WO2002029012A1 - Procede de multiplication par clonage de cellules souches hepathiques - Google Patents

Procede de multiplication par clonage de cellules souches hepathiques Download PDF

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WO2002029012A1
WO2002029012A1 PCT/US2000/027428 US0027428W WO0229012A1 WO 2002029012 A1 WO2002029012 A1 WO 2002029012A1 US 0027428 W US0027428 W US 0027428W WO 0229012 A1 WO0229012 A1 WO 0229012A1
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hepatic
progenitors
cells
progeny
mixtures
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PCT/US2000/027428
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Hiroshi Kubota
Lola M. Reid
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University Of North Carolina
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Priority to IL15538700A priority Critical patent/IL155387A0/xx
Priority to CA2424779A priority patent/CA2424779C/fr
Priority to CN00820051A priority patent/CN1461341A/zh
Priority to PCT/US2000/027428 priority patent/WO2002029012A1/fr
Priority to KR1020037004803A priority patent/KR100887623B1/ko
Priority to AU7753500A priority patent/AU7753500A/xx
Priority to JP2002532583A priority patent/JP2004510434A/ja
Priority to AU2000277535A priority patent/AU2000277535B2/en
Priority to EP00967317A priority patent/EP1325111A1/fr
Publication of WO2002029012A1 publication Critical patent/WO2002029012A1/fr
Priority to HK03106718.5A priority patent/HK1054570A1/zh

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the present invention relates to novel conditions for clonal growth of mammalian hepatic progenitors, including pluripotent cells, stem cells, and other early hepatic progenitor cells.
  • the invention relates to methods of propagating hepatic progenitor cells using defined culture medium and feeder cells in co-cultures.
  • the invention relates to the cells used as feeders and capable of sustaining hepatic progenitor cell growth.
  • Progenitor cell populations are ideal targets for gene therapy, cell transplantation and for tissue engineering of bioartificial organs (Millar, AD. 1992 Nature 357, 455; Langer, R. and Vacanti, J. P. 1993 Science 260, 920; Gage, F.H. 1998 Nature 392, 18).
  • tissue-specific, "determined" stem cells or progenitors having high growth potential and/or pluripotentiality is readily apparent from studies on hematopoietic stem cells (Spangrude, G.J. et al.
  • neuronal stem cells (Davis, A. A., and Temple, S. 1994 Nature 372, 263; Stemple, D. L., and Anderson, D. J. 1992 Cell 71, 973) and epidermal stem cells (Jones, P. H. and Watt, F. M. 1993 Cell 73, 713), each having been identified clonally by using the particular methods appropriate for that tissue.
  • progenitors are regarded as the cells responsible for normal hematopoietic, neuronal or epidermal tissue homeostasis and for regenerative responses after severe injury (Hall, P. A., and Watt, F. M. 1989 Development 106, 619).
  • the mammalian adult liver has a tremendous capacity to recover after either extensive hepatotoxic injury or partial hepatectomy (Fishback, F. C. 1929 Arch. Pathol. 7, 955); (Higgins, G. M. and Anderson, R. M. 1931 Arch. Pathol. 12, 186), even though the liver is usually a quiescent tissue without rapid turnover.
  • Data from recent studies in the mouse have been interpreted to suggest that adult parenchymal cells have an almost unlimited growth potentiality as assayed by serial transplantation experiments (Overturf et al. 1997 Am. /. Pathol. 151, 1273); (Rhim, J. A. et al. 1994 Science 263, 1149).
  • hepatic cells are intrinsically sensitive to developmental stress stimuli or that the particular microenvironment in fetal liver per se causes such destructive effects (Doi, T. S. et al 1999 Proc. Natl. Acad. Sci. USA 96, 2994).
  • the basic architecture of adult liver is dependent on the appearance of the initial cylinder of bile duct epithelium surrounding the portal vein (Shiojiri, ⁇ . 1997 Microscopy Res. Tech. 39, 328).
  • the first sign of the differentiation of intrahepatic bile duct epithelial cells is the expression of biliary- specific cytokeratin (CK).
  • CK proteins the cytoplasmic intermediate filament (IF) proteins of epithelial cells
  • IF intermediate filament
  • CK19 is one of the most remarkable biliary markers, because adult hepatocytes don't express CK19 at all, whereas adult biliary epithelial cells do express this protein.
  • Only CK8 and CK18 are expressed through early hepatic cells to adult hepatocytes (Moll, R. et al. Cell 1982, 37, 11.
  • U.S. Patent No. 5,559,022 to Naughton et al claims liver reserve cells that bind Eosin Y, a stain that was used to characterize the "reserve cells", but did not use well-established markers for liver cells, nor provided methods for clonal expansion, nor provided markers by which to isolate viable liver reserve cells.
  • methods for clonal growth of hepatic progenitors Clonal growth is essential as a clear and rigorous distinction and identification of pluripotent hepatic progenitors.
  • U.S. Patent 5,405,772 to Ponting claims a culture medium for cell growth. The
  • Patent No. 5,405,772 requires the use of 3-30 ⁇ g/ml cholesterol, 5-30 ⁇ g/ml nucleosides, and either 2-100 ⁇ g/cm 2 collagen IV or 0.5 - 100 ⁇ g/cm 2 fibronectin. There is a need for a culture medium that is specific for, and optimized for, hepatic progenitor cell growth.
  • U.S. Patent No. 4,914,032 to Kuri-Harcuch et al. claims a process for culturing hepatocytes.
  • Patent No. 4,914,032 fails to teach either the culture of hepatic progenitors or clonal growth conditions for hepatic cells.
  • Patent 5,030,105 to Kuri-Harcuch et al. claims methods of assessing agents by treating hepatocyte cultures. There is an unfilled need for clonal growth conditions so that defined populations of cells may be used for testing and also for methods for the culture of hepatic progenitors.
  • the 5,858,721 patent is limited, however, by the requirement for a framework of biocompatible, non-living material.
  • the instant invention by contrast, there is an unfilled need for growth conditions that do not require a synthetic meshwork.
  • the present inventors have recognized the inadequacy of growing mature liver cells, such as hepatocytes, rather than the far more useful hepatic progenitors. They have carefully defined the isolation parameters for hepatic progenitors and requirements for clonal growth.
  • the progenitor cells and the methods for selecting and culturing the progenitors have many uses, including utility in medicine for treatment of patients with liver failure, and utility for evaluation of toxicity agents, and utility for evaluation of drags.
  • US Patent Nos. 5,576,207 and 5,789,246 to Reid, et al. teach the need for feeders and a hormone-supplemented defined medium.
  • embryonic liver stromal cells in combination with defined extracellular matrix substrata, and a serum-free, hormonally defined medium as conditions for expansion of hepatic progenitors.
  • the defined medium used was more complex than the one used by the instant invention; the cells were plated onto purified matrix substrata (type IV collagen and laminin), whereas here they are plated directly onto the feeders (that supply that matrix); and the embryonic stromal cells were prepared as primary cultures of embryonic livers and were not established as cell lines.
  • the feeder cells are provided by a far easier, more practical and more reproducible means of supporting the cells.
  • the STO feeders will not restrict support to just hepatic progenitors but can be used for progenitors from multiple tissue types.
  • the prior patent the hepatic progenitor cultures were seeded at high cell densities and expansion of them was observed as colony formation, meaning that the aggregates of the cells, not clones of cells, were induced to proliferate.
  • the present invention relates to a method of propagating progenitors, their progeny, or mixtures thereof.
  • the present invention relates to a method of propagating endodermally-derived progenitors, their progeny, or mixtures thereof.
  • the cells are derived from endodermal tissue.
  • the endodermally-derived progenitors, their progeny, or mixtures thereof are cultured on a layer comprising feeder cells in a culture medium.
  • the progenitors, their progeny, or mixtures thereof can be vertebrate cells.
  • the progenitors, their progeny, or mixtures thereof can express the phenotype ICAM or ICAM-1 positive and classical MHC class I antigen negative.
  • the classical MCH class I antigen is also termed MHC class la antigen.
  • the present invention also relates to a method of culturing hepatic stem and other progenitor cells using a serum-free, hormone-supplemented, defined medium and feeder cells. Also, the invention relates to a method of culturing the progeny of progenitor cells, or combinations of progenitor cells and progenitor progeny. Preferably, the progenitor cells are hepatic progenitors. Likewise, the present invention relates to a method of cloning hepatic pluripotent progenitor cells using specific culture conditions. Preferably, the invention relates to a method of cloning hepatic pluripotent progenitor cells.
  • the hepatic pluripotent progenitor cells may be derived from any invertebrate or vertebrate species and more preferably mammalian. Even more preferably, the hepatic pluripotent progenitor cells are human, primate, pig, dog, rat, rabbit or mouse in origin. Most preferably the pluripotent progenitor cells are human in origin.
  • the invention teaches particular culture conditions that are required for the ex vivo expansion of hepatic progenitor cells, and their progeny.
  • the invention also teaches use of embryonic feeder cells, such as STO mouse embryonic cells, as feeder cells for hepatic progenitors.
  • the feeder cells are used in combination with a novel serum-free, hormonally defined medium (HDM) taught in the invention.
  • HDM hormonally defined medium
  • the invention relates to methods of cloning feeder cells capable of sustaining propagation of hepatic progenitor cells, and their progeny.
  • the invention also relates to specific cell lines that, when used as feeders, support hepatic progenitor cell growth.
  • the invention additionally relates to methods of cloning hepatic progenitor cells.
  • the invention teaches the use of the hepatic cell lines and the HDM-STO co- culture system for development of an in vitro colony forming assay (CFA) for defining clonal growth potential of freshly isolated hepatic progenitors.
  • CFA colony forming assay
  • progenitors from E13 rat livers corresponding to El 1.5 in the mouse, and with high growth potential have the same phenotype as classical MHC class I (RT1A 1 ) " , OX18 (pan-MHC class I) du11 , and intracellular adhesion molecule 1 (ICAM-1) + .
  • the invention additionally relates to the culture medium capable of sustaining clonal hepatic cell growth.
  • the culture medium features several specific hormones and nutrients and an absence of serum.
  • the invention relates to the culture of heptic progenitors in medium with feeder cell biosynthetic products.
  • the invention further relates to methods of inducing hepatic cell differentiation, including production of hepatocyte and biliary cell phenotypes.
  • Epidermal growth factor (EGF) is taught in this invention to influence both growth of the progenitor colonies and their fates as either hepatocytes or biliary epithelial cells.
  • Figure 1 is a characterization of hepatic cell lines from day 15 fetal rat liver.
  • Figure 2 is an assay of colony formation on fibroblast feeder cells.
  • Figure 3 is an expression of rat cell surface antigens on various hepatic cell lines in adult liver cells.
  • Figure 4 depicts phenotypic analysis of day 13 fetal rat livers.
  • Figure 5 depicts characterization of hepatic colonies in the absence and presence of EGF.
  • Figure 6 depicts induction of CK19 expression on RT1 A 1" hepatic cells.
  • Figure 7 is a schematic representation of hepatic colony formation on STO5 feeder cells.
  • the instant invention is a process for propagation and use of stem cells.
  • Various tissues are appropriate sources of progenitors, including tissues of ectoderm, mesoderm and endoderm origin.
  • the ectoderm tissues can include skin tissue, brain tissue and other nerve tissue.
  • the mesoderm tissues can include muscle, the blood and hemopoietic systems.
  • the endoderm tissues can include the gut, stomach, pancreas thyroid and glands associated with the digestive system.
  • the instant invention is a process for the propagation of hepatic stem cells and of other hepatic progenitor cells.
  • the process involves exposing populations of isolated hepatic stem cells and/or hepatic progenitor cells and/or their progeny, to growth conditions capable of sustaining clonal growth, that is, growth at very low cell densities.
  • the process involves using a serum-free, hormone-supplemented, defined medium to support the propagation of hepatic progenitor cells on a layer of feeder cells.
  • the function of the feeder cells is multi-fold, including supplying nutrients, supplying an attachment surface, and secreting into the medium certain growth factors and extracellular matrix components needed for survival, growth and/or differentiation of the hepatic progenitor cells.
  • the process involves selecting for cells that are capable of sustaining the growth of hepatic stem and hepatic progenitor cells.
  • the feeder cells may be from reptiles, birds, crustaceans, fish, annelids, molluscs, nematodes, insects, or mammals, preferably human.
  • the feeder cells derive from embryonic tissues.
  • the feeder cells derive from embryonic tissue.
  • the feeder cells can derive from embryonic liver tissue.
  • the feeder cells may be genetically modified.
  • the process involves cloning feeder cells that optimally sustain hepatic cells.
  • any method of isolating hepatic stem and hepatic progenitor cells is acceptable, including by affinity-based interactions, e.g. affinity panning, immunosurgery in combination with complement, by flow cytometry, by centrifugal elutriation, by differential centrifugation, etc.
  • the isolated hepatic stem and progenitor cells have the capacity to express some or all of the phenotype markers (classical MHC class I " , ICAM-1 + , OX18 du11 , alpha-fetoprotein + , or albumin + ). It is another embodiment of the invention that the hepatic progenitors express a growth pattern in the colonies characterized by formation of piled-up cells as aggregates, colonies or clusters.
  • hepatic cells be selectively grown in a serum-free, hormone-supplemented, defined medium (HDM).
  • the composition of HDM comprises a nutrient medium including, but not limited to a mixture of Dulbecco's modified Eagle's medium and Ham's F12 to which is added up to about 40 ng/ml EGF, up to about 5-10 ⁇ g/ml insulin, up to about 10"° " M Dexamethasone or other glucocorticoid hormone, up to about 10 ⁇ g/ml iron- saturated transferrin, up to about 5 x lO' ⁇ M nicotinamide, up to about 2% bovine serum albumin, up to about 5 x 10' ⁇ M 2-mercaptoethanol or equivalent reducing agent, up to about 8 ⁇ eq/1 free fatty acid, up to about 2 x 10' ⁇ M glutamine, up to about 1 x 10 "6 M CuSOzj., up to about 3 x 10 -8 M
  • a nutrient medium
  • Antibiotics can include penicillin, streptomycin, gentamycin, and others common in the art, and combinations thereof.
  • other nutrient media e.g. Ham's F-10, Medium 199, or one of the MCDB series including MCDB 151 and MCDB 302, can, after minimal testing, be used in place of DMEM/F12.
  • the most minimal conditions for cell expansion are use of the feeders in the absence of any hormones; and the most critical of the hormonal requirements listed above are glucocorticoids, insulin, transferrin, and EGF constituting the strict hormonal mitogens for progenitor cell expansion.
  • Other hormonal factors can be added and might have secondary growth effects but do not replace the critical requirements noted above.
  • changes in the hormone constituents such as can be made by one of ordinary skill in the art, are within the scope of the instant invention.
  • Preferable ranges include 10-50 ng/ml EGF, 2-10 ug/ml insulin, 5xl0 "7 M to 5xl0 “6 M dexamethasone (9 ⁇ -fluoro-16 -methyl-prednisolone), 5-20 ug/ml iron- saturated transferrin, 2-8 x 10 "3 M nicotinamide, 0.05 - 0.5% serum albumin, 2-8 x 10 " 5 M 2-mercaptoethanol, 5-10 ueq free fatty acid mixture, 1-3 x 10 "3 M glutamine, 0.5 - 2x 10 "6 M CuSO 4 , l-5x 10 "8 M H 2 SeO 3 , 1-5 uM palmitic acid, 0.1 - 0.4 uM palmitoleic acid, 0.5-1.2 uM stearic acid, 0.5 - 2 uM oleic acid, 1-5 uM linoleic acid, and 0.2 - 0.8 uM linolenic
  • the serum- free, hormonally defined culture medium of the invention is suitable for the clonal growth of hepatic cells.
  • This HDM contains a basal medium that can be any of a number of options such as Dulbecco's modified Eagle's medium (DME), Ham's F12, RPMI 1640, Williams E medium, etc.
  • DME Dulbecco's modified Eagle's medium
  • Ham's F12 Ham's F12
  • RPMI 1640 RPMI 1640
  • Williams E medium etc.
  • a preferred embodiment is a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, from, for example GIBCO/BRL, Grand Island, NY).
  • the basal medium is supplemented with epidermal growth factor, EGF (from, for example, Collaborative Biomedical Products) at a preferred concentration of 10 ng/ml, insulin (from, for example, Sigma) at a preferred concentration of 5 ⁇ g/ml, lO' ⁇ M Dexamethasone
  • EGF epidermal growth factor
  • insulin from, for example, Sigma
  • the growth factors secreted by the feeder cells including but not limited to insulin-like growth factors (IGFs), interleukin (E )-6 family, hepatocyte growth factors (HGFs), and fibroblast growth factors (FGFs), can be added to the culture medium to augment feeder effects but have not been found to replace feeder effects when added singly or in various combinations, meaning that the feeder cells are producing other signals, yet unidentified that are needed alone or in combination with these growth factors. It is a still further embodiment of the invention that the hepatic progenitor cells are propagated from a single progenitor cell, that is, that the cells are cloned.
  • IGFs insulin-like growth factors
  • E interleukin-6 family
  • HGFs hepatocyte growth factors
  • FGFs fibroblast growth factors
  • progenitor cells may be cloned with the use of cloning rings, by selective ablation, by dilute culture on microparticles, by single-cell sorting using flow cytometry, by picking individual cells with micropipet or optical tweezers, and by agar. It is a yet further embodiment of the invention that many of the cloned progenitor cells are capable of mitosis. It is preferred that the progenitor cells are capable of a least one cycle of mitosis and even more preferred that the progenitor cells are capable of at least ten cycles of division.
  • hepatic progenitor cells and their progeny are propagated in medium supplemented with metabolic and biosynthetic products of feeder cells.
  • the supplement can take the form of conditioned medium, that is, medium previously incubated with living feeder cells.
  • the supplementing can take the form of isolating from feeder cell- conditioned medium those factors including proteins, peptides, lipids, carbohydrates, and metabolic regulators that sustain and enhance the growth of hepatic progenitors and their progeny.
  • the proteins can include soluble and insoluble components of extracellular matrix and growth factors including epidermal growth factor and insulin-like growth factors.
  • hepatic cells be selectively grown in culture using a layer of feeder cells, where those feeder cells are embryonic or adult cells or other suitable cells.
  • the feeder cells are stromal cells or fibroblasts.
  • the fibroblasts or other suitable cells may be genetically modified, e.g. by transfection. It is preferred that the fibroblasts or other suitable cells be human, non-human primate, pig, dog, rabbit, rat, or mouse mesodermal cells, and other mammalian and avian mesodermal cells are also suitable.
  • the fibroblasts can be cloned and selected for the ability to support hepatic progenitor cells.
  • isolated hepatic progenitor cells be committed to a hepatocyte or biliary cell lineage by the selective application, or absence, of epidermal growth factor (EGF), or other differentiation signal.
  • isolated stem cells and other hepatic progenitor cells be used as a component of a bioartificial liver that can be used as an extracorporeal liver assist device.
  • the bioartificial liver containing isolated hepatic progenitor cells and their progeny be used to support the life of a patient suffering from liver malfunction or failure.
  • Pregnant Fisher 344 rats are obtained from Charles River Breeding Laboratory (Wilmington, MA). For timed pregnancies, animals are put together in the afternoon, and the morning on which the plug is observed is designated day 0. Male Fisher 344 rats (200-250g) are used for adult liver cells.
  • Fetal livers are prepared from day 15 of the gestation. Single cell suspensions are obtained by incubating the livers with 0.05% trypsin and 0.5mM EDTA or lOunits/ml thermolysin (Sigma, St. Louis, MO) and lOOunits/ml deoxyribonuclease I (Sigma) for at 37 °C. The cells are overlayed on Ficoll-paque (Pharmacia Biotech, Uppsala, Sweden) for gradient density centrifugation at 450g for 15 min. The cells from the bottom fraction are inoculated into tissue culture dishes coated with 17 mg/ml collagen type IV (Collaborative
  • the serum-free hormonally defined culture medium, HDM is a 1 : 1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, GIBCO/BRL, Grand Island, NY), to which is added 20 ng/ml EGF (Collaborative Biomedical Products), 5 ⁇ g/ml insulin
  • STO Sublines One hundred cells of parent STO from ATCC are cultured in 100mm culture dishes for 7 days in DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum, 2 x 10' ⁇ M glutamine, 5 x lO' ⁇ M 2-mercaptoethanol and antibiotics. Four subclones are selected for further characterization according to the cell morphology and the growth speed. Although CFA for rter ⁇ is performed in the four subclones, one of them, STO6, does not persist in attaching to culture plates after mitomycin C-treatment. One subclone, STO5, is transfected with pEF-Hlx-MClneo or pEF-MClneo kindly provided from Dr. J. M.
  • Fetal livers are dissected into ice-cold Ca ++ free HBSS with lO M HEPES, 0.8mM MgSO4 and ImM EGTA (pH7.4).
  • the livers are triturated with 0.2% type IV collagenase (Sigma) and 16.5 units/ml thermolysin (Sigma) in HBSS prepared with lOmM HEPES, 0.8mM MgSO4, and ImM CaCl2- After incubation at 37 °C for 10 min, the cell suspension is digested with 0.025% trypsin and 2.5mM EDTA (Sigma) for 10 min. Trypsin is then quenched by addition of lmg/ml trypsin inhibitor (Sigma).
  • the cells are treated with 200 units/ml deoxyribonuclease I (Sigma). In all experiments, 3-5 x lO ⁇ cells per liver are obtained. Isolation of adult liver cells. The two step liver perfusion method is performed to isolate liver cells . After perfusion, the cells are centrifuged for 1 min at 50g twice to enrich for large parenchymal cells. Cellular viability is >90% as measured by trypan blue exclusion.
  • Flow cytometric analysis Cells are analyzed on a FACScan (Becton-Dickinson, Mountain View, CA) and sorted using a Moflow Flow Cytometer (Cytomation, Fort Collins, CO). The cell suspensions from E13 fetal liver are incubated with HBSS, containing 20% goat serum (GIBCO/BRL) and 1% teleostean gelatin (Sigma), on ice to prevent nonspecific antibody binding. After rinsing, the cells are resuspended with FITC-conjugated anti rat RTlA a >W antibody B5 (Pharmingen, San Diego, CA) and PE-conjugated anti-rat ICAM-1 antibody 1A29 (Pharmingen).
  • the cells are stained with biotinylated anti-rat monomorphic MHC class I antibody OX18 (Pharmingen) followed by a second staining with streptavidin-red670 (GIBCO/BRL) for 3 color staining. All stainings are performed with ice-cold Ca ++ free HBSS containing lOmM HEPES, 0.8mM MgSO4, 0.2mM EGTA, and 0.2%
  • BSA bovine serum-derived autoantibody serum
  • FTO-2B rat hepatoma cell line
  • WB-F344 rat liver epithelial cell line
  • adult liver cells are stained to compare with the fetal hepatic cell lines.
  • the cell lines are kind gifts of Dr. R.E.K. Founder, Fred Hutchinson Cancer Research Center, Seattle, WA, and Dr. M.-S. Tsao, University of North Carolina, Chapel Hill, NC, respectively.
  • FITC-conjugated B5 OX18, PE-conjugated 1 A29 or anti FITC-conjugated rat integrin ⁇ i antibody Ha2/5 (Pharmingen).
  • FITC-conjugated anti mouse IgG is used for OX18.
  • Cell suspensions of three fetal hepatic cell lines are stained with biotinylated anti-mouse CD98 followed by a second staining with streptavidin-red670 as well as anti-rat moAb to gate out mouse cell populations.
  • antigens are expressed in different relative numbers by cells.
  • the level of expression of a particular antigen can be NO expression, a low level of expression, a level of expression that is normal or regular for many antigens, and a high level of expression.
  • the term "low is used interchangeably with a weak or dull. More detailed description of the level of expression can, alternatively, be made, but these four levels suffice for many purposes. It should be clear that measurement of antigen expression by, for example, flow cytometry, provides a continous range for antigen expression.
  • CFA for hepatic cell lines, sorted cells, and adult liver cells.
  • the hepatic cell lines are plated in triplicate at 500 cells per 9.6 cm ⁇ on mitomycin C-treated STO feeder layer with the same HDM as used for maintaining each cell line. Before plating, cell are trypsinized and fractionated by Percoll density gradient centrifugation to remove feeder cells. The cultures are incubated for 10 to 14 days with medium changes every other day. Double immunofluorescence staining of alpha-fetoprotein and albumin is then performed. 100 colonies per well are analyzed by the colony morphology, P or F type, and the expression of alpha-fetoprotein and albumin.
  • the colonies are stained using Diff-Quick (Baxter, McGaw Park, IL) to count the number of the colonies per well.
  • Diff-Quick Biller, McGaw Park, IL
  • the plating cell number is changed as described.
  • the culture period is expanded to between 14 and 17 days, and the concentration of dexamethasone is increased to 10 ⁇ 6M. All other procedures are performed as above.
  • small numbers of clumps of liver cells are not eliminated from the cell suspension after the preparation. Therefore, an undefined number of the colonies might be produced from the clumps.
  • double immunofluorescence staining of albumin and CK19 of the colonies is performed at 5 days each of the culture in the presence or absence of EGF.
  • any colony with more than one CK19 + cell is counted as a CK19+ colony.
  • colonies containing multiple clusters of two CK19+ cells or one cluster of more than three CK19+ cells are counted as a CK19+ colony.
  • About 100 colonies per well are counted. Each point represents the mean ⁇ SD from triplicate-stained cultures.
  • hepatic cell lines from independent experiments are selected by the morphological criteria of either P-type or F-type colonies.
  • Rhel4321 (Fig. la) consists mostly of packed small cells, P-type colonies, whereas thl 120-3 (Fig. lc) makes only a flattened monolayer of F-type colonies.
  • Rter6 (Fig. lb) is an intermediate phenotype of these two. Interestingly, the heterogeneity of rter6 is still observed after three rounds of sequential cloning of the flattened colony.
  • Fig. 2a, 2b, 2c, 2d, 2e, and 2f shows the results, hi the cell lines, rhel4321 (Fig. 2b) and rter6 (Fig. 2c), and in the original cell population prior to cloning (Fig.
  • the efficiency of rter6 and rhel4321 is 45.7% ( ⁇ 1.3% SD) and 36.4% ( ⁇ 1.1% SD), respectively.
  • the thl 120-3 cells tightly attach to each other along their lateral borders making preparation of single cell suspensions difficult. However, the thl 120-3 cells do not produce piled up clusters.
  • the culture system has to be able to support cell expansion at clonal seeding densities and with conservation of critical original hepatic functions, albumin and alpha-fetoprotein are two of the most significant markers for early hepatic development .
  • the culture conditions optimizing P type colonies should be the best, since P type, but not F type, colonies maintain the expression of alpha-fetoprotein and albumin during clonal expansion. Therefore, STO subclones are compared in their support of P type colonies of rter ⁇ .
  • One of the clones, STO5 supports the P type colony formation more than any of the other sublines and more than the parent line (Fig. 2d).
  • the CFA of rhel4321 also confirms that STO5 is a more effective feeder than the parent STO (Fig. 2e).
  • the mouse Hlx gene product expressed in the mesenchymal cells lining digestive tract from El 0.5 , is essential for fetal hepatic cell expansion.
  • the mRNA expression for the Hlx gene is analyzed in all the STO subclones, there is no significant difference in its expression among the subclones (data not shown).
  • the stable transfectants of mouse Hlx in STO5 do not result in an improvement in the colony formation assays (Fig. 2f).
  • One clone of the transfectants is used for further experiments, because the transfectant supports a more stable persistence of the original morphology of STO5 at relatively high passages.
  • Hepatopoiesis and massive amounts of hematopoiesis co-exist in the fetal liver. So far, the antigenic profile of hematopoietic progenitors has extensively been analyzed, whereas studies of early hepatic progenitors are still in their infancy. The antigenic profile of hepatic cells is analyzed using the three hepatic cell lines established in this study, an adult hepatocarcinoma cell line (FTO-2B), an epithelial cell line from adult rat liver (WB-F344), and freshly isolated adult liver cells.
  • FTO-2B adult hepatocarcinoma cell line
  • WB-F344 epithelial cell line from adult rat liver
  • rhel4321 Compared with FTO-2B, WB-F344, and adult liver cells, the pattern of the most immature of the fetal hepatic cell lines, rhel4321, is quite unique in that there is no expression of classical MHC class I (RTl A 1 ) (Fig. 3a - 3x).
  • the cell line, thl 120-3 (Fig. 3i-31) is similar to rhel4321 (Fig. 3a-3d) in the pattern of RT1A 1 , OX18 (pan- MHC class I), and ICAM-1, whereas rter ⁇ (Fig. 3e-3h) has relatively high expression of RTl A 1 and OX18 (Fig.3).
  • RTl A 1_ another cell line from a different experiment, which has an identical morphology to rhel4321, is also RTl A 1_ , OX18 du11 , and ICAM-1 + . Integrin bi expression is similar in all the cell lines, while the pattern of RTl A a ' b ' 1 and ICAM-1 is unique among them.
  • the antigenic profile of adult liver cells is RT1A 1+ , OX18 + , and ICAM-1 + . Since, in the adult rat, all bone marrow cells except mature erythrocytes strongly express MHC class I molecules, the fetal hepatic population can be separated from the hemopoietic cell populations by MHC class I expression.
  • Fig. 4a shows the 2 color-staining pattern of RTl A 1 and ICAM- 1.
  • Fig. 4b represents the result of resorting of the five fractions after sorting.
  • the hepatic cell colonies defined by expression of albumin and alpha-fetoprotein, are distinguishable also morphologically, enabling one to count the number of hepatic colonies per well. The majority of the hepatic colonies are detected in the gate RTlA ldu11 and ICAM-f (Table 1, Fig.
  • gate 2 shows a much lower number of the colonies, and the other fractions contain negligible numbers of cells with colony forming ability.
  • gates 1 and 2 the expression of both alpha-fetoprotein and albumin is confirmed in all the hepatic colonies. Some of the colonies, derived from cells in gate 2, are larger than others.
  • SSC sidescatter
  • SSC Sidescatter
  • Table 1 The Frequency of hepatic colonies from sorted El 3 fetal liver based on the expression of RTl A and ICAM-1.
  • EGF has long been known as a potent growth factor for adult liver cells. Therefore, the effects of EGF for colony formation of sorted hepatic cells are investigated.
  • the colony-size of the RTl A l ⁇ OX18 dul1 ICAM-1 + hepatic cells becomes bigger in the absence of EGF, whereas adult liver cells yielded colonies only in the presence of EGF (Fig. 6 c).
  • the morphology of the colonies derived from adult liver cells is the typical F type, whereas all RTl A 1" hepatic cells produce P type colonies without EGF.
  • RTl A 1 After 3 weeks of culture, when growth seems to reach a maximum, the expression of RTl A 1" , OX18, and ICAM-1 is assessed. As shown in Fig. 5b-5d, the expression of RTl A 1 is not induced, while that of OX18 is reduced. The level of ICAM-1 does not change. Furthermore, the average cell number of single colony is calculated from the recovered cell number, the percentage of rat hepatic cells and the colony efficiency. The estimated cell number reaches 3 to 4 x 10 3 (Table 2). This indicates that the single cell forming the colonies divided approximately 11-12 times on average under this culture condition. Table 2. Calculation of the cell number in single hepatic colony.
  • the hepatic cells are thought to have a bipotent precursor giving rise to the mature hepatocyte and bile duct epithelium.
  • the colonies are stained by anti-CK19 as a specific marker for biliary epithelial cells. CK19 is expressed in the bile duct epithelial precursors after day 15.5 in the fetal rat liver at which time the expression of albumin disappears in the cells.
  • the sorted RT1A 1" ICAM-1 + cells are cultured in the presence or absence of EGF, and their fates are monitored by the expression of CK19 and albumin after 5 days of culture. After the first 5 days, the CK19 + colonies are negligible in the cultures treated with EGF, whereas a few colonies containing CK19 + cells occurred in those in the absence of EGF. Although the intensity of the CK19 expression is fairly weak, the CK19 + cells show reduced albumin expression. At the 10th day of the culture, some colonies apparently express only CK19 or albumin and others have dual positive expression. The pattern of the CK19 + and albumin "1" cells in a single colony is reciprocal.
  • Fresh embryonic tissue or frozen tissue (e.g. liver, lung, kidney, muscle, intestine) from pig, beagle, rabbit, mouse or monkey is minced in calcium-free, phosphate-buffered saline (PBS). After rinsing with PBS a couple of times, the cell suspension is incubated with 0.25% trypsin for 10 min at 37° or for 60 min at room temperature with agitating using a magnetic stirrer. The remaining cell chunks are removed by filtering the suspension thorough mesh. The cells are then cultured on tissue culture dishes with a basal medium (e.g. Eagle's MEM) supplemented with serum (e.g. 10% fetal calf serum) and with any of various growth supplements (e.g.
  • a basal medium e.g. Eagle's MEM
  • serum e.g. 10% fetal calf serum
  • any of various growth supplements e.g.
  • Plastic substratum and serum supplemented medium are generic conditions that permit expansion of a cell population that is a candidate as support cells ("feeder cells"), most commonly being mesodermally-derived (e.g. stromal cells), and that provide factors supporting the survival, growth and/or functions of another cell type (e.g. progenitor cells).
  • the feeder cells are subcultured with 0.05% trypsin when they become confluent or almost confluent. After several rounds of subculture, expanded cells are prepared as frozen stocks and stored as such until use.
  • An alternative source of feeder cells can be commercially available primary cultures of feeder cells or feeder cell lines. In any case, the following criteria are needed to identify the appropriate feeder cells:
  • MHC class I antigen In the field, classical MHC class I antigen is also known as MHC class la antigen. Non-classical MHC class I antigen is also known as MHC class lb antigen.
  • the MHC antigens have different designations in different species: RTl in rat, H-2 in mouse, and HLA in humans, for example.
  • the hepatic progenitors are plated at 500 cells per 9.6 cm ⁇ on growth-arrested, i.e. cells treated to prevent proliferation, feeder cells.
  • the feeder cells are growth-arrested by treating them with mitomycin C or by irradiating (3000-5000 rads depending upon cell type).
  • the growth-arrested feeder cells and progenitor cells are fed with a serum- free HDM.
  • HDM for the rodent cells is a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 with added 10 ng/ml EGF, 5 ⁇ g/ml insulin, 10 " 6M Dexamethasone, 10 ⁇ g/ml iron-saturated transferrin, 4.4 x lO' ⁇ M nicotinamide, 0.2% bovine serum albumin, 5 x lO' ⁇ M 2-mercaptoethanol , 7.6 ⁇ eq/1 free fatty acid, 2 x 10" 3 M glutamine, 1 x 10" 6 M CuSO4, 3 x 10 ⁇ 8 M H2Se ⁇ 3 and antibiotics. The cultures are incubated for 10 to 14 days with medium changes every other day.
  • Double immunofluorescence staining of alpha-fetoprotein, albumin, and/or CK19 is then performed for identifying the fate of the progeny. About 100 colonies are analyzed by the expression of alpha-fetoprotein and albumin. Furthermore the colony morphology, P or F type, could be useful identification of the relevant progeny.
  • the ideal combination of feeder cells and hepatic progenitors are those that originated from the identical species.
  • the feeder cells are from the same tissue and same species as the hepatic progenitors.
  • mixing of feeders from one species and progenitors from another is possible.
  • rodent feeder cells can be used for human hepatic progenitors.
  • Soluble and insoluble factors help the clonal growth of hepatic stem cells or hepatic progenitors.
  • the source of the factors is : 1) Conditioned medium from the cultured feeder cells of the optimal species and tissue.
  • the feeder cells can be of any cell type, not just stromal cells.
  • the critical factor(s) are known, one can also replace the feeder cells altogether by supplementing the medium with those signals, whether they be proteins, peptides, carbohydrates, lipids, glycopeptides, glycoproteins, lipoproteins, glycolipids, or a combination of these constituting the signals derived from optimal feeder cells active for hepatic progenitors.
  • signals whether they be proteins, peptides, carbohydrates, lipids, glycopeptides, glycoproteins, lipoproteins, glycolipids, or a combination of these constituting the signals derived from optimal feeder cells active for hepatic progenitors.

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Abstract

L'invention concerne un procédé permettant de propager des progéniteurs mammaliens d'origine endodermique, tels que les progéniteurs hépatiques, leur descendance, ou des mélanges de ceux-ci sur une couche de cellules nourricières embryonnaires mammaliennes dans un milieu de culture. Le milieu de culture peut être complété par une ou plusieurs hormones et par d'autres agents de croissance. Ces hormones et autres agents de croissance peuvent comprendre l'insuline, la dexaméthasone, la transferrine, la nicotinamide, la sérumalbumine, le β-mercaptoéthanol, l'acide gras libre, la glutamine, le CuSO4 et le H2SeO3. le milieu de culture peut également comprendre des anticorps. Le milieu de culture ne comprend pas de sérum. Le procédé décrit dans cette invention comprend des moyens permettant d'inciter la différentiation des progéniteurs de leurs formes adultes, telle que la différentiation des cellules souches hépatiques des hépatocytes ou des cellules biliaires, par ajout ou par exclusion du facteur de croissance épidermique, respectivement. Le procédé de production de progéniteurs mammaliens est utile en ce que les progéniteurs peuvent être utilisés ultérieurement dans un ou plusieurs des procédés suivants: l'identification des facteurs de croissance et de différentiation, les études toxicologiques, la mise au point de médicaments, les études antimicrobiennes, ou la préparation d'un organe extra-corporel, tel qu'un foie bioartificiel.
PCT/US2000/027428 2000-10-03 2000-10-03 Procede de multiplication par clonage de cellules souches hepathiques WO2002029012A1 (fr)

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IL15538700A IL155387A0 (en) 2000-10-03 2000-10-03 Processes for clonal growth of hepatic progenitor cells
CA2424779A CA2424779C (fr) 2000-10-03 2000-10-03 Procede de multiplication par clonage de cellules souches hepathiques
CN00820051A CN1461341A (zh) 2000-10-03 2000-10-03 肝祖先细胞克隆生长的方法
PCT/US2000/027428 WO2002029012A1 (fr) 2000-10-03 2000-10-03 Procede de multiplication par clonage de cellules souches hepathiques
KR1020037004803A KR100887623B1 (ko) 2000-10-03 2000-10-03 간 전구세포의 클론 증식방법
AU7753500A AU7753500A (en) 2000-10-03 2000-10-03 Processes for clonal growth of hepatic progenitor cells
JP2002532583A JP2004510434A (ja) 2000-10-03 2000-10-03 肝前駆細胞のクローン増殖方法
AU2000277535A AU2000277535B2 (en) 2000-10-03 2000-10-03 Processes for clonal growth of hepatic progenitor cells
EP00967317A EP1325111A1 (fr) 2000-10-03 2000-10-03 Procede de multiplication par clonage de cellules souches hepathiques
HK03106718.5A HK1054570A1 (zh) 2000-10-03 2003-09-19 克隆培養肝祖先細胞的方法

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JP2007523638A (ja) * 2004-01-14 2007-08-23 ノーバヘップ アーベー ヒトの肝臓前駆細胞およびその使用方法
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US8211696B2 (en) 2001-03-27 2012-07-03 Vertex Pharmaceuticals Incorporated Method useful for HCV RNA898 infection
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HK1054570A1 (zh) 2003-12-05
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AU7753500A (en) 2002-04-15
CN1461341A (zh) 2003-12-10
KR100887623B1 (ko) 2009-03-11
AU2000277535B2 (en) 2007-03-29
JP2004510434A (ja) 2004-04-08
KR20040047732A (ko) 2004-06-05

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