Hepatocytes with high regenerative capacity for liver repair
11065280 · 2021-07-20
Assignee
Inventors
Cpc classification
C07K16/28
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
Abstract
The invention provides purified mammalian hybrid hepatocyte (HybHP) cells, compositions comprising HybHP cells, methods for purifying HybHP cells, methods for in vitro culture of HybHP cells, and methods for using HybHP cells to repopulate and/or treat the liver of a subject in need thereof.
Claims
1. A method for purifying a hybrid hepatocyte (HybHP) cell from a normal mammalian liver, said method comprising, a) preparing a single-cell suspension from said normal mammalian liver, b) combining said single-cell suspension with i) at least one first antibody that specifically binds to a plasma membrane first protein marker of liver ductal (DC) cells, and ii) at least one second antibody that specifically binds to a second protein marker of conventional hepatocyte (cHP) cells, wherein said second protein marker of cHP cells is underexpressed in said HybHP cells compared to cHP cells, wherein said combining is under conditions for specific binding of said at least one first antibody to said first protein marker, and of said at least one second antibody to said second protein marker, and wherein said specific binding produces a first composition that comprises a first antibody-HybHP cell-second antibody conjugate, and c) isolating said first antibody-HybHP cell-second antibody conjugate from said single-cell suspension, thereby producing a second composition that comprises a purified HybHP cell.
2. A method for purifying a hybrid hepatocyte (HybHP) cell from a normal mammalian liver, said method comprising, a) preparing a single-cell suspension from said normal mammalian liver, b) substantially removing ductal cells from said single-cell suspension to obtain a first population of cells that contains conventional hepatocyte (cHP) cells and HybHP cells, c) combining said first population of cells with at least one first antibody that specifically binds to a plasma membrane first protein marker of liver ductal (DC) cells, wherein said combining is under conditions for specific binding of said at least one first antibody to said first protein marker, and wherein said specific binding produces a first composition that comprises a first-antibody-HybHP cell conjugate, and d) isolating said first antibody-HybHP cell conjugate from said first population of cells, thereby producing a second population of cells that comprises a purified HybHP cell.
Description
DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DEFINITIONS
(18) “Hepatocyte cells” are highly polarized epithelial cells and form cords. Hepatocytes account for approximately 50% of total liver cells and 80% of the volume of the organ. Their basolateral surfaces face fenestrated sinusoidal endothelial cells, facilitating the exchange of materials between hepatocytes and blood vessels. Tight junctions formed between hepatocytes create a canaliculus that surrounds each hepatocyte. Bile secreted from mature hepatocytes is exported sequentially through bile canaliculi surrounded by the apical membrane of neighboring hepatocytes, intrahepatic bile ducts, extrahepatic bile ducts, and, finally, the duodenum. The bile duct is formed by a specialized type of epithelial cell called a cholangiocyte. The term “hepatocyte” cell includes conventional hepatocyte (cHP) cells, and the invention's newly discovered hybrid hepatocyte (HybHP) cells.
(19) “Conventional hepatocyte cell” and “cHP cell” interchangeably refer to a hepatocyte cell that expresses at least one protein marker encoded by the genes in text that is neither in bold nor in italics in Table 2. The cHP cells co-migrate with HybHP cells, and differentially migrates from ductal cells, on density gradients (Examples 1, 2).
(20) “Ductal cell” and “DC cell” interchangeably refer to a liver cell that expresses at least one protein marker encoded by the genes in bold italic text in Table 1 and Table 3. DC cells differentially migrates from cHP cells and from HybHP cells on density gradients (Examples 1, 2). Ductal cells include oval cells (OCs) and bile duct (BD) cells.
(21) “Oval cell” is an ovoid cell observed in animal models of liver carcinogenesis (Farber, E. (1956) Cancer Res. 16, 142-148), and is sometimes introduced in literature as a synonym for liver stem cells. Oval cells have been extensively characterized histologically, which cumulatively suggests that they have bipotential differentiation capability toward both hepatocytes and cholangiocytes (Miyajima et al., 2014).
(22) “Bile duct” cells (also referred to as “BD” cells) are located in the bile duct.
(23) “Density gradient” is a spatial variation in density over an area. “Density gradient centrifugation” is a procedure for separating cells in which the sample is placed on a preformed gradient (e.g., percoll, Stractan, etc.). Upon centrifugation either by rate zonal or equilibrium procedures, the cells are banded in the gradient and can be collected as a purified fraction.
(24) “Purified,” “purify,” “isolate,” and grammatical equivalents thereof when in reference to a desirable component (such as cell type, protein, nucleic acid sequence, etc.) refer to the reduction in the amount of at least one undesirable component (such as another cell type, protein, nucleic acid sequence, etc.) from a sample. This reduction may be by any numerical percentage of from 5% to 100%, such as, but not limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, from 90% to 100%, from 95% to 100%, from 99% to 100%, and most preferably by 100%. Thus, purification results in “enrichment” (i.e., an increase) in the amount of the desirable component relative to one or more undesirable component. For example, “purified HybHP cells” refers to HybHP cells that are purified from cHP cells and from DC cells. Preferably, the purified HybHP cells are substantially free from cHP cells and from DC cells. In another example, “purified first antibody-HybHP cell-second antibody conjugate” and “purified first-antibody-HybHP cell conjugate” refer to conjugates containing purified HybHP cells. Methods for purifying of single cells are known in the art, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), etc. Single-cell purification methods (such as FACS, MACS, etc.) may employ direct labeling of the antigen of interest using a primary antibody that specifically binds to the antigen of interest. Alternatively, purification methods employ indirect labeling of the antigen of interest using a secondary antibody that specifically binds to the Fc portion of the primary antibody, biotin, and/or to fluorescein isothiocyanate (FITC). Indirect labeling may also use Streptavidin, instead of the secondary antibody, which binds to biotin on the primary antibody.
(25) “Fluorescence-activated cell sorting” and “FACS” interchangeably refer to a method for sorting a heterogeneous mixture of biological cells based upon the specific light scattering and fluorescent characteristics of each cell, thus purifying cells of interest (e.g., HybHP cells). FACS is particularly preferred for isolating a target cell population (e.g., HybHP cells) at high purity, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is a particularly desired for technique to isolate cells based on internal staining and/or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with a fluorescently-tagged primary antibody that recognizes specific surface markers on the desired cell population. Negative selection of unstained cells is also possible. Basu et al. (2010) “Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)” J Vis Exp. (41): 1546. doi: 10.3791/1546.
(26) “Magnetic-activated cell sorting” and “MACS” interchangeably refer to a method for purifying various cell populations depending on their surface antigens. In one embodiment, MACS uses nanoparticles conjugated to an antibody against an antigen of interest (e.g., on the surface of HybHP cells). In another embodiment, MACS uses magnetic nanoparticles coated with anti-fluorochrome antibodies that are incubated with the fluorescent-labelled antibodies against the antigen of interest for cell separation with respect to the antigen (such as by using anti-immunoglobulin MicroBeads, anti-biotin MicroBeads, streptavidin MicroBeads, and anti-fluorochrome MicroBeads).
(27) The term “population of cells” refers to a plurality of cells, i.e., more than one cell. The population may be a pure population comprising one cell type. Alternatively, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise.
(28) Cell “marker molecule” refer to a molecule (such as protein, nucleotide sequence, etc.) that is present on, and/or is produced by, a particular type of cell (such as cancer cell, epithelial cell, fibroblast cell, muscle cell, synovial cell, stem cell, embryonic cell, etc.), at a different level (e.g., a higher level or lower lever, preferably a higher level) than other types of cells. Cell marker molecules may be used to distinguish one cell type of one or more other cell types.
(29) “Mammal” includes a human, non-human primate, murine (e.g., mouse, rat, guinea pig, hamster), ovine, bovine, ruminant, lagomorph, porcine, caprine, equine, canine, feline, ave, etc. In one preferred embodiment, the mammal is murine. In another preferred embodiment, the mammal is human.
(30) A subject “in need” of treatment with the invention's methods and/or compositions includes a subject that is “suffering” from liver damage (i.e., a subject that is experiencing and/or exhibiting one or more symptoms of liver damage), and subject “at risk” of liver damage. A subject “in need” of treatment includes animal models of liver damage. Subject “at risk” of liver damage refers to a subject that is not currently exhibiting liver damage symptoms and is predisposed to expressing one or more symptoms of the disease. This predisposition may be based on family history, genetic factors, environmental factors such as exposure to detrimental compounds present in the environment, etc.). It is not intended that the present invention be limited to any particular signs or symptoms. Thus, it is intended that the present invention encompass subjects that are experiencing any range of disease, from sub-clinical symptoms to full-blown disease, wherein the subject exhibits at least one of the indicia (e.g., signs and symptoms) associated with the disease.
(31) The term “conjugate” when in reference to a cell and antibody refers to a cell that is linked (directly or indirectly) to an antibody via an antigen that is expressed on the cell surface. Antibodies bind antigens through weak chemical interactions, and bonding is essentially non-covalent, such as electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions. For example a “First-antibody-HybHP cell conjugate” refers to a HybHP cell that is linked to a first antibody via an antigen that is expressed on the surface of the HybHP cell. Also, a “HybHP cell-first antibody-second antibody conjugate” refers to a HybHP cell that is linked to a first antibody via a first antigen that is expressed on the surface of the HybHP cell, and also linked to a second antibody via a second antigen that is expressed on the surface of the HybHP cell.
(32) The terms “specifically binds,” “specific binding,” and grammatical equivalents, when made in reference to the binding of antibody to a molecule (e.g., peptide) refer to an interaction of the antibody with one or more epitopes on the molecule where the interaction is dependent upon the presence of a particular structure on the molecule. For example, if an antibody is specific for epitope “A” on the molecule, then the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled “A” and the antibody will compete with, and thus reduce, the amount of labeled A bound to the antibody.
(33) “Overexpression,” “upregulation,” and grammatical equivalents, when used in reference to a protein in a cell of interest (such as a HybHP cell, cHP cell, DC cell, etc.) refers to the presence of a higher level of the protein, and/or its encoding mRNA, in the cell of interest compared to another cell (such as a control HybHP cell, cHP cell, DC cell, etc.).
(34) “Underexpression,” “downregulation,” and grammatical equivalents, when used in reference to a protein in a cell of interest (such as a HybHP cell, cHP cell, DC cell, etc.) refers to the presence of a lower level of the protein, and/or its encoding mRNA, in the cell of interest compared to a another cell (such as a control HybHP cell, cHP cell, DC cell, etc.).
(35) The terms “increase,” “elevate,” “raise,” and grammatical equivalents (including “higher,” “greater,” etc.) when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, and/or phenomenon (e.g., level of expression of a gene, disease symptom, level of binding of two molecules such as binding of a ligand to a receptor, specificity of binding of two molecules (such as an antigen and antibody), affinity of binding of two molecules, disease symptom, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) in a first sample (or in a first subject) relative to a second sample (or relative to a second subject), mean that the quantity of the molecule, cell, and/or phenomenon in the first sample (or in the first subject) is higher than in the second sample (or in the second subject) by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the quantity of the molecule, cell, and/or phenomenon in the first sample (or in the first subject) is at least 10% greater than, at least 25% greater than, at least 50% greater than, at least 75% greater than, and/or at least 90% greater than the quantity of the same molecule, cell, and/or phenomenon in the second sample (or in the second subject). This includes, without limitation, a quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) that is at least 10% greater than, at least 15% greater than, at least 20% greater than, at least 25% greater than, at least 30% greater than, at least 35% greater than, at least 40% greater than, at least 45% greater than, at least 50% greater than, at least 55% greater than, at least 60% greater than, at least 65% greater than, at least 70% greater than, at least 75% greater than, at least 80% greater than, at least 85% greater than, at least 90% greater than, and/or at least 95% greater than the quantity of the same molecule, cell, and/or phenomenon in the second sample (or in the second subject). In one embodiment, the first sample (or the first subject) is exemplified by, but not limited to, a sample (or subject) that has been manipulated using the invention's compositions and/or methods. In a further embodiment, the second sample (or the second subject) is exemplified by, but not limited to, a sample (or subject) that has not been manipulated using the invention's compositions and/or methods. In an alternative embodiment, the second sample (or the second subject) is exemplified by, but not limited to, a sample (or subject) that has been manipulated, using the invention's compositions and/or methods, at a different dosage and/or for a different duration and/or via a different route of administration compared to the first subject. In one embodiment, the first and second samples (or subjects) may be the same, such as where the effect of different regimens (e.g., of dosages, duration, route of administration, etc.) of the invention's compositions and/or methods is sought to be determined on one sample (or subject). In another embodiment, the first and second samples (or subjects) may be different, such as when comparing the effect of the invention's compositions and/or methods on one sample (subject), for example a patient participating in a clinical trial and another individual in a hospital.
(36) The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” and grammatical equivalents (including “lower,” “smaller,” etc.) when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, and/or phenomenon (e.g., level of expression of a gene, disease symptom, level of binding of two molecules such as binding of a ligand to a receptor, specificity of binding of two molecules (such as an antigen and antibody), affinity of binding of two molecules, disease symptom, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) in a first sample (or in a first subject) relative to a second sample (or relative to a second subject), mean that the quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) is lower than in the second sample (or in the second subject) by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity of the same molecule, cell, and/or phenomenon in the second sample (or in the second subject). In another embodiment, the quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) is lower by any numerical percentage from 5% to 100%, such as, but not limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, and from 90% to 100% lower than the quantity of the same molecule, cell, and/or phenomenon in the second sample (or in the second subject). In one embodiment, the first sample (or the first subject) is exemplified by, but not limited to, a sample (or subject) that has been manipulated using the invention's compositions and/or methods. In a further embodiment, the second sample (or the second subject) is exemplified by, but not limited to, a sample (or subject) that has not been manipulated using the invention's compositions and/or methods. In an alternative embodiment, the second sample (or the second subject) is exemplified by, but not limited to, a sample (or subject) that has been manipulated, using the invention's compositions and/or methods, at a different dosage and/or for a different duration and/or via a different route of administration compared to the first subject. In one embodiment, the first and second samples (or subjects) may be the same, such as where the effect of different regimens (e.g., of dosages, duration, route of administration, etc.) of the invention's compositions and/or methods is sought to be determined on one sample (or subject). In another embodiment, the first and second samples (or subjects) may be different, such as when comparing the effect of the invention's compositions and/or methods on one sample (subject), for example a patient participating in a clinical trial and another individual in a hospital.
(37) The terms “alter” and “modify” when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, and/or phenomenon (e.g., level of expression of a gene, disease symptom, level of binding of two molecules such as binding of a ligand to a receptor, specificity of binding of two molecules (such as an antigen and antibody), affinity of binding of two molecules, disease symptom, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) refer to an increase and/or decrease.
(38) “Substantially the same,” “without substantially altering,” “substantially unaltered,” and grammatical equivalents, when in reference to the level of any molecule (e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.), cell, and/or phenomenon (e.g., level of expression of a gene, disease symptom, level of binding of two molecules such as binding of a ligand to a receptor, specificity of binding of two molecules (such as an antigen and antibody), affinity of binding of two molecules, disease symptom, specificity to disease, sensitivity to disease, affinity of binding, enzyme activity, etc.) means that the quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) is neither increased nor decreased by a statistically significant amount relative to the second sample (or in a second subject). Thus in one embodiment, the quantity of molecule, cell, and/or phenomenon in the first sample (or in the first subject) is from 90% to 100% (including, for example, from 91% to 100%, from 92% to 100%, from 93% to 100%, from 94% to 100%, from 95% to 100%, from 96% to 100%, from 97% to 100%, from 98% to 100%, and/or from 99% to 100%) of the quantity in the second sample (or in the second subject).
(39) The term “composition,” such as when made in reference to a composition comprising a desired component (such as HybHP cell, first-antibody-HybHP cell conjugate, first antibody-HybHP cell-second antibody conjugate, etc.) refers to any container, receptacle, and/or medium that can receive and/or contain the desired component. Thus, the term composition includes aqueous solutions (such as culture medium), three-dimensional matrix (such as matrigel, collagen, agar, etc.), flask, petri dish, multi-well dish, etc.
(40) The terms “propagating,” “culturing,” and “growing,” when made in reference to target cells, are interchangeably used to refer to increasing the number of the target cells.
(41) The term “in vivo” refers to the natural environment (e.g., within an organism, tissue, and/or a cell).
(42) The term “ex vivo” refers to an environment outside an organism.
(43) The term “in vitro” refers to an ex vivo environment that includes manipulation under artificially-created conditions (e.g., culture medium, cell culture, transfection, assay) and/or using laboratory equipment (e.g., flasks, test tubes, petri dishes, multiwell plates, etc.).
(44) The term “culture medium” refers to a nutritive substance that is suitable to support maintenance and/or growth of cells in vitro (i.e., cell cultures). A culture medium includes, for example, liquid media, and three-dimensional media. A culture medium includes salts (e.g., sodium chloride), carbohydrates (e.g., sugar), proteins (e.g., serum), etc.
(45) “Three-dimensional media,” “3D media,” and “three-dimensional matrix” interchangeably refer to an artificially-created environment (e.g., matrigel, collagen, agar, etc.) in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. This is in contrast to growing cells in “two-dimensional” (2D) monolayers (e.g., on a petri dish) because the 3D model more accurately models the in vivo cell environment.
(46) The term “treating” liver damage interchangeably refers to delaying, reducing, palliating, ameliorating, stabilizing, preventing and/or reversing one or more symptoms (such as objective, subjective, pathological, clinical, sub-clinical, etc.) of liver damage. Symptoms of liver damage may be assessed by, for example, biopsy and histology, and blood tests to determine levels of relevant enzymes (e.g., enzymes involved in metabolism, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, 5′ Nucleotidase, gamma-glutamyl transpeptidase (GGT)), levels of proteins involved in normal blood clotting (e.g., prothrombin time (PT) using international normalized ratio (INR) and/or partial thromboplastin time (PTT)), levels of albumin and/or bilirubin, levels of circulating antigen and/or antibody, and imaging tests (e.g., to detect a decrease in the growth rate or size of hepatocellular carcinoma).
(47) The terms “therapeutic amount,” “pharmaceutically effective amount,” and “therapeutically effective amount,” are used interchangeably herein to refer to an amount that is sufficient to achieve a desired result, such as treating liver damage, and/or repopulating the liver.
(48) “Antibody” refers to an immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.) and/or portion thereof that contain a “variable domain” (also referred to as the “F.sub.V region”) that specifically binds to an antigen. More specifically, variable loops, three each on the light (V.sub.L) and heavy (V.sub.H) chains are responsible for binding to the antigen. These loops are referred to as the “complementarity determining regions” (“CDRs”) and “idiotypes.” In one embodiment, the invention's antibodies are “monoclonal antibodies” (“MAbs”) produced by a single clone of hybridoma cells.
DESCRIPTION OF THE INVENTION
(49) The invention provides the discovery of the existence of hybrid hepatocyte (HybHP) cells. These cells are superior to any known cell type in repairing diseased and/or injured liver. This new and distinct population of hepatocytes is located at the periportal region that expresses several bile duct enriched genes. Hybrid Hepatocytes expand upon liver injury and form larger and more stably differentiated clones than other hepatocytes, and can also transdifferentiate into bile duct cells. Data herein demonstrate that in Fah.sup.−/− mice, which suffer chronic and fatal liver injury (Grompe et al., 1995), HybHP show higher regenerative capacity than normal hepatocytes and are far superior to oval cells. Neither HybHP nor oval cells serve as the origin for HCC in three independent mouse models, including two models of NASH-driven HCC (Fujii et al., 2013; Nakagawa et al., 2014b) (
(50) The invention's compositions and methods are useful in the treatment of liver damage, liver tissue engineering, transplantation back into the donor subjects to generate a HLA compatible HybHP cell biobank and/or to treat other subjects.
(51) The invention is further described under A) Purified hybrid hepatocyte (HybHP) cells and compositions comprising the, B) Methods for isolating HybHP cells, C) Methods for in vitro culture of HybHP cells, and D) Methods for repopulating the liver and treating liver damage.
(52) A) Purified Hybrid Hepatocyte (HybHP) Cells and Compositions Comprising them
(53) Compensatory proliferation triggered by liver damage is required for liver regeneration and maintenance of mass and function, but is also a potent tumor promoter giving rise to hepatocellular carcinoma (HCC). Despite extensive investigation, the nature of the cells responsible for liver regeneration or HCC development remains obscure.
(54) Data herein demonstrate that the primary cell type responsible for regeneration of the liver parenchyma is the newly described HybHP. Yet, despite their high regenerative potential and extensive expansion during chronic liver injuries, which promote HCC development (Hui et al., 2007; Maeda et al., 2005; Nakagawa et al., 2014b), data herein show that HybHP are not the preferential origin for HCC in the 3 different liver cancer models we have examined herein. Thus, in contrast to the skin and the intestine, the liver does not contain a specialized compartment of adult or reserve stem cells that maintains tissue homeostasis and expands upon liver damage to restore tissue mass. A recent analysis of relative cancer rates at different tissue sites revealed a correlation with rates of cell division in most tissues and gave rise to the suggestion that the majority of cancer originates from adult tissue stem cells (Tomasetti and Vogelstein, 2015). This does not seem to apply to liver, in which HybHP generate new hepatocytes only upon liver damage but rarely give rise to cancer.
(55) i) Hybrid Hepatocytes as a New Hepatocyte Subpopulation
(56) Hepatocyte diversity has been long appreciated, especially in the context of metabolic zonation across the portal to central axis (Jungermann and Katz, 1989). However, it has been difficult to analyze the distinct properties of different hepatocyte populations under physiological conditions, although uni-zonal livers were generated through genetic manipulation of the Wnt-β-catenin signaling pathway (Benhamouche et al., 2006). Using the ductal transcription factor Sox9 as a marker, we identified a subpopulation of periportal hepatocytes located at the limiting plate, which express low amounts of Sox9 and normal amounts of HNF4α, a hepatocyte transcription factor. These cells were termed HybHP, a term fully supported by subsequent transcriptomic and immunohistochemical analyses, which show expression of hepatocyte-specific genes along with a small number of genes that are preferentially expressed in bile duct cells. Using a Sox9-Cre.sup.ERT line that is sensitive to low amounts of Sox9 gene transcription, we labeled HybHP and traced their fate under different conditions. HybHP are kept quiescent in the unchallenged liver for at least 9 months after birth, but during chronic liver damage, they proliferate and serve as a source for a large fraction of the new hepatocytes that repopulate the liver. Interestingly, in the two injury models we employed, the manner in which HybHP re-seed the liver appears to be different. In MUP-uPA mice, extensive damage is randomly produced throughout the parenchyma consequent to ER stress (Nakagawa et al., 2014b; Weglarz et al., 2000). In these mice, HybHP clones radiate from the portal area, covering as much volume as possible. Chronic CCl.sub.4 administration, on the other hand, results in necrotic damage to pericentral zone 3 hepatocytes. In this case, HybHP clones expand along the portal-central axis, following hepatic chords. These observations suggest that tissue polarity and polarized cell-cell interactions determine the pattern of tissue repair.
(57) Many of the genes that are over-expressed in HybHP relative to conventional hepatocytes are also expressed in bile duct cells and are functionally related to cell adhesion and tubule formation, suggesting that HybHP and bile duct cells may originate from embryonic ductal plate progenitors (Carpentier et al., 2011). Perhaps ductal plate progenitors give rise to mature bile duct cells and HybHP which remain attached to duct cells via homotypic interactions, in clear contrast to parenchymal hepatocytes which originate from hepatoblasts (Miyajima et al., 2014). Interestingly, some of the genes that are underexpressed in HybHP relative to conventional hepatocytes are involved in drug metabolism and immune responses, categories that represent specialized functions of more differentiated hepatocytes that allow them to detoxify xenobiotics and products of the gastrointestinal microbiome that reach the liver via the portal vein. The underexpression of such genes may make HybHP more resistant to toxic insults and inflammatory stress. At this point, however, we do not know whether this specific transcriptomic profile is hardwired or not. Plausibly, it may be maintained by the specific location at which HybHP reside. Indeed, HybHP descendants upregulate certain metabolic genes, such as GS, once they reach the central zone. In normal human liver, a weak signal for Sox9 and Opn can be detected, reminiscent of what was observed in mice, suggesting that HybHP are also present in humans. However, markers that are uniquely expressed in HybHP remain to be identified.
(58) “Hybrid hepatocyte” cell and “HybHP” cell interchangeably refer to a hepatocyte cell that has similar morphology to a conventional hepatocyte cell, and that expresses at least one marker gene that is specific to liver ductal (DC) cells (exemplified by genes in bold italic text in Tables 1 and Table 3), as well as at least one marker gene that is specific to conventional hepatocyte (cHP) cells (exemplified by the genes in text that is neither in bold nor in italics in Table 2) (Also see
(59) Data herein demonstrate that HybHP cells are located in the liver (including the portal area of the liver) (
(60) Data herein (
(61) Data herein demonstrate that, in one embodiment, HybHP cells display elevated basal Sox9 promoter activity and other ductal markers.
(62) Data herein demonstrate that, in one embodiment, HybHP cells express markers Sox9 and Opn that are not expressed by conventional hepatocytes.
(63) Data herein demonstrate that, in one embodiment, HybHP express Sox9 and HNF4α (
(64) Data herein demonstrate that, in one embodiment,
(65) Data herein demonstrate that there is not a single marker that is uniquely expressed only by HybHP (
(66) Data herein demonstrate that, in one embodiment, specific expression of Sox9, Opn, Agxt2l1 and Aqp4 in HybHP by immunostaining (
(67) In one embodiment, HybHP cells are negative for the bile duct marker CK19 (
(68) In one embodiment, expression of TLR5 and TLR8 is downregulated to very low levels in HybHP compared to HP.
(69) In one embodiment, HybHP cells are negative for the bile duct marker CK19.
(70) In one embodiment, HybHP cells are positive for the hepatocyte marker HNF4a.
(71) The observed differences in gene expression between conventional hepatocytes and HybHP cannot be explained by contamination. Data herein also rule out that differences in gene expression could result from contamination of HybHP with bile duct (BD) cells using immunostaining (
(72) Data herein demonstrate that HybHP cells multiply in number in vitro under conditions for growth of ductal cells (Example 8) to form organoids. This is unlike cHP cells that do not grow in vitro. Also, HybHP cells can be cultured indefinitely in vitro under growth conditions similar to those for ductal cells.
(73) Data herein also show that HybHP cells transdifferentiate in vivo into ductal cells (including bile duct cells and oval cells) (Example 3, Example 5).
(74) Upon in vivo transplantation of HybHP cells, the transplanted HybHP cells repopulate all liver tissue zones, including zone 1, zone 2, and zone 3 and differentiate and transdifferentiate into progeny hepatocytes and DC cells that express the correct liver zonation cell markers (Example 2, Example 3, Example 5). Data herein demonstrate that the in vitro expanded HybHP cells can be reverted to the hepatocyte phenotype for transplantation, and that HybHP cells expand upon liver injury and form larger and more stably differentiated clones than other hepatocytes (Example 2, Example 3, Example 5).
(75) Data herein show that repopulation of diseased liver by HybHP is more efficient than by Pluripotent Stem Cells (iPSCs) or ductal cells. For example, following in vivo transplantation of 45,000 HybHP cells, the transplanted HybHP cells repopulated 50% of the liver after 3 months (Example 2, Example 3, Example 5,
(76) Data herein also demonstrate that the repopulation, i.e., the contribution of HybHP to new hepatocytes, in the CCl4 chronic injury model is around 65%. This means that starting with 5% of total hepatocytes, after 6 weeks of CCl4 treatment, HybHP expand 13-fold to give rise to 65% of all hepatocytes. At the same time, conventional hepatocytes decline from 95% to 35% of all hepatocytes.
(77) The results clearly show that HybHP expand during the course of repetitive CCl4 injury whereas single or acute doses barely produce any effect on the HybHP population (
(78) Data herein demonstrate that that HybHP are superior to conventional hepatocytes in repopulating an injured liver (
(79) Not only do HybHP cells repopulate liver, but they also treat liver damage. Data herein show that animals suffering from liver damage that are transplanted with HybHP do not die from otherwise terminal liver damage (Example 5). Following transplantation of 14,000-50,000 HybHP cells, much less than the 500,000-1,000,000 cells commonly used in such studies, all HybHP transplanted animals were still alive compared to the death of 90% of control animals and of 50% of cHP-transplanted mice (Example 5;
(80) Furthermore, transplanted HybHPs repopulate the liver without giving rise to hepatocellular carcinoma (HCC) (Example 6)].
(81) ii) HybHP Provide a New Framework for Understanding Liver Regeneration
(82) Liver regeneration has been most extensively studied in the context of partial (⅔.sup.rd) hepatectomy, in which all liver cells undergo limited rounds of cell division to restore organ mass. However, controversy had dominated studies of liver homeostasis and regeneration after injury giving rise to contrasting hypotheses such as the streaming liver hypothesis (Fellous et al., 2009; Furuyama et al., 2010), the liver stem cell hypothesis (Dorrell et al., 2011), and more recent findings that question the ability of oval cells to give rise to hepatocytes (Español-Suñer et al., 2012; Malato et al., 2011; Rodrigo-Torres et al., 2014; Schaub et al., 2014; Tarlow et al., 2014a; Yanger et al., 2014) and suggest that hepatocytes can transdifferentiate into oval cells (Michalopoulos et al., 2005; Sekiya and Suzuki, 2014; Tanimizu et al., 2014; Tarlow et al., 2014b; Yanger et al., 2013). However, it was also shown that cultured bile duct cells can transdifferentiate into hepatocytes (Huch et al., 2013; Huch et al., 2015). The discovery of HybHP allows us to propose a model of liver regeneration that reaffirms the fundamental role of the portal-central axis in liver physiology, with the portal tract being the organizing center and source of cells responsible for the majority of parenchymal regeneration. We suggest that HybHP are mainly activated under conditions when the damaged parenchyma is more efficiently repopulated by hepatocytes that originate from the limiting plate. When the damage is mild and scattered, it may be more efficient to restore lost cells by a simple division of neighboring hepatocytes. But when many hepatocytes are continuously destroyed, the highly complex network of hepatic sinusoids and bile canaliculi is compromised, and the most effective way to properly regenerate the liver is to induce expansion of hepatocytes that are already connected to bile duct cells from the portal tract outwards, a task that can be most easily fulfilled by HybHP.
(83) Despite their expansion in MUP-uPA mice, oval cells are incapable of differentiating into hepatocytes. Notably, oval cell expansion is mainly detected in areas where MUP-uPA mice have lost HybHP and a similar pattern was detected in the STAM liver and in mice fed with CDE diet. These observations suggest that the oval cell response is activated when HybHP fail to expand. Nonetheless, since oval cells do not give rise to differentiated hepatocytes the logic underlying their expansion remains enigmatic. One possible function for oval cells could be restoration of the bile canaliculi network and liver polarity. Supporting these lines, inking the ductal tree has allowed visualization of its structure in different models of liver injury with oval cell expansion (Kaneko et al., 2015). Such studies show that oval cells are connected, representing extensions of the preexisting ductal tree, further challenging the old view according to which oval cells are liver progenitor cells that seed the parenchyma to generate new hepatocytes. More likely, oval cells participate in remodeling of the ductal tree, rather than functioning as bipotential stem cells, at least in mice (Schaub et al., 2014; Tarlow et al., 2014a; Yanger et al., 2014).
(84) Data herein demonstrate the use of genetic lineage tracing to identify cells responsible for liver repair following injury and then queried their roles in three distinct HCC models. Data herein teaches the discovery of HybHP cells, a new cell type that is a hybrid between a hepatocyte and a duct cell. Data herein show that the unique HybHP cells are low Sox9 expressing, periportal hepatocytes that also express other bile duct enriched genes (hence Hybrid Hepatocytes) and are the primary cells that mediate liver injury repair. Surprisingly, despite their high regenerative potential and multiple division cycles after tumor promoting injuries, data herein show that HybHP cells are not the preferred origin for HCC. Thus, the invention demonstrates that the inventors' newly discovered HybHP cells represent a unique mechanism for tissue repair that avoids excessive tumorigenesis by using a specialized set of differentiated cells rather than adult stem cells. The invention's cells can be used to treat liver injury and/or disease via transplantation.
(85) Thus, in one embodiment, the invention provides a purified mammalian HybHP cell, and/or purified population of mammalian HybHP cells, that expresses at least one first protein marker of liver ductal (DC) cells, and express at least one second protein marker of conventional hepatocyte (cHP) cells.
(86) The invention also provides a purified mammalian first antibody-HybHP cell conjugate wherein the first antibody specifically binds to a first protein marker of liver ductal (DC) cells. Also provided by the invention is a composition comprising the purified first antibody-HybHP cell conjugate.
(87) The invention further provides a purified mammalian first antibody-HybHP cell-second antibody conjugate, wherein said first antibody specifically binds to a first protein marker of liver ductal (DC) cells, and wherein said second antibody specifically binds to a second protein marker of conventional hepatocyte (cHP) cells. Also provided by the invention is a composition comprising the purified first antibody-HybHP cell-second antibody conjugate.
(88) The invention further provides a mammalian first antibody-HybHP cell-second antibody conjugate isolated by any of the methods described herein. The invention additionally provides a composition comprising the mammalian first antibody-HybHP cell-second antibody conjugate isolated by any of the methods described herein.
(89) B) Methods for Isolating HybHP Cells
(90) The invention also provides methods to propagate the newly discovered HybHP cells ex vivo. In a first approach, purifying a hepatocyte (HybHP) cell (and/or a population of hepatocyte (HybHP) cells) from a mammalian liver, comprises a) preparing a single-cell suspension from the liver (e.g., by collagenase digestion of liver tissue (Example 1)), b) combining the single-cell suspension with i) at least one first antibody that specifically binds to a first protein marker of liver ductal (DC) cells, and ii) at least one second antibody that specifically binds to a second protein marker of conventional hepatocyte (cHP) cells, wherein the combining is under conditions for specific binding of the at least one first antibody to the first protein marker and of the at least one second antibody to the second protein, and wherein the specific binding produces a first composition that comprises a first antibody-HybHP cell-second antibody conjugate, and c) isolating the first antibody-HybHP cell-second antibody conjugate from the single-cell suspension, thereby producing a second composition that comprises a purified HybHP cell, and/or a purified population of HybHP cell.
(91) In one embodiment, the “isolating” step may be achieved using a second antibody that specifically binds to the one or both of the first antibody-second antibody.
(92) In one embodiment, the first protein marker of liver ductal (DC) cells is encoded by one or more gene shown in bold italic text in Table 1 and Table 3.
(93) TABLE-US-00001 TABLE 1 HybHP marker genes that are upregulated in HybHP compared to cHP. Bold italics text (numbered 1-193 in the first column) reflect genes that are also expressed in ductal cells. Mouse gene Mouse Human name Entrez gene ID Entrez gene ID 1 67082 #N/A 2
69862 #N/A 3
74430 #N/A 4
229722 #N/A 5
320939 #N/A 6
74525 #N/A 7
319951 #N/A 8
27416 10057 9
54608 11057 10
632263 11096 11
23794 11096 12
108897 83543 13
107765 27063 14
74251 122416 15
101772 55107 16
69787 312 17
80287 #N/A 18
11820 351 19
11856 395 20
230972 27237 21
11931 481 22
56336 9334 23
57278 4059 24
83675 80114 25
107771 #N/A 26
54648 90060 27
381175 80323 28
68339 440193 29
100048780 4345 30
17470 4345 31
12484 #N/A 32
12512 967 33
12550 999 34
12614 9620 35
12671 1131 36
11443 1140 37
54419 284620 38
53624 1366 39
12837 1295 40
26427 90993 41
30785 83992 42
13131 1600 43
195208 #N/A 44
13193 1641 45
73284 115265 46
12305 780 47
26757 10570 48
13614 1906 49
13618 1910 50
268780 133584 51
259300 30846 52
13661 26298 53
13717 2006 54
13723 133418 55
13730 2012 56
13800 55740 57
13821 #N/A 58
13824 #N/A 59
17075 4072 60
13838 2043 61
56226 83715 62
18612 2118 63
59056 2121 64
68525 132884 65
14063 2150 66
71721 220965 67
245050 729085 68
68659 51313 69
76886 145773 70
239463 84985 71
14115 2199 72
241639 55612 73
14182 2260 74
14183 2263 75
399558 23768 76
15223 2302 77
242022 341640 78
83554 10272 79
14537 2650 80
635918 2650 81
14613 2702 82
226075 169792 83
208166 #N/A 84
14697 10681 85
234267 2823 86
215798 57211 87
232431 9052 88
64297 51704 89
15131 #N/A 90
15132 #N/A 91
100044263 #N/A 92
380975 613227 93
393082 613227 94
554292 613227 95
216019 80201 96
545370 83872 97
319765 10644 98
29817 3490 99
50905 55540 100
57890 132014 101
106347 286676 102
57444 3669 103
16403 3655 104
320910 3696 105
67374 58494 106
100044693 10008 107
57442 10008 108
16525 3775 109
16531 3778 110
16560 547 111
16582 3801 112
16669 3880 113
110310 3855 114
16776 3911 115
16779 3913 116
16782 3918 117
23928 10319 118
16826 9079 119
192198 64101 120
16979 57633 121
17153 4118 122
26415 5603 123
19099 9479 124
73608 91862 125
14013 2122 126
230971 1953 127
17304 4240 128
30060 4241 129
208943 55930 130
100044475 8440 131
621446 8440 132
17974 8440 133
18049 4803 134
223473 79815 135
67122 441478 136
18212 4915 137
244723 93145 138
79196 114879 139
214230 56924 140
210622 25891 141
236899 9468 142
71785 80310 143
170676 23089 144
56421 5214 145
18767 5569 146
19041 5493 147
70673 63976 148
116847 5549 149
19126 8842 150
217138 79170 151
19249 5783 152
19250 5784 153
80718 5874 154
19415 8437 155
237504 9182 156
631094 8434 157
53614 8434 158
244058 56963 159
68585 57142 160
50524 6297 161
219151 51435 162
20250 #N/A 163
110876 #N/A 164
20348 10512 165
20356 9037 166
73251 80854 167
27428 57619 168
171286 84561 169
20536 6508 170
20537 6523 171
58994 55512 172
100340 27293 173
20677 6659 174
20673 6659 175
20682 6662 176
20732 6692 177
20750 6696 178
20779 6714 179
20606 6752 180
19143 6768 181
76630 57559 182
20849 6775 183
74051 261729 184
21808 7042 185
21859 7078 186
229302 7104 187
319832 730130 188
56277 55076 189
216350 7103 190
226654 100131187 191
22371 7450 192
22417 54361 193
72720 #N/A 194 Aim1l 230806 55057 195 Aldh1b1 72535 219 196 Apoa4 11808 337 197 Apoc2 11813 344 198 Aqp4 11829 361 199 Arhgap10 78514 79658 200 Atp4a 11944 495 201 Bdh2 69772 56898 202 Cbs 12411 875 203 Cib3 234421 117286 204 Clic5 224796 53405 205 Cryl1 68631 51084 206 Ctnnbip1 67087 56998 207 Ctsc 13032 1075 208 Cyp2f2 13107 #N/A 209 Defb1 13214 1672 210 Efna3 100046031 1944 211 Efna3 13638 1944 212 Fbp1 14121 2203 213 Gas2 14453 2620 214 Gldc 104174 2731 215 Gls2 216456 27165 216 Gucy2c 14917 2984 217 Hal 15109 3034 218 Hsbp1l1 66255 440498 219 Hsd17b13 243168 345275 220 Hsd17b6 27400 8630 221 Kcp 333088 375616 223 Mrgpre 244238 116534 224 Ncam2 17968 4685 225 Olfm3 229759 118427 226 Osbp2 100044882 23762 227 Osbp2 74309 23762 228 Plxna2 18845 5362 229 Ptgds 19215 5730 230 Ptges 64292 9536 231 Sdsl 257635 113675 232 Serpina12 68054 145264 233 Sulf2 72043 55959 234 Tbc1d30 74694 23329 235 Tmprss4 214523 56649 236 Ugt2b38 100559 #N/A 237 Xirp1 22437 165904 238 From Mouse Human 239 1700011H14Rik 67082 #N/A 240 1810064F22Rik 69862 #N/A 241 4930452B06Rik 74430 #N/A 242 5330417C22Rik 229722 #N/A 243 5930403L14Rik 320939 #N/A 244 8430419L09Rik 74525 #N/A 245 A230001M10Rik 319951 #N/A 246 Abcc5 27416 10057 247 Abhd2 54608 11057 248 Adamts5 632263 11096 249 Adamts5 23794 11096 250 Aif1l 108897 83543 251 Ankrd1 107765 27063 252 Ankrd9 74251 122416 253 Ano1 101772 55107 254 Anxa13 69787 312 255 Apobec3 80287 #N/A 256 App 11820 351 257 Arhgap6 11856 395 258 Arhgef16 230972 27237 259 Atp1b1 11931 481 260 B4galt5 56336 9334 261 Bcam 57278 4059 262 Bicc1 83675 80114 263 Bmyc 107771 #N/A 264 Ccdc120 54648 90060 265 Ccdc68 381175 80323 266 Ccdc88c 68339 440193 267 Cd200 100048780 4345 268 Cd200 17470 4345 269 Cd24a 12484 #N/A 270 Cd63 12512 967 271 Cdh1 12550 999 272 Celsr1 12614 9620 273 Chrm3 12671 1131 274 Chrnb1 11443 1140 275 Cldn6 54419 284620 276 Cldn7 53624 1366 277 Col8a1 12837 1295 278 Creb3l1 26427 90993 279 Cttnbp2 30785 83992 280 Dab1 13131 1600 281 Dcdc2a 195208 #N/A 282 Dcx 13193 1641 283 Ddit4l 73284 115265 284 Ddr1 12305 780 285 Dpysl4 26757 10570 286 Edn1 13614 1906 287 Ednrb 13618 1910 288 Egflam 268780 133584 289 Ehd2 259300 30846 290 Ehf 13661 26298 291 Eln 13717 2006 292 Emb 13723 133418 293 Emp1 13730 2012 294 Enah 13800 55740 295 Epb4.1l1 13821 #N/A 296 Epb4.1l4a 13824 #N/A 297 Epcam 17075 4072 298 Epha4 13838 2043 299 Espn 56226 83715 300 Etv4 18612 2118 301 Evc 59056 2121 302 Evc2 68525 132884 303 F2rl1 14063 2150 304 Fam13c 71721 220965 305 Fam198a 245050 729085 306 Fam198b 68659 51313 307 Fam81a 76886 145773 308 Fam83a 239463 84985 309 Fbln2 14115 2199 310 Fermt1 241639 55612 311 Fgfr1 14182 2260 312 Fgfr2 14183 2263 313 Flrt2 399558 23768 314 Foxj1 15223 2302 315 Frem2 242022 341640 316 Fstl3 83554 10272 317 Gcnt1 14537 2650 318 Gcnt1 635918 2650 319 Gja5 14613 2702 320 Glis3 226075 169792 321 Gm609 208166 #N/A 322 Gnb5 14697 10681 323 Gpm6a 234267 2823 324 Gpr126 215798 57211 325 Gprc5a 232431 9052 326 Gprc5b 64297 51704 327 Hbb-bh1 15131 #N/A 328 Hbb-bh1 15132 #N/A 329 Hbb-bh1 100044263 #N/A 330 Higd1c 380975 613227 331 Higd1c 393082 613227 332 Higd1c 554292 613227 333 Hkdc1 216019 80201 334 Hmcn1 545370 83872 335 Igf2bp2 319765 10644 336 Igfbp7 29817 3490 337 Il17rb 50905 55540 338 Il17re 57890 132014 339 Ildr1 106347 286676 340 Isg20 57444 3669 341 Itga6 16403 3655 342 Itgb8 320910 3696 343 Jam2 67374 58494 344 Kcne3 100044693 10008 345 Kcne3 57442 10008 346 Kcnk1 16525 3775 347 Kcnma1 16531 3778 348 Kif1a 16560 547 349 Kifc3 16582 3801 350 Krt19 16669 3880 351 Krt7 110310 3855 352 Lama5 16776 3911 353 Lamb2 16779 3913 354 Lamc2 16782 3918 355 Lamc3 23928 10319 356 Ldb2 16826 9079 357 Lrrc4 192198 64101 358 Lrrn1 16979 57633 359 Mal 17153 4118 360 Mapk13 26415 5603 361 Mapk8ip1 19099 9479 362 Marveld3 73608 91862 363 Mecom 14013 2122 364 Megf6 230971 1953 365 Mfge8 17304 4240 366 Mfi2 30060 4241 367 Myo5c 208943 55930 368 Nck2 100044475 8440 369 Nck2 621446 8440 370 Nck2 17974 8440 371 Ngf 18049 4803 372 Nipal2 223473 79815 373 Nrarp 67122 441478 374 Ntrk2 18212 4915 375 Olfm2 244723 93145 376 Osbpl5 79196 114879 377 Pak6 214230 56924 378 Pamr1 210622 25891 379 Pcyt1b 236899 9468 380 Pdgfd 71785 80310 381 Peg10 170676 23089 382 Pfkp 56421 5214 383 Pkia 18767 5569 384 Ppl 19041 5493 385 Prdm16 70673 63976 386 Prelp 116847 5549 387 Prom1 19126 8842 388 Prr15l 217138 79170 389 Ptpn13 19249 5783 390 Ptpn14 19250 5784 391 Rab27b 80718 5874 392 Rasal1 19415 8437 393 Rassf9 237504 9182 394 Reck 631094 8434 395 Reck 53614 8434 396 Rgma 244058 56963 397 Rtn4 68585 57142 398 Sall2 50524 6297 399 Scara3 219151 51435 400 Scd2 20250 #N/A 401 Scn2a1 110876 #N/A 402 Sema3c 20348 10512 403 Sema5a 20356 9037 404 Setd7 73251 80854 405 Shroom3 27428 57619 406 Slc12a8 171286 84561 407 Slc4a3 20536 6508 408 Slc5a1 20537 6523 409 Smpd3 58994 55512 410 Smpdl3b 100340 27293 411 Sox4 20677 6659 412 Sox4 20673 6659 413 Sox9 20682 6662 414 Spint1 20732 6692 415 Spp1 20750 6696 416 Src 20779 6714 417 Sstr2 20606 6752 418 St14 19143 6768 419 Stambpl1 76630 57559 420 Stat4 20849 6775 421 Steap2 74051 261729 422 Tgfb2 21808 7042 423 Timp3 21859 7078 424 Tm4sf4 229302 7104 425 Tmem229a 319832 730130 426 Tmem45a 56277 55076 427 Tspan8 216350 7103 428 Tstd1 226654 100131187 429 Vwf 22371 7450 430 Wnt4 22417 54361 431 Zfp248 72720 #N/A 432 Aim1l 230806 55057 433 Aldh1b1 72535 219 434 Apoa4 11808 337 435 Apoc2 11813 344 436 Aqp4 11829 361 437 Arhgap10 78514 79658 438 Atp4a 11944 495 439 Bdh2 69772 56898 440 Cbs 12411 875 441 Cib3 234421 117286 442 Clic5 224796 53405 443 Cryl1 68631 51084 444 Ctnnbip1 67087 56998 445 Ctsc 13032 1075 446 Cyp2f2 13107 #N/A 447 Defb1 13214 1672 448 Efna3 100046031 1944 449 Efna3 13638 1944 450 Fbp1 14121 2203 451 Gas2 14453 2620 452 Gldc 104174 2731 453 Gls2 216456 27165 454 Gucy2c 14917 2984 455 Hal 15109 3034 456 Hsbp1l1 66255 440498 457 Hsd17b13 243168 345275 458 Hsd17b6 27400 8630 459 Kcp 333088 375616 460 Mrgpre 244238 116534 461 Ncam2 17968 4685 462 Olfm3 229759 118427 463 Osbp2 100044882 23762 464 Osbp2 74309 23762 465 Plxna2 18845 5362 466 Ptgds 19215 5730 467 Ptges 64292 9536 468 Sdsl 257635 113675 469 Serpina12 68054 145264 470 Sulf2 72043 55959 471 Tbc1d30 74694 23329 472 Tmprss4 214523 56649 473 Ugt2b38 100559 #N/A 474 Xirp1 22437 165904
(94) TABLE-US-00002 TABLE 2 HybHP marker genes that are downregulated in HybHP cells compared to cHP cells. Bold italics text (numbered 1-118 in the first column) reflect genes that are specifically downregulated in HybHP compared to cHP and BD. Text that is neither in bold nor in italics (numbered 119-573 in the first column) reflect genes that are downregulated both in HybHP and BD compared to cHP. Mouse gene Mouse Human name Entrez gene ID Entrez gene ID 1 231014 #N/A 2
26897 641371 3
432530 107 4
11629 199 5
26360 23452 6
71085 84986 7
26362 558 8
26879 8706 9
12162 655 10
12229 695 11
12259 712 12
12260 713 13
12262 714 14
319996 113201 15
20305 #N/A 16
12443 595 17
93671 9332 18
217305 100131439 19
100046097 100131439 20
12505 960 21
12508 963 22
11801 922 23
16149 972 24
12524 942 25
14007 10659 26
18636 5199 27
12738 9075 28
73149 #N/A 29
51811 165530 30
56620 #N/A 31
56644 64581 32
12831 1289 33
107684 7464 34
12978 1436 35
13034 1510 36
13040 1520 37
13058 1536 38
242484 #N/A 39
210293 55619 40
668212 22979 41
13733 2015 42
52614 #N/A 43
71946 23052 44
66540 83641 45
14131 #N/A 46
246256 #N/A 47
14133 #N/A 48
26382 221472 49
14190 10875 50
329977 114827 51
14276 2350 52
23880 2533 53
14675 9630 54
237175 10149 55
14960 #N/A 56
14968 #N/A 57
14998 #N/A 58
15162 3055 59
15277 642546 60
212032 3101 61
80719 10261 62
16408 3683 63
16414 3689 64
239217 115207 65
16792 7805 66
16854 3958 67
232801 353514 68
14728 11006 69
14727 11006 70
16956 4023 71
669888 4023 72
17105 #N/A 73
17476 219972 74
17969 653361 75
105855 3071 76
17988 10397 77
230103 4882 78
18227 4929 79
70021 64943 80
258571 #N/A 81
70839 64805 82
170721 89932 83
54635 56034 84
18768 5570 85
66350 81579 86
27226 7941 87
104759 122618 88
56193 5341 89
29858 5372 90
50706 10631 91
18751 5579 92
19264 5788 93
19354 5880 94
53901 10231 95
56739 9985 96
19699 5649 97
622473 92129 98
20970 9672 99
20612 6614 100
83382 #N/A 101
18173 6556 102
216867 162515 103
69309 201232 104
66859 220963 105
20514 6510 106
21810 7045 107
21814 7049 108
210757 387357 109
53791 7100 110
170744 51311 111
29820 55504 112
94185 27242 113
28240 7226 114
21807 8848 115
22177 7305 116
24108 10537 117
22359 7436 118
278180 11326 119 1500017E21Rik 668215 #N/A 120 1810058I24Rik 67705 #N/A 121 2210417A02Rik 70138 #N/A 122 Abcb1b 18669 #N/A 123 Abcg8 67470 64241 124 Abi3 66610 51225 125 Acsm5 272428 54988 126 Adam11 11488 4185 127 Adamts13 279028 11093 128 Adrb3 11556 155 129 Ahr 11622 196 130 Akr1c6 83702 #N/A 131 Aldh1a1 11668 216 132 Aldh3a2 11671 224 133 Ang 11727 283 134 Aqp9 64008 366 135 Asns 27053 440 136 Atp6v0d2 242341 245972 137 Avpr1a 54140 552 138 AW549542 100993 #N/A 139 Axin2 12006 8313 140 Blvrb 233016 645 141 Cela1 109901 1990 142 Chic1 12212 53344 143 Chrna2 110902 1135 144 Cib2 56506 10518 145 Csad 246277 51380 146 Csrp3 13009 8048 147 Cxcl9 17329 4283 148 Cyp1a2 13077 1544 149 Cyp27a1 104086 1593 150 Cyp2a22 233005 #N/A 151 Cyp2a22 13085 #N/A 152 Cyp2a5 330491 #N/A 153 Cyp2a5 13087 #N/A 154 Cyp2c29 13095 #N/A 155 Cyp2c37 13096 #N/A 156 Cyp2c37 100046484 #N/A 157 Cyp2c38 13097 #N/A 158 Cyp2c39 13098 #N/A 159 Cyp2c40 100048323 #N/A 160 Cyp2c40 545288 #N/A 161 Cyp2c40 13099 #N/A 162 Cyp2c40 100043108 #N/A 163 Cyp2c50 107141 #N/A 164 Cyp2c54 404195 #N/A 165 Cyp2c54 639023 #N/A 166 Cyp2c54 100044352 #N/A 167 Cyp2c67 100048323 #N/A 168 Cyp2c67 545288 #N/A 169 Cyp2c67 13099 #N/A 170 Cyp2c67 100043108 #N/A 171 Cyp2c69 100048323 #N/A 172 Cyp2c69 545288 #N/A 173 Cyp2c69 13099 #N/A 174 Cyp2c69 100043108 #N/A 175 Cyp2e1 13106 1571 176 Cyp2g1 13108 #N/A 177 Cyp7a1 13122 1581 178 Dcn 13179 1634 179 Dntt 21673 1791 180 E2f8 108961 79733 181 Ecm1 13601 1893 182 Egln3 112407 112399 183 Esrrg 26381 2104 184 Fam89a 69627 375061 185 Fam89a 100047808 375061 186 Fam89a 677631 375061 187 Fbxl21 213311 26223 188 Fitm1 68680 161247 189 Fmo3 14262 2328 190 Gabrb3 14402 2562 191 Gas7 14457 8522 192 Gbp10 626578 #N/A 193 Gbp10 76074 #N/A 194 Gbp10 634650 #N/A 195 Glul 240219 2752 196 Glul 14645 2752 197 Gsta3 14859 2940 198 Gstm1 433943 2944 199 Gstm1 100043965 2944 200 Gstm1 14862 2944 201 Gstm2 14863 2946 202 Gstm2 626327 2946 203 Gstm3 14864 2947 204 Gstm6 14867 #N/A 205 Gulo 268756 #N/A 206 H2-Q1 15006 #N/A 207 H2-Q1 15018 #N/A 208 H2-Q1 110558 #N/A 209 H2-Q1 100044307 #N/A 210 H2-Q1 68395 #N/A 211 H2-Q1 676708 #N/A 212 H2-Q1 15019 #N/A 213 H2-Q1 100044021 #N/A 214 H2-Q1 100044020 #N/A 215 H2-Q1 110557 #N/A 216 H2-Q1 100044019 #N/A 217 H2-Q1 15013 #N/A 218 Hamp2 66438 #N/A 219 Hand2 15111 9464 220 Hcrtr2 387285 3062 221 Hhip 15245 64399 223 Hsd3b7 101502 55124 224 Kif26b 269152 55083 225 Lama1 16772 284217 226 Lect1 16840 11061 227 Lect2 16841 3950 228 Lgr5 14160 8549 229 Lhpp 76429 64077 230 Lifr 16880 3977 231 Mab21l2 23937 10586 232 Meg3 17263 55384 233 Mirg 100040724 #N/A 234 Mycn 18109 4613 235 Nkd1 634379 85407 236 Nkd1 93960 85407 237 Notum 77583 147111 238 Nr1i3 12355 9970 239 Nrn1 68404 51299 240 Nt5e 23959 4907 241 Ntn1 672215 9423 242 Ntn1 18208 9423 243 Oat 18242 4942 244 Olig1 50914 116448 245 Paqr9 75552 344838 246 Pcp4l1 66425 654790 247 Phospho1 237928 162466 248 Pir 69656 8544 249 Plbd1 66857 79887 250 Plbd1 100045163 79887 251 Pon1 18979 5444 252 Pparg 19016 5468 253 Prrx1 18933 5396 254 Rap2a 76108 5911 255 Rarres1 109222 5918 256 Rdh9 103142 #N/A 257 Rfx4 71137 5992 258 Rgn 19733 9104 259 Rhbg 58176 57127 260 Rian 75745 #N/A 261 Rnase4 58809 6038 262 Robo1 19876 642132 263 Sema5b 20357 54437 264 Serpina7 331535 6906 265 Sh2d7 244885 646892 266 Slc13a3 114644 64849 267 Slc13a4 243755 26266 268 Slc16a10 72472 117247 269 Slc1a2 20511 6506 270 Slc1a4 55963 6509 271 Slc22a1 20517 6580 272 Slc22a3 20519 6581 273 Slc25a21 217593 89874 274 Slc26a4 23985 5172 275 Slco1a1 28248 #N/A 276 Slco1b2 28253 #N/A 277 Sp5 64406 389058 278 Susd4 96935 55061 279 Tbx3 21386 6926 280 Tlr12 384059 #N/A 281 Tmeff2 56363 23671 282 Tnfaip8l1 66443 126282 283 Tomm40l 641376 84134 284 Ttc7b 104718 145567 285 Ube2e2 218793 7325 286 Vnn1 22361 8876 287 From mouse human 288 9330182L06Rik 231014 #N/A 289 Acot1 26897 641371 290 Adcy1 432530 107 291 Aif1 11629 199 292 Angptl2 26360 23452 293 Arhgap19 71085 84986 294 Axl 26362 558 295 B3galnt1 26879 8706 296 Bmp7 12162 655 297 Btk 12229 695 298 C1qa 12259 712 299 C1qb 12260 713 300 C1qc 12262 714 301 Casc4 319996 113201 302 Ccl6 20305 #N/A 303 Ccnd1 12443 595 304 Cd163 93671 9332 305 Cd300ld 217305 100131439 306 Cd300ld 100046097 100131439 307 Cd44 12505 960 308 Cd53 12508 963 309 Cd51 11801 922 310 Cd74 16149 972 311 Cd86 12524 942 312 Celf2 14007 10659 313 Cfp 18636 5199 314 Cldn2 12738 9075 315 Clec4a3 73149 #N/A 316 Clec4f 51811 165530 317 Clec4n 56620 #N/A 318 Clec7a 56644 64581 319 Col5a1 12831 1289 320 Coro2a 107684 7464 321 Csf1r 12978 1436 322 Ctse 13034 1510 323 Ctss 13040 1520 324 Cybb 13058 1536 325 D630039A03Rik 242484 #N/A 326 Dock10 210293 55619 327 Efr3b 668212 22979 328 Emr1 13733 2015 329 Emr4 52614 #N/A 330 Endod1 71946 23052 331 Fam107b 66540 83641 332 Fcgr3 14131 #N/A 333 Fcgr4 246256 #N/A 334 Fcna 14133 #N/A 335 Fgd2 26382 221472 336 Fgl2 14190 10875 337 Fhad1 329977 114827 338 Folr2 14276 2350 339 Fyb 23880 2533 340 Gna14 14675 9630 341 Gpr64 237175 10149 342 H2-Aa 14960 #N/A 343 H2-Aa 14968 #N/A 344 H2-DMa 14998 #N/A 345 Hck 15162 3055 346 Hk2 15277 642546 347 Hk3 212032 3101 348 Igsf6 80719 10261 349 Itgal 16408 3683 350 Itgb2 16414 3689 351 Kctd12 239217 115207 352 Laptm5 16792 7805 353 Lgals3 16854 3958 354 Lilra5 232801 353514 355 Lilrb4 14728 11006 356 Lilrb4 14727 11006 357 Lpl 16956 4023 358 Lpl 669888 4023 359 Lyz2 17105 #N/A 360 Mpeg1 17476 219972 361 Ncf1 17969 653361 362 Nckap1l 105855 3071 363 Ndrg1 17988 10397 364 Npr2 230103 4882 365 Nr4a2 18227 4929 366 Nt5dc2 70021 64943 367 Olfr1033 258571 #N/A 368 P2ry12 70839 64805 369 Papln 170721 89932 370 Pdgfc 54635 56034 371 Pkib 18768 5570 372 Pla2g12a 66350 81579 373 Pla2g7 27226 7941 374 Pld4 104759 122618 375 Plek 56193 5341 376 Pmm1 29858 5372 377 Postn 50706 10631 378 Prkcb 18751 5579 379 Ptprc 19264 5788 380 Rac2 19354 5880 381 Rcan2 53901 10231 382 Rec8 56739 9985 383 Reln 19699 5649 384 Ripply1 622473 92129 385 Sdc3 20970 9672 386 Siglec1 20612 6614 387 Siglece 83382 #N/A 388 Slc11a1 18173 6556 389 Slc16a11 216867 162515 390 Slc16a13 69309 201232 391 Slc16a9 66859 220963 392 Slc1a5 20514 6510 393 Tgfbi 21810 7045 394 Tgfbr3 21814 7049 395 Themis 210757 387357 396 Tlr5 53791 7100 397 Tlr8 170744 51311 398 Tnfrsf19 29820 55504 399 Tnfrsf21 94185 27242 400 Trpm2 28240 7226 401 Tsc22d1 21807 8848 402 Tyrobp 22177 7305 403 Ubd 24108 10537 404 Vldlr 22359 7436 405 Vsig4 278180 11326 406 1500017E21Rik 668215 #N/A 407 1810058I24Rik 67705 #N/A 408 2210417A02Rik 70138 #N/A 409 Abcb1b 18669 #N/A 410 Abcg8 67470 64241 411 Abi3 66610 51225 412 Acsm5 272428 54988 413 Adam11 11488 4185 414 Adamts13 279028 11093 415 Adrb3 11556 155 416 Ahr 11622 196 417 Akr1c6 83702 #N/A 418 Aldh1a1 11668 216 419 Aldh3a2 11671 224 420 Ang 11727 283 421 Aqp9 64008 366 422 Asns 27053 440 423 Atp6v0d2 242341 245972 424 Avpr1a 54140 552 425 AW549542 100993 #N/A 426 Axin2 12006 8313 427 Blvrb 233016 645 428 Cela1 109901 1990 429 Chic1 12212 53344 430 Chrna2 110902 1135 431 Cib2 56506 10518 432 Csad 246277 51380 433 Csrp3 13009 8048 434 Cxcl9 17329 4283 435 Cyp1a2 13077 1544 436 Cyp27a1 104086 1593 437 Cyp2a22 233005 #N/A 438 Cyp2a22 13085 #N/A 439 Cyp2a5 330491 #N/A 440 Cyp2a5 13087 #N/A 441 Cyp2c29 13095 #N/A 442 Cyp2c37 13096 #N/A 443 Cyp2c37 100046484 #N/A 444 Cyp2c38 13097 #N/A 445 Cyp2c39 13098 #N/A 446 Cyp2c40 100048323 #N/A 447 Cyp2c40 545288 #N/A 448 Cyp2c40 13099 #N/A 449 Cyp2c40 100043108 #N/A 450 Cyp2c50 107141 #N/A 451 Cyp2c54 404195 #N/A 452 Cyp2c54 639023 #N/A 453 Cyp2c54 100044352 #N/A 454 Cyp2c67 100048323 #N/A 455 Cyp2c67 545288 #N/A 456 Cyp2c67 13099 #N/A 457 Cyp2c67 100043108 #N/A 458 Cyp2c69 100048323 #N/A 459 Cyp2c69 545288 #N/A 460 Cyp2c69 13099 #N/A 461 Cyp2c69 100043108 #N/A 462 Cyp2e1 13106 1571 463 Cyp2g1 13108 #N/A 464 Cyp7a1 13122 1581 465 Dcn 13179 1634 466 Dntt 21673 1791 467 E2f8 108961 79733 468 Ecm1 13601 1893 469 Egln3 112407 112399 470 Esrrg 26381 2104 471 Fam89a 69627 375061 472 Fam89a 100047808 375061 473 Fam89a 677631 375061 474 Fbxl21 213311 26223 475 Fitm1 68680 161247 476 Fmo3 14262 2328 477 Gabrb3 14402 2562 478 Gas7 14457 8522 479 Gbp10 626578 #N/A 480 Gbp10 76074 #N/A 481 Gbp10 634650 #N/A 482 Glul 240219 2752 483 Glul 14645 2752 484 Gsta3 14859 2940 485 Gstm1 433943 2944 486 Gstm1 100043965 2944 487 Gstm1 14862 2944 488 Gstm2 14863 2946 489 Gstm2 626327 2946 490 Gstm3 14864 2947 491 Gstm6 14867 #N/A 492 Gulo 268756 #N/A 493 H2-Q1 15006 #N/A 494 H2-Q1 15018 #N/A 495 H2-Q1 110558 #N/A 496 H2-Q1 100044307 #N/A 497 H2-Q1 68395 #N/A 498 H2-Q1 676708 #N/A 499 H2-Q1 15019 #N/A 500 H2-Q1 100044021 #N/A 501 H2-Q1 100044020 #N/A 502 H2-Q1 110557 #N/A 503 H2-Q1 100044019 #N/A 504 H2-Q1 15013 #N/A 505 Hamp2 66438 #N/A 506 Hand2 15111 9464 507 Hcrtr2 387285 3062 508 Hhip 15245 64399 509 Hsd3b7 101502 55124 510 Kif26b 269152 55083 511 Lama1 16772 284217 512 Lect1 16840 11061 513 Lect2 16841 3950 514 Lgr5 14160 8549 515 Lhpp 76429 64077 516 Lifr 16880 3977 517 Mab2112 23937 10586 518 Meg3 17263 55384 519 Mirg 100040724 #N/A 520 Mycn 18109 4613 521 Nkd1 634379 85407 522 Nkd1 93960 85407 523 Notum 77583 147111 524 Nr1i3 12355 9970 525 Nrn1 68404 51299 526 Nt5e 23959 4907 527 Ntn1 672215 9423 528 Ntn1 18208 9423 529 Oat 18242 4942 530 Olig1 50914 116448 531 Paqr9 75552 344838 532 Pcp4l1 66425 654790 533 Phospho1 237928 162466 534 Pir 69656 8544 536 Plbd1 66857 79887 537 Plbd1 100045163 79887 538 Pon1 18979 5444 539 Pparg 19016 5468 540 Prrx1 18933 5396 541 Rap2a 76108 5911 542 Rarres1 109222 5918 543 Rdh9 103142 #N/A 544 Rfx4 71137 5992 545 Rgn 19733 9104 546 Rhbg 58176 57127 547 Rian 75745 #N/A 548 Rnase4 58809 6038 549 Robo1 19876 642132 550 Sema5b 20357 54437 551 Serpina7 331535 6906 552 Sh2d7 244885 646892 553 Slc13a3 114644 64849 554 Slc13a4 243755 26266 555 Slc16a10 72472 117247 556 Slc1a2 20511 6506 557 Slc1a4 55963 6509 558 Slc22a1 20517 6580 559 Slc22a3 20519 6581 560 Slc25a21 217593 89874 561 Slc26a4 23985 5172 562 Slco1a1 28248 #N/A 563 Slco1b2 28253 #N/A 564 Sp5 64406 389058 565 Susd4 96935 55061 566 Tbx3 21386 6926 567 Tlr12 384059 #N/A 568 Tmeff2 56363 23671 569 Tnfaip8l1 66443 126282 570 Tomm40l 641376 84134 571 Ttc7b 104718 145567 572 Ube2e2 218793 7325 573 Vnn1 22361 8876
(95) TABLE-US-00003 TABLE 3 HybHP marker genes for isolation of HybHP cells, that are located on the plasma membrane, and that are upregulated in HybHP compared to cHP. Bold italics text (numbered 1-64 in the first column) reflects genes that are also expressed in ductal cells. Mouse gene Mouse Human name Entrez gene ID Entrez gene ID 1 27416 10057 2
101772 55107 3
69787 312 4
11820 351 5
11931 481 6
57278 4059 7
100048780 4345 8
17470 4345 9
12484 #N/A 10
12512 967 11
12550 999 12
12614 9620 13
12671 1131 14
11443 1140 15
54419 284620 16
53624 1366 17
30785 83992 18
12305 780 19
13618 1910 20
268780 133584 21
259300 30846 22
13800 55740 23
17075 4072 24
13838 2043 25
56226 83715 26
14063 2150 27
241639 55612 28
242022 341640 29
14613 2702 30
14697 10681 31
215798 57211 32
232431 9052 33
64297 51704 34
545370 83872 35
50905 55540 36
106347 286676 37
16403 3655 38
320910 3696 39
67374 58494 40
100044693 10008 41
57442 10008 42
16531 3778 43
16669 3880 44
17153 4118 45
19099 9479 46
17304 4240 47
30060 4241 48
18212 4915 49
19041 5493 50
19126 8842 51
80718 5874 52
631094 8434 53
53614 8434 54
244058 56963 55
68585 57142 56
110876 #N/A 57
27428 57619 58
20537 6523 59
20779 6714 60
20606 6752 61
19143 6768 62
74051 261729 63
56277 55076 64
22371 7450 65 Aqp4 11829 361 66 Arhgap10 78514 79658 67 Atp4a 11944 495 68 Clic5 224796 53405 69 Cyp2f2 13107 #N/A 70 Efna3 100046031 1944 71 Efna3 13638 1944 72 Hsd17b6 27400 8630 73 Mrgpre 244238 116534 74 Ncam2 17968 4685 75 Ptges 64292 9536 76 Sulf2 72043 55959 77 Xirp1 22437 165904 78 From mouse human 79 Abcc5 27416 10057 80 Ano1 101772 55107 81 Anxa13 69787 312 82 App 11820 351 83 Atp1b1 11931 481 84 Bcam 57278 4059 85 Cd200 100048780 4345 86 Cd200 17470 4345 87 Cd24a 12484 #N/A 88 Cd63 12512 967 89 Cdh1 12550 999 90 Celsr1 12614 9620 91 Chrm3 12671 1131 92 Chrnb1 11443 1140 93 Cldn6 54419 284620 94 Cldn7 53624 1366 95 Cttnbp2 30785 83992 96 Ddr1 12305 780 97 Ednrb 13618 1910 98 Egflam 268780 133584 99 Ehd2 259300 30846 100 Enah 13800 55740 101 Epcam 17075 4072 102 Epha4 13838 2043 103 Espn 56226 83715 104 F2rl1 14063 2150 105 Fermt1 241639 55612 106 Frem2 242022 341640 107 Gja5 14613 2702 108 Gnb5 14697 10681 109 Gpr126 215798 57211 110 Gprc5a 232431 9052 111 Gprc5b 64297 51704 112 Hmcn1 545370 83872 113 Il17rb 50905 55540 114 Ildr1 106347 286676 115 Itga6 16403 3655 116 Itgb8 320910 3696 117 Jam2 67374 58494 118 Kcne3 100044693 10008 119 Kcne3 57442 10008 120 Kcnma1 16531 3778 121 Krt19 16669 3880 122 Mal 17153 4118 123 Mapk8ip1 19099 9479 124 Mfge8 17304 4240 125 Mfi2 30060 4241 126 Ntrk2 18212 4915 127 Ppl 19041 5493 128 Prom1 19126 8842 129 Rab27b 80718 5874 130 Reck 631094 8434 131 Reck 53614 8434 132 Rgma 244058 56963 133 Rtn4 68585 57142 134 Scn2a1 110876 #N/A 135 Shroom3 27428 57619 136 Slc5a1 20537 6523 137 Src 20779 6714 138 Sstr2 20606 6752 139 St14 19143 6768 140 Steap2 74051 261729 141 Tmem45a 56277 55076 142 Vwf 22371 7450 143 Aqp4 11829 361 144 Arhgap10 78514 79658 145 Atp4a 11944 495 146 Clic5 224796 53405 147 Cyp2f2 13107 #N/A 148 Efna3 100046031 1944 149 Efna3 13638 1944 150 Hsd17b6 27400 8630 151 Mrgpre 244238 116534 152 Ncam2 17968 4685 153 Ptges 64292 9536 154 Sulf2 72043 55959 155 Xirp1 22437 165904
(96) In some embodiments, the second protein marker of conventional hepatocyte (cHP) cells is overexpressed in the HybHP cells compared to conventional hepatocyte (cHP) cells. Such overexpressed second proteins are encoded by one or more gene shown in Table 1 and Table 3. In an alternative embodiment, the second protein marker of conventional hepatocyte (cHP) cells is underexpressed in the HybHP cells compared to conventional hepatocyte (cHP) cells. Such underexpressed second proteins are encoded by one or more gene as shown in Table 2.
(97) In some embodiments, the step of isolating the first antibody-HybHP cell-second antibody conjugate from the single-cell suspension comprises fluorescence-activated cell sorting (FACS).
(98) In a second approach, purifying a hepatocyte (HybHP) cell (and/or a population of hepatocyte (HybHP) cells) from a mammalian liver, comprises a) preparing a single-cell suspension from the liver, b) substantially removing ductal cells from the single-cell suspension to obtain a first population of cells that contains conventional hepatocyte (cHP) cells and HybHP cells, and c) combining the first population of cells with at least one first antibody that specifically binds to a first protein marker of liver ductal (DC) cells, wherein the combining is under conditions for specific binding of the at least one first antibody to the first protein marker, and wherein the specific binding produces a first composition that comprises a first-antibody-HybHP cell conjugate, and d) isolating the first antibody-HybHP cell conjugate from the first population of cells, thereby producing a second population of cells that comprises a purified HybHP cell (and/or a purified population of HybHP cell).
(99) In one embodiment, the “isolating” step may be achieved using a second antibody that specifically binds to the one or both of the first antibody-second antibody.
(100) In a further embodiment, removal of ductal cells can be done based on the differences in physical properties (such as size/granularity) of ductal cells on the one hand, and conventional hepatocyte (cHP) cells and HybHP on the other hand, by for example differential centrifugation (Example 1;
(101) C) Methods for In Vitro Culture of HybHP Cells
(102) The invention provides a method for propagating mammalian hybrid hepatocyte (HybHP) cells in vitro, comprising a) combining purified mammalian HybHP cells with culture medium that is suitable for in vitro growth of liver ductal (DC) cells, to produce a first culture composition, b) incubating the first culture composition in vitro under conditions for growth of liver ductal (DC) cells, thereby propagating the HybHP cells. This method is exemplified in Example 8 and
(103) Several culture methods are known for in vitro propagation of DC cells, including the use of matrigel (Example 8,
(104) In some embodiments, the step of combining purified mammalian HybHP cells with culture medium that is suitable for in vitro growth of liver ductal (DC) cells comprises introducing the purified HybHP cells into a three-dimensional matrix (e.g., matrigel) (Example 8 and
(105) D) Methods for Repopulating the Liver and Treating Liver Damage
(106) The invention further provides methods for successfully transplanting the isolated and/or ex vivo propagated HybHP cells into a mammalian subject for repair of liver damage.
(107) Currently, hepatocytes can be derived from iPSCs, however, this procedure does not generate fully functioning hepatocytes. Other approaches use ductal cells, which also does not generate fully functional hepatocytes. The inventors have overcome these issues by utilizing these newly discovered HybHP cells. Hybrid hepatocytes (HybHP) are morphologically similar to conventional hepatocytes but also express the bile duct gene expression program. These cells can repopulate a diseased mouse liver more efficiently than conventional hepatocytes. Unlike conventional hepatocytes, the HybHP can be efficiently grown in vitro with a ductal phenotype and expanded indefinitely. Later, these cells can be reverted to the hepatocyte phenotype for transplantation.
(108) Thus in one embodiment, the invention provides a method for repopulating the liver of a mammalian host subject in need thereof, comprising transplanting purified hybrid hepatocyte (HybHP) cells into the host subject to produce a treated subject that comprises the purified hybrid hepatocyte (HybHP) cells, wherein the transplanting is under conditions for repopulating the liver of the host subject.
(109) The terms liver “repopulation” and “regeneration” by a given cell type (e.g., HybHP cell) interchangeably refer to the proliferation (i.e., increase in number) of the given cell type in a recipient liver, and to its differentiation into other progeny cell types, such as into conventional hepatocyte (cHP) cells and liver ductal (DC) cells. Liver repopulation is assessed by methods disclosed herein, such as using two-dimensional histological sections to measure the relative area covered by HybHP derived cells compared with the total area of hepatocytes (Example 5,
(110) In one embodiment, liver “repopulation” is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and/or 90%. For example 40% repopulation by donor HybHP cells following transplantation of a host liver means that 40% of the total number of hepatocytes are derived from the donor HybHP, and 60% of the total number of hepatocytes are from the host liver. Methods for measuring the level of liver repopulation are known in the art including (Zhu et al., Nature (2014) 508(7494):93-7; Huch et al., Nature (2013) 494(7436):247-50; and Huch et al., Cell (2015) 160:299-312).
(111) While not intending to limit the invention to any particular degree of liver repopulation, in one embodiment, in one preferred embodiment, repopulation is at least 20%. Data herein demonstrate, in each of three independent mouse models, that HybHP show higher regenerative capacity than normal hepatocytes and are far superior to oval cells (Example 2, Example 3, and Example 5). For example, data herein demonstrate a 13 fold proliferation by HybHP cells following transplantation, which resulted in 65% repopulation of the liver.
(112) Also, data herein show that upon in vivo transplantation of HybHP cells, the transplanted HybHP cells differentiate into and/or revert to progeny hepatocytes that repopulate a host liver by 50% after 3 months following transplantation of 45,000 of the HybHP cells into a mammalian host (Example 2, Example 3, Example 5,
(113) While not intending to limit the invention to any particular degree of liver proliferation, in one embodiment, repopulating comprises at least 10 fold proliferation of the HybHP cells. Data herein demonstrate a 13 fold proliferation by HybHP cells following transplantation, which resulted in 65% repopulation of the liver.
(114) “Proliferation” of a given cell type (e.g., HybHP) after a particular treatment (e.g., following transplantation) refer to an increase in the number of the given cell type (e.g., HybHP cell) and/or the number of its progeny cell types (e.g., cHP cells and liver DC cells) after the particular treatment compared to prior to the particular treatment. In one embodiment, “proliferation” of HybHP cell results in at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 61 fold, 62 fold, 63 fold, 64 fold, 65 fold, 66 fold, 67 fold, 68 fold, 69 fold, 70 fold, 71 fold, 72 fold, 73 fold, 74 fold, 75 fold, 76 fold, 77 fold, 78 fold, 79 fold, 80 fold, 81 fold, 82 fold, 83 fold, 84 fold, 85 fold, 86 fold, 87 fold, 88 fold, 89 fold, 90 fold, 91 fold, 92 fold, 93 fold, 94 fold, 95 fold, 96 fold, 97 fold, 98 fold, 99 fold, and/or 100 fold increase in the number of HybHP cells and/or of its progeny cHP cells and/or of its progeny liver DC cells. Data herein demonstrate a 13 fold proliferation by HybHP cells following transplantation, which resulted in 65% repopulation of the liver.
(115) The invention's methods are useful wherein the host subject has liver damage.
(116) In one embodiment, liver repopulation by HybHP and its progeny treats the liver damage in the treated subject compared to the host subject. Data herein demonstrate that after transplantation of 14,000-50,000 HybHP cells, much less than the 500,000-1,000,000 cells commonly used in such studies, all HybHP transplanted animals were still alive compared to the death of 90% of control animals and of 50% of cHP-transplanted mice (Example 5;
(117) “Liver damage” refers to one or more undesirable change in the structure (e.g., fibrosis) and/or synthetic function and/or metabolic function of liver cells and/or liver tissue. Symptoms of liver damage may be assessed by, for example, biopsy and histology, and blood tests to determine relevant enzyme levels or circulating antigen or antibody, and imaging tests (e.g., to detect a decrease in the growth rate or size of hepatocellular carcinoma). For example, liver damage may be determined by assays of enzymes involved in metabolism (e.g., aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, 5′ Nucleotidase, gamma-glutamyl transpeptidase (GGT)), proteins involved in normal blood clotting (e.g., prothrombin time (PT) using international normalized ratio (INR) and/or partial thromboplastin time (PTT)), and/or assays of albumin and/or bilirubin. Liver damage is exemplified by liver disease and liver injury.
(118) “Liver disease” refers to a disorder of one or more of the liver functions. Some of the “symptoms” (i.e., signs) of liver disease include fatigue, flu-like symptoms, dark urine, pale stool, abdominal pain, loss of appetite, unexplained weight loss, and/or yellow skin and eyes (which may be signs of jaundice). Liver disease includes chronic liver diseases (such as cirrhosis and non-alcoholic steatohepatitis (NASH)), hepatocellular carcinoma (HCC), liver inflammation, fibrosis, and hepatitis.
(119) “Cirrhosis” (also known as “hepatic; fibrosis”) is a chronic disease of the liver marked by degeneration of cells, inflammation, and fibrous thickening of tissue. It is typically a result of alcoholism or hepatitis. Cirrhosis has also been increasingly defined by clinical outcomes. In this context, cirrhosis is distinguished between compensated and decompensated stages, with different features, prognoses and predictors of death. Within the compensated stage, two subpopulations have been identified based on the absence or presence of varices, each of which confers a distinct prognosis. Decompensated cirrhosis is defined by the development of clinically evident complications of portal hypertension (ascites, variceal hemorrhage, hepatic encephalopathy) or liver insufficiency (jaundice). The decompensated stage can be subclassified further into a more severe stage defined by the development of recurrent variceal hemorrhage, refractory ascites, hyponatremia and/or hepatorenal syndrome.
(120) “Non-alcoholic steatohepatitis” (“NASH”) is a chronic disease of the liver that develops in patients who are not alcoholic and is characterized by liver inflammation and damage caused by a buildup of fat in the liver, and is histologically indistinguishable from alcoholic hepatitis. NASH can get worse and cause scarring of the liver, which leads to cirrhosis. Laboratory findings of NASH include elevations in aminotransferase levels.
(121) “Hepatocellular carcinoma” (“HCC”), also called “malignant hepatoma,” is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis. Hepatocellular carcinoma may present with yellow skin, bloating from fluid in the abdomen, easy bruising from blood clotting abnormalities, loss of appetite, unintentional weight loss, abdominal pain especially in the right upper quadrant, nausea, vomiting, or feeling tired.
(122) “Liver inflammation” is a reaction that occurs when liver cells are attacked by a disease-causing microbe (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E), substance (e.g., alcohol), or autoimmune disorder.
(123) Liver “fibrosis” is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension.
(124) “Hepatitis” is a disease of the liver characterized by the presence of inflammatory cells in the tissue of the organ. Hepatitis may occur without symptoms, but can lead to jaundice (a yellow discoloration of the skin, mucous membranes, and conjunctiva of the eyes), poor appetite, and fatigue. Depending on the cause, hepatitis can manifest either as an acute or as a chronic disease. Worldwide, viral hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E) is the most common cause, followed closely by alcoholic liver disease and non-alcoholic liver disease (NAFLD). Other less common causes of hepatitis include autoimmune diseases, ingestion of toxic substances, certain medications (such as paracetamol), some industrial organic solvents, and plants.
(125) “Liver injury” refers to mechanical and/or functional damage to liver tissue. The liver can be damaged as a result of impact (for example, a motor vehicle crash), penetrating trauma (such as a knife or gunshot wound), and/or exposure to chemicals, drugs, etc. Injuries may range from relatively small collections of blood (hematomas) within the liver to large tears that go deep into the liver. Because the liver has many large blood vessels, the main problem resulting from liver injury is severe bleeding, particularly within the abdominal cavity. Subjects with liver injury and severe bleeding have symptoms of shock, including a rapid heart rate, rapid breathing, and cold, clammy, pale or bluish skin. Subjects also have abdominal pain and tenderness because blood in the abdomen irritates the abdominal tissue. When bleeding is severe, the abdomen may also be swollen. Liver injury may be diagnosed by computed tomography (CT), ultrasonography, and/or surgery to determine the extent of the injury and to stop the bleeding.
(126) While not intending to limit the type of donor subject that provides the source of the purified HybHP, and without limiting the type of host subject receiving the purified and/or in vitro cultured HybHP cells, in one embodiment, the host subject and the donor subject are different individuals, such as in heterologous liver cell therapy. In another embodiment, the host subject and the donor subject are the same individual (such as in heterologous liver cell therapy).
(127) In some embodiments, the HybHP cells are transgenic. “Transgenic” and “genetically engineered” cell refer to a cell whose genome has been manipulated by any molecular biological technique, including, for example, the introduction of a transgene, homologous recombination, knockin of a gene, and/or knockout of a gene. The term “transgene” as used herein refers to any nucleic acid sequence which is introduced into the cell by experimental manipulations. A transgene may be an “endogenous DNA sequence” that is present in the cell in nature, or a “heterologous DNA sequence” (also referred to as “foreign DNA”) that is not present in the cell in nature (including gene sequences that are found in that cell and that further contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring gene). For example, the transgene may be a marker to facilitate detection of the transgenic HybHP cells and/or its progeny. Alternatively, the transgene may be a therapeutic gene whose expression treats liver damage.
(128) In one embodiment, repopulating the liver does not produce hepatocellular carcinoma (HCC), i.e., the liver of the treated subject lacks HCC. In a further embodiment, the HybHP cells are purified and/or propagated in vitro prior to the transplanting.
(129) The term “transplanting” refers to introducing cells of interest (e.g., HybHP cells) into a tissue of a host subject. Transplanting may be accomplished by intrasplenic injection, injection into the portal vein, direct intrahepatic injection, and/or injection into lymph nodes.
(130) Thus, the invention provides therapeutic methods for the treatment of liver damage. In one embodiment, the invention provides a method for treating one or more of liver damage in a mammalian host subject in need thereof, comprising transplanting a therapeutically effective amount of purified hybrid hepatocyte (HybHP) cells into the host subject to produce a treated subject, wherein the transplanting is under conditions for repopulating the liver of the host subject, thereby treating the one or more of the liver damage.
(131) Data herein demonstrate that HybHP with their high repopulation potential are ideal candidates for liver repair. Data herein demonstrate that after transplantation of 14,000-50,000 HybHP cells, much less than the 500,000-1,000,000 cells commonly used in such studies, all HybHP transplanted animals were still alive compared to the death of 90% of control animals and of 50% of cHP-transplanted mice (Example 5;
EXPERIMENTAL
(132) The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Example 1
(133) Experimental Procedures
(134) Mice
(135) Mouse studies were performed in accordance with NIH guidelines for the use and care of laboratory animals and approved by the UCSD Institutional Animal Care and Use Committee, 500218. Sox9-Cre.sup.ERT2 mice were generated as described (Kopp et al., 2011). For the current study, we utilized a founder that induces recombination in cells expressing high and low levels of Sox9 and exhibits higher recombination efficiency than the founder described in Kopp et al. (2011). In contrast to the previously reported founder, the founder used in this study displays low levels of recombination in the absence of tamoxifen. Sox9-Cre.sup.ERT2 mice were backcrossed to C57Bl/6 mice for at least 8 generations. R26R.sup.YFP (Jax 006148), R26R.sup.tdTomato (Jax 007909), MUP-uPA (Weglarz et al., 2000), Ck19Cre.sup.ERT and Ttr-Cre.sup.ERT and Alb-Cre.sup.ERT (Schuler et al., 2004) mice were also maintained in the C57Bl/6 background. NZG (Jax 012429) mice were maintained in the FBV background and all experiments involving this line were in 50% FBV-057Bl/6 background. In all experiments at least 3 mice were included per experimental group.
(136) Tamoxifen (Sigma-Aldrich) was dissolved in corn oil and injected subcutaneously (s.c.). Mice were fed with 0.1% DDC (Sigma-Aldrich) in LabDiet #5015 (Testdiet) for 6 weeks. Mice were fed with a choline-deficient diet (MP Biomedicals) supplemented with 0.15% ethionine (CDE) in drinking water (MP Biomedicals) for 3 weeks. CCl.sub.4 (Sigma) was dissolved at 1:3 (25%) in corn oil and i.p. injected at 2 μl/gr or dissolved 1:2 (50%) and injected at 2 μl/gr for the acute model. HFD was composed of 59% fat, 15% protein, and 26% carbohydrates based on caloric content (BioServ).
(137) The STAM model of NASH-driven HCC was established as previously described (Fujii et al., 2013). Briefly, 2-day-old male mice were (s.c.) injected with 200 μg streptozotocin (Sigma), and then fed with HFD (Bio-Serv) beginning at 4 weeks of age. NASH-driven HCC in MUP-uPA mice was previously described (Nakagawa et al., 2014). DEN induced HCC was also described (Maeda et al., 2005).
(138) Adenovirus and AAV Vectors
(139) Recombinant adenovirus expressing FLP-recombinase (Ad-Flpo, Vector Biolabs #1775) was expanded and prepared at the Vector Core Developmental Lab at UCSD. Ad-Flpo was diluted in PBS and intravenously injected to mice (1×10{circumflex over ( )}9 plaque-forming units/mouse). AAV-TBG-FLPo was obtained from Vector Biolabs (#1728) and diluted in PBS and intravenously injected to mice (5×10{circumflex over ( )}11 genome copies/mouse).
(140) Immunofluorescence and Immunohistochemical Analysis
(141) Mice were intra-cardial perfused with Zn-Formalin (Polysciences) and excised livers further fixed o.n. in Zn-Formalin. Livers were washed with PBS and incubated for 2 hrs with 100 mM Tris pH 9.4-10 mM DTT. Livers were washed stepwise with PBS-15% and 30% sucrose at 4° C. embedded in Tissue Tek OCT compound (Sakura Finetek) and frozen. Tissue blocks were cut with a cryostat to 8 μm sections. Slides were washed in PBS and antigen retrieval was performed with citrate pH6 buffer at 96° C. for 1 hr. After cooling and washing the slides with PBS, they were incubated with PBS-0.1% Triton-X-100 for 20 min. After extensive washing with PBS, the slides were blocked with PBS-0.1% Tween-2% Donkey serum for 30 min. Antibodies were diluted in the same blocking buffer and incubated at 4° C. o.n. Slides were washed 3 times with PBS-0.1% Tween and incubated with corresponding secondary antibodies diluted in blocking solution for 2 hrs, followed by 3×PBS-0.1% Tween and 2×PBS washes. Slides were washed further with deionized water and 70% ethanol prior to incubation with 0.1% Sudan Black (Sigma) in 70% ethanol for 20-30 min. Extensive washing with PBS-0.2% Tween was performed before incubating the slides with DAPI for nuclear staining and mounting with Mowiol. Imaging was performed with a Zeiss Axioimager2 and Hamamatsu Nanozoomer. Images were processed using Zeiss ZEN and NDPview software.
(142) Antibodies used were: chicken α-GFP (Abcam #ab13970), 1:200 for YFP and 1:1250 for GFP. Rabbit α-Sox9 (Santa Cruz #sc-20095) 1:50. Goat α-Opn (R&D #AF808) 1:100. Goat α-Ck19 (Santa Cruz #sc-33111), 1:100. Mouse α-Lacz (Promega #Z3781), 1:50. Goat HNF-4α (Santa Cruz #sc-6556), 1:50. Mouse α-GS (BD #610517), 1:200. Rabbit α-Ki67 (Gentex #GTX16667), 1:50. Rabbit α-FAH (AbboMax #602-910), 1:200. Alexafluor fluorescent antibodies (Molecular Probes, Invitrogen), 1:800. Donkey Alexa-488 α-Chicken (Jackson), 1:800.
(143) For tyramide amplification signal (TSA), Life Technologies T-20925 kit was used following manufacturer instructions. Antibodies used with TSA were rabbit α-Sox9 (Millipore #5535), 1:20,000. Goat α-Opn (R&D #AF808) 1:2000. Rabbit α-Agxt2l1 (Sigma #HPA044546), 1:1000. Goat α-Aqp4 (Santa Cruz #sc-9888), 1:10000.
(144) Normal human liver paraffin sections were stained with rabbit α-Sox9 (Millipore #5535), 1:100 and Goat α-Opn (R&D #AF1433) 1:100. ImmPRESS-AP Anti-Rabbit Ig (alkaline phosphatase) Polymer Detection Kit and Immpact VECTOR Red Alkaline Phosphatase (AP) Substrate Kit were used for Sox9. ImmPRESS HRP Anti-Goat Ig (Peroxidase) Polymer Detection Kit and ImmPACT NovaRED Peroxidase (HRP) Substrate were used for Opn.
(145) Quantification of HybHP Labeling Efficiency
(146) To estimate the number of HybHP cells in the unchallenged liver, sections from Sox9-GFP mice livers were stained with GFP and β-Catenin antibodies to delineate the boundaries of hepatocytes. Stained slides including at least 4 lobes were scanned with Nanozoomer and for each slide 7-9 areas ranging from 0.05 to 0.25 mm.sup.2 were drawn. Hepatocytes contained in the areas were manually quantified Linear regression was then used to model the relationship between area and number of hepatocytes. We observed substantial variability in estimates of hepatocyte number obtained when applying models built from different slides (estimates were up to 20% different) suggesting that models did not generalize across slides. Thus we applied this procedure independently to every slide quantified. In all cases area explained most of the variability in hepatocyte count with R.sup.2>0.98. In parallel, different areas ranging from 4 to 10 mm.sup.2 were drawn from the same slides. Areas corresponding to CV and PV empty spaces were subtracted from the total area and the total number of hepatocytes was estimated using the slide-specific regression models. The number of HybHP (GFP.sup.+) within these larger areas was counted manually. The same procedure was repeated in Sox9-Cre.sup.ERT; R26R.sup.YFP mice treated with 100 mg/kg of tamoxifen. The labeling efficiency, estimated by the ratio between labeled HybHP (YFP.sup.+) and total HybHP (GFP.sup.+) estimated in Sox9-GFP mice was 51%.
(147) Clarity
(148) Whole livers from Zn-Formalin perfused Sox9-Cre.sup.ERT; R26R.sup.tdTomato mice were isolated and incubated in a polyacrylamide hydrogel solution as previously described (Chung et al., 2013, Yang et al., 2014) for 1 week. Samples were embedded in a polymerized hydrogel by raising the temperature to 37° C. for 3 hours, and clarified by incubating in a solution of 4% Sodium Dodecyl Sulfate (SDS, Amresco) in Sodium Borate buffer (200 mM, pH 8.5, Sigma) at 37° C. for 2-3 weeks, followed by washing for 2 days in PBS+0.1% Triton X-100 (PBST, Sigma). Individual lobes were isolated and stained with anti-CK19 antibody (TROMAIII Developmental Studies Hybridoma Bank) at 1:100 dilution in PBST, washed in PBST 4 times, and stained in donkey anti-rat secondary antibody (Jackson Immunoresearch) at 1:100 dilution in PBST, washed in PBST plus DAPI (1:5000 dilution), and incubated in FocusClear (CelExplorer) prior to imaging.
(149) For imaging, samples were incubated in FocusClear mounted on coverglass-bottom dishes (Wilco) as previously described (Chung et al., 2013) and visualized with a single-photon excitation scanning confocal microscope system (Olympus), using a 10×, 0.60NA, 3 mm WD water-immersion objective (Olympus). Images were taken with z-step size of 1 um and reconstructed using Imaris software (Bitplane).
(150) Liver Perfusion, Flow Cytometry and Cell Sorting
(151) For flow cytometry analysis, liver single cell suspensions were isolated by two step collagenase digestion and differential centrifugation. Single cell suspensions were analyzed using HNF-4α (Santa Cruz #sc-6556) and CK19 (TROMAIII Developmental Studies Hybridoma Bank) antibodies. Donkey anti-goat alexa-647 and donkey anti-goat alexa-405 (Molecular Probes, Invitrogen) were used as secondary antibodies. For intracellular staining Transcription Factor Staining Kit (BD Biosciences) was used. Fixable Viability Dye eFlour® 780 Dye or eFlour® 506 were used for exclusion of dead cells (eBioscience). Samples were measured on a CyAn™ ADP flow cytometer (Beckman Coulter, USA) and analyzed with FlowJo.8 software (Tree Star, USA) or a BD Influx for cell sorting.
(152) RNA Extraction, Sequencing and Data Analysis
(153) Sorted cells were pelleted and lysed with Trizol. Extracted aqueous phase was then purified with RNAeasy microkit (Qiagen) following manufacturer instructions. 200 ng of RNA with an RNA Integrity Number (RIN) of 8.5 or greater was used to generate libraries using Illumina's TruSeq Stranded mRNA Sample Prep Kit. The manufacturer's protocol was followed, with the exception that RNA was fragmented for 5 minutes. Libraries were multiplexed and sequenced with 50 basepair (bp) single end reads (SR) to a depth of approximately 20 million reads per sample on an Illumina HiSeq2000 using V3 chemistry.
(154) Fastq files were aligned to the mouse genome (mm9 release) with STAR v2.3.0e (Dobin et al., 2013). Differential gene expression was obtained using the Homer pipeline (Heinz et al., 2010). The analyzeRepeats.pl script was used to obtain integer read counts from the alignments. Read counts were aggregated across exons and condensed at the gene level. Log 2 fold change, p-values and false discovery rates (FDR) for differentially expressed genes were obtained with Homer's getDiffExpression.pl script using the R EdgeR software (Robinson et al., 2010). Genes achieving an FDR less than 0.05 were considered differentially expressed. Gene classes were defined by: 1—HybHP/BD>HP (n=49) and BD>HybHP>HP (n=140). 2—HP/BD>HybHP (n=61); BD>HP>HybHP (n=58) and HP>BD>HybHP (n=2). 3—HybHP>HP/BD (n=42) and HybHP>HP>BD (n=2). 4—HP>HybHP/BD (n=71) and HP>HybHP>BD (n=65). Differentially expressed genes were analyzed with DAVID (Default settings without PIR_Superfamily, SMART, COG Ontology, SP_PIR_KEYWORDS and UP_SEQ_FEATURE) (Huang da et al., 2009a, b) to identify biological processes enriched for each category.
(155) Transplantation
(156) Fah.sup.−/− mice (Grompe et al., 1993) were crossed with the Il2rg.sup.−/− (B6.129S4-Il2rgtm1Wjl/J) mice from the Jackson Laboratory and Rag2.sup.−/− mice (RagN12) from Taconic Farms. Genotyping was done with the same primers and conditions as described elsewhere (Grompe et al., 1993) or according to the protocols provided by vendors. Mice were kept on 7.5 mg/ml NTBC (100%) via drinking water. Mice were kept in temperature- and humidity-controlled animal quarters with a 12-hr light-dark cycle.
(157) Adult mice were transplanted as previously described (Ponder et al., 1991). Mice were anesthetized with isoflurane and kept on a heating pad during the whole procedure. Then a mid-abdominal incision was performed and the spleen was prepared for injection. The lower pole of the spleen was injected with the three different cell types—oval cells, cHP and the HybHP re-suspended in DMEM 15% FBS. Closing of the abdominal muscle layer was performed with 4-0 silk sutures, and the skin was stapled. NTBC was immediately withdrawn in a stepwise gradation with each step lasting a day (25%, 12%, 6% and 3% of 7.5 mg/ml colony maintenance concentration). After three weeks of the first cycle, the mice were put back on 100% NTBC containing water (7.5 mg/ml) for one week and then returned to 0% NTBC as described earlier in daily steps. The second cycle on regular water was continued for 4 weeks after which tissues were collected.
(158) For the survival study, animals were intrasplenically transplanted with 14000-40000 of the two cell types HybHP and cHP as described above. The animals went through two cycles of NTBC withdrawal (each comprising of 3 weeks on water followed by one week on NTBC) at the end of which they were maintained only on regular water (starting post-op day 68). The periods on NTBC and on regular water are schematically depicted in the
Example 2
(159) Periportal Hepatocytes with High Regenerative Capacity
(160) Since the portal area may be the organizing center for liver repair, harboring putative stem cell niches (Kuwahara et al., 2008), we aimed to analyze the regenerative capacity of cells in this region using transgenic mice expressing GFP from the Sox9 promoter, which is primarily active in bile duct cells and bipotential hepatobilliary progenitors (Gong et al., 2003). Immunofluorescence (IF) analysis indicated that bile duct cells (CK19.sup.+) showed high GFP expression (
(161) To examine the regenerative and differentiative capacity of Sox9.sup.+ periportal hepatocytes and determine whether they differ from other hepatocytes that are Sox9-GFP negative, we used a Sox9-Cre.sup.ERT transgenic line suitable for labeling low Sox9 expressing cells (see Experimental Procedures). When expressed in combination with a R26R.sup.YFP reporter line and treated with tamoxifen, the Cre.sup.ERT protein enters the nucleus and excises the STOP cassette that prevents YFP expression only in Sox9 expressing cells. Once recombination occurs, the cell and its progeny are permanently YFP labeled. In Sox9-Cre.sup.ERT; R26R.sup.YFP mice, some ductal cells (CK19.sup.+ HNF4α.sup.−) were labeled with YFP prior to tamoxifen administration (
(162) Next, we analyzed the behavior of YFP.sup.+ HybHP during the regeneration period that follows different types of liver injury. First, we examined MUP-uPA transgenic mice, which undergo liver damage due to ER stress induced by expression of urokinase-type plasminogen activator (uPA) in hepatocytes (Nakagawa et al., 2014b; Weglarz et al., 2000). MUP-uPA mice first display liver damage at 3 weeks of age, and after peaking at 5 weeks the damage dissipates by 13 weeks (Weglarz et al., 2000). Without tamoxifen treatment, 99.9% YFP.sup.+ cells were also CK19.sup.+ in 9 weeks old Sox9-Cre.sup.ERT; MUP-uPA; R26R.sup.YFP mice (6 weeks after damage onset;
(163) We also used repetitive CCl.sub.4 administration which causes pericentral damage that eventually gives rise to fibrosis (Wong et al., 1998) as an independent model of chronic liver injury. No oval cell expansion takes place and new hepatocytes are not derived from ductal cells in this model (Español-Suñer et al., 2012; Grompe, 2014; Rodrigo-Torres et al., 2014; Tarlow et al., 2014a; Yanger et al., 2014). After twice-weekly CCl.sub.4 administration for 6 weeks, 34.5%±2.2 of hepatocytes in Sox9-Cre.sup.ERT; R26R.sup.YFP mice previously treated with 100 mg/kg tamoxifen were YFP.sup.+, and hence derived from HybHP, with most cells extending along the hepatic cords, from the periportal region to the central vein (
(164) We confirmed the contribution of HybHP to newly produced hepatocytes using an independent genetic tool. We reasoned that by labeling hepatocytes sparsely, we could compare the clonal behavior of HybHP to that of parenchymal hepatocytes. We found that the TTR-Cre.sup.ERT driver (Tannour-Louet et al., 2002) can be used to randomly label hepatocytes in undamaged liver (
Example 3
(165) Clonally Labeled HybHP Produce New Hepatocytes and Transdifferentiate into Duct Cells
(166) To further examine the role of HybHP in regeneration after chronic liver damage, we used a system in which only HybHP are specifically and clonally labeled without a strict dependence on tamoxifen. We crossed Sox9-Cre.sup.ERT and NZG mice (Yamamoto et al., 2009), which contain a loxP-flanked STOP cassette upstream of a nuclear targeted LacZ marker as well as a downstream GFP marker whose expression is prevented by the LacZ cassette, which is flanked by Frt sites (
(167) Biphenotypic hepatocytes expressing ductal markers were reported in different mouse models of cholestatic liver injury (Sekiya and Suzuki, 2014; Tanimizu et al., 2014; Tarlow et al., 2014b; Yanger et al., 2013). These cells are thought to descend from mature hepatocytes during cholestatic liver damage. Virus-mediated hepatocyte labeling or transplantation of labeled hepatocytes into damaged livers (Tanimizu et al., 2014; Tarlow et al., 2014b; Yanger et al., 2013) showed that a few hepatocytes can trans-differentiate into duct cells. Other studies have shown that almost all duct cells arose from biphenotypic hepatocytes (Sekiya and Suzuki, 2014), but another report ruled out transdifferentiation of mature hepatocytes into ductal cells in the same cholestatic injury models (Malato et al., 2011). We reasoned that transdifferentiation of HybHP during cholestatic liver injury may explain the above discrepancy. We tested this possibility with the NZG dual recombinase system that specifically labels HybHP and triggered cholestatic injury with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) diet (Preisegger et al., 1999). After 6 weeks of DDC feeding, most labeled HybHP underwent pronounced morphological changes, with cell and nuclear sizes diminished and acquisition of strong expression of Sox9 and the ductal marker osteopontin (Opn) (
Example 4
(168) HybHP Exhibit a Unique Transcriptome
(169) To compare the molecular characteristics of HybHP to those of conventional hepatocytes (cHP) and bile duct cells (BD), we isolated the three populations by sorting collagenase liver digests from tamoxifen (100 mg/kg)-treated Sox9-Cre.sup.ERT; R26R.sup.tdTomato mice given an additional tamoxifen dose (20 mg/kg) 10 days later to maximize HybHP labeling (
(170) It was previously shown that normal human liver contains periportal hepatocytes with simultaneous HNF4α and HNF1β expression (Isse et al., 2013). We also detected hSox9 and hOpn in some periportal hepatocytes in human liver sections (
(171) Furthermore, we conducted a simulation study to determine whether HybHP-specific signals, could be due to BD contamination. The simulation identified a group of genes with large differences in expression between HybHP and cHP, which are extremely unlikely to have originated from a mixture of BD+cHP (P-value<0.001; Example 7;
Example 5
(172) Transplanted HybHP Display High Regenerative Capacity
(173) The high regenerative potential and plasticity of HybHP make them attractive candidates for liver disease cell therapy. We therefore performed transplantation experiments using Fah.sup.−/− mice, which due to FAH (fumarylacetoacetate hydrolase) deficiency, undergo spontaneous liver damage upon withdrawal of NTBC (2-nitro-4-trifluoromethylbenzoyl-1,3-cyclo-hexanedione), and if left untreated succumb to fatal liver failure within 1-2 months (Grompe et al., 1995). We isolated HybHP (tdTomato.sup.+) and cHP (tdTomato.sup.−) from tamoxifen-treated Sox9-Cre.sup.ERT; R26R.sup.tdTomato mice and oval cells (tdTomato.sup.+) from CDE diet-fed animals of the same genotype. Although it was reported that DDC-induced oval cells can repopulate the Fah.sup.−/− liver (Wang et al., 2003), considering the ability of HybHP to transdifferentiate into duct cells in DDC-fed mice (
Example 6
(174) Neither HybHP Nor Oval Cells Give Rise to HCC
(175) In many experimental models of hepatic carcinogenesis, oval cell responses precede the emergence of neoplasia, as has been observed in humans where ductular reactions precede cancer in cirrhotic livers (Roskams, 2006). Such observations led to the suggestion that oval cells could be the origin of a large fraction of liver cancers (Alison et al., 2009). Given the important role of liver damage and compensatory proliferation in liver tumorigenesis (Kuraishy et al., 2011; Maeda et al., 2005), the relationship between cell proliferation and cancer risk (Tomasetti and Vogelstein, 2015) and the high proliferation rate of HybHP during chronic liver injury, we examined the potential contribution of both oval cells and HybHP to hepatic carcinogenesis. We traced HybHPs and oval cells in three independent mouse models of HCC: DEN-induced HCC (Maeda et al., 2005), MUP-uPA mice fed with high fat diet (HFD) (Nakagawa et al., 2014b) and the STAM model of diabetes-promoted HCC (Fujii et al., 2013), using Sox9-Cre.sup.ERT; R26R.sup.YFP mice and the same labeling conditions as above. Whereas DEN is metabolically activated in pericentral/zone 3 hepatocytes and does not induce oval cell expansion, consumption of HFD, which induces liver damage and compensatory proliferation in both MUP-uPA and STAM mice, gives rise to substantial oval cell proliferation (
(176) We next analyzed the relationship between HybHP and the oval cell response. Whole slide scans of Sox9-Cre.sup.ERT; R26R.sup.YFP; MUP-uPA livers revealed that in the portal tracts where HybHP are expanding, only a few oval cells were present (
Example 7
(177) Statistical Analysis
(178) 1. Data Pre-Processing and Model Goodness-of-Fit Assessment
(179) In order to simulate expression data for the HybHP under various hypothesized relationships with cHP and BD, we considered five probability models (Poisson, Negative Binomial, Tweedie, Normal, Gamma) and assessed the degree to which they captured the nature of the observed RNA-seq data. For these analyses we used the 13,827 genes that had at least 100 read counts across all samples, in order to consider genes with relatively precise gene expression measurements.
(180) The first three models focus on the gene-level read counts, assuming that these arise from Poisson, Negative Binomial or Tweedie distributions (respectively). Esnaola et al (2013) pointed that, although commonly used for the analysis of RNA-seq data, the Poisson and Negative Binomial are often an inadequate description of RNA-seq counts and that a generalization based on the Tweedie distribution is needed. As suggested by the authors we used function gofTest from R package TweeDEseq to produce quantile-quantile plots (
(181) As an alternative, the Normal and Gamma models assume a continuous distribution for gene expression. As these would be inadequate for read counts, we computed log 2 reads per kilobase per million (RPKM) and applied quantile normalization to remove systematic biases across samples. We assessed goodness-of-fit of the Normal model using function qqnormGenomeWide from R package casper (Rossell et al., 2014), observing a departure from Normality (
(182) 2. Testing the Hypothesis that HybHP Arises as Noise from cHP
(183) We conducted an analysis to confirm that HybHP expression cannot be explained as simply a noisy version of cHP expression, i.e. we considered the null hypothesis that for all genes there are truly no differences between the HybHP and cHP groups.
(184) HybHP samples clustered separately from cHP samples both in a Principal Components (
(185) Pattern 0: BD=cHP=HybHP
(186) Pattern 1: cHP=HybHP≠BD
(187) Pattern 2: BD=HybHP≠HP
(188) Pattern 3: BD=cHP≠HybHP
(189) Pattern 4: BD≠HP≠HybHP
(190) GaGa estimated that 0.3% genes arose from Patterns 2-4, which correspond to configurations under which there truly are differences in expression between HybHP and cHP.
(191) To conduct a formal statistical test we simulated 1,000 genome-wide datasets under the null hypothesis (no differences between HybHP and cHP for any gene) and assuming that the data are either Gamma (for log 2-RPKM) or Tweedie (for read counts) distributed. For the Gamma simulations we relabeled HybHP and cHP samples as belonging to a single group and generated new samples from the posterior predictive distribution of the GaGa model (Rossell, 2009), as implemented in function simnewsamples in the R package gaga. For the Tweedie simulations we adapted the rPT function in R package TweeDEseq. Given that the numerical optimizer in rPT to find parameter estimates may fail to converge for certain genes, whenever this happened we used the Negative Binomial distribution instead.
(192) We produced a Principal Components plot showing the observed and simulated data (
(193) 3. Testing the Hypothesis that HybHP is a Mixture of cHP and BD
(194) We performed an analysis to rule out the hypothesis that the HybHP samples were generated by a mixture of cHP cells contaminated by a small fraction of BD cells. Under this hypothesis, the expression for any gene g should follow the pattern HybHP.sub.g=w BD.sub.g+(1−w) cHP.sub.g, where w is the proportion of contaminating BD cells (common across all genes), or equivalently the linear model HybHP.sub.g/cHP.sub.g=w BD.sub.g/cHP.sub.g+1−w. The common parameter w was estimated using robust linear regression as implemented in function rlm from R package MASS (Hampel et al., 1986; Venables and Ripley, 2011).
(195) To prevent outliers in the HybHP.sub.g/cHP.sub.g or BD.sub.g/cHP.sub.g fold changes from unduly biasing the estimate, we obtained an estimated w=0.8% with 95% bootstrap confidence intervals [0.1%, 2.8%].
(196) To test the mixture hypothesis we pre-screened candidate genes unlikely to arise from such a mixture by selecting the 770 genes with HybHP.sub.g/cHP.sub.g above 2 in both direction and read count >10 in the cHP group. We then simulated 10,000 datasets under the mixture hypothesis using the GaGa model (Algorithm 1).
(197) Algorithm 1. Simulation of Gene Expression Data Under the Mixture Hypothesis
(198) For b=1, . . . , 10000, do the following steps.
(199) 1. Simulate 3 new cHP.sup.(b) and HybHP.sup.(b) samples from the GaGa model posterior predictive under the pattern HybHP BD for the 770 genes. Center simulated data so that the grand average equals that in the observed data, i.e. 0.5(cHP.sub.g.sup.(b)+HybHP.sub.g.sup.(b))=0.5(cHP.sub.g+HybHP.sub.g).
2. Simulate a value of the proportion of BD cells w.sup.(b) from a bootstrap rlm fit.
3. Generate 3 new HybHP samples as HybHP.sub.g.sup.(b)=w.sup.(b) BD.sub.g.sup.(b)+(1−w.sup.(b)) cHP.sub.g.sup.(b).
Algorithm 1 incorporates the uncertainty both in the parameter estimates at the gene level and that in w, and uses a Gamma distribution for the observations as it provided a better fit to the observed data that the other four probability models described in Example 7, under the heading “Testing the hypothesis that HybHP arises as noise from cHP.” Under the mixture hypothesis, for each gene the proportion p.sub.g of simulated HybHP.sub.g.sup.(b)/cHP.sub.g.sup.(b) below the observed HybHP.sub.g/cHP.sub.g should follow a Uniform(0,1) distribution. Hence we computed a two-tailed P-value as min{p.sub.g,1−p.sub.g}, and applied Bonferroni's adjustment to determine statistically significant hits. To ensure that no hits were due to Monte Carlo error in the P-value estimate, we grouped the 770 genes into 15 subgroups according to their BD.sub.g/cHP.sub.g values and computed the interval containing 95% of the simulated fold changes (
(200) The results of the statistical analysis are shown in
Example 8
(201) Culture of Isolated HybHP In Vitro
(202) In practice, the therapeutic potential of HybHP will depend on the ability to maintain and expand them in culture. In recent years, there has been an explosion of three dimensional (3D) cell culture techniques, which have now been applied to a great variety of tissue and cell types. The liver has been no exception, and it was reported that ductal cells can form organoids that can be expanded for extended periods of time without compromising genome stability. Importantly, hepatocytes do not form organoids under the same conditions as ductal cells. Since HybHP retain a ductal character and even transdifferentiate into ductal cells in vivo in the setting of cholestatic injury, when cultured in the 3D culture conditions, HybHP could transdifferentiate and generate ductal organoids allowing their expansion.
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(205) Each and every publication and patent mentioned in the above specification is herein incorporated by reference in its entirety for all purposes. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.