Method for production of human living hepatic cells, including hepatic stem cells/precursor cells

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

 

The technical field to which the invention relates.

This invention relates to the field of transplantation of liver cells or cell therapy and provides the product for transplantation of liver cells or cell therapy pharmaceutical quality and methods of producing populations of functional hepatic cells with high viability, including hepatic stem cells/precursor cells.

The level of technology

The normal liver has the ability to regenerate through repair or replace damaged tissue. Despite this protection, as soon as a critical mass of liver cells die as a result of disease or damage, the liver may stop working, resulting in illness or death. Liver failure is a serious health problem. Every year there are 300,000 registered hospitalizations and 30,000 deaths in the United States due to chronic liver disease. Currently, the only treatment for many of these diseases the liver is a liver transplant. However, only approximately 5,000 donor disease become available each year in the United States. May 2002 18000 patients were in the waiting list of liver transplant; an increase of more than 100% in comparison with p is sleznimi four years and 1700 ten years ago. In addition, approximately 100,000 adults who currently suffer from severe cirrhosis and other forms of chronic liver failure in the United States, may become candidates for transplant.

Due to the shortage of donor organs potential awaiting liver transplant patients must often wait for years when the donor liver is available. Currently, transplantation of the liver in the form of the whole body demands a donor who has undergone brain death, but whose heart is still declining. This only happens about one or two percent of deaths in hospitals, which greatly limits the potential pool of donors. It is clear that the vast majority of patients with liver disease may not rely on liver transplantation as a solution. There is an urgent need for new technologies to support patients with damaged liver.

The regenerative ability of the liver suggests that transplantation of liver cells may provide a valuable alternative to orthotopic transplantation of the whole disease. Donor liver cells, infusione in patients with liver disease may be able to colonize the liver recipient (and/or spleen when infusion in this body and in ostanavlivat function. However, the potential survival and growth in the number for extended periods after transplantation in Mature hepatocytes remains uncertain.

Common sense suggests that all Mature adult liver cells (hepatocytes) are able to share a lot of times, allowing the body to regenerate after damage. However, there is a growing understanding about the range of the regenerative potential of parenchymal cells derived from the liver. Studies in rodent hepatocytes has been shown that Mature hepatocytes from pericentral zone may undergo limited, if any, can undergo cell division; periportal hepatocytes adults, sometimes called "small hepatocytes"have high regenerative ability, but still pass only through a limited number of divisions; and the greatest regenerative ability is present in a small population of diploid parenchymal cells with properties similar to stem cells or precursor cells that can multiply very rapidly and can give an increase in the number of Mature hepatocytes [Kubota H and Reid LM. 2000/ Clonogenic hepatoblasts, common precuesors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proceedings of the National Academy of Sciences (USA) 97: 12132-12137].

Hepatic stem cells/letterpressing are a population of immature cells, which commiteeman in the direction of the hepatic line of differentiation, but still do not show most of the features of the Mature liver cells.

However, they can rapidly proliferate and lead to the emergence of fully differentiated daughter cells, which do provide the function of the liver. Studies in rodent models have demonstrated the existence of stem cells/progenitor cells in fetal liver and adult liver, which are at least bipotential; that is, their offspring includes two types of cells, namely, hepatocytes and cells of the bile ducts [Kubota H and Reid LM. 2000/Clonogenic hepatoblasts, common precuesors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proceedings of the National Academy of Sciences (USA) 97:12132-12137]. It was shown that in the liver of adult stem cells/precursor cells are involved in liver regeneration and intensive re-inhabit the liver owners after certain types of liver damage that Mature hepatocytes recipient were destroyed or have impaired the ability of cell proliferation.

Over the past 30 years has generated a significant body of scientific literature demonstrating the ability induzirovannich hepatocytes isolated from a disease of adults, to settle down in the host tissue, proliferate, to function and participate in regen the administrative process. It has been shown that transplantation of hepatocytes into the spleen or liver corrected hereditary defects of metabolism in numerous models, completely re-inhabited the liver of the host under conditions in which liver cells were lost or had a reduced life expectancy (as in the model of FAH-deficient mice), provided liver function during acute liver failure induced by various injuries, improved liver function and prolonged survival in CCl4induced models of cirrhosis.

Studies of cases and case histories in the literature describe the introduction of hepatocytes more than 40 patients with various acute and chronic, hereditary and acquired diseases of the liver. [Strom SC, Chowdhury JR, and Fox IJ. 1999. Hepatocyte transplantation for the treatment of human disease (Review). Seminars in Liver Disease 19:39-48]. Data from a number of these messages suggest that these cells were indeed taken root, survived and functioned in the period up to several months. In one study, the synthetic ability of the liver showed improvement within four to six months after transplantation of the cells, as evidenced by improved levels of albumin and prothrombin time. One of the best posted messages, demonstrating engraftment and function of transplan the new hepatocytes, associated with 10-year-old girl with down syndrome criglernajjar Najjar syndrome, a hereditary disease in which the individual has lead to severe jaundice deficiency of the enzyme UDF-glucuronosyltransferase, which conjugates bilirubin (Fox et al., I.J., "Treatment of the Crigler-Najjar Sundrome Type I with hepatocyte transplantation, "New England Journal of Medicine, (1998) 338:1422-1426). Within 18 months after transplantation the patient experienced a significant increase in excretion of conjugated bilirubin in her bile, increased enzyme activity in the biopsy of her liver and a reduced need with regard phototherapy UV light. However, these previous experiments with transplanted hepatocytes led only to a temporary improvement. Limited proliferative capacity of Mature hepatocytes inevitably limits the effective duration of therapy among hepatocytes.

At present, it was found that the above-mentioned problems in previous attempts the treatment of liver diseases are overcome by using populations of cells of the present invention, which is enriched in viable, functional liver cells. High proliferative ability of cells of the present invention supports a maximum tissue regeneration and reduces the required dose of cells for a successful transplant. The presence of stem cleto is/precursor cells also provides increased effective duration of therapy in the liver cells due to their superior ability, in comparison with Mature hepatocytes, to survive, proliferate, to function and participate in the regenerative process.

If therapy liver cells have become a commercial reality and a viable therapeutic alternative for a significant number of patients, should be established sufficient liver tissue. It was found that the cells obtained by the methods of the present invention, can be obtained from certain disease of organ donors, which is not suitable for receipt of a transplant of a whole organ or, due to the time/transport restrictions, may not be used in a timely manner. Viable, functional liver cells can be isolated by the method of this invention of the disease, which, according to the generally accepted recommendations that are not suitable for orthotopic transplantation or to obtain large quantities of Mature hepatocytes for cell transplantation. The most important is that cleaning populations of cells in the human liver, including stem cells/precursor cells, the method of the present invention promises to dramatically increase the pool of donors for treatment using liver cells. Moreover, due to the relative stability of stem cells/progenitor cells to ischemic damage, which was found, these cells can be obtained in this way from many sistoliceski (i.e., not shrinking heart) donors.

The allocation method of the present invention provides for the inclusion of viable, functional liver cells, including hepatic stem cells/precursor cells from the liver donor cryopreserved mixture of cells. This method isolates the cell suspension with proportionally higher viability of the entire disease people-donors or resected parts (in comparison with crude preparations of liver) and eliminates dead cells and debris without excessive depletion, if at all, with any depletion of populations of small hepatic stem cells/precursor cells. The obtained cell population may contain more than 80% of cells viable before cryoconservation, more than 70% of cells viable after thawing, and more than 75% of these cells are hepatocytes.

In contrast, known methods for isolating hepatocytes using low-speed centrifugation, in many cases through the environment Percoll, for the enrichment of live hepatocytes (detected in the sediment after centrifugation). Although this method is very effective for large selection of viable hepatocytes, in particular larger heparin is ozimov, it leads to a significant depletion of hepatic stem cells/progenitor cells and even to a significant loss of small Mature hepatocytes.

This invention is directed to the above-mentioned needs and develops state of transplantation of liver cells or cellular therapy software product for transplantation of liver cells or cell therapy pharmaceutical quality and ways to obtain populations of functional hepatic cells with high viability, including hepatic stem cells/precursor cells, which could not be obtained previously accepted methods. Product for transplantation of liver cells or cell therapy of the present invention consists of a well-characterized mixture of liver cells containing hepatic stem cells/precursor cells, as well as other types of cells found in the liver.

Disclosure of inventions

This invention relates to a method of obtaining a population of cells enriched in viable liver cells, including:

the splitting of a human liver or resected part of the preparation of proteolytic enzymes to provide a split of a human liver or resected part; the dissociation of a split liver brow of the ESA or its resected part to ensure the suspension of cells; setting the density of the medium in which these suspended cells, whereby after centrifugation receive at least two stripes of cells divided by barrier density, and at least one band of these, at least two bands is a band lower density than the other strip of these, at least two strips; and this collection, at least one band lower density to obtain a population of cells enriched in viable cells human liver, including hepatic stem cells/cells-precursors.

In another embodiment of the present invention is provided a method of obtaining a population of cells enriched in viable liver cells, including hepatic stem cells/cells-precursors, including: splitting a human liver or resected part of the preparation of proteolytic enzymes to provide a split of a human liver or resected part; dissociation split a human liver or resected part to ensure the suspension of cells; determining the density of the medium in which these suspended cells, whereby after centrifugation receive at least one strip of cells, and this, at least one strip tile who is to strip lower density, than the precipitation cells or cellular debris; and this collection, at least one band lower density to obtain a population of cells enriched in viable cells human liver, including hepatic stem cells/cells-precursors. Other embodiments of the present invention include, but are not limited to, populations of cells with functional hepatocytes, functional biliary cells, functional hematopoietic cells or combinations thereof.

The following embodiment of the present invention ensures that the liver or resected part can be obtained from having a shrinking heart or sistoliceski neonatal, pediatric, juvenile or adult donors. In particular, cells may be obtained by the method of this invention from the liver donor, which was subjected to heat ischemia or was obtained from sistolicheskogo donor.

In addition, this invention relates to a composition comprising a population of liver cells enriched in viable, functional liver cells, and this population of cells contains functional hepatocytes and hepatic stem cells/precursor cells. In a specific embodiment of this enriched population of cells enriched in hepatic stem cells/cells of the predecessor, having a diameter in the range of about 9 to 13 microns, which are positive for the expression of ER-HIMSELF (also called GA733-2, S-1A, EGP40, KS1-4, KSA), CD133 or both.

In the following embodiment of this invention relates to a composition comprising a population of liver cells enriched relatively coarse suspension of cells obtained from the liver of a viable, functional hepatocytes and hepatic stem cells/cells-precursors. Another embodiment further comprises biliary cells. It was found that biliary cells of these cell populations of this invention are positive for the expression of cytokeratin-19 (CK) and are negative in relation to the expression of albumin.

This invention relates also to a method of treatment of liver disease, involving the administration of an effective amount of a population of cells enriched in viable, functional liver cells, including hepatic, stvolovymi cells/cells-precursors. Various routes of administration are defined by this method, including, but not limited to, the introduction through the splenic artery or the portal vein directly to the slurry liver under the capsule of the liver or directly into the spleen.

In another embodiment of this izobreteyonija to pharmaceutical compositions, contains a population of liver cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors, and a pharmaceutically acceptable carrier. In the following embodiment of the pharmaceutically acceptable carrier may include cryoconserved, such as HYPOTHERMOSOL™.

In another embodiment, the invention relates to a method for testing toxicity in vitro, providing exposure to the effect of the test agent population of liver cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors, and monitoring at least one action, if any, of the test agent on this population of liver cells (e.g., cell viability, cell function, or both). This invention also provides a method of conducting studies of drug metabolism in vitro, providing exposure of the population of liver cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors, the effect of the test agent and observing at least one change, if any, comprising the test agent after a specified test period. This, at least, one is changing may include, but not limited to, changes in the structure, concentration, or both in the structure and concentration of the test agent.

Another embodiment of the present invention relates to an auxiliary device for the liver, comprising a housing accommodating a population of liver cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors. These liver cells can include cells of human liver or liver cells pigs.

This invention relates also to a method of treatment of errors in gene expression, introducing into a population of cells of the human liver, which includes a viable, functional liver stem cells/precursor cells, a functional copy of a gene to provide a transformed population and introduction into the patient's liver, which needs to be functional copies of this gene, at least part of this transformed population. Another embodiment of the present invention is a composition of the present invention, applicable in the above-mentioned method.

Other methods provided by the invention include methods of enhancing regeneration of damaged or diseased liver, methods of testing effective agents for the treatment of infections of the liver, how is Holocene protein of interest and the methods of obtaining the interest of the vaccine.

In the way of testing for effective agents to treat infections of the liver cell population human liver infect interest infectious agent. Then this infected population is subjected to the action of a specified number of test agent and determine the effects, if any, of this action on infected population. In the method of obtaining a protein of interest functional gene that encodes a protein of interest, is introduced into a population of liver cells, including hepatic stem cells/precursor cells. Then, the resulting population is incubated under conditions effective for the passage of transcription, translation and, if necessary, posttranslational modification, and then collect interest protein. Also provides a method of obtaining a vaccine, wherein the recombinant virus particle or virion is introduced into a population of cells of the present invention, the virus particle or virion is able to infect at least some members of this population of cells, causing those infected members to Express the antigen, so that the induced immune response in the subject to be immunized against exposure to future action of the infectious agent associated with this antigen, after the introduction in tirovannyh members of this population in this subject.

The practice of this invention will be used, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, Microbiology, recombinant DNA and immunology, which are within the skill in this field. Such methods are described in the literature. See, for example. Molecular Cloning: A Laboratory Manual, Second Edition, ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Mullis et al. U.S. Pat. No: 4,683 195; Nucleic Acid Hybridization (B.D. Hames &S.J. Higgins, eds. 1984); And reduced Translation (B.D. Hames &S.J. Higgins, eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molrcular Cloning (1984); multi-volume edition of Methods in Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M.P. Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods in Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods in Cell And Molecular Biology (Mayer and Walker, eds.. Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986);

Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986); Applications and Products 2001: Density Gradient Media (Axis-Shield PoC AS, Oslo, Norway, 2001).

Other characteristics and advantages of this invention will become apparent from the following detailed description of the present invention, taken together with the accompanying drawings, which illustrate by way of examples the principles of the present invention.

To the capacity description drawings

Figure 1 shows the profile of the dimensions defined by means of a counter of Coulter, for the new method of fractionation OptiPrep, which helps to identify the 2 peak cells: cells that are named inventors "small" (usually in the range of about 9 to 13 μm), and relatively large cells (typically in the range of about 18-22 µm). The population of small cells contains stem cells/precursor cells, as these cells have a size of approximately 10 microns.

Figure 2 shows the profile of the dimensions defined by means of a counter of Coulter for the standard (conventional) method with Parallam, showing that the relative number of larger cells (18-22 μm) is higher in the sediment of Percoll (100 × g)than in the corresponding supernatant (300 × g).

Figure 3 shows the results of FACS analysis after immunostaining with an antibody specific to ER-man HIMSELF, showing that precipitate Percoll (100 × (g) contains 6 times less than the EP-CAM-positive stainable cells (left panel, 0,12% of the population (+) against EP-CAM)than the original material (right panel, 0,76% of the population (+) against EP-CAM).

Figure 4 shows the results of FACS analysis after immunostaining with an antibody specific to ER-man HIMSELF, indicating that fractionation OptiPrep, apparently, does not affect the total number of ER-SA is positively increased population (3,07% and 3.06% of the population, increased positively in relation to the EP ITSELF in fractionated and nefrackzionirovannam samples, respectively).

Figure 5 shows graphs showing the relative populations of the EP-CAM-positive cells detected in the cell allocation of the present invention (left panel) in comparison with the supernatant of the standard method (Central panel) and the precipitate standard method (right panel).

The results obtained for the nine-month donor.

6 shows graphs showing the relative populations of the EP-CAM-positive cells detected in the cell allocation of the present invention (left panel) in comparison with the supernatant of the standard method (Central panel) and the precipitate standard method (right panel).

The results obtained for 3-year-old donor.

7 shows graphs demonstrating the enrichment against EP-CAM-positive cells after immunoselection.

Fig shows graphs demonstrating the enrichment against EP-CAM-positive cells after immunoselection.

Fig.9 shows micrographs of colonies grown from single cells, under different conditions of staining, demonstrating that the cells isolated by the method of this invention are liver stem cells/cells-precursors.

Figure 10 shows mi is rephotography colonies, grown from single cells, under different conditions of staining, demonstrating that the cells isolated by the method of this invention are liver stem cells/cells-precursors.

11 shows a micrograph of human hepatocytes obtained using the present invention on the pellet-microsites in mice NOD-SCID.

Fig shows a micrograph taken at a lower resolution than micrograph 11, to visualize the big island of hepatocytes, which are vascularsurgery the owner, as evidenced by the red blood cells.

Fig results evidence that hepatic stem cells/precursor cells of the present invention is able to develop in hepatocytes and display a Mature phenotype, demonstrating positive staining for glycogen in their cytoplasm. Note the explicit organization of these cells in the strands.

Fig shows three hepatocytes obtained using the present invention, attached to Micronesia, injected into the host. A black rectangle is drawn around the boundary surface between two adjacent cells. Enlarged photograph of this area shows the structure of microboring characteristic gall tubules, is another marker of Mature hepatocytes.

F. g shows the micrograph, showing engraftment of cryopreserved liver cells of human of the present invention in the liver of mice NOD-SCID. Two hours after the transplantation of human cells can clearly be seen by in situ hybridization in the portal veins and hepatic sinusoidal capillaries of the liver. These cells have not yet moved from the vascular space into the hepatic parenchyma.

Fig shows a micrograph showing engraftment of cryopreserved liver cells of human of the present invention in the liver of mice NOD-SCID. 40 days after transplantation of human cells do not only remain in the liver, but engraftment in the cell plate of the liver, becoming fully integrated into the hepatic parenchyma.

The implementation of the invention

This invention relates, in particular, to a method of obtaining a population of cells enriched in viable, functional hepatocytes and hepatic stem cells/cells-precursors. Another, related, aspect of this invention is the identification of the hepatic stem cells/precursor cells. The following embodiment of the present invention will lead to important progress in the identification and allocation of hepatic stem cells/precursor cells from the liver of adults.

Identified several proteins on the cell surface, which expr serwotka hepatic stem cells/cells-precursors, isolated from fetal human liver. Discovered that the same surface antigens expressed by a small percentage of cells in the neonatal, pediatric liver disease and liver disease in adults. Method of magnetic sorting of cells used for significant enrichment of cells expressing one of these surface antigens. Cells isolated using this approach are, on average, much smaller than Mature hepatocytes, in contrast to previous studies of hepatic stem cells/precursor cells of rodents, which have identified a class of large (larger than Mature parenchymal cells), acidophilic liver cells as a reserve of the liver cells (U.S. patent number 5559022). In addition, the vast majority of the cells also Express a second antigen characteristic of fetal liver stem cells/precursor cells. Under cultivation in conditions that are strictly selected on the growth of hepatic stem cells/precursor cells of rodents and limit the growth of more Mature liver cells selected by the sorting cells of adults discover the enhanced growth potential. The most convincing argument is that the analysis of colonies grown from single cells selected by sorting the population, the demo is helpful expression of proteins characteristic lines of differentiation as hepatocytes and bile ducts, as is expected for bipotential hepatic stem cells/precursor cells.

Conspicuous in this invention is that cells expressing the characteristic surface antigens remain present in liver disease (from donors with a stopped heart), which suffered several hours of severe oxygen deprivation before collecting. In fact, hepatic stem cells/precursor cells are, apparently, much more resistant to ischemia than Mature hepatocytes. In addition, although total preparations of liver cells from sistoliceski donors usually contain a significantly higher number of cells associated with tissue damage and inflammatory reactions, is still possible in a strong enrichment of viable, functional liver cells, including hepatic stem cells/cells-precursors using the methods of the present invention. In preferred embodiments of this invention use methods of immunoselection and magnetic sorting for additional identification or delete the selected types of cells derived from the liver.

The methods of the present invention, which are used to enrich the LM is unable, functional liver cells may be applied directly to the total preparations of liver cells or drugs derived from resected parts of the liver. This procedure is fast, gives satisfactory yields of cells and satisfactory viability and can be scaled to process tens of billions of cells. The selected cells are easily cryoconserved and remain viable during thawing.

This invention demonstrates that viable liver cells can be isolated post-mortem from various hepatic sources, including liver donors with unbreakable heart, liver which cannot be used for transplantation of whole organs. Because populations of liver cells of this invention can be obtained from sistoliceski donors, the invention will dramatically expand the pool of donor organs, which are suitable for use in transplantation of the liver cells or cell therapy. Table 1 summarizes the outputs from donors with a shrinking heart and sistoliceski donors.

Cells were isolated from whole donor disease or resected parts perfusion tissue Liberase™purified form of collagenase, and collecting the resulting suspension of cells. Experienced two FPIC is BA compartments of living cells from dead cells. In the new method of this invention, an aliquot of the suspension of liver cells mixed with an equal volume of a solution iodixanol (OptiPrep™, 60% iodixanol in water, Axis-Shield, Norway) and centrifuged at 2000 rpm (approximately 500 × (g) in the washer for cells Kobe 2991™ (available from the Blood Component Technology, Lakewood, CO) for 15 minutes at room temperature as follows.

In a sterile vial for 500 ml add 208,5 ml OptiPrep™, 291,5 ml of RPMI-1640 medium without phenol red. This gives a 25% solution iodixanol, having a density of 1.12.

After calculating the volume of cells on a mass basis add a sufficient amount of RPMI-1640 medium without phenol red with bringing the final volume to 250 ml with the total number of cells 10 × 109(40 × 106cells/ml). Add 250 ml of 25% iodixanol and gently stirred for good mixing. Serves the resulting solution cells in iodixanol under the action of gravity into the bag to handle washer cells Kobe 2991™. Layer 100 ml of RPMI-1640 medium without phenol red to the surface of the solution cell in iodixanol using a peristaltic pump at a speed of 20 ml/min during centrifugation of the bag. Centrifuged at 2000 rpm (approximately 500 × g) for 15 minutes in General. The resulting strip of hepatic cells on the boundary surface between the concrete the cells in iodixanol and medium RPMI-1640 without phenol red is extracted separately from the precipitated material.

In separate experiments, leading to the conditions, described above, was determined by the density of raw materials and materials selected bands after centrifugation, including bands, designated as zone "Umix"band "gradient contents", and sediment. Found that interest strip, strip "Umix", has a density 1,0607. This density value is smaller than the value found for the original material (1,0792), the band "gradient contents" (1,0792) and sediment (1,1061). Then it is determined that 11,59% solution iodixanol necessary to ensure the gradient directly into which will be deposited interest of cells after centrifugation.

In one of the standard ways of obtaining preparations of liver cells an aliquot of the suspension of liver cells mixed with isotonic Perkola (Sigma, MO) to a final concentration of 22.5% of Percoll. After centrifugation at 100 × g for 5 minutes at 4°in the centrifuge Sorvall RC3B remove sediment. It should be noted that in the descriptions of conventional methods, the supernatant discarded due to the common belief that this supernatant contains cells lower viability and that it contains usually more cellular debris. For comparative purposes supernatant extract, diluted 5 times and centrifuged at 300 × g for 5 minutes at 4°and obtained the sediment extract. The other main standard way to obtain preparations of liver cells is the secretion of cell suspension of liver enzymatic splitting of the liver and then by loosening these cells by low-speed centrifugation, typically at about 50 g. Precipitated cells remain, and the cells in the supernatant discarded.

Exceptions Trypanosoma blue reveal that the sediment Percoll 100 × g enriched in viable cells (range 70-90%) in comparison with the supernatant Percoll (range 40-60%). In contrast to OptiPrep™, the gradient of this invention, the upper stripe of cells enriched in viable cells (80-90%) in comparison with the deposited material (usually less than 20%). Size analyses using a counter of Coulter revealed enrichment for larger cells (with a diameter of 18-22 μm) in the sediment of Percoll, in comparison with the supernatant Percoll, which contains a large population of cells with diameters ranging from 9 to 13 μm, than the precipitation. The upper band of the gradient of OptiPrep™ contains cells with a diameter of 18 to 22 μm, and the diameter 91-13 μm. Determination of the size distribution for sediment OptiPrep™ is problematic due to the large amount of debris. Analysis by the method of fluorescence-activated sorting of cells (FACS) after the EP-CAM-immunostaining of these glue is OK shows the sedimentation through Percoll leads to depletion of the EP-CAM-positive cells; these positive cells are left behind in the supernatant Percoll. The upper band of the OptiPrep gradient™ contains a population of EP-CAM-positive cells, similar to that of the supernatant Percoll. Analyses of the formation of colonies is not actually detect the formation of colonies of cells detected in the sediment of Percoll, whereas the supernatant of Percoll has a comparable level of colony-forming ability with the upper band of the OptiPrep gradient™. This ability to form colonies correlates with EP-CAM-positive staining, as enrichment against EP-CAM-positive cells also increases the ability to form colonies of this cell population.

As shown in figure 1, the profile size distribution defined by means of a counter of Coulter, for the new method of fractionation using OptiPrep™ showed 2 peaks of cells: cells, which the authors called relatively small (typically in the range of diameters from 9 to 13 μm), and large cells (typically in the range of diameters from 18 to 22 μm).

A small population of cells contains stem cells/precursor cells, as these cells have a size of approximately 10 microns. The relative size of these two populations varies depending on the donor the liver, as the average size in microns population peak.

Figure 2 shows that according to the standard method with Parallam relative population of larger cells (18-22 microns in diameter) is higher in the sediment of Percoll (100 × g)than in the corresponding supernatant (300 × g).

Figure 3 shows the results of FACS analysis after immunostaining with an antibody specific to ER-man HIMSELF, showing that precipitate Percoll (100 x g) contains 5 times less than the EP-CAM-positive stainable cells (left panel, 0,12% of the population (+) against EP-CAM)than the original material (right panel, 0,76% of the population (+) against EP-CAM).

In contrast, as shown in figure 4, fractionation with OptiPrep™apparently does not affect the total number of EP-CAM-positive increased population (3,07% and 3.06% of the population increased positively in relation to the EP ITSELF, fractionated and nefrackzionirovannam samples, respectively).

Using fractionated with OptiPrep™ cell (the upper band) as a starting material in the experiments using 2 different donor disease, the inventors also show that positive painted on the EP ITSELF, the cells remain in the supernatant after the standard way with Parallam. As shown in figure 5 and 6, the population of EP-CAM-pological the data cells is comparable to the source material fractionated with OptiPrep™ cells and supernatant Percoll, whereas the sediment Percoll has a 2-5-fold depletion in respect of these positive cells.

As a biological test for the presence of stem cells/precursor cells in preparations of cells after fractionation in OptiPrep™ and centrifugation in density gradient Percoll 20000 living cells per well were sown on layers-STO feeders, supported in an environment with a certain amount of hormones and evaluated in the points to form colonies after 2-week incubation. For additional confirmation that the EP-Cam immunoreactivity corresponds to the presence of stem cells/precursor cells, the authors reasonable way believed that by increasing the population of the EP-CAM-cells should increase the number of colony forming cells in this population. For this purpose, the inventors conducted enrichment immunoselected on these cells and included them in the analysis. As shown in Fig.7 and 8, the authors received the enrichment of 40 times (0,59% of the initial population are the EP-CAM-positive, but after immunoselection 24,7% of this population are positive for this marker).

Table 2 shows that the authors really enrich the education of the colonies during enrichment against EP-CAM-positive cells. In addition, fractionated with ptiPrep™ cells and supernatant of Percoll contain colony-forming cells, whereas the sediment Percoll does not contain such cells.

To ensure that these colonies arise from a single cell, was performed limiting dilution enriched EP-CAM-positive cells, and wells containing an average of 1 cell, immunoablative antibody to human albumin, indicating parenchymal cells, and SK, which interacts with biliary cells. As shown in figures 9 and 10, the presence of both of these cell types in a single colonies confirms bipotential cells, giving rise to this colony. This bipotential is the functional definition of stem cell/cell precursor.

These data clearly show that the new method of fractionation using OptiPrep™ the present invention separates the living cells from dead cells while maintaining a viable hepatic stem cells/precursor cells in the fraction of living cells. In contrast, the standard method in this field, centrifugation through a density gradient Percoll, eliminates these cells from the sediment. Although there are modifications in the conditions under which centrifuged gradients density Percoll, all of these modifications include short-centres is Pyromania (minutes) and low power g (50, 70, 88 x g). The purpose of this standard way, apparently, is the enrichment of viable Mature hepatocytes larger. As for the subsequent experiments using sediment Percoll (discard supernatant), this area is constantly used drugs in cells deprived of such proliferative stem cells/precursor cells. A new way authors present invention surely will change the types of experiments that recognize the crucial to be transplanted cells with the highest proliferative capacity, in order to have the greatest likelihood of recovery of liver function.

Immunoselected is only one means of enriching a population of hepatic stem cells/precursor cells of the present invention. In particular, monoclonal antibodies applicable to identify markers (surface membrane proteins, such as receptors)associated with specific lines of differentiation and/or stages of differentiation. Procedures for the Department considered the hepatic stem cells/precursor cells may include magnetic separation, using antibody-coated magnetic granules, affinity chromatography and panning" with antibody attached to a solid matrix, such as a tablet, or other convenient the ways. Ways providing accurate separation include fluorescence-activated sorting of cells, which may have varying degrees of complexity, such as multiple color channels, the channels of detection of light scattering by a small and obtuse angles, channels, impedance, etc.

You can also drain unwanted cell population of viable cells human liver of the present invention. In addition to hepatocytes and their immediate predecessors, the liver contains a number of additional cell types, including cells of the bile ducts, endothelial cells, tissue macrophages (Kupffer cells), stellate cells and lymphocytes. The cell suspension obtained from perfoirmance disease, is also likely to contain some residual blood cells from circulation. In the preparations of viable cells human liver of this invention, the majority of cells Express intracellular albumin and, therefore, are hepatocytes or stem cells/cells-precursors, which form the hepatocytes. Among the other cells in these preparations, which does not Express intracellular albumin, the most stained for surface marker CD45, the common leukocyte antigen, antigen, which is known that it is present on most or all of lymphocytic cells, voltarol/macrophage and granulocyte line differentiation, and on erythroid precursors. Important sets of cells resident in the adult liver, which, as you might expect, Express CD45, include T-lymphocytes, Kupffer cells (which make up approximately 80% of the tissue macrophages of the body) and possibly granulocytes.

Depletion of CD45-positive cells from viable cells human liver the present invention can be achieved by means of immunostained known to specialists in this field. For example, a monoclonal antibody, specific for CD45, may be due to magnetic microspheres. Then these microspheres are in contact with viable cells of the human liver. CD45-positive cells come in contact with the microspheres and can be removed by application of a magnetic field. One system suitable for the depletion of CD45-positive cells, is commercially available from Miltenyi Biotec (MiltenyiBiotec GmbH, Friedrich-Ebert-Ska e 68, D-51429 Bergisch Gladbach, Germany). In the system Miltenyi CD45-microspheres used in conjunction with a magnetic column (in such a device, or AutoMACS CliniMACS) for the binding and removal of CD45-positive cells. Using this or equivalent systems, at least about 90-95% of CD45-positive cells can be removed in a single round of exhaustion. Exhausted by SP-cell population remains as essentially all cells and the preparation of the liver, able to attach and grow in culture under conditions conducive to epithelial cells, including hepatocytes. Many of these cells can be attached to the coated collagen cups in serum-free medium, and some show the morphology of hepatocytes. In contrast, the obtained magnetic sorting CD45-population, which is removed from preparations of viable liver cells of the present invention using immunotoxin, contains few cells, which are attached and show the morphology of the parenchymal cells of the liver.

Other monoclonal antibodies can be easily selected by experts in the field for specific depletion of specific unwanted populations of cells. For example, the antibody to the cell surface marker CD3 may be used for removal of T-lymphocytes. Similarly, the antibody to the cell surface marker CD 14 can be used to remove the cells of the macrophage/monocytic line of differentiation, such as Kupfer cells.

The easy way can be provided antibodies conjugated form to facilitate cell division. Materials for conjugation include, but are not limited to: magnetic granules, which allow for direct separation; Biotin, which makes it possible mediated Department is through their binding to Avidya or streptavidin, attached to the carrier; fluorochromes, which can be used with fluorescence-activated by cell sorting device. Can be used any way that is not unduly detrimental to the viability of the cells.

Cells of this invention may be retained by the cryoconservation of using any of several methods of cryopreservation. Usually isolated cells (as described above) was diluted to the desired concentration in the aqueous mixture Hypothermosol™ (Biolife Solutions, NY) and subjected to controlled freezing to the desired storage temperature. Frozen cells of this invention can be stored in liquid nitrogen.

The functionality of the cells of this invention

After separation and cryopreservation of populations of liver cells of the present invention, including hepatic stem cells/precursor cells, characterize them by means of flow cytometry using cladosporioides monoclonal or polyclonal antibodies and fluorochrome-conjugated secondary antibodies for quantitative determination of present types of cells. In addition, the functionality of these cryopreserved cells evaluated on a number of in vitro and in vivo parameters.

For example, hepatic stem cells/precursor cells of the present invention can interact the SQL on ice with 100 μl of mouse monoclonal IgG-antibodies to polymorphic EP-CAM-antigens person, conjugated with fluoresceinisothiocyanate (FITC) (Serotec Inc, UK). Control cells treated only mouse IgG-FITC. Samples analyzed using a flow cytometer EPICS (Coulter Electronics, Hialeah, Fla), configured at a wavelength of 488 nm with a gain of fluorescence established to exclude 98% of the control cells. Window set around the various populations of cells using two-parameter histogram of direct light scattering (FLS) versus side scatter (SS)and determine the percentage of positive fluorescent events.

In vitro parameters include: the metabolism of 7-ethoxycoumarin, which measures how dependent microsome cytochrome P-450 oxidation of phase I and associated reactions of conjugation phase II; the formation of urea to assess the ability of cells to convert ammonia into urea (important function lost in hepatic failure); and proliferative potential.

In vivo, the inventors evaluated the ability of these cells to survive over time; to establish and maintain the phenotype of Mature hepatocytes and prejustice in the liver parenchyma.

These studies use mice and NOD-SCID who have severe combined immunodeficiency prevent rejection of the transplanted cells cheloveka these animals. In one study, the cells are thawed and incubated in vitro with Mironosetsky to which they are attached. Then these microsocial injected into the peritoneal cavity of mice. After one week conglomerates Micronesian-cells collected from the peritoneal cavity, make slices and stained for light microscopy. Also perform electron microscopy.

Figure 11 micrograph of human hepatocytes on the granules microsites in mice NOD-SCID can clearly see hepatocytes attached to microsites. Note their round nuclei and a large number of transparent cytoplasm. The right arrow indicates dual core cell. Cells in the far right side are the stromal cells of the host, probably, which fibroblasts from peritoneal lining mouse recipient.

Fig shows a micrograph taken at a lower resolution to visualize the big island of hepatocytes, which were vascularsurgery the owner, as evidenced by the erythrocytes. Especially notable is the visible organization of these cells in strands or rows of hepatocytes - structural organization, which can easily be observed on cross sections of the liver tissue.

Evidence that hepatic stem cells/precursor cells of the present invention deistvitel is but can Mature hepatocytes and display a Mature phenotype, shown in Fig showing positive staining for glycogen in their cytoplasm. Again, note the visible organization of these cells in the strands.

At the electron microscopic level, it is possible to distinguish on Fig three hepatocytes attached to Micronesia. A black rectangle is drawn around the boundary surface between two adjacent cells. Enlarged photograph of this area shows the structure of microboring, pointing to the bile canaliculi, another marker of Mature hepatocytes.

Two micrograph shown in Fig and 16 demonstrate engraftment of cryopreserved liver cells of human of the present invention in the liver of mice NOD-SCID. In this study, thawed 1 million cells injected into the spleen of the mouse. At different time points after transplantation, animals killed, and the presence of human cells detected by in situ hybridization using DNA probes to centromeres person, and PCR analysis. Two hours later after transplantation (Fig) human cells are clearly visible by in situ hybridization in the portal veins and hepatic sinusoidal capillaries. However, these cells have not yet moved from the vascular space into the hepatic parenchyma.

After 40 days after transplantation (Fig) human cells not only remain in the liver, but also engraftment in the cellular PLA is Tinku liver, becoming fully integrated into the liver parenchyma.

Transplantation of liver cells

The population target for the treatment of cells and using the method of the present invention are ambulatory patients with cirrhosis and end stage liver disease (ESLD), caused by different factors. Patients have an estimated life expectancy without a liver transplant more than six months but less than two years. Thus, most of these patients were considered to be placed in the waiting list for orthotopic liver transplantation (i.e. transplantation of intact donor organ). These patients experienced one or more complications of the disease, such as abdominal fluid (ascites), bleeding, confusion (hepatic encephalopathy), infections and other problems. It is assumed that all these considered, patients will receive immunosuppressive therapy to prevent rejection of the transplanted liver cells, as this would have taken place in the transplantation of intact disease. The purpose of therapy is to delay or eliminate the need transplantation of the whole liver, reducing hospitalizations due to complications of liver disease and improving the quality of life of the patient.

Estimate the baseline and at last the blowing medical supervision include routine laboratory and clinical evaluation of liver function, as well as specific quantitative biochemical evaluation of the ability of the damaged liver to remove toxins, metabolize drugs and synthesize proteins. Since it is expected that the transplanted liver cells colonize the liver and spleen, are conducted periodically in specific relation to liver cells of the spleen scan to monitor the engraftment and proliferation of transplanted liver cells. The transplanted cells secrete soluble antigens that are specific for donor cells. These soluble antigens, which can be measured in the blood are monitored as further evidence of the viability and function of transplanted cells.

Two weeks before admission to the hospital for cell transplantation patients are accepted by the researcher in the clinic. The researcher obtains informed consent and begins estimate the baseline, including ABO-typing of blood. The blood type of the patient must be compatible with the blood cells of the donor in the transplantation of the whole liver or liver cells. Two days before admission, starting immunosuppressive therapy. Cryopreserved cells is taken to the hospital, where they remain frozen until immediately re the transplantation.

The night before the transplant, the patient is admitted to a hospital. The next morning the patient was transferred to the Department of interventional radiology, where he/she get mind-altering sedative. In the femoral artery of the patient (in the groin) place the catheter and advance it into the splenic artery. Donor liver cells was thawed, diluted and delivered preferably via a syringe into the catheter into the splenic artery. Time will vary depending on the doses of from five to about 30 minutes. The catheter is removed and the patient was transferred back to his/her ward for further observation. Patient discharged from the hospital eight hours after the procedure.

Transplantation of hepatocytes and hepatic stem cells/precursor cells of the present invention can be used to perform replacement of the liver by injection of a quantity of viable liver cells, including hepatocytes and/or hepatic stem cells/precursor cells (contained in the environment for transplantation, such as saline solution), in a suitable anatomical site where these liver cells, including hepatocytes and/or hepatic stem cells/cells-the precursors are implanted at the target site, such as the parenchyma of the liver and/or spleen, and p is havlat functions differentiated liver, including functions of hepatocytes. Depending on the number of liver cells, including hepatocytes and/or hepatic stem cells/precursor cells, transplanted thus, various degrees of deficiency of the liver can be adjusted by replacement of liver function in these cell transplants. However, the most profitable cellular transplantation of hepatocytes and/or hepatic stem cells/precursor cells is in the treatment of liver disease caused by genetic defects that lead to the absence or reduced function of certain enzyme or other protein product. Such diseases include, for example, hyperlipidemia and deficiency of alpha-antitrypsin. Other liver diseases that can be treated using this invention include hepatitis, cirrhosis, inborn errors of metabolism, acute liver failure, acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, Alagille syndrome, a deficiency of alpha 1-antitrypsin deficiency, autoimmune hepatitis, biliary atresia, liver cancer, cystic liver disease, fatty liver, galactosemia, gall stones, Gilbert's syndrome, hemochromatosis, hepatitis a, hepatitis b, hepatitis C, then iriu, primary sclerosing cholangitis, Reye's syndrome, sarcoidosis, tyrosinemia, glycogenosis type 1 and Wilson's disease.

To perform the transplant procedure choose the injection site for transplantation of liver cells in the liver parenchyma. One way of doing this is by injection is the spleen of a person. After calculation of the coordinates of the location of the spleen injector is placed for injection transplant environment containing liver cells, including hepatocytes and/or hepatic stem cells/precursor cells in the spleen. Then migrated cells migrate through the splenic vein into the liver parenchyma [Cm. Gupta et al., Seminars in Liver Disease 12, 321 (1992)]. In another way images are branches of the portal vein, for example, scanning of the abdomen with the help of computerized axial tomography (CAT) after injection of a radiopaque medium. The coordinates of the location of branches of the portal vein supplying a separate lobe of the liver, can then be used for injection transplant environment in the branch of the portal vein and, therefore, the infusion of a specific lobe of the liver the liver cells. Such selective infusion makes possible continuous portal blood flow through another liver.

Alternatively, liver cells, including hepatocytes and/or hepatic stem cells/the entrances-the predecessors of the present invention, can be injected or infusorian directly in the pulp liver via the splenic vein or the portal vein, or under the capsule of the liver.

Suitable routes of administration of the cells of the present invention to subjects, particularly subjects-people, described in detail here, including injection or implantation of the cells into sites of target entities, or cells of this invention can be introduced into a delivery device which facilitates introduction by injection or implantation of these cells into subjects. Such the delivery device includes a tube, for example, catheters, injection of the cells and fluids in the body of the subject the recipient. In the preferred embodiment, these tubes have an additional needle, for example a syringe, through which the cells of this invention can be administered to the subject in the desired location. Liver cells, including hepatocytes and/or hepatic stem cells/precursor cells of the present invention, can be introduced into such a delivery device, such as a syringe, in different forms. For example, these cells can be suspended in solution or embedded in a matrix carrier when they are in such a delivery device. In this context, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the present invention remain isresponsible. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersing medium. The use of such carriers and diluents are well known in this field. This solution is preferably sterile and fluid to such an extent that provides ease of feeding by syringe. Preferably, this solution is stable in the conditions of preparation and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi, during its use, for example, through the use of parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and tpicture of the present invention can be prepared by the inclusion of viable, functional liver cells, described herein, in a pharmaceutically acceptable carrier or diluent and, if required, other of the above ingredients, followed by sterilization by filtration.

The matrices media which can be enabled or made viable, functional cells include matrices which are recipient-compatible and which are destroyed to products that are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are prioritary of matrices. Natural biodegradable matrices include clots plasma, for example derived from a mammal, and collagen matrices. Synthetic biodegradable matrices include synthetic polymers, such as polyanhydride, polyarteritis and polylactic acid. Other examples of synthetic polymers and methods for enabling or conclusions of the cells in these matrices are also known in this field. See, for example, U.S. patent No. 4298002 and U.S. patent No. 5308701. These matrices provide support and protection to liver cells in vivo and, therefore, are the preferred form in which liver cells injected into the subjects of the recipient.

Gene therapy of the present invention

The results of clinical trials of gene therapy have been generally disappointing for doctors and patients, often due to the inability to obtain stable expression of the target genes. Population of liver cells, such as the population of the present invention, which includes stem cells/precursor cells, due to their potential for intensive breeding, represent a promising cell population, in which you can get and maintain an effective gene expression. Gene therapy of the present invention in one embodiment is achieved by incorporation of exogenous gene in these cells and transplantation of these to etok in the patient. Rational disorders-drug targets for the disease resulting from the inability of the liver cells of the patient's right to make important protein, such as missing receptors low-density lipoprotein (LDL) in the NCEP and coagulation factors in hemophilia.

The main obstacle in existing attempts to achieve stable integration of foreign genes in eukaryotic cells are the owners of various organs is the inability of most of these cells to proliferate in vitro. This is particularly problematic for attempts to integrate foreign genes in liver cells as Mature hepatocytes do not undergo complete cell division in vitro or, at best, are 1-2 divisions. More recently, studies of gene transfer using hepatocytes isolated from hereditary hyperlipidemics Watanabe rabbits, which are widely used as an animal model of familial hypercholesterolemia person. Like their human counterparts cells Watanabe rabbits contain genetic deficiency in respect of receptor low-density lipoprotein (LDL), which leads to high cholesterol levels in the bloodstream and increased frequency of early coronary artery disease (Wilson et al., 1990, Proceedings National Academy of Sciences USA 87:8437). Hepatocytes Crawley is and infected with recombinant viruses, bearing a functional gene for the LDL receptor, and it was shown that he had caused the temporary weakening of hyperlipidemia in genetically defective rabbits after transplantation. The authors believe that the degree of success of this form of therapy can be further increased, if the gene of interest can achieve a more stable integration into a population of recipient cells, which are capable of substantial cell division. Because hepatic stem cells/precursor cells of the present invention proliferate in vitro, in particular, for longer periods of time, in the system described herein, in which the parenchymal cells are co-cultured with embryonic stromal cells, these cells may be ideal candidates as recipients for the introduction of exogenous genes in culture.

Various inborn errors of metabolism due to genetic deficiency of the liver cells. These diseases can be treated by transplantation of the liver cells of the present invention with functional copies of the correct genes. Briefly, this procedure involves the selection of liver cells, including hepatic stem cells/precursor cells, the present invention of patients affected by a specific disease, the transfer of functional genes in this the cells to correct this genetic defect common genetic transfer technology, confirm stable integration and expression of the desired products of these genes and transplantation of these cells in the liver of the same or another patient for recovery. This approach is particularly applicable in situations where a defect in a single gene is responsible for this disease, and this defective gene has been identified and molecularly cloned; however, it is not limited to these conditions. In addition to gene therapy in autologous conditions hepatic stem cells/precursor cells of the present invention, functional genes, can also be transplanted into allogeneic HLA-compatible individuals. Examples of target genes and associated liver diseases that are amenable to this form of therapy include, but are not limited to, the gene for the LDL receptor in familial hypercholesterolemia, genes factor for coagulation factors VIII and IX in hemophilia, gene alpha-1-antitrypsin in emphysema, phenylalanine hydroxylase gene in patients with phenylketonuria, gene infintessimally with hyperammonemia genes and proteins of complement in various forms deficiency of complement.

The liver is the center of production of many secretory proteins. It is anatomically connected with the circulatory system so that it is possible to effectively visualaid is of different proteins in the bloodstream. Thus, genes encoding proteins that have systemic effects, can be embedded in the liver cells of the present invention, as opposed to specific types of cells that normally produce them, particularly when it is difficult to integrate genes into these cells. For example, different genes hormones or genes specific antibodies can be embedded in the liver cells of this invention for their secretion of gene products into the bloodstream.

For use in practice of this invention liver cells of the present invention, highlighted above described procedures, used as recipients in experiments on gene transfer. These cells can be grown in culture before, during, or after introduction of an exogenous gene. The differentiation of these cells can be minimized by the addition of cytokines in a manner similar to the use of factor inhibiting leukemia in cultures of hematopoietic stem cells.

For the introduction of exogenous genes into cultured cells of the present invention any of the cloned gene can be transferred using conventional methods, including, but not limited to, microinjection, transfection and transduction. In addition, if the liver cells Express receptors for asialoglycoprotein, plasmids containing genes of interest can be conjugated with and what sialoglycoproteins and added to the cells for the induction of uptake and expression (Wu et al., 1991, Journal of Biological Chemistry 266: 14338). This procedure is more gentle on the cells of the recipient.

The preferred method of gene transfer uses recombinant viruses, such as retroviruses and adenoviruses. For example, when using adenovirus expression vectors, the coding sequence can be Legerova with the regulatory complex of the transcription/translation of adenovirus, e.g., the late promoter and consists of three parts leader sequence. Then this chimeric gene may be integrated into the genome of the adenovirus recombination in vitro or in vivo. Embedding in non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and able to Express the gene product in infected backup liver cells (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 3655-3659). Alternatively, it may be used by the promoter 7,5K of vaccinia virus (for example, see Mackett et al., Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al., Proc. Natl. Acad. Sci. USA 79:4927-4931). Of particular interest are vectors based on bovine papillomavirus that can replicate as extrachromosomal elements (Sarver, et al., 1981, Mol. Cell. Biol. 1:486). Soon after getting this DNA into cells, this plasmid is replicated to approximately 100-200 copies on Kletsel built this cDNA does not require integration of this plasmid into the chromosome of the host, that leads to high level expression. These vectors can be used for stable expression of the inclusion of breeding marker in this plasmid, such as, for example, gene peo. Alternatively, the retroviral genome can be modified for use as a vector capable to establish and regulate the expression of any gene of interest in the hepatic stem cells/cells-the precursors of the present invention (Cone &Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353). The high expression level can also be achieved using inducible promoters, including, but not limited to, promoter of metallothionein IIA and promoters of heat shock.

For long-term, high-producing recombinant proteins, it is preferable for stable expression. Instead use expressing vectors that contain viral sites of replication initiation, viable, functional liver cells, including hepatic stem cells/precursor cells, the present invention can be transformed with cDNA controlled by appropriate elements of the regulation of expression (e.g., promoter, enhancer sequences, transcription terminator, polyadenylation sites, etc. and breeding marker. Selective the first marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow with the formation of lesions, which, in turn, can be cloned and expanded into cell lines. For example, after the introduction of foreign DNA, engineered cells of the liver can be left to grow in enriched media and then transferred to selective medium. Can be used a number of breeding systems, including, but not limited to, gene timedancing of herpes simplex virus (Wigler, et al., 1977, Cell 11:223), gene hypoxanthineguanine (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and gene adrinfo.standortstr (Lowy, et al., 1980, Cell 22:817). Resistance to antimetabolites can also be used as the basis of selection for dhfr gene, which confers resistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt gene, which confers resistance to mycophenolate acid (Mulligan &Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); gene peo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro gene, which confers resistance to hygromycin (Santene, et al., 1984, Gene 30: 147), were Recently described additional breeding genes, namely trpB, which allows cells to utilize indole instead of tryptophan; hisD, which allows cells to use gastinel, instead of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047) and ODC (interdiscursivity), which confers resistance to the inhibitor Ornit is decarboxylase, 2-(deformity)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Labotatory ed.).

Liver cells of the present invention, which has integrated specific gene, as measured by their expression product of this gene through methods such as Northern blotting and ELISA, can be transplanted as described above, in the patients from which these cells were originally obtained, or HLA-compatible individual. For HLA-compatible allogeneic transplantation backup liver cells may not necessarily require gene transfer prior to transplantation. For example, backup liver cells obtained from a donor who has a functional gene encoding coagulation factor VIII, can be used directly using transplantation of HLA-compatible hemophilic patient. These transplanted cells will be expected to proliferate and to form Mature PC that performs the normal functions of the liver, including the production of coagulation factor VIII.

In addition to using the liver cells of the present invention for correction of defects in the genes of the liver, these cells can be used to fill the liver parenchyma in the case of cirrhosis of the liver, as mentioned above, or they can be designed against specific infectious diseases of the liver. For example, uninfected safety net to the major stem cells/precursor cells can be obtained from a patient with early stage hepatitis and used as recipients for genes coding antisense RNA that is complementary critical associated with replication of the genetic elements of the hepatitis virus. Then these cells can be transplanted into patients to combat the spread of the virus and restore normal liver function.

Application of genomics and research

Technology hepatic cells of the present invention has application as a tool for identification of new drugs and the development and testing of medicines. Hepatic stem cells/precursor cells can be forced to grow and differentiate into Mature liver cells. The definition of the expression profiles of genes at various stages of the lines of differentiation of the liver provides genomic information to discover drugs. For example, this information can be used to identify new targets for detection of drugs or for the identification of proteins that perform biological functions that can be used in therapy.

As a tool for testing and drug development of liver cells and their progeny could be used to assess changes in gene expression profiles caused by drugs considered for development. Change the value in the profile of gene expression, called potential drugs that could be compared with changes caused by drugs, about which it is known that they act on the liver. This would allow pharmaceutical companies to conduct the screening of compounds for their effects on the liver in an earlier period of time in the development process that will save time and money. Full line of differentiation of liver cells from precursors to Mature cells could also be used to test drug toxicity to the liver and to investigate how this drug is metabolized. Currently, pharmaceutical companies are experiencing difficulty in obtaining a constant supply of liver cells for testing toxicity. The methods of the present invention satisfy this need.

Auxiliary device for the liver

Technology hepatic stem cells/precursor cells of the present invention has application in the development of assistive devices for the liver ("LAD"). LAD are designed to provide treatment for patients with acute liver failure by ensuring liver in a short period of time (7-30 days), to allow sufficient time for recovery of the native liver of the patient of the week is statusnote or to provide a bridge to transplant.

In attempts to clinically applicable LAD by other researchers, used the pig hepatocytes or poorly differentiated liver cells from human tumors in bioreactors of different types. These devices have been promising, but all used cells with defects in that the cells of the authors of the present invention must overcome. The pig hepatocytes, although easily obtained, had serious flaws: for example, the immune response to secreted proteins pigs, limited lifetime and viruses are not human. Tumor cells of the liver can easily be grown, but retain only a part of the functions of normal cells of the liver and cause concerns about their safety. The functioning of the cells of the human liver from a donor organs were not an alternative due to lack of donor disease. LAD using precursor cells human liver of this invention is to overcome many of the problems that have been encountered to date. Proteins secreted by these cells will be of human origin, so that the immune response should be minimized. Precursor cells can actively divide in culture, the cells from one donor liver will be able to provide numerous LAD. The most important is the fact that e and cells should show a wide range of liver, required for clinical application.

Example LAD suitable for cells of the present invention, is described in international publication registration number PCTUS00/15524.

Preparation of the vaccine of the present invention

Liver cells of this invention can also be used to produce vaccines. For example, the lack of replication of the virus (for example, lentivirus, see Nadini et al. Science 272:263-267, 1996) can be used to infect liver cells of humans, and the virus was further modified in order to carry genes encoding one or more specific protein antigens. These specific protein antigens selected depending on the type of the desired immune response. Basically, the liver cells of the present invention infect recombinant virus. Then these infected cells Express a protein antigen, against which the induced immune response. It is expected that this immune response (antibody or cell-based) directed against an infectious agent, such as hepatitis C. Subjects, subjected to the action of these infected cells, are then protected against this infectious agent. Cm. Blister et al., Tomashevskaya et al. 83 (Pt. 2): 369-381,2002 in respect of the description of the recombinant virus Semliki forest encoding nonstructural be the OK of the hepatitis C virus, for the induction of cellular immune response.

Examples

A brief description of the method

All processing of the liver is performed in a laminar Boxing class 100, located in the room of class 10000, in accordance with aseptic techniques and in accord with good manufacturing processes. All components that come into contact with the liver, bought in sterile or collected and subjected to sterilization by gas or by autoclaving.

Initial processing

The liver get immersed in VIASPAN™ (see http://www.viaspan.com/viaspan/pclf), in three bags in the refrigerator on wet ice. In the biological safety Cabinet (BSC) liver was weighed and documented its macroscopic appearance. Take samples of VIASPAN to test sterility (VIASPAN™ applicable as hypothermic solution for washing and storage organs). Liver move in a sterile bucket and kept immersed in antibiotic wash solution (0.1 mg/ml gentamicin and 5 mg/ml Cefazolin) for 5 minuten turn from the top side to the bottom during this procedure to ensure that both sides were immersed.

The liver is taken out and washed twice with a total volume of 2 l of sterile normal saline solution over the bucket. Then the liver is transferred into another sterile bucket. Vena cava compress using sterile the s disposable plastic umbilical cord clamps, and the portal vein and/or hepatic artery kanyoro pre-sterilized cannula, made of plastic transition couplings/connectors of different sizes. Take a small biopsy (from the front edge of the shares) for histological examination. The liver is transferred into a perfusion reservoir and perfusion warm (<37° (C) chelating buffer for 15 minutes at a speed that makes it possible for the maximum swelling of the liver (usually 120-240 ml/min). At the end of the perfusion period, the buffer is drained into the drain through the hole to drain located at the bottom of the perfusion reservoir.

Perfusion and cleavage

Then the liver break down (digest) the perfusion solution containing LIBERASE™ CI (enzyme preparation containing collagenase and elastase) for 30 minutes at 28°-37°C. At the end of cleavage containing LIBERASE™ the buffer is drained, and liver perfusion cold, containing serum buffer to collect to stop splitting enzyme. After the last perfusion buffer is drained into a waste container, and tank refill new, containing serum, buffer Assembly. The capsule of the liver is cut using a sterile surgical scalpel stainless steel tissue massage (for a period of not more than 20 minutes) to facilitate the dissociation of cells. When all the cells of the split tissue,apparently, are disocyanate in the buffer, the obtained cell suspension is passed through a prefilter and a number of 1000, 500, 250 and 150 micron pre-sterilized sieves of stainless steel and then collected in a 4-liter bag for blood chilled on ice. From this coarse cell suspension take samples for testing in the process of vitality, concentration, total number of cells, output per gram of tissue and sterility.

Subsequent processing

Coarse cell suspension is aseptically transferred into an appropriate number of bags for blood to 600 ml and concentrated by centrifugation at 800 × g. Concentrated cell suspension enriched living cells by mixing equal volumes of the concentrate cells and OPTIPREP solution™ (25% iodixanol) using the washer for the cells of the OWL 2991. After centrifugation at 2000 rpm for 15 minutes, the desired cell population moves up and form a band. These bands aseptically collect and distribute the appropriate number of bags for blood to 600 ml in volume, preferably not more than 200 ml per bag. Then the volume in the bag dilute to 500 ml of RPMI medium 1640. The bag is centrifuged at 800 × g for 10 minutes and the supernatant udalyayushiy residue is weighed, add a sufficient amount of medium RPMI 1640 obtained for the I final volume of 500 ml and centrifuged at 800 × g for 10 minutes. After removal of the supernatant precipitate after washing and weigh and take samples to determine the number of cells and viability and resuspending in HTS with obtaining the concentration of 6X107cells/ml. If you conduct multiple runs using the OWL because of the large number of cells, strips collected from each run, unite.

Filling and storage

These cells then manually placed (if the filling volume of 1.5 ml) in marked fluoroplastic kreolski 33 ml and then mixed with an equal volume of tributary (HTS:DMSO: serum human 60:20:20) to obtain a final concentration of 3x107cells/ml, 10% DMSO and 10% human serum. These bags are frozen using a programmable freezer Cryomed, and the frozen cells are stored in freezers in the vapor of liquid nitrogen. After at least 24 hours after freezing the samples from the freezer unite and transported to the designated testing institution for industrial testing.

Technological scheme of the process

Process diagram describing process of manufacturing, is presented below.

Introduction to clinical conditions

Clinical supplies will be transported in a clinical setting in certified containers for the transport in the vapour phase of liquid nitrogen, which supports ribaut temperature -120° With or at a lower temperature. Kreolski containing cell suspension will remain in the container until the patient is ready. Before using the product removed from the container, quickly thawed at 37°C and placed on ice. Then remove the outer packaging and, using standard aseptic hospital procedures, the cell suspension was diluted ten times with cold Plasma-Lyte®; And in creamest before the introduction of the patient. This procedure eliminates the need for washing cells before infusion and minimizes the risk of compromising the sterility. In one embodiment of the present invention, which will be introduced to a patient contains 3x106cells/ml, and optionally contains DMSO (1%), serum AB (1%), HypoThermosol®;

(4%-8%) and RPMI without phenol red (0%-4%) in PLASMA-Lyte®.

The selection of the liver cells pigs

Follow the procedure for filtering and collecting cell suspension, as described above, to obtain samples of pig liver.

Sample test for viability, density and output. After all the calculations take 10 billion cells. If the density is lower than 25 million cells per ml, the cells are concentrated by centrifuge Sorval RC3B, Sorval centritech or cell processor OWL 2991. Sediment resuspended in 250 ml of RPMI medium 1640 without phenol is wow red. The cell suspension is transferred into a bag for the blood of 600 ml and add 25% iodixanol (OptiPrep™see http://www.nycoined-diagnostics.com/gradmed/optiprep/optil.htmr)diluted in RPMI 1640 without phenol red. These two solutions are mixed thoroughly together and store in the cold.

The cell processor OWL 2991 collected using one set for processing. Cell suspension is served by gravity using red tube kit. As soon as the toroidal chamber filled, begin centrifugation at 2000 rpm for 15 minutes beginning centrifugation 100 ml of medium RPMI 1640 layer on top of the gradient using a peristaltic pump at a rate of 20 ml/min to act as a buffer for the blending of the strip. After 15 minutes this upper buffer "decanted" at a rate of 100 ml/min in the bag for waste, and the upper line of cells is collected in the collection bag. Sediment also collect, if it is desired, for subsequent analysis. The upper line of cells is placed on the ice and take the test on the viability and yield. This process is repeated until you process all nefrackzionirovannam porcine hepatocytes. After collecting all the strips and join them together, the combined collected stripes cells washed with dilution buffer for collection and centrifugation at 3000 rpm for 10 minutes using centrif the guy Sorval RC3B. The precipitate resuspended in the buffer for cryopreservation at a density of 30 million cells/ml. Aliquots, if necessary, placed in bags and/or bottles. Final preparations of porcine cells stored in the regulated freezer over liquid nitrogen.

This invention should not be limited to the amount provided as examples of embodiments, which are intended to illustrate certain aspects of the present invention. Indeed, various modifications of the present invention, in addition to the shown and described herein will become apparent to experts in this field from the previous description and accompanying drawings. It is assumed that such modifications fall within the scope of the attached claims.

1. A method of obtaining a population of cells enriched in viable cells human liver, including hepatic stem cells/cells-precursors, where this population of cells contains functionally active hepatocytes and biliary cells expressing cytokeratin 19 (CK) and not expressing albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers of the EP-CAM, CD 133, or both, including:

(a) splitting a human liver or resected part of the prep is the atom of proteolytic enzymes to provide a split of a human liver or resected portion;

(b) mechanical dissociation split a human liver or resected part of cell suspension;

(c) the suspension of a suspension of cells in a solution of 25% (wt./about.) iodixanol;

(d) centrifuging the mixture according to (C) to obtain at least one band is enriched in viable cells human liver; and

(e) collecting at least one strip to obtain a population of cells enriched in viable cells human liver, including hepatic stem cells/cells-precursors.

2. The method according to claim 1, wherein the population of cells enriched in viable cells human liver, and additionally includes functional hepatocytes.

3. The method according to claim 1, wherein the population of cells enriched in viable cells human liver, and additionally includes functional biliary cells.

4. The method according to claim 1, wherein the population of cells enriched in viable cells human liver, and additionally includes functional hematopoietic cells.

5. The method according to claim 1, in which stage (a) provides at least one of the following:

(e) perfezione a human liver or obtained by resection of part of the chelating buffer;

(f) splitting the price is Oh human liver or resected part of the preparation of enzymes containing collagenase and at least one other proteolytic enzyme, at approximately 37°to provide split liver; and

(g) perfezione split liver buffer to collect temperature of 4-15°C.

6. The method according to claim 5, in which the preparation of enzymes includes at least one neutral protease.

7. The method according to claim 5, in which the preparation of enzymes includes the elastase.

8. The method according to claim 5, in which the product contains enzymes LIBERASE™.

9. The method according to claim 1, in which the specified dissociation includes mechanical dissociation by cutting, surowka, combing through over the net of the liver.

10. The method according to claim 1, wherein stage (C) provides for at least one of the following:

(h) filtering the cell suspension to remove debris and aggregates of cells;

(i) collect the resulting filtered cell suspension in the first bag;

(j) optionally, determining the concentration of cells in the filtered cell suspension;

(k) making available, if necessary, the concentration of cells to provide the original cell suspension;

(1) mixing an aliquot of the original cell suspension with an equal volume of 25% solution iodixanol in a liquid medium to provide a mixture; and

(m) be the W, at least part of this mixture, which is layered a predetermined volume of a liquid medium, centrifugation to obtain at least one band is enriched in viable cells human liver.

11. The method according to claim 1, wherein stage (d) provides at least one of the following:

(n) collecting at least one strip in the container on ice;

(o) determining the viability and cell concentration;

(b) washing of these cells by centrifugation and resuspending

buffer for cryopreservation to obtain a final cell

suspension;

(q) the exposure of the final cell suspension freezing with variable speed to ensure the frozen cell suspension and

(r) this storage frozen cell suspension in the freezer with liquid nitrogen.

12. The method according to claim 5, in which the buffer for the collection contains medium RPMI 1640 with 10% human or bovine serum.

13. The method according to claim 10, in which the specified stage filtration involves passing the specified cell suspension through the filter cartridge

14. The method according to claim 10, in which the specified liquid medium contains medium RPMI 1640 containing no phenol red.

15. The method according to claim 10, in which the specified centrifugation is carried out in the course of AP is sustained fashion for 15 minutes at approximately 500 × g.

16. The method according to claim 11, wherein said container includes a bag for collection.

17. The method according to claim 11, in which the buffer for cryopreservation contains a mixture containing Na+To+, CA2+Mg2+CI-H2PO4-, HCO3-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, glutathione, or a combination of both.

18. The method according to 17, in which the buffer for cryopreservation further comprises serum and dimethylsulfoxide.

19. The method according to p, in which the mixture of the serum and the dimethyl sulfoxide is present in a ratio of about 80:10:10./about./about.

20. The method according to p, in which serum includes human serum, bovine serum, or a combination of both.

21. The method according to claim 1, in which the density of the medium set using an aqueous solution iodixanol.

22. The method according to item 21, in which an aqueous solution iodixanol contains sterile 60% (wt./about.) iodixanol in the water.

23. The method according to claim 1, wherein the enriched population of cells includes hepatic precursor cells/stem cells having a diameter in the range of 9-13 MKN, which are positive for the expression of EP-CAM, CD 133, or both.

24. The method of obtaining an enriched population of viable cells human liver, and this population of cells contains options is online active hepatocytes and biliary cells, expressing cytokeratin 19 (CK) and not expressing albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13

μm and expressing markers of the EP-CAM, CD 133, or both, providing

(a) providing a human liver or resected part of the neonatal, pediatric, juvenile, adult or cadaveric donor;

(b) perfezione a human liver or resected part chelating buffer;

(c) splitting of a human liver or resected part of the preparation of enzymes to ensure cell suspension;

(d) if necessary, mechanical dissociation of a liver or resected part of cell suspension;

(e) if necessary, remove debris and aggregates of cells;

(f) mixing the cell suspension with an equal volume of a solution iodixanol;

(g) the exposure of the mixture, which is layered a predetermined volume of culture medium, centrifugation to obtain at least two bands of cells divided by barrier density, and at least one strip has a lower density than the other band/other strip; and

(h) collecting at least one band lower density.

26. Composition for the treatment of patients suffering from liver disease, contains a population of cells enriched in viable, functional liver cells, including hepatic, stvolovymi cells/cells-precursors, where this population of cells contains functionally active hepatocytes and biliary cells expressing cytokeratin 19 (CK) and not expressing

albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers of the EP-CAM, CD 133, or both.

27. The composition according to p, in which liver cells are liver cells or liver cells of a mammal.

28. The composition according to item 27, which further comprises hepatocytes, biliary cells, hematopoietic cells, or combinations thereof.

29. The composition according to p, in which liver cells contain cells that are positive for the expression of ER-HIMSELF, CD 133, or both.

30. The composition according to p, in which liver cells contain stem cells/precursor cells with a diameter of AP is sustained fashion 9-13 MKN.

31. Composition for the treatment of patients suffering from liver disease, contains a population of liver cells, enriched, relatively coarse suspension cells derived from liver, viable, functional hepatocytes and hepatic stem cells/cells-precursors, where this population of cells contains functionally active hepatocytes and biliary cells expressing cytokeratin 19 (CK) and not expressing albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers ER-HIMSELF, CD 133, or both.

32. The composition according to p, in which liver cells are cells of the human liver.

33. The composition according to p, in which the population of liver cells further comprises biliary cells that are positive for the expression of cytokeratin 19 (CK) and negative in relation to the expression of albumin.

34. The composition according to p, in which stem cells/precursor cells are positive for the expression of ER-HIMSELF, CD 133, or both.

35. The composition according to p in which this population further comprises cells of the monocytic/macrophage line of differentiation (e.g. Kupffer cells), lymphocytes (e.g. T cells), endothelial cells, or combinations thereof.

36. Compo is ice on p, in which the population of liver cells depleted in the cells of the immune system.

37. The composition according to p, in which the population of liver cells depleted in the cells of the immune system through the use of negative immunoselection.

38. The composition according to clause 37, in which negative selection uses depletion for CD45, CD3, CD lIb or CD 14 or their combinations to remove hematopoietic cells.

39. The composition according to p in which cells of the immune system, which depleted, include cells of the monocytic/macrophage line of differentiation (e.g. Kupffer cells), lymphocytes (e.g. T-lymphocytes), or both.

40. The composition according to p in which donor cells were subjected to a period of thermal ischemia.

41. The composition according to p, in which liver cells obtained from sistolicheskogo donor.

42. The composition according to p, in which at least 75% of the population of liver cells contains hepatocytes and hepatic stem cells/precursor cells.

43. Composition for the treatment of patients suffering from liver disease, contains a population of cells

liver enriched in viable, functional biliary cells, and hepatic stem cells/cells-precursors, where this population of cells

contains functionally active hepatocytes and biliary cells, e is preservada cytokeratin 19 (CK) and not expressing albumin, as well as hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers ER-HIMSELF, CD 133, or both.

44. The composition according to item 43, in which liver cells are cells of the human liver, the liver cells pigs or mixtures thereof.

45. The composition according to item 43, in which liver cells obtained from sistolicheskogo donor or were subjected to a period of thermal ischemia.

46. The composition according to item 43, in which the biliary cells are positive for the expression SK and negative in relation to the expression of albumin.

47. A method of treating liver disease, involving the administration of an effective amount of a population of cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors, where this population of cells contains functionally active hepatocytes and biliary cells expressing cytokeratin 19 (CK) and not expressing albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers ER-HIMSELF, CD 133, or both.

48. The method according to p, in which the introduction is carried out by introduction through the splenic artery or portal vein.

49. The method according to p, in which the introduction is carried out by introducing directly itulu liver.

50. The method according to p, in which the introduction is carried out by introduction into the capsule of the liver.

51. The method according to p, in which the introduction is carried out by injection directly into the spleen.

52. The method according to p, in which the introduction is by infusion or injection.

53. The method according to p, in which liver disease include hepatitis, cirrhosis, congenital metabolic disorders, acute liver failure, acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, Alagille syndrome, a deficiency of alpha-1-antitrypsin deficiency, autoimmune hepatitis, biliary atresia, liver cancer, cystic liver disease, fatty liver, galactosemia, gall stones, Gilbert's syndrome, hemochromatosis, hepatitis a, hepatitis b, hepatitis and infection With other hepatitis viruses, porphyria, primary sclerosing cholangitis, Reye's syndrome, sarcoidosis, tyrosinemia, glycogenosis type 1 or Wilson's disease.

54. Pharmaceutical composition for the treatment of patients suffering from liver disease, contains a population of liver cells enriched in viable, functional liver cells, including hepatic stem cells/cells-precursors, where this population of cells contains functionally active hepatocytes and biliary glue the key, expressing cytokeratin 19 (CK) and not expressing albumin and hepatic stem cells/precursor cells having a diameter in the range from 9 to 13 μm and expressing markers of the EP-CAM, CD 133, or both, and a pharmaceutically acceptable carrier.

55. The composition according to item 54, in which liver cells are cells of the human liver, the liver cells pigs or mixtures thereof.

56. The composition according to item 54, in which liver cells obtained from sistolicheskogo donor or were subjected to a period of thermal ischemia.

57. The composition according to item 54, in which stem cells/precursor cells are positive for the expression of ER-HIMSELF, CD 133, or both.

58. The composition according to item 54, in which stem cells/precursor cells have a diameter of approximately 9-13 MKN.

59. The composition according to item 54, in which the pharmaceutically acceptable carrier includes HYPOTHERMOSOL™.

60. The composition according to item 54, in which the pharmaceutically acceptable carrier additionally includes human serum and dimethylsulfoxide.



 

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