WO2005068612A2 - Human hepatic progenitor cells and methods of use thereof - Google Patents
Human hepatic progenitor cells and methods of use thereof Download PDFInfo
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- WO2005068612A2 WO2005068612A2 PCT/IB2005/000660 IB2005000660W WO2005068612A2 WO 2005068612 A2 WO2005068612 A2 WO 2005068612A2 IB 2005000660 W IB2005000660 W IB 2005000660W WO 2005068612 A2 WO2005068612 A2 WO 2005068612A2
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Definitions
- the invention relates to progenitor cells.
- BACKGROUND OF THE INVENTION Acute liver failure remains an important problem with high mortality. Despite the high incidence of diseases that result in liver dysfunction and failure, major advances in medical therapies are currently limited to the prevention and treatment of certain forms of viral hepatitis. The acute and chronic liver diseases are still treated with supportive rather than curative approaches. Orthotopic liver transplantation has so far been the only available therapy for patients with end-stage liver failure. Unfortunately, the availability of donor organs is limited and many patients die each year waiting for liver transplants. Recently, transplantation of healthy hepatocytes into diseased liver has been used as an alternative therapy.
- the invention is based on the discovery a liver progenitor cell.
- the progenitor cell is multipotent.
- the progenitor cell is capable of differentiating, e.g., in-vivo or in-vitro, into a hepatic cell, e.g., a hepatocyte, a cholangiocyte or a liver endothelial cell ( i.e., asinusoidal cell).
- the invention features a liver progenitor cell culture, e.g., an in-vitro culture.
- the culture is an adhesion culture.
- the cells in the culture are in suspension.
- the cell is derived from liver tissue, such as fetal liver tissue.
- the tissue is from a mammal such as human, a primate, mouse, rat, dog, cat, cow, horse, pig.
- the cell is immunoreactive for CD117 and CD34 and non-immunoreactive with Lin.
- the cells proliferate in vitro.
- the cells are capable of doubling 2, 3, 4, 5, 6,7, 8, 9 10, 11,12, 15, 20, 25 or more times and maintain there ability to differentiate into hepatocytes, cholangiocytes or a liver endothelial cells.
- Also provided are methods of producing a hepatocyte, a cholangiocyte or a liver endothelial cell by differentiating the liver progenitor cell cultures in a culture medium containing one or more differentiation factors.
- Differentiation factors include for example vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF).
- VEGF vascular endothelial growth factor
- HGF hepatocyte growth factor
- EGF epidermal growth factor
- the invention further features a method of transplanting the progenitor cell or the progenitor cell progeny in a host, e.g., mammal such as human, a primate, mouse, rat, dog, cat, cow, horse, pig by providing a human CD117 "1" , CD34 + , and Lin " liver progenitor cells and transplanting the cell into the host.
- liver stromal or mesenchymal cells are co-transplanted in the host.
- the host is administers hepatocyte growth factor prior to, after or concomitantly with the progenitor cells.
- the host is suffering from a hepatic disorder or hepatic tissue damage.
- the subject is suffering from hepatitis, cirrhosis, liver cancer, fatty liver disease, Reye syndrome, glycogen storage disease, liver cysts or Wilson's disease.
- Transplantation confers a clinical benefit, e.g. alleviating one or more symptoms of the particular liver disorder.
- Liver disorders are diagnosed by a physician using methods know in the art.
- Compounds which effect proliferation, differentiation or survival of liver cells are identified contacting the liver progenitor cell culture with a test compound and determining if the compound has an effect on proliferatio , differentiation or survival of the cells. Similarly, the metabolite of a test compound is determined.
- Metabolites are identified by screening the culture medium after contacting the culture with the test compound. Metabolites are identified by methods know in the art such a HPLC, Mass spectroscopy or gel electrophoresis.
- Anti-viral activity of a test compound is determined by introducing a virus to a progenitor cell culture in the presence or absence of a test compound and determining the survival rate of the cells. An increase in survival rate in the presence of the test compound compared to the absence of the test compound indicates that the test compound has anti-viral activity.
- Infectivity of a virus is determined by contacting a progenitor cell culture with a virus and determining the effect of proliferation or survival of the cells.
- the virus is differentiated prior to contacting the culture with a test compound. Proliferation and or survival is determined by methods know in the art such as BrdU assay. Differentiation is determined morphologically or histologically by determining hepatic cell surface markers.
- the virus is a liver trophic virus such as hepatitis virus A, hepatitis virus B, or hepatitis virus C.
- Fig. 1 is a series of F ACS analysis and photographs showing the characterization of human hepatic progenitor cells, a, Magnetically sorted FL cells immediately after isolation were double-stained for GDI 17 and CD34, but negative for CD90, CD45, albumin and cytokeratin 19 (CK19). b, CD117+/CD34+/Lin- cells when cultured grew in colonies and the first marker to be expressed was the hepatocyte growth factor receptor (c- Met). Magnification 40x.
- FIG. 2A is a bar chart showing proliferation of hepatic progenitors and their progeny. Use of 20% conditioned medium (CM) significantly increased the proliferation of hepatic progenitor cells and could be passaged several times as compared to without CM (p ⁇ Q.00l, students t test).
- Figure 2B is a bar chart showing the results of flow cytometric analysis demonstrating that, a high proliferation (BrdU+ cells) was observed in albuminCK19+, albumin+CK19- and albumin-CK19+ cells, while, double negative (albumin- CK19-) cells were more quiescent.
- Figure 2C is a series of photographs showing liver progenitor cells in various passages, fluorescence stained for albumin (green) and CK19 (red) and enzymatically for detection of BrdU incorporation, showing proliferative capacity in the various subpopulations. Magnification 40x.
- Figure 3 A- 3N are a series of photographs Localization of human hepatic progenitor cells in the mouse liver, a-b, In situ hybridization with human centromere probe, showing nuclear signals in human liver (DAB) but not in the mouse liver, c-d, using immunohistochemistry, similar results were obtained with an anti-human nuclei antibody (DAB-Ni).
- e-j Freshly isolated cells (e-g), sixth and twelfth passage cells when transplanted into D-galactosamine-treated (GalN) mice showed differentiation into hepatocytes cholangioytes and endothelial cells (DAB-Ni- arrow heads), k, Transplanted cells were observed in the livers, I-m, but not in the spleens, lungs of GalNtreated mice, n, Livers of sham-transplanted mice were used as controls. Magnification 60x.
- Fig. 4A-J is a series of photographs showing the in vivo fate of human hepatic progenitor cells.
- a-d 5 Human hepatic progenitor cells one month after transplantation into GalN-treated mice contained glucose-6-phosphatase (brown), glycogen (pink), dipeptidyl peptidase IV (red-brown), gamma glutamyl transpeptidase (brown).
- Double-staining with the anti-human nuclei antibody visualised human nuclei (black), e, Regeneration of a whole mouse tissue segment by human hepatic progenitors (black nuclei, arrow heads), f, Expression of cytokeratin 19 (red-brown) and g-j, human albumin (green) was not observed in the sham-transplanted mice but was observed (arrow heads) in the livers of GalN-treated mice that were transplanted with freshly isolated, sixth passage and twelfth passage human hepatic progenitor cells, a-j, (Magnification 60x). f, (Magnification 200x).
- Figure 4K is a photograph showing transcription of human liver-specific genes in the mouse liver.
- Human cytokeratin 19, ⁇ -fetoprotein and albumin were detected in the livers of GalN-treated mice that received human hepatic progenitor cells but not in the sham- transplanted mice. However, ⁇ l antitrypsin was slightly amplified also in the control. Glucose-6-phosphate dehydrogenase was used as the housekeeping gene.
- Figure 5 is a series of photographs demonstrating the effects of co-transplantion of fetal liver stromal cells and fetal liver progenitor cells. A, Normal human liver section stained with anti-human nuclei antibody shows positive staining (black/brown).
- B Normal mouse liver section showed no positive staining with the same antibody, demonstrating the specificity of the antibody.
- C Mice treated with retrorsine (30mg/kg), followed by partial hepatectomy and subcutaneously injected with the hepatocyte growth factor (HGF) followed by fetal liver progenitor cell transplantation did not result in high engraftment of cells.
- D Mice treated with retrorsine (30mg/kg), followed by partial hepatectomy and injected with stromal cells isolated from fetal livers did not result in high engraftment of cells.
- HGF hepatocyte growth factor
- E-H Mice treated with retrorsine (30mg/kg), followed by partial hepatectomy and transplanted with a mixture of fetal liver stromal and progenitor/stem cells resulted in high engraftment of stem cells.
- I-K Mice treated with retrorsine (30mg/kg), followed by partial hepatectomy and subcutaneously injected with the hepatocyte growth factor (HGF) followed by fetal liver stromal and progenitor cell transplantation resulted in high engraftment of cells.
- Figure 6 is a chart showing the detection levels of human factor VIII in normal nude C57 black mice and C57 black mice treated in various ways.
- Figure 7 is a chart showing expression of various hematopoietic, hepatic and pancreatic cell surface markers on fetal and adult livers.
- Figure 8 is a series of photographs showing hepatic markers in human fetal and adult liver.
- Figure 9A is a series of FACS analysis showing the characterization of human hepatic progenitor cells.
- Figure 9B-F is a series of photographs showing morphology of human hepatic progenitor cells on different matrixes.
- Figure 10A is a photograph of a NorthernBlot showing gene expression of hepatic markers in human hepatic progenitor cells.
- Figure 10 B is a chart demonstrating gene expression of hepatic markers in human hepatic progenitor cells.
- the present invention is based upon the unexpected discovery of a defined population of non-hematopoietic progenitor cells within human fetal liver that expand in vitro for several passages and maintain a progenitor phenotype. These cells, when transplanted into animals with acute liver injury exhibited functional differentiation into hepatocytes, cholangiocytes and sinusoidal cells. Liver diseases include a wide spectrum of both acute and chronic conditions associated with significant morbidity and mortality world-wide. Hepatocyte transplantation has tremendous therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue.
- hepatocytes in vitro from adult or fetal liver cell progenitors or identification of a progenitor population which in vivo can generate mature liver cells would solve this problem.
- the data described herein demonstrate the identification a defined population of cells from human fetal livers that are successfully expanded ex vivo for several passages and when transplanted into animals with acute liver • injury exhibited functional differentiation into hepatocytes, cholangiocytes and sinusoidal cells.
- Successful in vitro expansion and differentiation of liver progenitor cells are useful for hepatic cell transplantation, metabolic and toxicity testing of candidate therapeutic drugs, and a vehicle for gene therapy.
- the present invention provides methods for inducing multipotent human hepatic progenitor cells from human fetal liver tissue to proliferate in vitro or in vivo (i.e. in situ), to generate large numbers of multipotent human progenitor cell progeny capable of differentiating into hepatocytes, cholangiocytes and sinusoidal cells. Methods for differentiation of the human hepatic progenitor cells progeny are also provided.
- Human Liver Progenitor Cells The invention provides a human liver progenitor cell (refered to herein as LPC) A
- LPC cell is an undifferentiated cell that can be induced to proliferate using the methods of the present invention.
- the LPC is capable of self-maintenance, such that with each cell division, at least one daughter cell will also be a LPC cell.
- LPC are capable of being expanded 100, 250, 500, 1000, 2000, 3000, 4000, 5000 or more fold. Phenotyping of LPCs reveal that these cells do not express any committed hematopoietic markers, however the cells express stem cell markers.
- a LPC is immunoreactive for both CD117 and CD34, and non immunoractive for Lineage surface antigen (Lin).
- the LPC is a multipotent progenitor cell.
- mutipotent progenitor cell is meant that the cell is capable of differenctiating into more that one cell type.
- the cell is capable of differentiating into a hepatocyte, acholangiocyte or a sinusoidal cell.
- CD117 is also known as c-kit, steel factor receptor or stem cell factor receptor.
- CD117 is a e 145 kD cell surface glycoprotein belonging to the class III receptor tyrosine kinase family. It is expressed on the majority of hematopoietic progenitor cells, including multipotent hematopoietic stem cells as well as on committed myeloid, erythroid and lymphoid precursor cells.
- CD117 is also expressed on a few mature hematopoietic cells, e.g. mast cells.
- CD34 is a 1 lOkD single chain transmembrane glycoprotein expressed on human lymphoid and myeloid hematopoietic progenitor cells.
- Lineage surface antigen is a mixture if Thirteen to 14 different cell-surface proteins that are markers of mature blood cell lineages LPCs are obtained from embryonic liver tissue.
- the liver tissue can be obtained from any animal that has liver tissue such as , fish, reptiles, birds, amphibians, and mammals, e.g. preferably rodents , and such as mice and humans.
- the tissue is obtained from a fetus that is at least 4, 5, 6, 7, 8, 9, 10 or more weeks of age.
- LPCs represent approximately 0.5 0.7% of whole fetal livers.
- LPCs can be maintained in vitro in long-term cultures.
- the LPCs are capable of being passed in culture 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or more times.
- Prior to transplantation freshly isolated LPCs (CD117+/CD34+/Lin- cells) did not express albumin and CK19. However, after transplantation, these cells differentiated into mature hepatocytes and cholangiocytes as determined by the expression of human albumin CK19, G-6-P, GGT, DPPIV and glycogen and had high proliferative capacity.
- liver progenitor cells expressed the two hematopoietic associated markers c-kit and CD34, but not CD45, the marker that distinguishes hematopoietic cells from non-hematopoietic cells.
- the LPCs with extensive proliferative capacity described herein are not of hematopoietic origin.
- the hepatic progenitors from fetal livers have high proliferative capacity.
- the ALB-CK19- cell subset were highly quiescent in vitro, however, in vivo the ten-fold increase in the number of cells transplanted in P0 passage indicate that these cells may have a high proliferative capacity. Despite the high proliferative capacity of liver progenitor cells no tumors were observed in vivo four weeks after human cell transplantation. Interestingly, CD117+/CD34+ Lin- cells from early fetal livers when grown in medium containing VEGF differentiated not only into hepatocytes and cholangiocytes but also into sinusoidal endothelial cells.
- LPCs are proliferated using the methods described herein.
- Cells are obtained from donor tissue by dissociation of individual cells from the connecting extracellular matrix of the tissue.
- Tissue from fetuses are removed using a sterile procedure, and the cells are dissociated using any method known in the art including treatment with enzymes such as trypsin, collagenase and the like, or by using physical methods of dissociation such as with a blunt instrument or homogenizer.
- Dissociation of fetal cells can be carried out in tissue culture medium. For example, dissociation of cells can be carried out in 0.1% trypsin and 0.05%
- Dissociated cells are centrifuged at low speed, between 200 and 2000 rpm, usually between 400 and 800 rpm, and then resuspended in a culture medium.
- the hepatic cells can be cultured in suspension or on a fixed substrate.
- Dissociated cell suspensions are seeded in any receptacle capable of sustaining cells, particularly culture flasks, culture plates or roller bottles, and more particularly in small culture flasks such as 25 cm 2 culture flasks. Cells cultured in suspension are resuspended at approximately 5 x 10 4 to 2 x 10 5 cells/ml (for example, 1 x 10 5 cells/ml).
- the dissociated hepatic cells can be placed into any known culture medium capable of supporting cell growth, including HEM, DMEM, RPMI, F-12, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and proteins such as transferrin and the like.
- the culture medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like.
- the culture medium may contain serum derived from bovine, equine, chicken and the like. Conditions for culturing should be close to physiological conditions.
- the pH of the culture medium should be close to physiological pH. (for example, between pH 6-8, between about pH 7 to 7.8, or at pH 7.4).
- Physiological temperatures range between about 30°C to 40°C.
- LPCs are cultured at temperatures between about 32°C to about 38°C (for example, between about 35°C to about 37°C).
- the culture medium is supplemented with at least one proliferation-inducing ("mitogenic") growth factor.
- a "growth factor” is protein, peptide or other molecule having a growth, proliferation-inducing, differentiation-inducing, or trophic effect on LPCs.
- Proliferation-inducing growth factors are trophic factor that allows LPCs to proliferate, including any molecule that binds to a receptor on the surface of the cell to exert a trophic, or growth-inducing effect on the cell.
- Proliferation-inducing growth factors include EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGF ⁇ ), VEGF and combinations thereof.
- Growth factors are usually added to the culture medium at concentrations ranging between about 1 fg/ml to 1 mg/ml. Concentrations between about 1 to 100 ng/ml are usually sufficient.
- Simple titration assays can easily be performed to determine the optimal concentration of a particular growth factor.
- the biological effects of growth and trophic factors are generally mediated through binding to cell surface receptors.
- the receptors for a number of these factors have been identified and antibodies and molecular probes for specific receptors are available.
- LPCs can be analyzed for the presence of growth factor receptors at all stages of differentiation. In many cases, the identification of a particular receptor provides guidance for the strategy to use in further differentiating the cells along specific developmental pathways with the addition of exogenous growth or trophic factors.
- the proliferating LPCs by aspirating the medium, and adding fresh medium to the culture flask.
- the aspirated medium is collected, filtered and used as a condition medium to subsequently passage LPCs.
- the 10%, 20%, 30%, 40% or more condition medium is used.
- the LPC cell culture can be easily passaged to reinitiate proliferation.
- the culture flasks are shaken well and LPCs are then transferred to a 50 ml centrifuge tube and centrifuged at low speed, he medium is aspirated, the LPCs are resuspended in a small amount of culture medium
- the cells are then counted and replated at the desired density to reinitiate proliferation. This procedure can be repeated weekly to result in a logarithmic increase in the number of viable cells at each passage.
- LPCs and LPC progeny can be cryopreserved by any method known in the art until they are needed. (See, e.g., United States patent 5,071,741, PCT International patent applications WO93/14191, WO95/07611, WO96/27287, WO96/29862, and WO98/14058, Karlsson et al, 65 Biophysical J. 2524-2536 (1993)).
- the LPCs can be suspended in an isotonic solution, preferably a cell culture medium, containing a particular cryopreservant.
- cryopreservants include dimethyl sulfoxide (DMSO), glycerol and the like.
- LPCs can be differentiated into hepatocytes, cholangiocytes or sinusoidal cells. LPCs can be differentiated into hepatocytes, or cholangiocytes by culturing the LPCs on a fixed substrate in a culture medium with HGF and EGF.
- LPCs can be differentiated into and sinusoidal cells by culturing the LPCs on a fixed substrate in a culture medium with VEGF. Differentiation of the LPCs can also be induced by any method known in the art which activates the cascade of biological events which lead to growth, which include the liberation of inositol triphosphate and intracellular Ca 2+ , liberation of diacyl glycerol and the activation of protein kinase C and other cellular kinases, and the like. Treatment with phorbol esters, differentiation-inducing growth factors and other chemical signals can induce differentiation.
- differentiation-inducing growth factors can be added to the culture medium to influence differentiation of the LPCs.
- Other differentiation inducing growth factors include platelet derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor betas (TGF,s), insulin-like growth factor (IGF-1) and the like.
- PDGF platelet derived growth factor
- TRH thyrotropin releasing hormone
- TGF transforming growth factor betas
- IGF-1 insulin-like growth factor
- Differentiated hepatocytes, cholangiocytes or sinusoidal cells are detected using immunocytochemical techniques know in the art. Immunocytochemistry (e.g.
- dual-label immunofluorescence and immunoperoxidase methods uses antibodies that detect cell proteins to distinguish the cellular characteristics or phenotypic properties of hepatic cells
- Cellular markers for hepatocytes and cholangiocytes include albumin and CK10, whereas cellular markers for sinusoidal cells includes Flk.
- Other suitable markers include glucose 6 phosphatase, glycogen, dipepidyl peptidase IV, gamma glutaryl transpeptidase
- Immunocytochemistry can also be used to identify hepatic cellss, by detecting the expression of hepatic genes responsible for liver function such as albumin, alpha 1- antitrpsin, CK-19, alpha fetal protein or human factor VIII.
- In situ hybridization histochemistry can also be performed, using cDNA or RNA probes specific for the hepatic gene mRNAs. These techniques can be combined with immunocytochemical methods to enhance the identification of specific phenotypes. If necessary, the antibodies and molecular probes discussed above can be applied to Western and Northern blot procedures respectively to aid in cell identification.
- Transplantation of Human Liver Progenitor Cells Transplantation of new cells into the damaged liver has the potential to repair damaged liver tissue, thereby restoring hepatic function.
- fetal stromal cells and or HGF are co-transplanted with the LPCs. However, the absence of suitable cells for transplantation purposes has prevented the full potential of this procedure from being met.
- Suitable cells are cells that meet the following criteria: (1) can be obtained in large numbers; (2) can be proliferated in vitro to allow insertion of genetic material, if necessary; (3) capable of surviving indefinitely and facilitate hepatic repair on transplantation in the liver; and (4) are non-immunogenic, preferably obtained from a patient's own tissue or from a compatible donor.
- the use of LPCs in the treatment of liver disorders can be demonstrated by the use of animal models.
- LPCs are administered to any animal with abnormal liver or liver failure symptoms.
- LPCs can be prepared from donor tissue that is xenogeneic to the host. For xenografts to be successful, some method of reducing or eliminating the immune response to the implanted tissue is usually employed.
- LPCs recipients can be immunosuppressed, either through the use of immunosuppressive drugs such as cyclosporin, or through local immunosuppression strategies employing locally applied immunosuppressants. Local immunosuppression is disclosed by Gruber, 54 Transplantation 1-11(1992). United States patent 5,026,365 discloses encapsulation methods suitable for local immunosuppression.
- LPCs can be encapsulated and used to deliver factors to the host, according to known encapsulation technologies, including microencapsulation (see, e.g., United States patents 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference) and macroencapsulation (see, e.g., United States patents 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and PCT International patent applications WO 92/19195 and WO 95/05452, each incorporated herein by reference).
- microencapsulation see, e.g., United States patents 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference
- macroencapsulation see, e.g., United States patents 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and PCT International patent applications WO 92/19195 and WO 95/05452, each incorporated herein by reference.
- Macroencapsulation is described in United States patents 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and PCT International patent application WO 95/05452, each incorporated herein by reference.
- Cell number in the devices can be varied; preferably each device contains between 10 3 - 10 9 cells (for example, 10 5 to 10 7 cells).
- Multiple macroencapsulation devices can be implanted in the host.
- LPCs prepared from tissue that is allogeneic to that of the recipient is tested for use by the well-known methods of tissue typing, to closely match the histocompatibility type of the recipient. LPCs can sometimes be prepared from the recipient's own liver (e.g., in the case of tumor removal biopsies).
- the LPCs can be generated from dissociated tissue and proliferated in vitro using the methods described above. Upon suitable expansion of cell numbers, the LPCs may be harvested, genetically modified if necessary, and readied for direct injection into the recipient's liver. LPCs are administered to the hepatic region can form a hepatic graft, so that the cells form normal connections with neighboring hepatic cells , maintaining contact with transplanted or existing hepaticcells. Thus the transplanted LPCs re-establish the liver tissue which have been damaged due to disease and aging.
- LPCs provide an additional way to retrieve and expand liver cells for use as vehicles in ex vivo gene therapy trials. Genetic Modification of Liver Progenitor Cells Although the LPCs are non-transformed primary cells, they possess features of a continuous cell line.
- the LPCs continuously divide and are thus targets for genetic modification.
- the genetically modified cells are induced to differentiate into hepatocytes, cholangiocytes or sinusoidal cells by any of the methods described above.
- the term “genetic modification” refers to the stable or transient alteration of the genotype of a LPCs by intentional introduction of exogenous DNA.
- DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences.
- the term “genetic modification” as used herein is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like. Any useful genetic modification of the cells is within the scope of the present invention.
- LPCs may be modified to produce or increase production of a biologically active substance such as a growth factor or the like.
- the biologically active substance is a transcription factor such as a transcription factor that modulates genetic differentiation.
- the biologically active substance is a non-mitogenic proliferation factor, e.g. v-myc, SV-40 large T or telomerase.
- the genetic modification is performed either by infection with viral vectors (retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, and the like) or transfection using methods known in the art (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like) (see, Maniatis et al, in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1982)).
- the chimeric gene constructs can contain viral, for example retroviral long terminal repeat (LTR), simian virus 40 (SV40), cytomegalovirus (CMV); or mammalian cell-specific promoters such as tyrosine hydroxylase (TH, a marker for dopamine cells), DBH, phenylethanolamine N-methyltransferase (PNMT), ChAT, GFAP, NSE, the NF proteins (NE-L, NF-M, NF-H, and the like) that direct the expression of the structural genes encoding the desired protein.
- the vectors can include a drug selection marker, such as the E.
- LPCs can be genetically modified using transfection with expression vectors.
- vector DNA containing the genes are diluted in 0.1X TE (1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 ⁇ g/ml. 22 ⁇ l of the DNA is added to 250 ⁇ l of 2X HBS (280 mM NaCl, 10 mM KC1, 1.5 mM Na 2 HPO 4 , 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 ml plastic tube.
- 2X HBS 280 mM NaCl, 10 mM KC1, 1.5 mM Na 2 HPO 4 , 12 mM dextrose, 50 mM HEPES
- 31 ⁇ l of 2 M CaCl 2 is added slowly and the mixture is incubated for 30 minutes (min) at room temperature. During this 30 min incubation, the cells are centrifuged at 800 g for 5 min at 4°C. The cells are resuspended in 20 volumes of ice-cold PBS and divided into aliquots of lxl 0 7 cells, which are again centrifuged. Each aliquot of cells is resuspended in 1 ml of the DNA-CaCl 2 suspension, and incubated for 20 min at room temperature. The cells are then diluted in growth medium and incubated for 6-24 hr at 37°C in 5%-7% CO 2 .
- LPCs are also genetically modified using calcium phosphate transfection techniques.
- the cells are mechanically dissociated into a single cell suspension and plated on tissue culture-treated dishes at 50% confluence (50,000-75,000 cells/cm 2 ) and allowed to attach overnight.
- the modified calcium phosphate transfection procedure is performed as follows: DNA (15-25 ⁇ g) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440 ⁇ L with TE, and 60 ⁇ L of 2 M CaCl 2 (pH to 5.8 with IM HEPES buffer) is added to the DNA/TE buffer. A total of 500 ⁇ L of 2x HeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KC1, 1.4 mM Na 2 HPO 4 , 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is added dropwise to this mix.
- 2x HeBS HPES-Buffered saline; 275 mM NaCl, 10 mM KC1, 1.4 mM Na 2 HPO 4 , 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92
- the mixture is allowed to stand at room temperature for 20 min.
- the cells are washed briefly with lx HeBS and 1 ml of the calcium phosphate precipitated DNA solution is added to each plate, and the cells are incubated at 37°C for 20 min.
- 10 ml of medium is added to the cells, and the plates are placed in an incubator (37°C, 9.5% CO 2 ) for an additional 3-6 hours.
- the DNA and the medium are removed by aspiration at the end of the incubation period, and the cells are washed 3 times and then returned to the incubator.
- the substance can be one that is useful for the treatment of a given liver disorder.
- LPCs are be genetically modified to express a biologically active agent, such as growth factors, growth factor receptors. For example, it may be desired to genetically modify cells so they secrete a proliferation-inducing growth factor or a differentiation-inducing growth factor. Growth factor products useful in the treatment of liver disorders include HGF, VEGF, FGF-1, FGF-2, EGF, TGF ⁇ , TGF,s, PDGF, IGFs, and the interleukins.
- the genetically modified LPCs can be implanted for cell therapy or gene therapy into the CNS of a recipient in need of the biologically active molecule produced by the genetically modified cells. Transplantation techniques are detailed below.
- the genetically modified LPCs can be subjected to various differentiation protocols in vitro prior to implantation. Once the cells have differentiated, they are again assayed for expression of the desired protein. Cells having the desired phenotype can be isolated and implanted into recipients in need of the protein or biologically active molecule that is expressed by the genetically modified cell. Methods for screening effects of drugs on Liver Progenitor Cells LPCs cultures can be used for the screening of potential therapeutic compositions. For example LPCs are used to identify compounds that effect proliferation, differentiation or survival of liver cells. In addition LPCs are used to identify anti-viral compounds, determine the infectivity of a virus or to identify metabolites of a test compound.
- test compositions can be applied to cells in culture at varying dosages, and the response of the cells monitored for various time periods. Physical characteristics of the cells can be analyzed by observing cell growth and morphology with microscopy. The induction of expression of new or increased levels of proteins such as enzymes, receptors and other cell surface molecules, or of neurotransmitters, amino acids, neuropeptides and biogenic amines can be analyzed with any technique known in the art which can identify the alteration of the level of such molecules. These techniques include immunohistochemistry using antibodies against such molecules, or biochemical analysis.
- Such biochemical analysis includes protein assays, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, and radioimmune assays (RIA).
- Nucleic acid analysis such as Northern blots can be used to examine the levels of mRNA coding for these molecules, or for enzymes which synthesize these molecules.
- LPCs can be used in methods of determining the effect of a biological agent on liver cells.
- biological agent refers to any agent, such as a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug or other substance that may have an effect on neural cells whether such effect is harmful, beneficial, or otherwise.
- Biological agents that are beneficial to hepatic cells are referred to herein as "hepatic agents", a term which encompasses any biologically or pharmaceutically active substance that may prove potentially useful for the proliferation, differentiation or functioning of hepatic cells or treatment of hepatic disease or disorder.
- a potential biological agent on hepatic cells from a a culture of LPCs can be obtained from Hvertissue or, alternatively, from a host afflicted with a liver disease or disorder.
- the choice of culture conditions depends upon the particular agent being tested and the effects one wants to achieve.
- the effects of a biological agent or combination of biological agents on the differentiation and survival of LPCs and their progeny can be determined. It is possible to screen LPCs which have already been induced to differentiate prior to the screening. It is also possible to determine the effects of the biological agents on the differentiation process by applying them to LPCs prior to differentiation.
- the biological agent can be solubilized and added to the culture medium at varying concentrations to determine the effect of the agent at each dose.
- the culture medium may be replenished with the biological agent every couple of days in amounts so as to keep the concentration of the agent somewhat constant. Changes in proliferation are observed by an increase or decrease in the number of cells.
- a "regulatory factor” is a biological factor that has a regulatory effect on the proliferation of LPCs.
- a biological factor would be considered a "regulatory factor” if it increases or decreases the number of LPCs that proliferate in vitro in response to a proliferation-inducing growth factor (such as EGF).
- a proliferation-inducing growth factor such as EGF
- the number of LPCs that respond to proliferation-inducing factors may remain the same, but addition of the regulatory factor affects the rate at which the LPCs proliferate.
- a proliferation-inducing growth factor may act as a regulatory factor when used in combination with another proliferation-inducing growth factor. Using these screening methods, one of skill in the art can screen for potential drug side-effects on hepatic cells by testing for the effects of the biological agents on hepatic cell proliferation and differentiation or the survival and function of differentiated hepatic cells.
- the proliferated LPCs are typically plated at a density of about 5-10 x 10 6 cells/ml. If it is desired to test the effect of the biological agent on a particular differentiated cell type or a given make-up of cells, the ratio of hepatocytes and cholangiocyte cells obtained after differentiation can be manipulated by separating the different types of cells. The effects of the biological agents are identified based upon significant differences relative to control cultures with respect to criteria such as the ratios of expressed phenotypes, cell viability and alterations in gene expression. Physical characteristics of the cells can be analyzed by observing cell morphology and growth with microscopy.
- the induction of expression of new or increased levels of proteins can be analyzed with any technique known in the art which can identify the alteration of the level of such molecules. These techniques include immunohistochemistry using antibodies against such molecules, or biochemical analysis. Such biochemical analysis includes protein assays, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, and radioimmune assays (RIA). Nucleic acid analysis such as Northern blots and PCR can be used to examine the levels of mRNA coding for these molecules, or for enzymes which synthesize these molecules.
- biochemical analysis includes protein assays, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, and radioimmune assays (RIA).
- Nucleic acid analysis such as Northern blots
- the factors involved in the proliferation of LPCs and the proliferation, differentiation and survival of LPCs progeny, and their responses to biological agents can be isolated by constructing cDNA libraries from LPCs or LPC progeny at different stages of their development using techniques known in the art.
- the libraries from cells at one developmental stage are compared with those of cells at different stages of development to determine the sequence of gene expression during development and to reveal the effects of various biological agents or to reveal new biological agents that alter gene expression in liver cells.
- genetic factors may be identified that play a role in the cause of dysfunction by comparing the libraries from the dysfunctional tissue with those from normal tissue. This information can be used in the design of therapies to treat the disorders.
- probes can be identified for use in the diagnosis of various genetic disorders or for use in identifying hepatic cells at a particular stage in development.
- the present invention is further illustrated, but not limited, by the following examples.
- EXAMPLE 1 GENERAL METHODS
- FL was dissected and placed in a sterile tube containing RPMI 1640 medium (Gibco, Invitrogen Corp. UK). The liver was then disintegrated into a single cell suspension by passage through a 70 ⁇ m metal mesh. The single cell suspension was centrifuged at 200 g for 10 min to pellet the cells. All women donating fetal tissue had been serologically screened for syphilis, toxoplasmosis, rubella, HIV-1, cytomegalovirus, hepatitis B and C, paro virus and herpes simplex types 1 and 2.
- the recovered progenitor cells were immediately analysed by the flow cytometer to make sure that no contaminating lin+ cells were present and to confirm the progenitor phenotype (CD117+/CD34+/lin-) of the cells.
- the recovered progenitor cells at the end of the procedure were tested for viability, afterwhich the cells were seeded in plastic petri dishes coated with collagen type I (Biocoat, Becton and Dickinson, New Jersey, USA) and cultivated in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO, Invitrogen, Sweden) containing, 10% inactivated fetal calf serum, penicillin and streptomycin, 5%L-glutamine, 5%minimum essential amino acids, 50ng/ml HGF (R&D Systems, Abingdon, England), 20ng/ml EGF (R&D Systems) and 10 ng ml basic fibroblast growth factor (R&D Systems).
- DMEM Dulbecco's Modified Eagle Medium
- CM conditioned medium
- Cells were washed and stained for albumin, cytokeratin 19 and BrdU using, a FITC-conjugated goat antibody against human albumin (Natutec, Frankfurt, Germany), a non-conjugated anti-human cytokeratin 19 (Neomarker, USA), and a non-conjugated anti-BrdU antibody (Sigma, Sweden).
- GalN Sigma Chemicals Co., Sweden
- mice were anaesthetized under ether and typically 1x105 freshly isolated cells (P0) and lxlO ⁇ cells in passages 6 and 12 suspended in 200ul of DMEM medium were injected into the spleen over approximately 10-15s.
- mice were sham-transplanted with just DMEM medium. After securing hemostasis, the abdominal incision was closed and the animals were monitored closely until recovery.
- DAB sees brown colour staining
- DAB-Ni black
- Vector NovaRed kit for doublestainings, combinations of DAB (gives brown colour staining) and/or DAB-Ni (black) and/or the Vector NovaRed kit were used.
- Other primary antibodies used were, a FITC-conjugated goat antibody against human albumin (not cross-reactive with mouse) (Natutec, Frankfurt, Germany), a non-conjugated anti-human cytokeratin 19 (Neomarker, USA), and a nonconjugated mouse-anti-human CD26 (detects dipeptidyl peptidase IV) (Pharmingen, USA).
- GGT , G-6-P and glycogen were demonstrated in situ as described earlier (39, 40). Sections were counterstained with hematoxylin and mounted in mounting media (ImmunKemi, Sweden).
- RNA isolation and reverse transcription 25 ⁇ L.
- Each reaction contained 2.5 ⁇ L of cDNA, 12.5 ⁇ L SYBR Green Master Mix (Applied Biosystems), and 500nM of each primer. Positive and negative controls were included in all runs.
- Thermal cycling conditions were 2 min at 50°C initially and 10 min at 95 °C, as recommended by the manufacturer. Cycle conditions were 40 cycles at 95°C for 15s and at 60°C for 1 min.
- the housekeeping gene, G6PD was included as endogenous normalization control, which was used to confirm successful RNA isolation and reverse transcription, and the total amount of RNA in every sample.
- liver cell markers including Flk-1 known to be expressed on fetal liver endothelial progenitors, revealed four populations of cells a) endothelial cells expressing the receptor Flk-1 (-50%), b) hepatocytes (-13%), c) cholangiocytes ( ⁇ 17%)(Fig. Id), and d) a cell population that did not express any of these markers (-20%) (data not shown), the sinusoidal phenotype of the Flk-1+ cells was confirmed by using a vast panel of antibodies (Table 1). Human umbilical vein endothelial cells were used to demonstrate the phenotypic differences between vascular and sinusoidal endothelial cells (Table 1).
- Electron microscopic analysis revealed the presence of fenestrae and absence of a basement membrane characteristic of sinusoidal endothelial cells (data not shown). These data show that nonhematopoietic primitive progenitor cells from early developing livers can differentiate in vitro into hepatocytes, cholangiocytes and endothelial cells.
- EXAMPLE 4 LIVER PROGENITOR CELLS CAN BE PASSAGED EXTENSIVELY IN VITRO
- CM conditioned medium
- HUVEC Human umbilical vein endothelial cells.
- EXAMPLE 5 EXPANDED HUMAN FETAL PROGENITOR CELLS SUCCESSFULLY ENGRAFT AND DIFFERENTIATE INTO MATURE HEPATOCYTES, CHOLANGIOCYTES AND SINUSOIDS IN THE LIVERS OF GALN-TREATED MICE
- P0 CD117+/CD34+/Lin- cells
- P6 and PI 2 cells expanded in culture
- This protocol induces acute liver injury and facilitates hepatic regeneration (21).
- mice in the control and two in the test group died within 24 hrs after treatment. Results from mice surviving at four weeks after cell transplantation are presented. After intrasplenic transplantation, primary P0 cells, as well as P6 and P12 subpassaged cells, survived in the GalN-treated mouse liver. Experiments to compare the results obtained using a human centromere probe and an antihuman nuclei antibody to localize transplanted cells gave similar results. The species specificity of the human DNA probe (Fig. 3a&b) and the anti-human 1 nuclei antibody is demonstrated by the positive result with human liver (Fig. 3 c) and the negative immunohistochemistry with sham- transplanted livers from nude mice (Fig. 3d).
- the number of transplanted cells in clusters of three sizes i.e. cells arranged singly or in clusters of ⁇ 5-20 or >20 cells was determined.
- the analysis showed that there was no significant difference in the number of transplanted cells arranged singly or in clusters of ⁇ 2-20 cells between the animals receiving P0, P6 and P12 cells.
- No clusters of >20 cells were observed in mice transplanted with P0 cells, but were observed in mice that received cells in P6 and P12 (Table 3) (p ⁇ 0.00l, ANOVA).
- mice transplanted with P0 cells we detected a ten-fold increase in the number of human transplanted cells while in mice injected with P6 and P12 cells a six-fold increase was detected (Table 3).
- EXAMPLE 6 TRANSCRIPTION OF HUMAN L ⁇ VER-SPECIFIC GENES IN MICE TRANSPLANTED WITH HUMAN HEPATIC PROGENITOR CELLS
- the engraftment of the transplanted cells was conformed by determining the expression of human genes in the transplanted mice. Livers of the mice sacrificed were analyzed one month after transplantation of human fetal hepatic progenitor cells by RT-PCR using primers specific for human liver-specific genes, including albumin, ⁇ l-antitrypsin, CK19, and AFP. RNA from the liver of a sham-transplanted nude mouse resulted in amplification for G6PD which was used as a control for the integrity of the RNA.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992004033A1 (en) | 1990-08-30 | 1992-03-19 | The General Hospital Corporation | Methods for inhibiting rejection of transplanted tissue |
US9924630B2 (en) | 2014-11-14 | 2018-03-27 | Luitauras Kairys | Device for connecting two rakes |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101955906A (en) * | 2000-01-19 | 2011-01-26 | 查珀尔希尔-北卡罗莱纳大学 | Liver tissue source |
AU2000277535B2 (en) * | 2000-10-03 | 2007-03-29 | University Of North Carolina At Chapel Hill | Processes for clonal growth of hepatic progenitor cells |
IL155249A0 (en) * | 2000-10-03 | 2003-11-23 | Univ North Carolina | Methods of isolating bipotent hepatic progenitor cells |
JP4344231B2 (en) * | 2001-05-16 | 2009-10-14 | 財団法人神奈川科学技術アカデミー | Method for detecting and separating undifferentiated hepatocytes using dlk |
WO2003033697A1 (en) * | 2001-10-18 | 2003-04-24 | Ixion Biotechnology, Inc. | Conversion of liver stem and progenitor cells to pancreatic functional cells |
EP2430923A1 (en) * | 2002-06-05 | 2012-03-21 | Genentech, Inc. | Compositions and methods for liver growth and liver protection |
-
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992004033A1 (en) | 1990-08-30 | 1992-03-19 | The General Hospital Corporation | Methods for inhibiting rejection of transplanted tissue |
US9924630B2 (en) | 2014-11-14 | 2018-03-27 | Luitauras Kairys | Device for connecting two rakes |
Non-Patent Citations (2)
Title |
---|
SMITHIES ET AL., NATURE, vol. 317, 1985, pages 230 - 234 |
ZHENG ET AL., PROC. NATL. ACAD. SCI., vol. 88, 1991, pages 8067 - 8071 |
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CN101365800B (en) * | 2005-08-17 | 2014-07-02 | Dx4U有限公司 | Composition and method for determination of CK19 expression |
US9593374B2 (en) | 2005-08-17 | 2017-03-14 | Lianidou Evriklia | Composition and method for determination of CK19 expression |
EP2295537A1 (en) * | 2008-05-14 | 2011-03-16 | Public University Corporation Yokohama City University | Human hepatic stem cell, method for preparation of the same, method for induction of differentiation of the same, and method for utilization of the same |
EP2295537A4 (en) * | 2008-05-14 | 2011-11-30 | Public Univ Corp Yokohama City | Human hepatic stem cell, method for preparation of the same, method for induction of differentiation of the same, and method for utilization of the same |
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WO2011070001A1 (en) * | 2009-12-09 | 2011-06-16 | Fresenius Medical Care Deutschland G.M.B.H. | Adult stem cell derived conditioned medium and/or adult stem cells for use in the therapeutic treatment of a tumor disease |
CN102686723A (en) * | 2009-12-09 | 2012-09-19 | 弗雷森纽斯医疗护理德国有限责任公司 | Adult stem cell derived conditioned medium and/or adult stem cells for use in the therapeutic treatment of a tumor disease |
US9034335B2 (en) | 2009-12-09 | 2015-05-19 | Fresenius Medical Care Deutschland Gmbh | Adult stem cell derived conditioned medium and/or adult stem cells for use in the therapeutic treatment of a tumor disease |
CN102686723B (en) * | 2009-12-09 | 2016-11-23 | 弗雷森纽斯医疗护理德国有限责任公司 | For treating the derivative conditioned medium of the adult stem cell processing tumor disease and/or adult stem cell |
WO2023060186A1 (en) * | 2021-10-07 | 2023-04-13 | Hepatx Corporation | Methods of tracking donor cells in a recipient |
Also Published As
Publication number | Publication date |
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EP1711597A2 (en) | 2006-10-18 |
WO2005068612A3 (en) | 2006-03-30 |
JP2011103884A (en) | 2011-06-02 |
CA2553303A1 (en) | 2005-07-28 |
JP5588587B2 (en) | 2014-09-10 |
EP1711597B1 (en) | 2012-03-07 |
CA2553303C (en) | 2014-06-10 |
JP2007523638A (en) | 2007-08-23 |
ATE548447T1 (en) | 2012-03-15 |
AU2005205240A1 (en) | 2005-07-28 |
AU2005205240B2 (en) | 2011-04-21 |
US20060040386A1 (en) | 2006-02-23 |
ES2385248T3 (en) | 2012-07-20 |
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