US20150166953A1 - Generation of hepatocytes from pluripotent stem cells - Google Patents

Generation of hepatocytes from pluripotent stem cells Download PDF

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US20150166953A1
US20150166953A1 US14/409,234 US201314409234A US2015166953A1 US 20150166953 A1 US20150166953 A1 US 20150166953A1 US 201314409234 A US201314409234 A US 201314409234A US 2015166953 A1 US2015166953 A1 US 2015166953A1
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hepatoblasts
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hesc
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Sanjeev Gupta
Sriram Bandi
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Albert Einstein College of Medicine
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • liver occupies a central position in life due to its crucial metabolic, synthetic, storage, and drug or toxin disposal functions. Isolated liver cells are extremely useful for developing disease models, as well as for toxicological testing and drug development. Moreover, because many proteins are made in liver cells, cell/gene therapy directed at the liver is of extensive interest for a long list of genetic or acquired conditions. However, shortages of donor organs have proved to be an insurmountable hurdle for therapeutic and other applications of liver cells. Therefore, alternative means to generate hepatocytes, e.g., from pluripotent stem cells, is of great interest. This requires understanding into the processes by which pluripotent stem cells may transition and differentiate, first into immature and then into mature hepatocytes.
  • hESC human embryonic stem cells
  • iPS induced pluripotent stem cells
  • available differentiation protocols to generate hepatocytes from hESC or iPS, etc. are inefficient and generate cells of indeterminate developmental or maturational stages.
  • the convention of generating hepatocytes from aggregation of hESC or other types of pluripotent stem cells to form embryoid bodies is not only inefficient, but yields complex lineage mixtures at various developmental stages or maturity that pose difficulties in isolating cells of interest, which may be additionally altered or damaged by cell separation procedures (1).
  • Directed differentiation of stem cells into hepatocytes could overcome these problems, (2, 3), but this accomplishment has generally been elusive.
  • the present invention addresses the need for directed differentiation of stem cells into hepatocytes.
  • a method of producing a differentiated cell from a pluripotent stem cell comprising maintaining the pluripotent stem cell in a medium comprising conditioned medium from immortalized fetal hepatoblasts, for a time sufficient to produce the differentiated cell.
  • a composition for differentiating stem cells into a differentiated cell of interest, the composition comprising conditioned medium obtained from a culture of hepatoblasts cultured in a medium comprising a basal medium.
  • a composition for differentiating stem cells into a differentiated cell of interest, the composition comprising components identified in conditioned medium obtained from a culture of human fetal hepatoblasts cultured in a medium comprising a basal medium without those components.
  • a method for treating a liver disorder in a subject comprising administering to the subject an amount of the described compositions, or an amount of the described differentiated cells, in an amount effective to treat a liver disorder.
  • FIG. 1A-1H Morphology of hESC cultured with FH-CM.
  • A Undifferentiated hESC with clusters of small cells.
  • B Primary embryonic/fetal hepatocyte-like cells (eFHLC) (P0) after 14 d with fetal hepatocyte-conditioned medium (FH-CM). Note larger cell size and epithelial morphology. Inset, higher magnification showing binucleation, common to hepatocytes.
  • C-F eFHLC with glycogen, G6P, glucagon and vimentin.
  • G-H eFHLC subpassaged once (P1) or twice (P2). Orig. Mag., ⁇ 200.
  • FIG. 2A-2C Characterization of differentiating eFHLC.
  • B RT-PCR for gene expression in hESC, d14 eFHLC and freshly isolated fetal human hepatocytes. Arrowheads indicate endodermal and mesodermal markers in eFHLC.
  • C Temporal gene expression profile by qRT-PCR in eFHLC over d0, d3 and d14.
  • eFHLC Cluster analysis of global gene expression in hESC, eFHLC, freshly isolated fetal human hepatocytes (FH-PP) or cultured fetal human hepatocytes (FH-P3), and adult human hepatocytes (AH), showed convergence of eFHLC most towards FH-P3.
  • FIG. 3A-3C Microarray analysis of gene expression in eFHLC versus hESC and freshly isolated fetal human hepatocytes (FH-PP). Panels A, B show global differences in gene expression. Data were from total gene sequences called as present, range, 48,942 to 51,031.
  • 505 or 2123 genes were uniquely upregulated versus hESC and FH-PP, respectively, and 236 or 1514 genes were uniquely downregulated versus hESC and FH-PP, respectively.
  • 1504 genes were uniquely upregulated and 1387 genes were uniquely downregulated in FH-PP cells versus hESC cells. This indicated that eFHLC were closer to hESC compared with FH-PP.
  • Table in 3C provides the overall distribution of differentially expressed genes and fractions representing major gene ontology groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Changes in TGF-0 signaling and BMP signaling in eFHLC were observed compared with FH-PP cells. The data indicated TGF-3 and BMP signaling were more active in eFHLC. This was in agreement with similar findings after FH-PP had been cultured.
  • FIG. 4A-4F Secretory, synthetic, metabolic and hepatoprotective functions in cultured eFHLC.
  • A-C Studies with hESC, d14 eFHLC and HepG2 hepatoma cells for albumin secretion, urea synthesis and CYP450 activity showing gain of these functions in eFHLC.
  • D Assay of TNF- ⁇ cytotoxicity in primary mouse hepatocytes showing eFHLC-CM protected cells. Asterisks indicate p ⁇ 0.05 versus hESC (A-C), or untreated controls (D).
  • E-F Show regulation of ataxia telangiectasia mutated (ATM) signaling in cells.
  • FIG. 5A-5C RayBiotech array analysis of 507 proteins in eFHLC-CM.
  • Basal medium not exposed to cells Basal medium not exposed to cells.
  • B eFHLC-CM harvested after 24 h. Boxes marked by (+) and ( ⁇ ) in A and B indicate positive and negative controls. Each protein was represented twice in arrays.
  • C Proteins found in eFHLC-CM are listed.
  • FIG. 6A-6C RayBiotech array analysis of proteins in FH-CM.
  • A Shows basal medium containing various additives but without exposure to cells.
  • B FH-CM harvested after 24 h. Boxes marked by (+) and ( ⁇ ) in A and B indicate positive and negative array controls. Each protein was represented twice in arrays.
  • C Shows categorization of proteins in FH-CM according to density of array spots to indicate higher or lower levels.
  • FIG. 7A-7D RayBiotech array analysis of receptor tyrosine kinase (RTK) expression in undifferentiated hESC.
  • RTK receptor tyrosine kinase
  • A Untreated control hESC with basal phosphorylation of several receptor tyrosine kinases (RTKs).
  • B and C hESC stimulated for 1 h (B) or 6 h (C) with FH-CM before analysis of phosphorylated RTKs. Data showed no differences from untreated controls in (A). Each protein was spotted twice. Boxes marked by (+) and ( ⁇ ) in A-C indicate positive and negative array controls.
  • D List of phosphorylated RTKs in hESC irrespective of basal or FH-CM-stimulated conditions.
  • FIG. 8A-8B Differentiation of hESC over 3 d with FH-CM altered by protracted heating to degrade proteins or passage through Amicon membrane of 3 Kd cut-off size.
  • A hESC after culture with heat denatured FH-CM showing switch to epithelial morphology.
  • B hESC cultured with FH-CM passed through Amicon membrane showing epithelial morphology. Orig. Mag., ⁇ 100.
  • FIG. 9A-9F Hepatic differentiation of hESC with group of 7 CP. Panels on left show undifferentiated hESC, panels in middle show hESC cultured with FH-CM and panels on right show hESC cultured with combination of 7 CP, which were most effective for this purpose.
  • A Phase contrast microscopy showing undifferentiated hESC with small size of cells (left), whereas hESC cultured with FH-CM (middle) or 10 ⁇ m amounts of 7 CP showed larger size and epithelial morphology (on right).
  • B-D Immunofluorescence staining of hESC under conditions indicated for OCT4, albumin and vimentin.
  • E & F eFHLC derived from hESC by either FH-CM or CP showed urea synthesis (E) as well as cytochrome P450 activity with conversion of ethoxyresorufin to resorufin (E).
  • Human HepG2 cells were included for comparisons in E & F.
  • FIG. 10A-10D Hepatic differentiation of iPSC with group of 7 CP. Panels on left show undifferentiated iPSC, panels in middle show iPSC cultured with FH-CM and panels on right show iPSC cultured with combination of 7 CP, which were most effective.
  • A Phase contrast microscopy showing undifferentiated iPSC with small size of cells (left), whereas iPSC cultured with FH-CM (middle) or 10 ⁇ m amounts of 7 CP showed larger size and epithelial morphology (on right).
  • B-D Immunofluorescence staining of iPSC under conditions indicated for OCT4, albumin and vimentin.
  • E & F eFHLC derived from iPSC by either FH-CM or CP showed urea synthesis (E) as well as cytochrome P450 activity with conversion of ethoxyresorufin to resorufin (E).
  • Human HepG2 cells were included for comparisons in E & F.
  • a method of producing a differentiated cell from a pluripotent stem cell comprising maintaining the pluripotent stem cell in a medium comprising isolated conditioned medium from hepatoblasts, for a time sufficient to produce the differentiated cell.
  • a method of producing a differentiated cell from a pluripotent stem cell comprising maintaining the pluripotent stem cell in a medium comprising isolated conditioned medium from fetal hepatoblasts, for a time sufficient to produce the differentiated cell.
  • Also provided is a method of producing a differentiated cell from a pluripotent stem cell comprising maintaining the pluripotent stem cell in a medium comprising conditioned medium from hepatoblasts, or a medium comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl, for a time sufficient to produce the differentiated cell.
  • the method comprises maintaining the pluripotent stem cell in a medium comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl.
  • the differentiated cell is a hepatocyte. In an embodiment, the differentiated cell exhibits a meso-endodermal phenotype of a fetal human hepatocyte. In an embodiment, the differentiated cell exhibits ureagenesis and/or albumin synthesis and/or vimentin expression. In an embodiment, the pluripotent stem cell is an inducible pluripotent stem cell or is an embryonic stem cell.
  • the hepatoblasts are immortalized.
  • the hepatoblasts have been immortalized by contact with a telomerase.
  • the hepatoblasts have been immortalized by expression of telomerase, or maintenance of telomerase expression.
  • hepatoblasts are human.
  • hepatoblasts are fetal.
  • hepatoblasts are immortalized human fetal hepatoblasts.
  • hepatoblasts are isolated.
  • the medium does not comprise serum.
  • the conditioned medium from immortalized human fetal hepatoblasts comprises medium obtained from a culture of immortalized human fetal hepatoblasts cultured in a medium comprising a basal medium.
  • the immortalized fetal hepatoblasts are human immortalized fetal hepatoblasts.
  • the hepatoblasts are mammalian, but are not human.
  • the hepatoblasts are human, but are not fetal.
  • the medium comprising a basal medium further comprises L-glutamine, one or more non-essential amino acids, and an antibiotic.
  • the basal medium is a Dulbecco's Modified Eagle's Medium (DMEM).
  • the antibiotic is penicillin-streptomycin.
  • the non-essential amino acids are glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.
  • the medium comprising a basal medium further comprises an artificial serum replacement.
  • a conditioned medium is a medium in which the cell culture has been maintained.
  • the conditioned medium has been exposed to the cells being cultured for 1 or more hours, 2 or more hours, 6 or more hours, 12 or more hours, 24 or more hours, one week or more or two weeks or more.
  • the medium further comprises one or more of L-cysteinglutathione disulfide, ⁇ -Glu-Cys, DL-kynurenine, D-penicillamine disulfide, and tetracaine HCl.
  • the medium does not comprise tetracaine HCl.
  • the medium comprises L-cysteinglutathione disulfide, ⁇ -Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl.
  • the medium comprises retinoic acid and/or dexamethasone.
  • the medium comprises L-glutamine.
  • the medium comprises a selenium compound.
  • the medium comprises sodium selenite.
  • the medium comprising a selenium compound also comprises one or more of an albumin, transferrin, insulin, progesterone, putrescine, biotin, 1-carnitine, corticosterone, ethanolamine, d(+)-galactose, glutathione (reduced), linolenic acid, linoleic acid, retinyl acetate, selenium, T3 (triodo-1-thyronine), dl- ⁇ -tocopherol, dl- ⁇ -tocopherol acetate, catalase, and superoxide dismutase.
  • proteins and enzymes of the medium are isolated from human or are recombinant with a human sequence.
  • a composition for differentiating stem cells into a differentiated cell of interest, the composition comprising isolated conditioned medium obtained from a culture of hepatoblasts cultured in a medium comprising a basal medium.
  • Basal media are widely-known in the art, and as used herein are understood to encompass cell-growth media (for example, un-supplemented) used for culturing mammalian cells.
  • a composition comprising isolated conditioned medium obtained from a culture of hepatoblasts cultured in a medium comprising a basal medium, or a basal medium further comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl.
  • a composition for differentiating stem cells into a differentiated cell of interest, the composition comprising conditioned medium obtained from a culture of hepatoblasts cultured in a medium comprising a basal medium, or a basal medium further comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl.
  • the medium comprising a basal medium from which the conditioned medium is obtained, or (ii) the basal medium further comprising two or more of phenacetin, phytosphingosine HCl, and pyridoxal HCl also further comprises L-glutamine, non essential amino acids, and an antibiotic.
  • the medium comprising a basal medium further comprises L-glutamine, non essential amino acids, and an antibiotic.
  • the basal medium is a DMEM.
  • the antibiotic is penicillin-streptomycin.
  • the non-essential amino acids are glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.
  • the medium comprising a basal medium further comprises an artificial serum replacement.
  • the medium further comprises one or more of L-cysteinglutathione disulfide, ⁇ -Glu-Cys, DL-kynurenine, D-penicillamine disulfide, and tetracaine HCl.
  • the medium does not comprise tetracaine HCl.
  • the medium comprises L-cysteinglutathione disulfide, ⁇ -Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl.
  • the aforelisted components are, independently, present at a concentration of 10 ⁇ M or less, of 7.5 ⁇ M or less, 5 ⁇ M or less, of 2.5 ⁇ M or less, or 1 ⁇ M or less. In an embodiment, the aforelisted components are, independently, present at a concentration of greater than 0.01 ⁇ M.
  • the medium comprises retinoic acid and/or dexamethasone. In an embodiment, the medium comprises L-glutamine. In an embodiment, the medium comprises a selenium compound. In an embodiment, the medium comprises sodium selenite.
  • the medium comprising a selenium compound also comprises one or more of an albumin, transferrin, insulin, progesterone, putrescine, biotin, 1-carnitine, corticosterone, ethanolamine, d(+)-galactose, glutathione (reduced), linolenic acid, linoleic acid, retinyl acetate, selenium, T3 (triodo-1-thyronine), dl- ⁇ -tocopherol, dl- ⁇ -tocopherol acetate, catalase, and superoxide dismutase.
  • proteins and enzymes of the medium are isolated from human or are recombinant with a human sequence.
  • the hepatoblasts are immortalized.
  • the hepatoblasts are human hepatoblasts.
  • the hepatoblasts are fetal hepatoblasts.
  • the hepatoblasts are immortalized human fetal hepatoblasts.
  • the hepatoblasts are mammalian, but are not human.
  • the hepatoblasts are human, but are not fetal.
  • the specified components are present in amounts consistent with the health and/or growth of the cells in the medium.
  • a hepatocyte is an epithelial parenchymatous cell of the liver.
  • the hepatocyte is capable of secreting bile.
  • the hepatocyte is polygonal.
  • Induced pluripotent stem cells are adult cells, typically somatic cells, that have been genetically reprogrammed to an embryonic stem cell-like state by being caused to express genes and factors important for maintaining the defining properties of embryonic stem cells.
  • the induced pluripotent stem cells are mammalian.
  • the adult cell from which the induced pluripotent stem cell is induced is a human adult cell.
  • Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst.
  • the embryonic stem cells are mammalian.
  • the embryonic stem cells are non-human.
  • the embryonic stem cells are human. Basal media are serum-free media widely available in the art.
  • a method for treating a liver disorder in a subject comprising administering to the subject an amount of the described compositions, or an amount of the described differentiated cells obtained by any of the methods described hereinabove, in an amount effective to treat a liver disorder.
  • a liver disorder is a disorder or pathology of the mammalian liver which impairs the proper functioning of the liver as compared to a healthy liver. Such disorders are widely known in the art.
  • pluripotent stem cell-derived hepatocytes expressed repertoires of hepatobiliary and mesenchymal genes, distinct microRNA profiles, as well as synthetic and metabolic hepatic functions, e.g., albumin secretion, urea production, and xenobiotic disposal, which recapitulated properties of fetal hepatocytes. Therefore, these hESC-derived cells were designated “embryonic/fetal hepatocyte-like cells” (eFHLC).
  • CM Conditioned medium
  • a multi-step protocol was developed that included culture of hESC in FH-CM alone for first 3 d followed over the next 3 d by culture of hESC with FH-CM plus three known soluble factors and then further culture of hESC for a total of 14 d with FH-CM plus another known soluble factor. None of the soluble factors added to FH-CM was essential for initiating hepatic differentiation in hESC. However, in the presence of these known soluble factors, hESC adhered better in plastic culture dishes.
  • hESC in primary culture gained epithelial morphology within 3 d ( FIG. 1A-B ).
  • the eFHLC expressed liver/pancreas foregut endoderm markers i.e., glycogen, glucose-6-phosphatase (G6P), glucagon, and others, and the mesenchymal marker, vimentin (VIM) ( FIG. 1C-F ).
  • G6P glucose-6-phosphatase
  • VIM mesenchymal marker
  • FIG. 1C-F mesenchymal marker
  • 0.50 ⁇ 0.07 ⁇ 10 6 hESC originated 1.94 ⁇ 0.05 ⁇ 10 6 eFHLC, which constituted a 4-fold gain in cell numbers.
  • population doublings of eFHLC during P1 and P2 cultures ( FIG. 1G-1H ), produced >20-fold gains in their cell numbers, which indicated the ability of differentiating cells to continue proliferating, and was consistent with highly efficient generation of differentiated cells from pluripotent stem cells.
  • eFHLC were characterized by morphology, gene expression and functional assays. eFHLC acquired more cytoplasm, larger nuclei, even binucleated cells, similar to hepatocytes ( FIG. 2A ). Cytoplasmic complexity with lysosomes, microperoxisomes and vacuoles, resembled that in hepatocytes.
  • RT-PCR Reverse transcription-polymerase chain reactions
  • AFP ⁇ -fetoprotein
  • AAT albumin
  • CK cytokeratin
  • metabolic enzymes e.g., CYP-1B1, -3A4, -2E1, and -1A1
  • mesenchymal markers i.e., VIM, ⁇ -smooth muscle actin (aSMA) ( FIG. 2B ), confirming conjoint meso-endodermal phenotype of natural fetal hepatocytes (6,7).
  • E-cadherin ECAD
  • eFHLC gene expression ontology and regulation of cytokine signaling trended toward fetal hepatocytes ( FIG. 3 ).
  • Global microRNA (miRNA) profiling showed several similarities between eFHLC and fetal hepatocytes when array analysis of cellular miRNA in eFHLC versus hESC and freshly isolated fetal human hepatocytes (FH-PP) was performed.
  • eFHLC Protein studies verified mRNA findings in eFHLC as stem cell markers declined (OCT4, NANOG, TRA-1-80) and hepatic (FOXA2, ALB, G6P and glycogen) and biliary (GGT) properties increased.
  • OCT4 was lost with gain of ECAD, VIM, FOXA2, and ALB expression.
  • eFHLC contained hepatobiliary markers, glycogen, G6P and GGT.
  • ECAD epithelial
  • VIM mesenchymal
  • asialoglycoprotein receptor (ASGPR1), which is specific to adult hepatocytes, indicated many eFHLC were maturing along the hepatic lineage. Flow cytometry showed 27% eFHLC expressed ASGPR1, marking mature hepatocytes.
  • eFHLC expressed hepatic synthetic, metabolic and xenobiotic disposal functions in vitro After 14 d of differentiation, eFHLC secreted albumin, synthesized urea and converted a xenobiotic, ethoxyresorufin, to resorufin ( FIG. 4A-4C ). Such functions are important for hepatic support in liver failure (7, 11). Similarly, paracrine factors may protect hepatocytes from injury (14). This was confirmed since CM from eFHLC protected mouse hepatocytes from tumor necrosis factor (TNF)- ⁇ toxicity ( FIG. 4D ).
  • TNF tumor necrosis factor
  • telangiectasia mutated (ATM) signaling 11
  • ATM ataxia telangiectasia mutated
  • Huh-7 cells were prepared (from human hepatocellular carcinoma) to express the tdTomato reporter gene under control of cloned human ATM promoter (15).
  • Cis-P cis-platinum
  • eFHLC rescued mice with drug-induced ALF: Identification of hepatic functions and potential for paracrine signaling suggested eFHLC could support recovery of the damaged liver. This was facilitated by a NOD/SCID mouse model of ALF (11), where rifampicin (Rif), phenytoin (Phen) and monocrotaline (MCT) caused dysregulation of Atm signaling, leading to severe oxidative stress, DNA damage, hepatic necrosis, liver test abnormalities, coagulopathy, encephalopathy, and 90-100% mortality. Intraperitoneal transplantation of mature hepatocytes with microcarrier scaffolds rescued mice with ALF.
  • Rif rifampicin
  • Phhen phenytoin
  • MCT monocrotaline
  • transplanted hepatocytes remained in peritoneal cavity without migrating to the liver. Moreover, reseeding of the liver with cells was unnecessary.
  • eFHLC mice survived (50%) versus only 1 mouse in the vehicle group (10%), p ⁇ 0.001.
  • encephalopathy was absent or less severe
  • sham-treated mice developed severe encephalopathy (grade 3-4), p ⁇ 0.05.
  • Liver tests improved in the eFHLC group versus the vehicle group.
  • ALT serum alanine aminotransferase
  • Transplanted eFHLC were absent from the native liver, as was expected. Livers of vehicle-treated mice were edematous, hemorrhagic and necrotic with extensive expression of phosphorylated histone H2AX, confirming oxidative DNA damage, and only interspersed Ki-67+ cells, indicating limited liver regeneration. In eFHLC-treated mice, liver necrosis and H2AX expression decreased, while the prevalence of Ki-67+ cells increased. Histological grading showed 5.4-fold less liver injury in eFHLC-treated mice after 7d, 0.7 ⁇ 0.3 versus 3.8 ⁇ 0.4, p ⁇ 0.05.
  • eFHLC did not proliferate, and no tumors were observed in NOD/SCID mice over 3 months. Undifferentiated hESC generated teratomas as expected (not shown) (5-7).
  • FH-CM hepatic differentiation of hESC
  • cytokines and growth factors were examined, and 62 of 507 such proteins were found to be present in FH-CM ( FIG. 6 ), including regulators of cell differentiation, e.g., activinA, FGFs, or transforming growth factors (TGF) (12, 13).
  • regulators of cell differentiation e.g., activinA, FGFs, or transforming growth factors (TGF) (12, 13).
  • TGF transforming growth factors
  • FH-CM receptors of 12 ligands present in FH-CM
  • FH-CM was degraded by heating to 100° C., and passed FH-CM through Amicon membranes to remove >3 kilodalton size proteins.
  • Protein-depleted FH-CM still induced epithelial differentiation in hESC, including changes in morphology and gene expression, including in expression of pluripotency (Nanog, Sox-2), epithelial (AFP), or mesenchymal (VIM) genes within 3 d ( FIG. 8 ). It was concluded that nonprotein molecules in FH-CM were involved.
  • FH-CM induced hepatic differentiation in hESC by the steps of endoderm specification followed by maturation to fetal stage. This reproduced paracrine effects of proteins during differentiation of mouse stem cells in vitro (17). However, hepatic differentiation induced by FH-CM lacking proteins was singularly different from cell differentiation induced by cytokine/chemokine/growth factor-based protocols.
  • putative differentiation-inducing molecules included those affecting differentiation in stem/progenitor cells, e.g., 2-aminoadipic acid (26).
  • Use of specific matrices and synthetic surfaces could help in further expanding eFHLC or other stem cell-derived cell types.
  • stem cell-derived hepatocytes may engraft and express liver functions in animals, although often largely at mRNA level (5-7, 22, 28)
  • proliferation of transplanted cells is critical (22, 28). In treating genetic conditions permanently, extensively modified cells, e.g., those reprogrammed with multiple transcription factors, will be less desirable than cells differentiated simply by extracellular soluble signals.
  • Fetal livers were from Human Fetal Tissue Repository at Einstein. Ep-CAM+ liver cells were isolated and cultured as previously described (5, 6). hTERT-FH-B cells were cultured in DMEM with 10% FBS (12). To obtain CM, hTERT-FH-B were cultured for 24 h in DMEM/F12 medium with 2% Knock-out Serum Replacer (KSR), 2 mM L-glutamine, 0.1 mM MEM Non Essential Amino Acids (NEAA), 1% penicillin-streptomycin (Invitrogen Corp., Carlsbad, Calif.).
  • KSR Knock-out Serum Replacer
  • NEAA Non Essential Amino Acids
  • penicillin-streptomycin Invitrogen Corp., Carlsbad, Calif.
  • hESC Culture of hESC.
  • WA-01 hESC were passaged weekly on matrigel-coated dishes in DMEM/F12 medium, 1% B27 supplement, 1% N2 supplement, 2 mM L-glutamine, 0.1 mM NEAA, 1% penicillin-streptomycin (Invitrogen Corp.) and 50 ng/ml basic FGF (R&D Systems, Minneapolis, Minn.).
  • DMEM/F12 medium 1% B27 supplement, 1% N2 supplement, 2 mM L-glutamine, 0.1 mM NEAA, 1% penicillin-streptomycin (Invitrogen Corp.) and 50 ng/ml basic FGF (R&D Systems, Minneapolis, Minn.).
  • hESC were washed with DMEM/F12, and cultured in FH-CM.
  • Cytotoxicity assays For effects of CM from hFHLC on TNF- ⁇ -induced cytotoxicity, 1.5 ⁇ 10 5 primary mouse hepatocytes were isolated by collagenase perfusion (11), and plated in 24-well dishes in RPMI 1640 medium with 10%/FBS and antibiotics. After overnight culture, cells were switched to CM plus 10 ng/ml TNF- ⁇ (Sigma) for 16-18 h, followed by thiazolyl blue viability assays, as described previously (14). For cisP toxicity assays, Huh-7 cells were used after transduction by a lentiviral vector to express human ATM promoter-driven tdTomato gene (15).
  • albumin cell culture medium harvested after 3 h was analyzed by human albumin immunoassay (Bethyl Laboratories, Montgomery, Tex.).
  • ureagenesis cells were incubated with 2.5-7.5 mM ammonium chloride for 12 h and urea content was analyzed as previously described (27).
  • CYP450 activity cells were induced overnight with phenobarbital, 7-ethoxyresorufin and ⁇ M dicumarol were added for 12 h at 37° C., and resorufin was measured, as described previously (30).
  • Human cytokine arrays Human cytokine arrays. Conditioned medium was analyzed by human antibody array I membrane for 507 human proteins and cell lysates were analyzed by Human RTK Phosphorylation Antibody Array 1 (RayBiotech, Norcross, Ga.), according to manufacturer.
  • 4-6 ⁇ 10 6 hESC-derived cells differentiated for 14 d were transplanted i.p. with 1 ml Cytodex 3TM microcarriers (Amersham Biosciences Corp., Piscataway. N.J.). Sham-treated animals received microcarriers. Encephalopathy was graded from 0 (absent) to 3 (coma). Mice were observed for 2 weeks. In some studies, hESC-derived cells were injected subcutaneously or i.p. for tumor formation over 3 months.
  • TRITC-conjugated goat anti-mouse IgG (1:50, Sigma) or anti-rabbit IgG (1:100) were added for 1 h with 4′-6-diamidino-2-phenylindole (DAPI) (Invitrogen) counterstaining. In negative controls, primary antibodies were omitted. Glycogen, G-6-P, and GGT were stained as described (4-7).
  • Electron microscopy Cells were fixed in 2.5% glutaraldehyde in cacodylate butter, postfixed in osmium tetroxide, and stained with 1% uranyl acetate before embedding in plastic. Ultrathin sections were examined under JEOL 1200 electron microscope.
  • RNA samples total RNA isolated by TRIzol reagent (Invitrogen) was analyzed by LC Sciences (Houston, Tex.) with probe set based on Sanger miRBase, v 9.0. Later, transcripts with ⁇ 500 arbitrary signals were excluded as these are not identified by qRT-PCR. Data were transformed to log 2 followed by clustering of transcripts (Cluster3, Stanford University) and heatmaps were drawn (JavaTree1.1.6r2).
  • Tissue studies Tissue samples were frozen to ⁇ 80° C. in methylbutane. Cryostat sections were prepared. Tissue morphology was analyzed by H&E stained sections. Tissue injury was graded as previously described (38). For hepatic function in transplanted cells, glycogen and G-6-P were stained (35). For Ki67 and histone H2AX, tissues were fixed in 4% PAF followed by rabbit anti-Ki67 (1:750, Vector Laboratories, Burlingame, Calif.) or rabbit anti-phosphoS139 H2AX (1:300, ab2893: Abcam, Cambridge, Mass.), respectively, and secondary anti-Rabbit Alexa Fluor 546 (1:500, Molecular Probes), followed by counterstaining with DAPI (7, 11). Transplanted cells were identified by in situ hybridization for alphoid satellite sequences in centromeres (27).
  • LC-MS Liquid chromatography-mass spectroscopy
  • Serological studies Sera were stored at ⁇ 20° C. and analyzed for ALT and bilirubin as previously described. Human albumin was measured by immunoassay (Bethyl Laboratories).
  • hESC human embryonic stem cells
  • FH-CM fetal hepatocytes
  • the culture medium contained additives, including retinoic acid, dexamethasone. Amounts of >10 micromolar of the compounds caused toxicity with cell death and detachment of adherent hESC from surfaces of cell culture dishes. Further analysis indicated culture of hESC with CP individually or in groupings of up to 6 CP at one time in 1, 5 or 10 micromolar amounts, did not alter morphology of cultured hESC and these continued to display characteristic small sizes and cluster formation (not shown). By contrast, culture of hESC with a group of 7 CP in 5 or 10 micromolar concentrations for 2 weeks generated large cells with uniform morphology of epithelial cells ( FIG. 9A ).
  • additives including retinoic acid, dexamethasone. Amounts of >10 micromolar of the compounds caused toxicity with cell death and detachment of adherent hESC from surfaces of cell culture dishes. Further analysis indicated culture of hESC with CP individually or in groupings of up to 6 CP at one time in 1, 5
  • CP1+CP2 The following combinations of CP were ineffective in hESC differentiation: CP1+CP2; CP1+CP2+CP3; CP1+CP2+CP3+CP4.
  • the following combinations of CP were partially effective because both undifferentiated hESC and differentiated cells were present in culture dishes: CP5+CP6; CP6+CP7; and CP5+CP6+CP7.
  • 7 or 8 CP were combined together, only differentiated cells were present in culture dishes.
  • the following group of CP was most effective: CP1+CP2+CP3+CP4+CP5+CP6+CP7.
  • iPSC were utilized that were obtained by reprogramming of normal human fibroblasts with non-integrating Sendai virus vectors from the Pluripotent Stem Cell Core at Albert Einstein College of Medicine.
  • the differentiation protocol with FH-CM and CP was identical to studies with hESC described above.
  • iPSC cultured with FH-CM became larger with epithelial morphology, whereas undifferentiated iPSC were smaller and were arranged in clusters ( FIG. 10A ).
  • iPSC cultured with above-described combination of 7 CP became larger with epithelial morphology.
  • Expression of OCT4 was lost in iPSC cultured with either FH-CM or combination of 7 CP ( FIG. 10B ).
  • Differentiated cells contained albumin as shown by immunostaining, whereas undifferentiated iPSC were negative for albumin staining ( FIG. 10C ). Moreover, similar to differentiation of hESC under these conditions, we found differentiated cells expressed vimentin. These iPSC-derived hepatocytes synthesized urea and metabolized ethoxyresorufin to resorufin ( FIG. 10E , 10 F). This substantiated that this combination of 7 CP generated hepatocytes from iPSC.
  • WA-01 hESC were passaged on matrigel-coated dishes in DMEM/F12 medium, 1% B27 supplement, 1% N 2 supplement, 2 mM L-glutamine, 0.1 mM NEAA (Life Technologies) and 50 ng/ml basic FGF (R&D Systems).
  • the iPSC were generated from normal human fibroblasts with CytoTune®-iPS Sendai Reprogramming Kit (Life Technologies, Cat #A1378001).
  • the iPSC were cultured on matrigel-coated dishes in DMEM/F12 medium, 1% B27 supplement, 1% N 2 supplement, 2 mM L-glutamine, 0.1 mM NEAA (Life Technologies) and 50 ng/ml basic FGF (R&D Systems).
  • hTERT-FH-B cells were cultured for 24 h in DMEM/F12 medium with 2% Knock-out Serum Replacer (KSR), 2 mM L-glutamine, 0.1 mM MEM Non Essential Amino Acids (NEAA), 1% penicillin-streptomycin (Life Technologies).
  • Hepatic differentiation For differentiation, hESC/iPSC were washed with DMEM/F12 and cultured for 2 weeks in FH-CM and CP in DMEM/F12 with 2% Knock-out Serum Replacer (KSR), 1% B27 supplement, 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids (NEAA), 1% penicillin-streptomycin (Life Technologies).
  • KSR Knock-out Serum Replacer
  • NEAA Non-Essential Amino Acids
  • NEAA penicillin-streptomycin
  • TRITC-conjugated goat anti-mouse IgG (1:50, Sigma) cells were counterstained for 1 h with 4′-6-diamidino-2-phenylindole (DAPI) (Life Technologies). In negative controls, primary antibody was omitted.
  • Hepatic functions For ureagenesis, cells were incubated with 5 mM ammonium chloride for 12 h and analyzed as described (Cho et al., 2004, (31)). For CYP450 activity, cells were analyzed for 7-ethoxyresorufin conversion, as described (Gupta et al., 1999, (32)).

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