the BDL model of biliary cirrhosis.

Example 5: Senescence and senotherapies in biliary atresia and biliary cirrhosis, human samples

[0253] 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.

[0254] 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. [0255] Biliary atresia (BA) 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. Despite the fact that 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.

[0256] In brief, premature senescence worsens the prognosis of adult chronic hepatobiliary diseases and some evidence suggests that this phenomenon could also occur in BA, which is the leading cause of liver transplantation in children. The aim of this example is to investigate premature senescence in BA and to assess senotherapies in a preclinical model of biliary cirrhosis.

Materials and Methods

[0257] For BDL animal model, [3-galactosidase activity, Immunohistochemistryimmunofluorescence assays: see Example 1.

[0258] For Cell transplantation: see Example 2.

[0259] Antibodies used in immunohistrochemistry- immunofluorescence assays listed in table 2.

Primary antibody (target) Company Cat. No Species Ag retrieval Dilution p21 WAFl/Cipl (H) Agilent M7202 M mAb T l hour 98C IHC 1/400 p21 WAFl/Cipl (R) Agilent M7202 M mAb T 900W 4 min - IHC 1/50

90W 15 min - 900W 1.5 min T Retriever 2100 IF 1/50 pl6 INK4a (H) Abeam abl08349 Rb mAb T l hour 98C IHC 1/1000 gamma H2A.X (Serl39) (H) Abeam ab81299 Rb mAb C 35 min 98C IHC 1/500

C Retriever 2100 IF 1/500 gamma H2A.X (Serl39) (H) Merck 05-636 M mAb C 5 min 450W IF 1/1000

TRF2 (H) Novus NB110- Rb pAb C 5 min 450W IF 1/2000

57130 HNF4a (H-R) R&D PP-H1415 M mAb C Retriever 2100 IF 1/400

System

CK19 (H) Dako M0888 M mAb C 35 min 98C IHC 1/100

C Retriever 2100 IF 1/100 CK19 (R) Abeam ab220193 M mAb C Retriever 2100 IF 1/200

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.

Human samples:

[0260] Patients who underwent hepatoportoenterostomy procedure (n = 5) or liver transplantation (n = 30) for BA were prospectively recruited in the Pediatric Gastroenterology and Hepatology Unit of Cliniques Universitaires Saint-Luc between 2018 and 2022. A liver biopsy or a fragment of the explanted liver was collected for each patient and obtained from the Cliniques Universitaires Saint-Luc Hepatic Biobank. Biochemical data were obtained the day before the procedure. Control livers (n=10) were obtained from the Cliniques Universitaires Saint-Luc biobank when consent for research purposes was given. Cryopreserved material was available for 5/10 controls. This project was approved by the Ethics Committee of Cliniques Universitaires Saint-Luc (registration number B403201938739). Written informed consent was obtained for all study participants. Senescence was investigated through digital spatial profiling whole transcriptome analysis, SA-p-gal activity, pl6 and p21 gene/protein expression, y-H2AX protein expression and senescence-associated secretory phenotype (SASP)-related gene expression.

Imaging and quantification of staining in whole tissue sections:

[0261] Quantification of staining was assessed as previously described (30). Briefly, stained slides were digitalized using a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany) or Axio Scan.Zl scanner for immunofluorescence (Zeiss, Oberkochen, Germany) (x20 magnification). For selected experiments, high magnification images (xlOO) with z-stack imaging were obtained through spinning disk confocal microscopy (Zeiss). Scanned slides were then analyzed using the image analysis tool Author version 2017.2 (Visiopharm, Horsholm, Denmark) for computer-assisted quantification of histological staining. 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:

[0262] 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.

Gene of interest (specie) Company Reference

CDKNIA (H) Thermo Fisher Scientific Hs00355782_ml

CDKN2A (H) Thermo Fisher Scientific Hs00923894_ml

CXCL8 (H) IDT Hs.PT.58.38869678. g

TGFB1 (H) IDT Hs.PT.58.39813975

COL1A1 (H) Thermo Fisher Scientific Hs00164004_ml

KRT19 (H) Thermo Fisher Scientific Hs00761767_sl

HNF4A (H) Thermo Fisher Scientific Hs00230853_ml

TBP (H) Thermo Fisher Scientific Hs99999910_ml

PPIA (H) Thermo Fisher Scientific Hs99999904_ml

Cdknla (R) IDT Rn.PT.58.12154239

Cdkn2a (R) Thermo Fisher Scientific Rn00580664_ml lllb (R) IDT Rn.PT.58.38028824

Tgfbl (R) IDT Rn.PT.58.6690138

Collal (R) IDT Rn.PT.58.7562513

Sox9 (R) IDT Rn.PT.58.29440750

Hnf4a (R) IDT Rn.PT.58.34229621.g

Gpxl (R) IDT Rn.PT.58.7204268. g

Gapdh (R) Thermo Fisher Scientific Rn01775763_gl

B2m (R) Thermo Fisher Scientific Rn00560865_ml

Table 3: Pre-designed TaqMan probes used for reverse transcription quantitative polymerase chain reaction. Digital spatial whole transcriptome of BA livers:

[0263] Digital spatial profiling (DSP) GeoMx slide preparation and analysis were performed by the Utrecht Sequencing Facility (USEQ) team (Utrecht, Netherlands). FFPE samples of BA late (n=2), BA early (n=2) and control (n=3) livers were processed and prepared according to the RNA FFPE Manual Slide Preparation Protocol section of the GeoMx NGS Slide Preparation User Manual (MAN-10115-05) by NanoString Technologies (Seattle, WA, USA). Briefly, FFPE slides were baked at 60C for 30 minutes immediately before deparaffinization and rehydration. The slides were then incubated in 100C Tris-EDTA for 15 min to achieve target retrieval. 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. 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. Only the cell type of interest was digitally selected in each ROI thanks to the pan-cytokeratin immunofluorescent staining (high intensity staining for cholangiocytes and low intensity staining for hepatocytes). ROI were validated only if containing at least 100 targeted cells in order to allow sufficient signal for subsequent detection. 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.

Analysis of DSP whole transcriptome data:

[0264] Two low-performing ROI segments with less than 10% of the genes detected were removed from the analysis (genes were considered as detected when their counts were higher than the LOQ value, defined as the negative probes geometric means + 2 standard deviations). Genes lowly detected across the dataset (in less than 10% of the segments) were removed, leaving 9146 genes for the differential expression analyses. Raw counts were normalized by the Q3 normalization method. Differential expression analyses were performed with DESeq2 Bioconductor package vl.32.0. Gene set enrichment analyses were done using clusterProfiler v4.0.5.

Statistical analysis: [0265] Statistical analysis was conducted using Graphpad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Continuous variables were presented as mean ± standard error of the mean (SEM) or median (range) and categorical variables as numbers and percentages. The non-parametric Mann-Whitney U test was used to compare continuous variables between subgroups. When more than two groups were included in the analysis, a non-parametric Kruskal-Wallis One-Way ANOVA with Dunn's post-hoc test was performed. A two-tailed p-value < 0.05 was considered to indicate statistical significance for all analyses.

Results at diagnosis in BA and until liver

[0266] 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).

Controls (n=10) BA early (n=5) BA Iate (n=30)

Median age 9.5 y (3 d - 41 y) 7 w (17 d - 2 m) 12 m (6 m - 15 y)

Gender (M/F) 5 (50%) / 5 3 (60%) / 2 15 (50%) / 15

AST (Ul/L) (< 80) NA 232 ± 78 228 ± 19

ALT (Ul/L) (< 35) NA 155 ± 57 123 ± 11 yGT (Ul/L) (< 40) NA 455 ± 78 221 ± 30

Total bilirubin (mg/dL) (< 1.2) NA 9.1 ± 1.7 17.5 ± 1.7

INR (0.8-1.2) NA 1 ± 0.04 1.5 ± 0.1

Histological fibrosis (Metavir score):

F0 10 (100%) 1 (20%) 0

Fl 0 0 0

F2 0 1 (20%) 0

F3 0 1 (20%) 0

F4 0 2 (40%) 30 (100%)

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.

[0267] Senescence was evidenced in BA at early and late stages through multiple markers and techniques, including SA-p-gal activity, pl6 protein and gene expression, yH2AX protein expression and SASP markers gene expression elevation (Fig. 7 & Fig. 8). Advanced premature senescence was already present in the BA early group, as suggested by an increased protein expression of pl6 but not p21, pl6 being described as a late marker of senescence (Fig. 7B & Fig. 8A). However, there was still a clear progression of senescence between the time of diagnosis and liver transplantation (Fig. 7C & 7E). Interestingly, senescence development was correlated to disease progression (Fig. 6B & 6C). Senescence was predominant in cholangiocytes and also present in surrounding perinodular hepatocytes (Fig. 7B, B & E, Fig. 9). Gene expression of SASP markers was elevated in BA livers (Fig. 7D). When we investigated the telomeric DNA damages in late stage BA, yH2AX-positive DNA damage foci were evenly distributed in the whole DNA sequence without any specific accumulation in telomeres (Fig. 8C). However, the absolute increase in the number of telomere damage foci might per se induce senescence in the affected cells.

[0268] Our results show that premature senescence was already advanced at diagnosis in BA and continued to progress until liver transplantation. Senescence was predominant in cholangiocytes and perinodular hepatocytes, corroborating the hypothesis of a paracrine transmission of senescence through elevated SASP markers. ana in BA livers confirms that senescence and

SASP predominate in cholangiocytes and progress with disease stage:

[0269] To further assess the localization and progression of cellular senescence, we performed a digital spatial whole transcriptomic analysis on BA and control liver samples. Both 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. Gene ontology (GO) enrichment analysis of biological processes (MSigDB Collection: C5 GO BP) and hallmark gene sets enrichment analysis (MSigDB Collection: H) were also performed on our dataset. GO terms that appeared to be the most significant when comparing genes differentially expressed between diseased cholangiocytes versus controls corresponded to "supramolecular fiber organization", "cell adhesion" and "ion transport" categories, in line with the disruption of apical-basal polarization described in BA cholangiocytes. [0270] Enrichment analysis of senescence gene lists available in the literature was then performed in our dataset (Fig. 10C). 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. In contrast, 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). Altogether, 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.