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WO2012005690A1 - Procédés de reconstitution d'hépatocytes humains et de cellules hématopoïétiques humaines chez des mammifères non humains - Google Patents

Procédés de reconstitution d'hépatocytes humains et de cellules hématopoïétiques humaines chez des mammifères non humains Download PDF

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Publication number
WO2012005690A1
WO2012005690A1 PCT/SG2011/000234 SG2011000234W WO2012005690A1 WO 2012005690 A1 WO2012005690 A1 WO 2012005690A1 SG 2011000234 W SG2011000234 W SG 2011000234W WO 2012005690 A1 WO2012005690 A1 WO 2012005690A1
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human
cells
human mammal
hpscs
liver
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PCT/SG2011/000234
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English (en)
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Qingfeng Chen
Maroun Khoury
Kok Yen Jerry Chan
Jianzhu Chen
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Massachusetts Institute Of Technology
National University Of Singapore
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Publication of WO2012005690A1 publication Critical patent/WO2012005690A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases

Definitions

  • the human liver is an important organ for pharmacological studies aimed at developing new human medicines.
  • Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver functions, diseases and hepatotropic viruses such as chronic hepatitis virus (HB V and HCV).
  • HB V and HCV chronic hepatitis virus
  • researchers have developed chimeric mice by partially repopulating the mouse liver with human mature hepatocytes and used these mice for HB V and HCV studies (Dandri, M., et al, Hepatology, 33:981-988 (2001); Tateno, C.Y., et al, Am J Pathol, 165:901-912 (2004)).
  • the existing models require uPA transgenic mice which are difficult to maintain as well as complicated surgical procedures.
  • liver-cell progenitors may play a role in recovery, proliferating, and subsequently differentiating into mature liver cells.
  • the proliferative potential that progenitor cells have is a major advantage for therapeutic exploitation compared to mature hepatocytes that have low proliferating activity. It has been reported that during rat fetal development, bipotential cells, known as hepatoblasts, are present in the liver (Shiojiri, N., et al, Cancer Res, 51:2611-2620 (1991)).
  • Described herein is the investigation of whether the human fetal liver is an enriched stem cell pool and a resource for hematopoietic stem cells (HSC) as well as non-hematopoietic stem cells including human hepatocyte progenitors.
  • HSC hematopoietic stem cells
  • non-hematopoietic stem cells including human hepatocyte progenitors.
  • a novel population of human hepatocyte progenitors were identified in fetal liver.
  • Human CD34 + stem cells were purified from total fetal liver cells by magnetic selection. Similar to cells from human cord blood, engraftment of these CD34 + cells into sub-lethally irradiated new born NSG mice led to the reconstitute human hematopoietic cells in the mice. However, human albumin hepatocytes were found only in the livers from fetal liver cell reconstituted mice.
  • CD34 + CD133 to cells are hepatocyte progenitors, referred to herein as hepatic progenitor/stem cells (HPSCs), that gave rise to hepatocytes following engraftment in NSG recipient mice
  • HPSCs hepatic progenitor/stem cells
  • CD34 + CD133 ta cells are hematopoietic stem cells (HSCs) that gave rise to hematopoietic cells when engrafted in NSG recipient mice.
  • HSCs hematopoietic stem cells
  • isolated mammalian e.g., human
  • hepatic progenitor/stem cells HPSCs
  • mammalian hepatic hematopoietic stem cells HSCs
  • methods of reconstituting human hepatocytes and optionally, human blood cell lineages in a non-human mammal such as a mouse
  • uses of such non-human mammals e.g., humanized mammalian models
  • methods of culturing human hepatocytes e.g., human
  • the invention is directed to an (one or more) isolated mammalian hepatic progenitor/stem cell (HPSC) which expresses CD34 + CD133 l0 .
  • HPSC can further express CD117, CD44, CD45, CD73, vimentin, EpCAM, a-fetoprotein (AFP), albumin, CD 166, CD24 or a combination thereof.
  • the HPSC is a human HPSC.
  • the HPSC is a fetal HPSC.
  • the isolated mammalian HPSC is a human fetal HPSC.
  • HSC hepatic hematopoietic stem cell
  • the HSC can further express CD45, CD44, CD117, CD 166, CD90, CD 105, CD97 or a combination thereof.
  • the invention is directed to a method of reconstituting human hepatocytes in a non-human mammal.
  • the method comprises introducing human hepatic progenitor/stem cells (HPSCs) which express CD34 + CD133 k> into an immunodeficient non-human mammal, and maintaining the non-human mammal under conditions in which the non-human mammal's liver is reconstituted with human hepatocytes, thereby reconstituting human hepatocytes in a non-human mammal.
  • HPSCs human hepatic progenitor/stem cells
  • the invention is directed to a method of reconstituting human hepatocytes and human blood cell lineages in a non-human mammal.
  • the method comprises introducing human hepatic progenitor/stem cells (HPSCs) which express CD34 + CD133 l0 and human hematopoietic stem cells (HSCs) which express
  • CD34 + CD133 W into an immunodeficient non-human mammal, and maintaining the non- human mammal under conditions in which the non-human mammal's liver is reconstituted with human hepatocytes and the non-human mammal's blood is reconstituted with human blood cells, thereby reconstituting human hepatocytes and human blood cell lineages in a non-human mammal.
  • the invention is directed to method of producing a non-human mammal for use as a model of human liver disease.
  • the method comprises introducing human hepatic progenitor/stem cells (HPSCs) which expresses CD34 + CD133 l0 into an immunodeficient non-human mammal; and mamtaining the non-human mammal under conditions in which the non-human mammal's liver is reconstituted with human hepatocytes.
  • HPSCs human hepatic progenitor/stem cells
  • One or more agents that produce a human liver disease are also introduced into the non-human mammal; and the non-human mammal is maintained under conditions in which the human liver disease develops in the non-human mammal;
  • Non-human mammals produced by the methods described herein are also provided.
  • the invention is directed to a method of identifying one or more agents or treatment protocols that can be used to treat a human liver disease.
  • the method comprises administering the one or more agents or treatment protocols to a non- human mammal that is a model of a human liver disease produced as described herein, and determining whether the human liver disease in the non-human mammal is alleviated. If the human liver disease is alleviated in the non-human mammal, then the one or more agents or treatment protocols can be used to treat the human liver disease.
  • the invention is directed to a method of identifying one or more effects of one or more agents on a human liver.
  • the method comprises
  • the invention is directed to a method of identifying how one or more agents are metabolized by a human liver.
  • the method comprises administering the one or more agents to a non-human mammal which comprises human hepatocytes produced as described herein and mamtaining the non-human mammal under conditions in which the one or more agents are metabolized by the human liver cells in the non- human mammal, thereby producing one or more metabolites in the non-human mammal.
  • the one or more metabolites are detected and/or analyzed in the non-human mammal, thereby identifying how one or more agents are metabolized by a human liver.
  • the invention is directed to an in vitro method of obtaining mammalian (e.g., human) hepatocytes.
  • the method comprises contacting mammalian (e.g., human) hepatic progenitor/stem cells (HPSCs) which express CD34 + CD133 l0 with hepatocyte differentiation medium, thereby producing a culture and maintaining the culture under conditions in which the HPSCs differentiate into mammalian (e.g., human) hepatocytes, thereby obtaining mammalian (e.g., human) hepatocytes.
  • mammalian e.g., human
  • HPSCs hepatic progenitor/stem cells
  • the invention is also directed to mammalian (e.g., human) hepatocytes (e.g., cells, cell lines, cell cultures) produced by the methods provided herein.
  • mammalian e.g., human
  • hepatocytes e.g., cells, cell lines, cell cultures
  • Fig. 1 shows isolation of three CD34 + populations by fluorescence activated cell sorting (FACS).
  • Fig. 2 shows a comparison of albumin levels in mice injected with CD133 to cells via intra-liver (i.l) and intra-cardiac (i.c) injection using ELISA.
  • Fig. 3 shows the results of cell surface phenotyping of CD133 to cell population.
  • Figs. 4A-4B show CD34 + cells from human fetal liver gave rise to
  • PBMC peripheral blood mononuclear cells
  • hCD45 human CD45
  • mCD45 mouse CD45
  • Representative plots are shown. The numbers indicate percentages of human and mouse CD45 + cells among total live cells. Paraffin sections of the livers were stained with antibody specific for human albumin. Representative stains are shown. Magnification, 100X.
  • Figs. 5A-5G show identification of HSCs and HPSCs among CD34 + fetal liver cells.
  • 5 A Comparison of CD34 + cells from human cord blood (left) and fetal liver (right) for CD133 expression. Purified CD34 + cells were stained for CD133 and analyzed by flow cytometry. Representative CD34 versus CD 133 staining profiles are shown from 5 cord blood and 3 fetal liver samples.
  • 5B CD133 hi , CD133 10 and
  • CD133 n3 ⁇ 4 subsets were isolated from CD34 + fetal liver cells by cell sorting. Purified cells were reanalyzed for purity.
  • Sera were analyzed for the level of human albumin by ELSIA.
  • 5E and 5F Livers from the mice injected with CD34 + CD133 bi and CD34 + CD133 l0 cells were harvested. Paraffin sections were stained with antibody specific for the human albumin (5E) or specific for human CK7 (5F). Arrow points to CK7 stained biliary duct cells.
  • Figs. 6A-6E show in vitro differentiation of the three subpopulation of CD34 + fetal liver cells.
  • Figs. 7A-7D show CD34 + CD133 to fetal liver cells exhibit long-term self-renewal properties.
  • 7 A Single cell suspensions of hepatocytes of primary recipient mice stained for human CD34 and CD 133 followed by flow cytometry. Representative staining profile of CD34 versus CD133 is shown for one of eleven mice. The number indicates percentage of human cells within the gated region.
  • 7B Human CD34 + cells were purified by magnetic cell sorting. The purified cells were stained for human CD34, CD133, CD44 and EpCAM. Staining profiles of CD34 versus CD133 and EpCAM versus CD44 are shown.
  • the purified CD34+ cells were injected into sublethally irradiated newborn NSG pups (10 5 cells per recipient). Eight weeks later, liver paraffin sections were stained for human albumin (7C) and the level of human albumin in the sera was quantified by ELISA (7D). Adult non-reconstituted NSG mice were used as negative control. One dot represents one mouse in 7D.
  • Figs. 8A-8F show transcription profiles of HPSCs are more closely related to those of HSCs from both fetal liver and cord blood than to mature hepatocytes.
  • 8 A Comparison of surface phenotype of selected markers among HPSCs and HSCs from fetal liver and HSCs from cord blood. Staining profiles of human stem cell-related markers CD44, CD117, EpCAM, CD90, CD105, CD97, CD24, CD73, CD166 and CD4S are shown as histograms. Dark line, stained with specific antibodies; thin line, isotype controls.
  • CD34 + CD133 l0 , CD34 + CD133 W and CD34 + CD133 neg cells were from three biological samples.
  • Fig. 9 shows a comparison of cell surface phenotype of human hepatoma cell lines with that of HPSCs and mature hepatocytes.
  • Human CD34 + CD 133 l0 fetal liver cells, human mature hepatocytes, Hep 3B cells, Hep G2 cells and SNU-423 cells were stained for CD133, CD34, CD44, CD117, EpCAM, CD90, CD105, CD97, CD24, CD73, and CD166. Expression of each marker is shown as histogram. Dark line, stained with specific antibodies; thin line, isotype controls.
  • Figs. lOA-lOC (10A) Fetal liver cells were stained for CD34 and CD133 after CD34 selection. Shown are cytometry FSC and SSC data and phase-contrast micrograph of the sorted three cell populations. (10B) Human specific CK19 antibody was used to stain human biliary epithelial cells in the liver sections. Black arrows indicate the bile ducts positive for human CD 19 while the white ones indicate the negative ducts. (IOC) Cy3 fluorescence conjugated probe against human pan centromere and DAPI were used to stain human cells in the liver sections. Human fetal liver sections were used as positive control; NSG mouse liver sections were used as negative control. White arrows in the liver sections prepared from mice injected with CD 1331o cells indicate positive cells for human pan-centromere stainings.
  • Figs. 12A-12C (12A) COU and CD133 10 cells were sorted by FACS sorter and fixed on slides by Cytospin. Antibodies against human albumin, AFP, CK7 and CK19 were used to stain the cells. DAPI. Albumin, AFP, CK7 and CK19. (12B) Principal component analysis (PCA) of the microarray data. Shown is 3 dimensional plot of PCA. Each data point represents individual sample/array, with all 12
  • PC 1 principal component 1 (X-axis); PC 2 principal component 2 (Y-axis); PC 3 principal component 3 (Z-axis).
  • CD133 neg CD133 111 and CB HSC samples/arrays aggregate together which signify their similarity in expression profile. However, hepatocyte were separated from the other.
  • Hepatic progenitor/stem cells are of great interest because of their potential applications in preclinical pharmaceutical testing and clinical transplantation.
  • hepatoblasts in fetal liver and oval cells in adult liver are known to exhibit hepatic progenitor cell properties as they can differentiate into both hepatocytes and biliary epithelial cells (cholangiocytes) in vitro as well as in vivo following adoptive transfer into recipient mice or rats (Strick-Marchand, H., and Weiss, M. C. (2002), Hepatology 36, 794-804; Walkup, M. H., and Gerber, D. A. (2006), Stem Cells 24, 1833-1840).
  • hepatoblasts and oval cells have also been reported in human fetal and adult livers, respectively, they are far less well characterized than their rodent counterparts.
  • HPSCs are distinct from HSCs by expressing hepatic and biliary cell markers such as a-fetalprotein (AFP), albumin and cytokeratin (CK) (Kubota and Reid (2000), Proc Natl Acad Sci USA, 97, 12132-12137; Minguet et al. (2003) J Clin Invest, 112(8), 1152-1163; Nierhoff et al. (2005),
  • AFP a-fetalprotein
  • CK cytokeratin
  • CD45 + CD34 + cells from mouse bone marrow were shown to give rise to hepatocytes following adoptive transfer into recipient mice (Lagasse, E., et al. (2000), Nat Med 6, 1229-1234; Theise, N. D. et al. (2000a), Hepatology 31, 235-240). This kind of trans-differentiation was also observed in the patients receiving bone marrow transplantation (Theise, N. D. et al. (2000b), Hepatology 32, 11-16). Although some of these observed trans- differentiations were later shown to result from fusion between hematopoietic cells and hepatocytes, confusion persists. Whether HPSCs and HSCs are separate lineages and can be uniquely isolated from human fetal liver remains unknown.
  • mice Construction of humanized mice by adoptive transfer of CD34 + cells from human fetal liver into sub-lethally irradiated NOD-SCID n2rg _ " (NSG) mice is described herein. It was found that the recipient mice have not only human
  • HPSCs hematopoietic cells in the lymphoid systems but also human hepatocytes in the mouse liver.
  • This technological breakthrough has enabled identification and isolation of HPSCs from human fetal liver that give rise to hepatocytes and biliary epithelial cells and HSCs that give rise to hematopoietic cells.
  • the isolated HPSCs express many HSC markers and are transcriptionally more closely related to HSCs than to mature hepatocytes.
  • some hepatoma cell lines exhibit the characteristic phenotype of HPSCs while others exhibit the surface phenotype of mature hepatocytes.
  • CD34 + CD133 10 fetal liver cells are HPSCs because they expanded and gave rise to both hepatocytes and biliary epithelial cells in primary recipients and were capable of self-renewal and secondary reconstitution.
  • CD34 + CD133 bi fetal liver cells gave rise to hematopoietic cells in recipient mice.
  • HPSCs were transcriptionally more closely related to HSCs from both fetal liver and cord blood than to mature human hepatocytes. Nevertheless, HPSCs expressed mesenchymal markers CD73 and progenitor hepatocyte markers a-fetoprotein, albumin, vimentin, CK18, and CK19. Furthermore, some human hepatoma cell lines shared similar surface phenotype and transcriptional profiles of HPSCs. The results provided herein unequivocally identified HPSCs in human fetal liver and revealed a close relationship between HPSCs and HSCs in the fetal liver. The simple and robust assay described herein facilitates both basic and applied research of human HPSCs in health and diseases.
  • the present invention is directed to mammalian HPSCs; methods of producing in vivo models containing livers repopulated with human hepatocytes, and optionally, a human hematopoietic system; the models produced by the methods; and uses of the models (e.g., to study the etiology and therapy of viral and nonviral human liver diseases, hepatocyte biology and human hepatomas). Also provided herein are abundant human hepatocyte progenitors which can be used in a variety of ways, as well as methods of culturing HPSCs.
  • the invention is directed to isolated HPSCs.
  • the invention is directed to compositions of mammalian fetal liver cells comprising, consisting essentially of, or consisting of HPSCs which express CD34 + CD133 to .
  • the composition comprises, consists of, or consists essentially of mammalian fetal HPSCs which express CD34 + CD 133 lo .
  • the invention is also directed to uses of such compositions.
  • the compositions can further comprise, consist essentially of, or consist of mammalian fetal
  • HSCs hematopoietic stem cells
  • the invention is directed to an (one or more) isolated mammalian (e.g., human) fetal HPSC wherein the cell expresses CD34 + CD 133 l °.
  • the fetal HPSC can further expresses CD117, CD44, CD45, CD73, vimentin, EpCAM, a-fetoprotein (AFP), albumin, CK7 and CK19 or a combination thereof.
  • the invention is also directed to isolated mammalian (e.g., human) fetal liver cells which comprise, consist essentially of, or consist of HPSC which express CD34 + CD133 k> and HSCs which express
  • CD34 + CD133 bi CD34 + CD133 bi .
  • isolated refers to substantially isolated with respect to the complex (e.g., cellular) milieu in which it (e.g., naturally) occurs (e.g., organ, body, tissue, blood, or culture medium).
  • the isolated material will form part of a composition (e.g., a crude extract containing other substances), buffer system, culture system or reagent mix. In other circumstances, the material can be purified to essential homogeneity.
  • Isolated mammalian fetal HPSCs can comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% (on a total cell number basis) of all cells present.
  • the invention is directed to isolated, or substantially isolated (or purified, substantially purified) mammalian fetal HPSCs described herein.
  • compositions of the invention can be used in a variety of ways.
  • the invention is directed to a method of reconstituting human hepatocytes in a non-human mammal.
  • the method comprises introducing human hepatic
  • HPSCs progenitor/stem cells which express CD34 + CD133 l0 into an immunodeficient non-human mammal, and maintaining the non-human mammal under conditions in which the non-human mammal's liver is reconstituted with human hepatocytes, thereby reconstituting human hepatocytes in a non-human mammal.
  • the invention is directed to a method of reconstituting human hepatocytes and human blood cell lineages in a non-human mammal.
  • the method comprises introducing human hepatic progenitor/stem cells (HPSCs) which express CD34 + CD133 to and human hematopoietic stem cells (HSCs) which express
  • CD34 + CD133 hi into an immunodeficient non-human mammal, and maintaining the non- human mammal under conditions in which the non-human mammal's liver is reconstituted with human hepatocytes and the non-human mammal's blood is reconstituted with human blood cells, thereby reconstituting human hepatocytes and human blood cell lineages in a non-human mammal.
  • the human HPSCs expand at least 200 fold, 300 fold, or 400 fold during differentiation into human hepatocytes in the non-human mammal.
  • HPSCs and/or HSCs can be obtained and purified as described herein, using routine methods known to those of skill in the art or from commercial sources.
  • HPSCs and or HSCs are obtained from a fetal (e.g., human) liver.
  • HSPSCs and/or HSCs are obtained from embryonic stem cells and/or cloned cells.
  • fetal liver e.g., single cell suspensions of human fetal liver
  • antibodies specific for the appropriate cell surface markers e.g., CD34 + CD133 lo ; CD34 + CD133 ⁇ ; CD117; CD44; CD45; CD73; vimentin; EpCAM; a-fetoprotein (AFP); albumin
  • FACS fluorescence activated cell sorting
  • the cells are further enriched for the HPSCs and/or HSCs prior to introduction into the non-human mammal. Methods for enriching an isolated cell population are routine and known to those of skill in the art.
  • the mammalian HPSCs and HSCs for use in the methods of the invention can be introduced into the non-human mammal directly as obtained (e.g., unexpended) or manipulated (e.g., expanded) prior to introduction into the non-human mammal.
  • the mammalian HPSCs and HSCs are not expanded prior to introducing the HSCs into the non-human mammal.
  • the mammalian HPSCs and HSCs are expanded prior to introducing the HSCs into the non-human mammal.
  • the mammalian HPSCs and HSCs for use in the methods can be obtained from a single donor or multiple donors.
  • the mammalian HPSCs and HSCs used in the methods described herein can be freshly isolated, preserved (e.g., cryopreserved) or combinations thereof.
  • HPSCs, and optionally, HSCs that are introduced into the non- human mammal will vary depending on a variety of factors such as the cells, the non- human mammal, the application for which the cells are being introduced. In one embodiment, about 0.1 x 10 5 HPSCs to about 10 x 10 s HPSCs are introduced into the non-human mammal.
  • about 1 x 10 5 HPSCs are introduced into the non-human mammal.
  • about 2 x 10 s HPSCs are introduced into the non-human mammal.
  • HPSCs and optionally HSCs, are introduced into a non-human mammal.
  • mammal and “mammalian” refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals).
  • HPSCs fetal
  • HSCs fetal
  • rodents e.g., rats, mice, guinea pigs
  • canines felines
  • ruminents e.g., cows, pigs, horses
  • the HPSCs and/or HSCs are obtained from the fetus of a (one or more) mammal.
  • the fetal HPSCs and/or HSCs are human fetal HPSCs and/or HSCs.
  • non-human mammalian species that can be used to reconstitute human hepatocytes, and optionally human blood cell lineages, include non-human primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs), canines, felines, and ruminents (e.g., cows, pigs, horses).
  • the non-human mammal is a mouse.
  • the non-human mammal used in the methods described herein can be adult, newborn (e.g., ⁇ 48 hours old; pups) or in utero. In particular embodiment, the non-human mammal is a newborn or pup.
  • the non-human mammal is an immunodeficient non-human mammal, that is, a non-human mammal that has one or more deficiencies in its immune system (e.g., NSG or NOD scid gamma (NOD. Cg-Prkdcscid H2rgtml Wjl/SzJ) mice) and, as a result, allows reconstitution of human hepatocytes and human blood cell lineages when fetal HPSCs and/or HSCs are introduced.
  • the non-human mammal lacks its own T cells, B cells, NK cells or a combination thereof.
  • the non-human mammal is an immunodeficient mouse, such as a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation (NOD/scid mouse); a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation and lacks a gene for the cytokine-receptor ⁇ chain (NOD/scid IL2R ⁇ -/- mouse); and a Balb/c rag-/- yc-/- mouse.
  • NOD/scid mouse a non-obese diabetic mouse that carries a severe combined immunodeficiency mutation and lacks a gene for the cytokine-receptor ⁇ chain
  • Balb/c rag-/- yc-/- mouse a Balb/c rag-/- yc-/- mouse.
  • immunodeficient mice include, but are not limited to, severe combined immunodeficiency (scid mice, non-obese diabetic (NOD)-scid mice, lL2rg ⁇ ' ⁇ mice (e.g., NOD/LySz-jciV/ IL2rg 'A mice, NOD/Shi- scid IL2rg ' mice (NOG mice), B ALB/c- Rag'lLlrg 1' mice, m -Rag'lL2rg'- mice), WS IRag ⁇ IUrg ⁇ mice.
  • severe combined immunodeficiency scid mice, non-obese diabetic (NOD)-scid mice, lL2rg ⁇ ' ⁇ mice (e.g., NOD/LySz-jciV/ IL2rg 'A mice, NOD/Shi- scid IL2rg ' mice (NOG mice), B ALB/c- Rag
  • the non-human mammal is treated or manipulated prior to introduction of the cells.
  • the non-human mammal can be manipulated to further enhance engraftment and or reconstitution of the cells.
  • the non-human mammal is irradiated prior to introduction of the cells.
  • one or more chemotherapeutics are administered to the non-human mammal prior to introduction of the cells.
  • the non-human mammal is not genetically engineered, and/or not treated to kill mouse liver cells and/or other mouse cells (e.g., immune cells) that would attack the human cells being introduced.
  • the non-human mammal is sublethally irradiated, but not genetically engineered and or not treated to kill mouse liver cells and/or other mouse cells (e.g., immune cells) that would attack the human cells being introduced, prior to introduction of the cells (e.g., HPSCs and/or HSCs).
  • mouse liver cells and/or other mouse cells e.g., immune cells
  • the cells there are a variety of ways to introduce the cells into a non-human mammal. Examples of such methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intrafemoral, intraventricular, intracranial, intrathekal, intravenous, intracardiac, intrahepatic, intra-bone marrow, subcutaneous, topical, oral and intranasal routes of administration. Other suitable methods of introduction can also include, in utero injection, hydrodynamic gene delivery, gene therapy, rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
  • the cells can be introduced in one or multiple injections. In a particular aspect, the cells are introduced in a single injection (e.g., a single injection of cells, for example, on the first day the non-human mammal is born).
  • HPSCs introduced into the non-human mammal will vary depending upon a variety of factors such as the HPSCs being introduced, the non- human mammal, the application for which the non-human mammal is produced and the like. In particular embodiments about 50,000; 75,000; 100,000; 125,000; 150,000;
  • HPSCs 175,000; 200,000; 250,000; 275,000; 300,000; 350,000; 375,000; 400,000; 450,000; 475,000; or 500,000 HPSCs are introduced into the non-human mammal.
  • the HPSCs and/or HSCs are not treated (e.g., not cultured) prior to introduction into the non-human mammal. That is, the HPSCs, and optionally the human HSCs, can be introduced directly (e.g., immediately) after isolation, or the cells can be treated prior to introduction. In a particular embodiment, the cells are introduced directly after isolation (e.g., uncultured HPSCs are introduced). That is, in this embodiment, the cells are not treated with any agents and are not cultured for any length of time (0 days).
  • the cells are cultured about 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days, 34 days, 36 days, 38 days, 40 days, 42 days, 44 days, 46 days, 48 days, 50 days, 52 days, 54 days, 56 days or 58 days. In other embodiments, the cells are cultured less than about one month or less than about two months.
  • the HPSCs introduced into the non-human mammal reconstitute the non-human mammal's liver with human hepatocytes
  • the human HSCs that are optionally introduced reconstitute the non-human mammal's blood with human blood cells.
  • the human hepatocytes and human HSCs are "mature” ( or “functional” or “biologically active") human hepatocytes and human blood cells.
  • mature cells refer to the fact that the cells (whether human liver cells and/or human blood cells) express one or more, and in some instances all, of the cell surface markers of the corresponding normal (wild type) cell found in humans, and as a result, function similarly in the non-human mammal as they function in a human.
  • the methods of producing the non-human mammals can further comprise assessing the reconstitution of human hepatocytes, the reconstitution of human blood cell lineages, or a combination thereof, in the non-human mammal.
  • reconstitution of human hepatocytes can be assessed by detecting human albumin, human CK7, human CK19 and combinations thereof in the non-human mammal (e.g., in the serum or the liver of the non-human mammal).
  • Assays for determining the function of human liver cells and human blood lineage cells in the non-human mammal are known to those of skill in the art and are described herein.
  • detection of human albumin in the non-human mammal indicates that the human liver cells are functional.
  • reconstitution of human blood cell lineages can be assessed by detecting human CD45+ blood lineage cells (e.g., in the blood, spleen, bone marrow, liver, or a combination thereof in the non-human mammal).
  • human CD45+ blood lineage cells e.g., in the blood, spleen, bone marrow, liver, or a combination thereof in the non-human mammal.
  • Non-human mammals produced by the methods described herein are also provided.
  • the non-human mammals produced by the methods described herein can be used in a variety of ways.
  • the non-human mammals can be used as models for pre-clinical testing of drugs, vaccines and human cell-based therapeutics prior to testing in the clinic.
  • the invention is directed to a method of identifying one or more agents or treatment protocols that can be used to treat a human liver disease.
  • the method comprises administering the one or more agents or treatment protocols to a non-human mammal that is a model of a human liver disease produced as described herein, and determining whether the human liver disease in the non-human mammal is alleviated. If the human liver disease is alleviated in the non-human mammal, then the one or more agents or treatment protocols can be used to treat the human liver disease.
  • liver diseases include a primary liver cancer, a secondary liver cancer or cirrhosis of the liver.
  • Specific examples of liver cancer include hepatocellular carcinoma, bile duct cancer, cholangiocarcinoma, angiosarcoma, or hepatoblastoma.
  • agents that produce the human liver disease are carcinogenic agents (e.g., alphotoxin), infectious agents (e.g., virus (e.g., hepatitis virus (hepatitis A, hepatitis B, hepatitis C) or human immunodeficiency virus (HIV)), bacteria, parasite, environmental agents (e.g., alcohol) or a combination thereof.
  • the method can further comprise reconstituting human blood cell lineages in the non-human mammal as described herein.
  • alleviation of a human liver disease includes removal of the disease, prolonging the life of the liver disease patient, or improving the quality of life of the liver disease patient or combinations thereof.
  • the method can further comprise determining whether the liver disease in the non-human mammal is alleviated as compared to a suitable control.
  • a suitable control is a non-human mammal that is a model of human liver disease and that has not received the one or more agents or treatment protocols.
  • the invention is directed to a method of identifying one or more effects of one or more agents on a human liver.
  • the method comprises
  • a non-human mammal which comprises human hepatocytes produced as described herein and determining the effect of the agent on the human liver cells of the non-human mammal, thereby identifying the effects of one or more agents on a human liver.
  • Methods for determining the effect of an agent on a human liver are apparent to those of skill in the art and include the agent's effect on albumin, alanine aminotransferase (ALT) (the ALT liver test), complement,
  • the agent can be, for example, a pharmaceutical composition, a drug, an environmental agent or a combination thereof.
  • the agent can have a therapeutic effect on the human liver cells, a toxic effect on the human liver cells, or no effect on the human liver cells.
  • the invention is directed to a method of identifying how one or more agents are metabolized by a human liver.
  • the method comprises administering the one or more agents to a non-human mammal which comprises human hepatocytes produced as described herein and maintaining the non-human mammal under conditions in which the one or more agents are metabolized by the human liver cells in the non- human mammal, thereby producing one or more metabolites in the non-human mammal.
  • the one or more metabolites are detected and/or analyzed (e.g., directly; indirectly) in the non-human mammal, thereby identifying how one or more agents are metabolized by a human liver.
  • One or more active metabolites, one or more toxic metabolites or a combination thereof can be detected.
  • the invention is directed to an in vitro method of obtaining mammalian hepatocytes (e.g., mature human hepatocytes).
  • the method comprises contacting mammalian hepatic progenitor/stem cells (HPSCs) which express
  • CD34 + CD133 l0 with hepatocyte differentiation medium thereby producing a culture and maintaining the culture under conditions in which the HPSCs differentiate into mammalian hepatocytes, thereby obtaining mammalian hepatocytes.
  • the invention is directed to an in vitro method of obtaining human hepatocytes (e.g., mature human hepatocytes).
  • the method comprises contacting human hepatic progenitor/stem cells (HPSCs) which express CD34 + CD133 l0 with hepatocyte differentiation medium, thereby producing a culture and maintaining the culture under conditions in which the HPSCs differentiate into human hepatocytes, thereby obtaining human hepatocytes.
  • HPSCs human hepatic progenitor/stem cells
  • the methods provided herein can be used to culture mammalian (e.g., human) hepatocytes and establish mammalian (e.g., human) hepatocytes (e.g., hepatic cells, hepatic cell cultures and hepatic cell lines).
  • the hepatocytes are cultured in vitro for days, months or years.
  • the hepatocytes can be cultured for about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more days; or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
  • the hepatocytes e.g., hepatocytes, hepatic cell liners produced by the methods
  • a suitable medium collagen in particular embodiments, about 0.5 x 10 5 , 1 x 10 5 , 1.5 x 10 5 , 2 x 10 5 , 2.5 x 10 s , 3 x 10 5 , 3.5 x 10 5 , 4 x 10 5 , 4.5 x 10 5 , 5 x 10 5 of the HPSCs are seeded on or in a suitable medium collagen and contacted with a hepatocyte differentiation medium.
  • a suitable mediums are known and available.
  • the medium can comprise a collagen, a plastic, a synthetic scaffold and the like.
  • a variety of mediums that promote hepatocyte differentiation are known and available.
  • the hepatocyte differentiation medium comprises fetal bovine serum (FBS) (e.g., about 5%), oncostatin, dexamethasone, insulin, transferring and selenous acid.
  • FBS fetal bovine serum
  • the method can further comprise assessing differentiation of the HPSCs into hepatocytes, for example, by detecting albumin in the culture.
  • the hepatocytes produced by the methods provided herein can be maintained as a fresh culture or can be preserved and/or stored (e.g., cryopreserved) for days, months or years.
  • the invention is also directed to mammalian (e.g., human) hepatocytes , cultures, and cell lines produced by the methods provided herein.
  • mammalian e.g., human
  • CD34 + CD133 Ugh The CD34 + cells were injected into sub-lethally irradiated NSG new born mice (200K/mouse). Eight weeks post injection, not only the reconstitution of human CD45 + blood lineage cells (blood), but also human albumin positive
  • CD34 + cells from human cord blood were used as a control. Most of cord blood CD34 + cells are CD133 ⁇ 8 * 1 cells which are shown to be HSC herein. In the mice reconstituted with cord blood cells, human blood lineage cells were present but no human hepatocyte was detected.
  • Purified 200K CD133 tow cells were injected into NSG mice either by i.l or i.c injection (5 mice in i.c group and 4 mice in i.l group). Eight weeks post injection, the human albumin levels in the serum were analyzed by ELISA. See Fig. 2.
  • CD34+ cells purified from fetal liver were stained with fluorescent antibodies. According to FSC and SSC, the CD 133 low cells were much bigger than the other two populations. When gated on the CD34+CD133 low cell population, it showed that CD133 low cells expressed CD44, EpCAM, CD31 and CD117. See Fig. 3.
  • CD 133 l0 cells can only attach and grow on the collagen I coated plates.
  • the sorted CD133 10 cells were seeded in human hepatocyte differentiation medium. On day 2, they began attaching to the bottom of the collagen I coated wells shown as single cells. Pictures were taken every week to show the expansion and differentiation of these cells. On day 28, the cultured cells were stained with human albumin specific antibody and DAPI. It showed that a lot of cells differentiated from the sorted CD133 10 cells were albumin positive. DAPI staining also showed that some of the cells were multinucleated cells, which is a typical characteristic of human hepatocyte.
  • Human fetal livers were obtained from aborted fetuses at 15-23 weeks of gestation in accordance with the institute ethical guidelines (Polkinhorne). All women have given written informed consent for the donation of fetal tissue for research. The fetuses were collected under sterile condition within 2 h of the termination of pregnancy. The liver tissue from the fetus was initially cut into small pieces, followed by digestion with 2 mg/ml coUagenase VI prepared in DMEM for 15 min at 37°C with periodic mixing. Then, single cell suspension was prepared by passing the digested tissue through 100 um cell strainer (BD Biosciences). Umbilical cord blood was obtained from the National Disease Research Interchange (NDRI) or the Singapore Cord Blood Bank.
  • NDRI National Disease Research Interchange
  • Singapore Cord Blood Bank the National Disease Research Interchange
  • CD34 + cells from both fetal liver and cord blood were purified with the CD34 positive selection kit (Stem Cell Technologies, Vancouver, BC). The purity of CD34 + cells was 90 to 99%. Viable cells were counted by excluding dead cells with trypan blue. Cell isolation procedures were carried out under sterile condition in class 100-biosafety cabinet.
  • NSG mice were obtained from the Jackson Laboratory and bred in the animal facilities at Nanyang Technological University and National University of Singapore. Pups within 24 hrs of birth were sub-lethally irradiated (100 rads) and engrafted with HSCs by intra-cardiac or intra-hepatic injection. 2 x 10 s CD34 + cells, or 1 x 10 s CD34 + CD133 U , CD34 + CD133 10 or CD34 + COm" cells were injected per recipient. All research with human samples and mice was performed in compliance with the institutional guidelines. Cells, cell culture and colony-forming assay
  • CD34 + CD133 W , CD34 + CD133 10 and CD34 + CD133 neg cells were seeded on collagen I (Sigma Chemical Co., St. Louis, MO)-coated tissue culture plates in IMDM medium supplemented with 10% fetal bovine serum (FBS), 20 ng mL epidermal growth factor (Peprotech Inc, Rocky Hill, NJ), 20 ng mL hepatocyte growth factor (HGF, Peprotech Inc), 10 ng/mL bFGF (Peprotech Inc) and 0.61 g/L nicotinamide (Sigma) for 14 days.
  • FBS fetal bovine serum
  • HGF epidermal growth factor
  • Peprotech Inc Rocky Hill, NJ
  • HGF hepatocyte growth factor
  • bFGF ng/mL bFGF
  • nicotinamide 0.61 g/L nicotinamide
  • Differentiation was induced by treating cells with maturation medium, consisting of IMDM supplemented with 5% FBS, 20 ng/mL HGF, 20 ng/mL oncostatin M (R&D Systems, Minneapolis, MN), 1 ⁇ /L dexamethasone (Sigma), and 50 mg/mL ITS + premix (Sigma).
  • maturation medium consisting of IMDM supplemented with 5% FBS, 20 ng/mL HGF, 20 ng/mL oncostatin M (R&D Systems, Minneapolis, MN), 1 ⁇ /L dexamethasone (Sigma), and 50 mg/mL ITS + premix (Sigma).
  • CD34 + COm , CD34 + CD133 10 and CD34 + COWTM* cells were plated with complete MethoCult® methylcellulose medium (Stem Cell Technologies) and cultured at 37°C in a 5% CO 2 incubator for 9 days.
  • Human hepatocytes were purchased from Invitrogen (Carlsbad, CA). Human hepatoma cell lines: Hep 3B, Hep G2, and SNU-423 were purchased from ATCC (Manassas, VA). All these cells were cultured according to the suppliers' instructions.
  • CD34 + CD133 l0 cells were injected intra-hepatically into newborn NSG pups. After 9 weeks, single cell suspensions were prepared from recipient mouse livers by a two step perfusion. Mouse livers were first perfused with prewarmed liver perfusion medium (Invitrogen) for 10 min at 0.7 mlVmin and then followed with prewarmed liver digestion medium (Invitrogen) for 10 min. Cells were washed with ice-cold DMEM medium. Cell viability after treatment exceeded 90% as assessed by Trypan blue dye exclusion. CD34 + cells were re-purified from the cell suspensions with the CD34 positive selection kit (Stem Cell Technologies). 1 x 10 s CD34 + cells were injected into sublethally irradiated newborn NSG pups. After another 8 weeks, the livers were harvested for histology and the sera were used for ELIS A.
  • Conjugated antibodies specific for EpCAM (9C4), CD44 (BJ18), CD34 (561), CD166 (3A6), CD105 (43A3), CD29 (TS2/16), CD24 (ML5), CD90 (5E10), CD73 (AD2), CD117 (104D2), CD97 (VIM3b), CD45 (HI30) were from BioLegend; and CD133 (EMK08) and mouse CD45.1 (A20) from eBioscience.
  • Cells were stained with appropriate antibodies in 100 ⁇ PBS containing 0.2% BSA and 0.05% sodium azide for 30 min on ice.
  • Flow cytometry was performed on a LSRII flow cytometer using the FACSDiva software (BD, Franklin Lakes, NJ). 10,000 to 1,000,000 events were collected per sample and analyzed using the Flowjo software.
  • the livers were removed, embedded in paraffin and 5-micrometer-thick sections were prepared. After blocking, deparaffinized sections were stained with optimal dilutions of rabbit anti-human albumin (AbCam), rabbit anti-human CK7 (Sigma) or rabbit anti-human CK19 (Sigma). Sections were developed with SuperPicture 3 Gen IHC Detection Kit (Invitrogen). For immunofluorescence staining, cells were fixed with pre-chilled methanol and then blocked with 1% BS A/PBS. After blocking, cells were stained with rabbit anti-human albumin antibody. Rhodamine conjugated goat anti- rabbit antibody (Santa Cruz) was served as the secondary antibody.
  • RT-PCR Reverse-transcription polymerase chain reaction
  • RNAs were extracted from CD34 + CD133 10 , CD34 + CD133 W , CD34 + CD133 neg cells and mature hepatocytes using RNeasy micro Kit (Qiagen, Valencia, CA) and transcripted into cDNA with iScriptTM Reverse Transcription Supermix (Bio-Rad, Hercules, CA). The primers used for quantitative PCR are listed in the Table. For qPCR, SsoFastTM EvaGreen® Supermix (Bio-Rad) and CFX96 Real-Time PCR Detection System (Bio-Rad) were used according to the manufacturer's instructions.
  • Human albumin levels in the mouse sera were measured by a sandwich enzyme- linked immunosorbent assay. Human specific albumin ELISA kit was purchased from Bethyl Laboratories, Inc (Montgomery, TX).
  • CD133 10 , CD34 + COm , CD34 + COm ⁇ cells were isolated using the Qiagen RNeasy micro kit (Qiagen). RNA quality and quantity was determined using
  • CD34 + cells from human fetal liver give rise to both hematopoietic cells and
  • CD34 + cells isolated from both cord blood and fetal liver were used as sources of stem cells.
  • 2 x 10 5 CD34 + cells ( ⁇ 95% purity) were engrafted into sublethally irradiated newborn pups of NSG mice by intracardic injection.
  • human CD45 + leukocytes were detected in the peripheral blood of recipient mice regardless the source of stem cells (Figs. 4A-4B).
  • the liver sections of recipient mice were stained for human albumin, no positive signal was detected in the recipients that were engrafted with cord blood CD34 + cells (Fig. 4A).
  • CD34 + CD133 l ° cells give rise to human hepatocytes whereas CD34 + CD133 hi cells give rise to hematopoietic cells
  • CD34 + cells from fetal liver were separated into three distinct populations when co-stained with anti-CD133: high, low and negative (Fig. 5A), whereas CD34 + cells from cord blood were uniformly high for CD133.
  • FSC forward scatter
  • CD34 + CD133'° cells were much larger in size than CD34 + CD133 hi cells and
  • CD34 + CD133 neg cells (Fig. 10A). Depending on the gestation stage of the fetal liver, the proportion of the CD34 + CD133 bi cells ranged from 10% to 25% whereas the proportion of the CD34 + CD133 l0 cells ranged from 8% to 18% (data not shown). The proportion of both CD34 + CD133 hi and CD34 + CD133 ,(> cells decreased with increasing gestation time of the fetus.
  • the total number of human hepatocytes is about 2xl0 7 per recipient mouse. Because only lxlO 5 CD34 + CD133 to cells were injected per recipient mice initially, there is a 200-fold expansion during the differentiation from
  • CD34 + CD133 l0 cells into human hepatocytes in the recipient mice CD34 + CD133 l0 cells into human hepatocytes in the recipient mice.
  • CD34 + human fetal liver cells can be separated into three subpopulations with different developmental potential: CD34 + CD133 hi cells likely contain HSCs as they give rise to hematopoietic cells in the recipient mice whereas CD34 + CD133 l0 cells likely contain HPSCs as they expand and give rise to both hepatocytes and biliary epithelial cells in recipient mice.
  • CD34 + CD133 lD cells are capable of differentiating into human hepatocytes in vitro When grown on collagen plates with hepatocyte differentiation medium (Lee et al., (2004) Hepatology 40, 1275-1284), CD34 + CD133 l0 cells adhered to the collagen substratum as a monolayer and gradually developed a typical polygonal morphology (Fig. 6A), with some of the cells becoming binucleated, a known characteristics of cultured hepatocytes (Fig. 9C). Development of hepatocytes were further confirmed by positive staining for human albumin on day 28. Cell numbers were counted on different days during the culture, which showed that the total cell numbers increased steadily (Fig.
  • CD34 + CD13 k> fetal liver cells are also capable of differentiating into hepatocytes in vitro.
  • CD34 + CD133 W , CD34 + CD133 te and CD34 + CD133 IM3 ⁇ 4 cells were analyzed by standard semi-solid colony forming assays.
  • CD34 + CD ⁇ 3 U cells proliferated rapidly and gave rise to mixed hematopoietic colonies containing granulocytic, erythroid, monocyte-macrophage, and megakaryocyte elements (CFU-GEMM, >95%), while CD34 + CD133 ne cells formed single colonies of granulocyte-macrophage progenitors (CFU-GM, ⁇ 25%) and erythroid precursor cells (BFU-E, >75%) (Fig. 6D and 6E).
  • CD34 + CD133 lc cells did not generate any hematopoietic colonies but attached on the bottom of the plates as single cell layer. Consistent with in vitro differentiation, CD3 + T cells, CD 19 + B cells, CD56 + NK cells, CD14 + monocyte/macrophages, CDllc + BDCA-1 + myeloid dendritic cells (DCs), and ILT7 + CD303 + plasmacytoid DCs were detected in the spleen of recipient mice engrafted with ⁇ 34 + ⁇ 133 ⁇ cells (Fig. 11). These results further support the in vivo observation showing that CD34 + CD133 1 ° cells contain HPSCs while CD34 + CD133 ⁇ cells contain the multipotent HSCs. CD34 + CD133 neg cells are likely hematopoietic progenitors which do not stably reconstitute mice with human blood lineage cells.
  • CD34 + CD133 1 ° fetal liver cells are capable of self-renewal
  • the stem cell property of CD34 + CD133 l0 fetal liver cells was further
  • the purified cells were injected into six secondary newborn NSG pups (1X10 5 cells per recipient). Eight weeks after secondary reconstitution, human albumin-expressing hepatocytes were detected in the liver sections and human albumin was detected in the sera of all six recipient mice (Fig. 7C and 7D).
  • some of the transferred CD34 + CD133 l0 cells can self-renew and maintain the ability to reconstitute human hepatocytes in the secondary recipient mice, indicating that CD34 + CD133 l0 cell population from the human fetal liver contains bona fide hepatic stem cells.
  • the CD34 + CD133 l0 fetal liver cells are referred to as hepatic progenitor/stem cells (HPSCs) herein.
  • HPSCs are transcriptionally more similar to HSCs man to mature hepatocytes
  • CD34 + CD133 l0 (HPSCs) cells from fetal liver were characterized using CD34 + CD133 hi HSCs from cord blood as a control.
  • CD34 + CD133 hi cells from both fetal liver and cord blood shared a very similar staining pattern, including high levels of CD45, CD44, CD117 and CD116, medium levels of CD90, CD105 and CD97, and negative for EpCAM, CD24 and CD73.
  • CD34 + CD133 hi cells from fetal liver stained more brightly than CD34 + CD133 hi cells from cord blood, but the background staining of the former was also higher.
  • CD34 + CD133 l0 cells from fetal liver exhibited a very different staining pattern.
  • HPSCs are not only positive for the mesenchymal stem cell markers such as EpCAM and CD73 but also some hematopoietic cell markers such as CD34, CD117 and even CD45.
  • CD34 + CD 133 to fetal liver cells expressed significant levels of hepatocyte and epithelial cell related transcripts, including CK18, AFP, albumin, CD31, c-met, vimentin, CK19, CK7 and CD29 (Fig. 8B).
  • CD34 + CD133 l0 HPSCs also expressed biliary cell markers such as CK19 and CK7.
  • mature human hepatocytes expressed a much higher level of albumin and were also positive for CK18, c-met and CD29 but not AFP, CD31 and vimentin.
  • FL HSC expressed all the hematopoietic genes including CD45, CD117, CD34, and CD 133, but the transcripts of albumin and AFP were also detected. Confirming the PCR results, immunofluorescence staining showed that all CD34 + CD133 l0 cells were positive for albumin, AFP, CK7 and CK19 while CD34 + CD133 hi cells were negative (Fig. 12A).
  • the uniform expression of both hepatic markers (albumin, AFP) and biliary markers (CK7, CK19) by CD34 + CD133 l0 cells indicates that they are a homogenous hepatic progenitor/stem cell population, capable of developing into both biliary cells and hepatocytes.
  • CD34 + CD133 , and CD34 + CD133 neg fetal liver cells as well as CD34 + CD133 hi HSCs from cord blood, mature human hepatocytes, and HEP 3B, a hepatitis B virus (HBV)- transformed hepatoma cell line, was performed. Comparison of data from surface staining, PCR analysis and microarray showed good correlation among the three different assays. For example, CD34, CD45, CD133, EpCAM, AFP and CK19 were detected in CD34 + CD133 l0 HPSCs by all three methods. Hierarchical clustering and principal component analysis (Fig.
  • HPSCs and HSCs from fetal liver shared 4636 genes that were up-regulated more than 5 fold as compared to hepatocytes, whereas they shared only 468 genes that were up-regulated 5 fold as compared to cord blood HSCs (Fig. 12C).
  • HPSCs were transcriptionally closer to HSC they did not express or had a much lower level of key HSC-specific transcriptional factors PBX1, SOX4, MEIS1 and HOXA9 (Fig. 8D). Instead, HPSCs expressed many endoderm specific genes (Seguin, C.
  • Some human hepatoma cells have similar surface marker expression pattern as HPSCs Stem cells have been considered as potential origin of various human cancers.
  • Hep 3B is a HBV-transformed human hepatoma cell line (Knowles, B. B., et al. (1980) Science 209, 497-499); Hep G2 is a widely studied human hepatoblastoma cell line and SNU-423 is a pleomorphic hepatocellular carcinoma cell line (Park, J. G. et al. (1995), Int J Cancer 62, 276-282).
  • Hep 3B and Hep G2 expressed the surface markers of HPSCs except that Hep 3B expressed high levels of CD133 and CD34 while Hep G2 expressed only a modest level of CD 133 and was negative for CD34 (Fig. 9).
  • the transcriptional profile of Hep 3B was more similar to HPSCs and HSCs from fetal liver than to mature hepatocytes.
  • the surface phenotype of SNU-423 was more similar to mature human hepatocytes, which were negative for all the progenitor/stem cell-related markers except for CD 166 and CD73.
  • SNU-423 was also positive for CD44.
  • the present method is simple without special genetic engineering, chemical treatment or surgical procedure of recipient mice.
  • a much higher degree of human liver chimerism can be achieved by transplanting mature human hepatocytes into immunodeficient mice, such as urokinasetype plasminogen activator (uPA) transgenic (Mercer, D. F. et al. (2001), Nat Med 7, 927- 933; Meuleman, ?.et al. (2005), Hepatology 41, 847-856) and fumarylacetoacetate hydrolase (FAH) knockout (Azuma, H., et al. (2007) Nat Biotechnol 25, 903-910) mice.
  • uPA urokinasetype plasminogen activator
  • these recipient mice are specially engineered and the endogenous mouse hepatocytes have to be ablated when human hepatocytes are transferred.
  • these recipient mice have not been used to assay developmental potential of human HPSCs.
  • human hepatic progenitor cells were assayed for their developmental potential in adult immunodeficient mice by intra-splenic injection (Dan, Y. Y., et al., (2006) Proc Natl Acad Sci USA 103, 9912- 9917; Malhi, H. et al. (2002), J Cell Sci 115, 2679-2688; Schmelzer, E., et al.
  • Another advantage of the methods provided herein is simultaneous reconstitution of both hematopoietic lineage cells and hepatocytes in the same recipient when CD34+ fetal liver cells are injected.
  • Such a humanized mouse could be especially useful for studying biological processes and disease mechanisms that requires interactions between human hepatocytes and immune system in the same recipient mice.
  • CD34 + CD133 +k human HPSCs Detailed analysis of CD34 + CD133 +k human HPSCs reveals that these cells express additional hematopoietic markers including CD117, CD44 and CD4S, mesenchymal cell markers such as CD73 and vimentin, and progenitor hepatocyte markers, such as EpCAM, AFP and albumin. Importantly, HPSCs uniformly expressed these different lineage markers when assayed by either flow cytometry or
  • EpCAM human fetal liver cells into hepatoblasts and HPSCs EpCAM human fetal liver cells into hepatoblasts and HPSCs. Hepatoblasts expressed AFP, albumin, CK19, CD133, and CD44 whereas HPSCs were positive for CK19, CD44, CD133, neural cell adhesion molecule (NCAM), and weakly positive for albumin, but negative for AFP. Moreover, their HPSCs and hepatoblasts are negative for hematopoietic markers CD34, CD45 and CD90. Weiss et al reported the use of CD90 to isolate human hepatic progenitor cells which are positive for hematopoietic markers CD34 and c-kit, biliary cell marker CK19 and hepatic marker HepParl.
  • HPSCs The physical and functional separation of HPSCs from HSCs in human fetal liver has prompted investigation of the controversial relationship between HPSCs and HSCs. Based on the expression of hematopoietic markers by HPSCs (Blakolmer, et al. (1995) Hepatology 21, 1510-1516; Lemmer, E. R. et al. (1998), J Hepatol 29, 450-454) and the controversial result of trans-differentiation from HSCs to hepatocytes in mice, it was hypothesized that HSCs can trans-differentiate into the hepatic lineage. By performing genome-wide transcriptional analysis, the relationship between HSCs and HPSCs in human fetal liver has been systematically investigated as described herein.
  • HPSCs are transcriptionally much closer to HSCs from both fetal liver and cord blood. While HPSCs transcribe many genes that are traditionally associated with hematopoiesis, HSCs from fetal liver, but not cord blood, also had a significant level of albumin and AFP transcript but not protein (Fig. 9A and 1 IB). Nevertheless, HPSCs lack the core transcriptional networks, such as PBX1 and HOXA9, that define HSCs. Because virtually all purified HPSCs from fetal liver expressed AFP, albumin, CK7 and CK19 by intracellular staining, the HPSCs described herein are unlikely to have significant contamination of HSCs.
  • the sharing of transcriptional profiles between HPSCs and HSCs indicate that the likelihood of trans-differentiation from HSCs to HPSCs during fetal development or more provocatively both HSCs and HPSCs originate from the same precursor in the fetal liver.
  • HPSCs hepatocellular carcinomas
  • CT (SEQ ID NO: 10) GGTGGTCCGGTAAATC
  • GAPDH Forward GATA4 Forward:
  • AAAGT SEQ ID NO: 12
  • CD45 Forward CD31 Forward:
  • AGA (SEQ ID NO: 18) TGTAAAACAGCACGTC
  • GCCTTTTCCGTGATCCA CCGGGCTCGAAGTTAA TTCA SEQ ID NO: 24
  • AATCC SEQ ID NO: 52

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  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des cellules souches/progénitrices hépatiques (HPSC) de mammifère ; des procédés de production d'un modèle in vivo contenant des foies recolonisés par des hépatocytes humains et facultativement un système hématopoïétique humain ; les modèles produits par les procédés ; et des utilisations des modèles (par exemple pour étudier l'étiologie et la thérapie des maladies du foie humaines virales et non virales, la biologie des hépatocytes et les hépatomes humains). La présente invention concerne également des progéniteurs abondants d'hépatocytes de mammifères (par exemple humains) qui peuvent être utilisés dans une diversité de façons et de procédés pour la mise en culture de HPSC.
PCT/SG2011/000234 2010-07-07 2011-07-04 Procédés de reconstitution d'hépatocytes humains et de cellules hématopoïétiques humaines chez des mammifères non humains WO2012005690A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013071053A1 (fr) * 2011-11-11 2013-05-16 The Christus Stehlin Foundation For Cancer Research Modèle thérapeutique de rongeur et procédés associés
WO2014196929A1 (fr) * 2013-06-05 2014-12-11 Agency For Science, Technology And Research Modèle de souris humanisé pour l'étude d'une infection par le virus de l'hépatite authentique et son utilisation
US11274279B2 (en) 2020-03-11 2022-03-15 Bit Bio Limited Method of generating hepatic cells

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2007071339A1 (fr) * 2005-12-21 2007-06-28 Universite Catholique De Louvain Cellules souches progenitrices hepatiques isolees

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2007071339A1 (fr) * 2005-12-21 2007-06-28 Universite Catholique De Louvain Cellules souches progenitrices hepatiques isolees

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Title
RAO, M. S. ET AL.: "Characterization of hepatic progenitors from human fetal liver during second trimester", WORLD JOURNAL OF GASTROENTEROLOGY, vol. 14, no. 37, 2008, pages 5730 - 5737, XP055187491, DOI: doi:10.3748/wjg.14.5730 *
SCHMELZER, E. ET AL.: "`Human hepatic stem cells from fetal and postnatal donors", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 204, no. 8, 2007, pages 1973 - 1987 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013071053A1 (fr) * 2011-11-11 2013-05-16 The Christus Stehlin Foundation For Cancer Research Modèle thérapeutique de rongeur et procédés associés
WO2014196929A1 (fr) * 2013-06-05 2014-12-11 Agency For Science, Technology And Research Modèle de souris humanisé pour l'étude d'une infection par le virus de l'hépatite authentique et son utilisation
EP3004330A4 (fr) * 2013-06-05 2016-11-23 Agency Science Tech & Res Modèle de souris humanisé pour l'étude d'une infection par le virus de l'hépatite authentique et son utilisation
US11274279B2 (en) 2020-03-11 2022-03-15 Bit Bio Limited Method of generating hepatic cells

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