Title of Invention	A METHOD OF ISOLATING PROGENITORS LIVER CELLS AND THEIR TRANS-DIFFERENTIATION THEREOF
Abstract	The present invention relates to a method of isolating liver stem celt -said method comprising steps suspending the hepatocytes in Oulbecco's Minimum Essential Medium (OM EM) with about 10% Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions; in addition, a method of treating diabetes Type I in a subject, said method comprising steps of transplanting transdifferentiated (TO) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels; further, a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture.

Title of Invention

A METHOD OF ISOLATING PROGENITORS LIVER CELLS AND THEIR TRANS-DIFFERENTIATION THEREOF

Abstract

The present invention relates to a method of isolating liver stem celt -said method comprising steps suspending the hepatocytes in Oulbecco's Minimum Essential Medium (OM EM) with about 10% Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions; in addition, a method of treating diabetes Type I in a subject, said method comprising steps of transplanting transdifferentiated (TO) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels; further, a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture.

Full Text	A METHOD OF ISOLATING PROGENITORS LIVER CELLS FROM FETAL LIVER CELLS AND THEIR TRANS-DIFFERENTIATION THEREOF Field of the present invention The present invention relates to a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks. Also, it relates to an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions and it use in treatment of diabetes Type I and acute liver failure. Background and prior art references of the present invention The term " Hepatic progenitors" refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitors having ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiated daughter cells. As used here, the term "progenitor cell" is also intended to encompass a cell which is sometimes referred to in the art as "stem cell". In the preferred embodiment, the term "progenitor cell" refers to a generalized mother cell whose descendents (progeny) specialize, often in different directions, by differentiation. The term " isolated hepatic progenitors" in the present invention refers to the single or cluster of the hepatic progenitors. Stem cells and early progenitors have long been known to exist in rapidly proliferating adult tissues such as bone marrow, gut and epidermis, but have only recently been thought to exist in quiescent tissues such as adult liver, an organ characterized by a long cellular life span. The ability of stem cells to self-replicate and produce daughter cells with multiple fates distinguishes them from committed progenitors. In contrast, committed progenitors produce daughter cells with only one fate in terms of cell type, and these cells undergo a gradual maturation process wherein differentiated functions appear in a lineage-position-dependent process. In adult organisms, stem cells in somatic tissues produce a lineage of daughter cells that undergo a unidirectional, terminal differentiation process. In all well-characterized ineage systems, such as hematopoiesis, gut and epidermis, stem cells have been identified by empirical assays in which the stem cells were shown to be capable of producing the full range of descendants. To date, no molecular markers are known which uniquely identify stem cells as a general class of cells, and no molecular mechanisms are known which result in the conversion of cells from self-replication and pluripotency to a commitment to differentiation and a single fate. The structural and functional units of the hepatic parenchyma is the acinus, which is organized like a wheel around two distinct vascular beds. Six sets of portal triads, each with a portal venule, a hepatic arteriole and a bile duct, form the periphery, and the central vein forms the hub. The parenchyma, which comprises the "spokes" of the wheel, consists of plates of cells lined on both sides by the fenestrated sinusoidal endothelium. Blood flows from the portal venules and hepatic arterioles at the portal triads, through sinusoids which align plates of parenchyma, to the terminal hepatic venules, the central vein. Hepatocytes display marked morphologic, biochemical and functional heterogeneity based on their acinar location (see Gebhardt, Pharmac. Ther., Vol. 53, pp. 275-354 (1990)). Comparatively, periportal parenchymal cells are small in size, midacinar cells are intermediate in size and pericentral cells are largest in size. There are acinar-position-dependent variations in the morphology of mitochondria, endoplasmic reticulum and glycogen granules. Of critical importance is that the diploid parenchymal cells and those with greatest growth potential are located periportally. In parallel, tissue-specific gene expression is acinar-position-dependent leading to the hypothesis that the expression of genes is maturation-dependent (see Sigal et al., Amer. J. Physiol., Vol. 263, pp. G139- G148(1993)). It is currently believed that the liver is a stem cell and lineage system which has several parallels to the gut, skin and hemopoietic systems (see Sigal et al., Amer. J. Physiol., Vol. 263, pp. G139-G148 (1993); Sigal et al. In Extracellular Matrix, Zern and Reed, eds, Marcel Dekker, NY., pp. 507-537 (1993); and Brill et al., Liver Biology and Pathobiology, Arias et al., 3d eds, Raven Press, NY (1994 in press)). As such, it is expected that there are progenitor cell populations in the livers of all or most ages of animals. A lineage model of the liver would clarify why researches have been unable to grow adult, mature liver cells in culture for more than a few rounds of division, have observed only a few divisions of mature, adult liver cells when injected in vivo into liver or into ectopic sites, and have had limited success in establishing artificial livers with adult liver cells. These impasses are of considerable concern in the use of isolated liver cells for liver transplantation, artificial livers, gene therapy and other therapeutic and commercial uses. The success of the above-listed procedures requires the use of hepatic progenitor cells which are found in a high proportion of liver cells in early embryonic livers and in small numbers located periportally in adult livers. Because it is desirable to isolate such hepatic progenitors , a need has arisen to develop a method of successfully isolating said hepatic progenitos (liver stem cells). Diabetes affects 16 million people in the U.S and is caused by the abnormal metabolism of insulin. Normally insulin is produced and secreted by the cellular structures called the islets of langerhans in the pancreas. Diabetes mellitus will frequently reach the stage of life threatening and severely disabling complications which cannot be kept under control by insulin alone. Since last two decades cell transplants programmes have been initiated by many centers around the world which has shown encouraging results. But, the shortage of the cell supply is the main hurdle for the success of cell based therapy. The knowledge of stem cell biology has revolutionized the area of cell biology. Stem cell transplantation appears to be the sole therapy available for the treatment of type I diabetes. Stem cells have two important characteristics that distinguish them from matured cells. Firstly, they can renew themselves for long periods through cell division. Secondly, is that under certain physiologic or experimental conditions, they can be induced to become other cell type. Other than the embryonic stem cells organ specific stem cells (adult stem cell) have shown much more plasticity than originally thought. Stem cells isolated from one tissue can differentiate into hematopoietic lineages. Similarly, bone marrow derived stem cells can differentiate into several non hemeatopoietic cell type including skeletal muscle, microglia, astroglia and hepatocytes. During embryogenesis, both liver and ventral pancreas appears to arise from the same cell population located with in the embryonic endoderm. Recently much attention has focused on the apparent plasticity of adult stem cells, especially the capability of such cells to transdifferentiate into cells of other organs. The well studied transdifferentiation mechanism is between liver to pancreas and vise versa. In various experimental conditions transdifferentaition of pancreas into liver cells has been reported. In hamsters, hepatocytes like ceils can be induced in a regenerating model based on methionine deficiency and the treatment with pancreatic carcinogen N-nitroso-bis (2-oxopropyl) amine. Hepatocytes can be produced in the rat pancreas either from feeding peroxisomal proliferators or in transgenic mice overexpressing keratinocyte growth factor in pancreas. Pancreatic epithelial cells transplanted into liver can also transdifferentiate to hepatocytes. The most reproducible method for inducing hepatocytes in the pancreas copper depletion of the diet in rats. Bartles et al. have shown that these pancreatic hepatocytes exhibit many of the characteristics of the normal hepatocytes; for example, at the level of electron microscope level, they form bile canaliculi between opposing cells, and the hepatocytes also express numerous liver specific proteins including albumin, dipeptidylpeptidase, and alpha 2 macroglobulim . Similarly, transdifferentiation of hepatic cells to pancreatic cells is well reported in literature. Yang et al. (2002) demonstrated that rat hepatic stem cells can transdifferentiate into functional pancreatic endocrine harmone producing cells after culture in high glucose concentration environment, insulin production was confirmed by immunocytochemistry and western blot analysis. These transdifferentiated cells, when transplanted in streptozotocin induced diabetic animal model. The animals received 200 islet -like clusters exhibited of similarly, Tosh et al. (2002) demonstrated the transdifferentiation of pancreatic cells to hepatocytes in cultures induced with dexamethasone. Further, these cells demonstrated all liver specific enzymes. Isolation of pancreatic progenitors from fetal source is of great clinical potential, because of higher proliferation rate and less imunogenecity. In present invention, isolated hepatic progenitors from fetal liver and were transdifferentiated to pancreatic cells. Tansdifferentiated cells produced insulin, an important cellular function pancreatic cell type. The efficacy of these TD cells have shown to normalize the glucose levels of streptozotocin induced diabetic animal model. The methods of the invention have been developed with embryonic livers in which there are significant numbers of pluripotent liver cells (liver stem cells) and committed progenitors (cells with a single fate to become either hepatocytes or bile duct cells). The present of separation isolated hepatic progenitors is on the basis of size and density. This is achieved by using centrifugation elutration system. The fraction with smallest size is separated from other subpopulation of cells. This method gives homogenous population of hepatic progenitors in a fraction. This method does not use antibody bound mediated separation. Objects of the present invention The main object of the present invention is to develop a method of isolating mammalian including human hepatic progenitors, also called liver stem cells. Another object of the present invention is to develop a method to proliferate liver stem cells in vitro culture. Yet another object of the present invention is to develop a method of differentiating liver stem cells in vitro culture condition. Still another object of the present invention is to develop a method of transdifferentiating liver stem cells into pancreatic progenitor cells. Still another object of this invention to develop a method of treating insulin dependent diabetes. Still another object of this invention to develop a method of treating acute liver failure, including fulminant hepatic failure. Summary of the present invention The present invention relates to a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 10% Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions; in addition, a method of treating diabetes Type I in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels; further, a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian including Human tetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture. Detailed description of the present invention The present invention relates to a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 10% Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions; in addition, a method of treating diabetes Type I in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels; further, a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian including human fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture. In an embodiment of the present invention, wherein a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps of: • obtaining pure hepatocytes from aborted fetuses of gestation period of about 14-20 weeks, • suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 8-12 % Fetal calf serum, • centrifuging the suspended cells at about 2200-2700 rpm preferably 2500 rpm at about 8-12°C, preferably at about 10°C, • obtaining 5 fractions at increasing pump flow rate of about 16 to 40 ml/minute, with step wise increase of a. about 16-20 ml/minutes, preferably 18 ml/minute; b. about 24-26 ml/minute, preferably 25 ml/minute; c. about 28-30 ml/minute, preferably 29 ml/minute; d. about 32-34 ml/minute, preferably 33 ml/minute; e. about 35-40 ml/minute; preferably 37 ml/minute; • obtaining 6th blow-out fraction (residue)with rotor stopped, • centrifuging each fraction in cold at about 475-525 X g preferably 500 X g to obtain pellet, • re-suspending the pellets in the DMEM, and • obtaining liver stem cells in fraction no. 5. In another embodiment of the present invention, wherein fetuses is from animals including humans. In yet another embodiment of the present invention, wherein the centrifugation is by using counterflow centrifugal elutriation system. In still another embodiment of the present invention, wherein an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions, said method comprising steps of: culturing liver stem cells in a culture comprising Dulbecco's Minimum Essential Media/HAM'SF-12, fungizone of concentration ranging between 20-30 \|ag/ml, penicillin of concentration ranging between 55-65 units/.ml, streptomycin of concentration ranging between 550-650 jxg/ml, and Fetal calf serum of concentration ranging between 7-13 %. • maintaining the culture at about 37°C in water saturated atmosphere of about 3-7 % preferably about 5% C02 and about 93-97% preferably about 95% air in a C02 incubator, and • obtaining transdifferentiated pancreatic cells. In still another embodiment of the present invention, wherein the fungizone is of concentration of about 23-27 \|ig/ml preferably about 25 (ig/ml. In still another embodiment of the present invention, wherein the penicillin is of concentration of about 57-63 units/ml preferably about 60 units/.ml. In still another embodiment of the present invention, wherein the streptomycin is of concentration of about between 575-625 \|ig/ml preferably 600 ng/ml. In still another embodiment of the present invention, wherein the fetal calf serum is of concentration of about 8-12% preferably about 10%. In still another embodiment of the present invention, wherein a method of treating Type I diabetes in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels. In still another embodiment of the present invention, wherein the glucose levels comes to normal in about 7 days. In still another embodiment of the present invention, wherein a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian including human fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture. In still another embodiment of the present invention, wherein injecting the hepatic stem cells twice or more. In still another embodiment of the present invention, wherein the transplanted liver attains normal architecture is about 25 days. In still another embodiment of the present invention, wherein the said method helps repopulation of the liver cells. In still another embodiment of the present invention, wherein the said method is used for treating Acute liver failures including fulminant hepatic failure. In still another embodiment of the present invention, wherein the success rate in acute liver failure is about 65-70%. Brief description of the accompanying drawings Fig 1 shows Scanning Electron Microscopy (SEM) figure of the single isolated Human Hepatic Progenitor Cell. The size of the isolated hepatic progenitor is around 3 micron. Fig 2 shows SEM figure of cluster of isolated human hepatic progenitors Fig 3 shows SEM figure of the differentiated proliferative biliary cell. The biliary is a important cell of hepatic lineage. These are characterized by specific perturbations. These proliferating cells arrange themselves either in a well defined tubular architecture or alternatively in an irregular, disorganized pattern sprouting into the parenchyma. Fig 4 shows SEM figure of the differentiated hepatocyte, round shape with smooth surface. Fig 5 shows SEM figure showing cluster of cells, Majority 'of the cells differentiated to hepatocyte (approximately 70%) with lesser number forming to biliary cell (approximately 30%). Fig 6 shows SEM figure also showing single differentiated pancreatic islet cell, characterized by the production of Insulin in the culture supernatant. Fig 7 shows Trans-differentiation of hepatic progenitors into pancreatic cells. Fig 8 shows Immuno cytochemical staining for insulin trans differentiated cells. Fig 9 shows decrease in the blood glucose level following TD cell transplantation In still another embodiment of the present invention, wherein this invention relates to methods for isolating hepatic progenitors (liver stem cells). The hepatic progenitors are committed progenitors for either hepatocytes or bile duct cells. The isolated hepatic progenitors of the invention may be used to treat liver of liver failure. In addition, the isolated hepatic progenitors of the invention may be used for research, therapeutic and commercial purposes which require the use of populations of functional liver cells. Unlike mature liver cells, the hepatic progenitors of the invention generate daughter cells that can mature through the liver lineage and offer the entire range of liver functions, many of which are lineage-position specific. Further, the hepatic progenitors of the invention have a greater capacity for proliferation and long-term viability than do mature liver cells. As a result, the hepatic progenitors of the invention are better for research, therapeutic and commercial uses than mature liver cells. In still another embodiment of the present invention, wherein this invention relates to method of isolating hepatic progenitors from developing fetal liver and transdifferentaiting (TD) to pancreatic cells invitro culture. The TD cells produce pancreatic specific harmone (insulin). This invention provides a method for the treatment of diabetes type-I by transplantating the cells in intrahepatically. Isolation of hepatocytes by in situ liver perfusion In the present method aborted fetuses with the gestation period of 13-24 weeks were taken. The time interval between fetal death (invitro or in vivo) and the commencement of liver perfusion is less than two hours. The fetuses are weighed and its crown crump length is measured in the lateral position. The chest and the abdomen of the fetuses are prepared with antiseptics and the procedure is carried out under strict aseptic conditions. The fetus is draped in supine position exposing the chest and the upper abdomen. A thin polythene catheter of 22-24 guage is introduced into umbilical cord vein and passed down for about 3-5 cms. The cord is then ligated with the catheter in situ. 2 ml of normal saline (containing one drop of 1 in 5000 heparin) is gassed continuously with 100% oxygen and warmed to 37 C. Initially 10-30ml is perfused until the skin of the epigastrum is stetched. The chest is opened transversely from the 9th costochondrial joint of one side to the other. Longitudinal incisions are taken on both sides along the costochondral junction to reach the level of manubrium sterni. This flap containing the sternum is now reflected towards the head. The pericardium is cut open with scissors and extended to both sides to expose the heart completely which is turned towards the left so the inferior vena cava can be seen entering the right atricum. A second plastic catheter is passed into the vena cava downwards to reach the tip of the hepatic vein and a ligature applied to the inferior vena cava with the catheter in situ. The inferior vena cava proximal to catheter is divided. The perfusate from the liver now flows out through this outlet catheter. The liver is irrigated continuously with the buffer until the outflow fluid becomes pale to clear. Simultaneously, the abdomen is opened using an upper paramedian incision with care taken not to injure the liver capsule beneath. The abdominal inferior vena cava is clamped. The incision is extended along the subcostal margin towards the flanks on both the sides which gives full exposure of the liver. The initial rate of perfusion is increased from 20-30 ml./min. to 30-50 ml./min. The outflow fluid becomes clear on perfusion with 300-500 ml. The perfusion is stopped and the liver is gently pressed to express as much irrigating fluid as possible. The inferior vena cava catheter is clamped with artery forceps and collagenase solution (0.050%) in Hank's buffer is perfused until the liver is swollen. The cord catheter is clamped and the enzyme solution kept in the liver for 10 minutes then the umbilical vein at its entrance into the liver is clamped and cut. The thoracic inferior vena cava is clamped near the diaphragm and cut and the liver is separated from all its attachments and removed. The liver is placed in a sterlized Petri-dish in a laminar flow chamber. The liver capsule is removed and tissue is freed of connective and vascular tissue using a scalpel blace. More collaeenase is added to the freed tissue and incubated at 37°C with constant stirring for 20 minutes so that the cell dispersion is complete. It is then filtered serially through nylon meshes of 250, 100 and 40 micron size to remove the vascular and connective tissue. The cells in suspension are washed with chilled Hank's washing medium containing calcium and magnesium salts" and kept for gravity sedimentation taking the following precautions (a) the pH of the washing solution is kept at 7.4 (b) the Hank's medium is always previously gassed with pure 100% oxygen before being added to the cell suspension. The supernatant containing non-hepatocytic cells is pipetted out. The viable hepatocytes form a light brown colour sediment at the bottom of the flask. The cell suspension is repeatedly washed (3-6 times) with the Hank's medium till a pure hepatocyte suspension is obtained. Medium 199 is then added to the cell suspension which is stored at 4°C. Isolation of hepatic progenitors (liver stem cells) The liver cell suspension obtained by collagenase digestion were separated according size and density using centrifugal elutration system. The equipment used was Beckman JE-6 elutration rotor with standard Beckman separator chamber(Beckman Instruments, Palo Alto California). The isolated cells were suspended in Dulbecco's Minimum Essential Medium with 10% Fetal Calf Serum was used as elutration medium and the sample was run at 2500rpm at IOC. Approximately 9ml of the cell suspension were injected into the mixing chamber five fraction of 100ml each were collected at increasing pump flow rate 24 ml /minute. Sixth blow out fraction was collected with the rotor stopped. The eutriated cellls from each fraction were centrifuged at 500xg 10 in cold high speed centrifuge and the pellets were resuspended in Dulbecco's Minmum Essential Medium. The ceils were fixed in 2.5% gluteraldehyde and processed for Scanning Electron Microscopy as shown in Figure 1. Please find below details of the composition of the buffers used in the experimentations. HANKS BUFFER Sodium Chloride Potassium Chloride Sodium Phosphate Dibasic (anhydrous) Potassium Phosphate Dibasic (anhydrous), and optionally D-GIucose, and Calcium Chloride2H20 Dulbecco, s Minimum Essential Medium Components Calcium Chloride anhydrous Cupric Sulphate .5H20 Feme Nitrare .9H20 Ferrous Sulphate.7 H20 Magnesium Chloride Magnesium Sulphate (anhydrous) Potassium Chloride Sodium Chloride Sodium Phosphate Dibasic (anhydrous) Sodium Phosphate Monobasic (anhydrous) L- Alanine L- Arsinine.HCL L- Asparagine L-Aspartic Acid L-Cystine.HCL.H20 L-Cystine.2HCL L-Glutamic Acid L-GIutamine Glycine L-Histidine . HCL.H20 L-Isoleucine L- Leucine L- Lysine .HCL L-methionine L- Phenylalanine L- proline L- Serine L-Threonine L- Tryptophan L-Tyrosine.2Na2H20 L- Valine D-Biotin Choline Chloride Foilic Acid Myo- inositol Niacinamide D-Pantothenic Acid (hemicalcium) Pyridoxal .HCL Pyridoxin .HCL Riboflavin Thiamine.HCL Vitamin B-12 D-Glucose HEPES Hypoxanthin Lionic Acid Phneol Red.Na Putrescine.HCL Pyruvicacid. Na Thiocitic Ac Characterization isolated mammalian including human hepatic progenitors (liver stem cells) Characterizing stem cells or their progeny in human liver has of course been more difficult. A number of surface determinants are shared between haematpoietic derived progenitors and hepatic progenitors (liver stem cells), including c-kit, CD34, and Thy-1 in rodents and c-kit and CD34 in humans. In the present invention the isolated progenitors were found positive for CD34. Scanning Electron Microscopy (SEM) of the isolated hepatic progenitors . The elutriated sample were fixed 2.5% gluteraldehyde and viewed under the Scanning Electron Microscope, the cells were viewed as small oval shaped cells. The size was found to be less than 3 micron as shown in figure No. 1. In the present invention the isolated progenitors CD34 was used as one of the marker to identify hepatic progenitors. The cells were analysed in Flow Cytometer (FACS) using commercially available CD34 (Becton Dickinson USA). In the present invention the cells were put in invitro culture and the AFP production was assessed by ELISA. Further, AFP secretion decreased from 5th day and disappeared by 10th day. Albumin production was observed from 3rd day of culture and continued. Morphological studies were carried by SEM after isolation and subsequent to a culture. The homogenous population of the 5 fraction of centrifugal elutriated sample was viewed under the Scanning Electron Microscope, the cells were viewed as small oval shaped within size range of 3-5 jaM as shown in figure Nos 1 and 2. Characterising stem cells or their progeny in mammalian including human liver has of course been more difficult. A number of surface determinants are shared between haematpoietic derived progenitors and hepatic progenitors (liver stem cells), including c-kit, CD34, and Thy-1 in rodents and c-kit and CD34 in humans. Alfafeto production (AFP): A liver specific marker AFP is a liver specific marker and is also marker of proliferation. AFP also has been postulated as a transient marker of liver stem cells. In the present invention the cells were put in invitro culture and the AFP production was assessed by Enzyme Liked Immuno Sorbent Assay (ELISA) The isolated hepatic progenitors continued to produce high level of AFP in vitro culture condition (Figure 1 ) GammaGlutamy Transpeptidase: A proliferative marker Gammaglutamyl transpeptidase is a marker of proliferation. In the present invention GGT has been used as one of the marker to assess the proliferative property of the isolated human hepatic progenitors. The isolated hepatic progenitors have shown to have high level of GGT levels in the isolated human hepatic progenitors during invitro culture condition (Figure 2) Differentiation of hepatic progenitors in invitro culture Invitro culture The hepatic progenitors (liver stem cells), may be bipotential or multipotential. They can differentiate into hepatocyte or cholangiocyte or they may produce other type of cell like pancreatic cell in a specified culture environment as shown in figure nos. 3 and 4. In the present invention majority of the hepatic progenitors differentiated to hepatocyte, with lesser number differentiating to cholangiocytes as shown in figure nos. 5 and 7. Clinical application of isolated hepatic progenitors Intrahepatic transplantation of Hepatic progenitors in fulminant hepatic failure animal model Hepatocyte transplantation is an attractive therapy for most liver diseases. Our earlier experience showed that the Intraperitoneal transplantation of human fetal hepatocytes in fulminant hepatic failure has reversed the disease condition of >50% of the hepatocyte transplantated cases (Habeebullah et al. Transplantation 1994). However, the success requires injection of large number of cells (-20 billion cells) and matured cells have limited growth potential in vivo. Further, the introduction of substantial numbers of large matured hepatocytes with a diameter 20-25 micron is very complicated. Intrahepatic transplantation of hepatic progenitors has several advantages over other ectopic sites 1 Transplanting cells into the unique hepatic architecture allows interaction with other hepatocytes and nonparenchymal cells; 2. Proximity to hepatocyte-specific growth and differentiation factors creates an environment particularly conducive to hepatocyte engraftment. 3. Locally released mitogens and portal-born hepatotrophic factors can increase transplanted cell numbers. 4.The liver may be an immunological privileged site. 5, Only in the liver would hepatocytes be able to secrete bile into the biliary tree. In the present invention, differentiated cells were transplanted intrahepatically for the treatment of acute liver failure animal model. • Massive acute necrosis of liver cells leads to FHF a condition which is characterized by a sudden onset of progressive jaundice, decrease in size of the liver, hepatic encephalopathy within eight weeks of acute illness. • The mortality rate ranges from 50-90 % primarirly because of associated cerebral edema, sepsis and multiorgan failure. • The major problem in FHF is the loss of hepatocytes rtather than a deficiency of proliferation of surviving hepatocytes Therefore salvaging the injured liver in FHF requires liver repopulation. • Orthotopic Liver Transplantation is the alternative sources for FHF. • Only 10 % of patients with acute liver failure receive OLT. • Different approaches to treat acute Hover failure include cross circulation, plasma exchange, hemoperfusion, hemodialysis, hemofilteration and exvivo isolated liver perfusions without any significant improvement of survival. • The potential successful option might be hepatocyte transplantation. • If temporary metabolic support is provided for a short period the damaged liver could regenerate. If sufficient amounts of viable liver cells can successfully transplanted to the ALF patient a bridge- to - time can be created to obtain wither sufficient regenaration of the host liver or to stabilize the patient expecting OLT in the near future. Acute Liver Failure animal model (rat) Acute liver failure injury animal model (rat) was developed injecting single dose of 875 mg/Kg body wt intraperitoneal^. Animals were divided into three groups Group I Only saline Group II D-Galactosamine only Group III Cell transplantation* after 24 hours of D-Gal induction Site of transplantation: Intrahepatic, by multiple injections at three points. Dosage: 10 X 10A6 million were transplanted intrahepatically after 24 hours of D-Gal induction. Results 1. Survival following transplantation of Hepatic progenitors in acute liver failure animal model • None of the animals died in group -I, • In group-II, 63% survival was observed, which received the cells intrahepatically, • All the animals died in Group-II, which did not receive cell therapy following liver • Injury • Histopathology of the liver following transplantation in D-Gal injured liver was taken which shows marked improvement in the pathology of the liver. By 25th day liver regained normal architecture as shown in figures 4, 5, and 6. Conclusion • Liver provides necessary conditions for survival differentiation and proliferation for the transplanted. • >60% (around 70%) survival in Gr-III shows that transplanted cells provides necessary support to the failing liver. Transdifferentiation of hepatic progenitors in specified culture condition as shown in figure no. 6 Culture Condition: The isolated hepatic progenitors were cultured in Dulbecco's Minimum Essential Media/HAM'SF-12 (Sigma, USA) was supplemented with fungizone (25 jig/ml), pencillin (60units/.ml), streptomycin (600 \|J.g/ml) (Hi Media Laboratories, India ) with 10% Fetal calf serum. The culture condition were maintained at 37C in water saturated atmosphere of 5%C02 and 95% air in a C02 incubator. In cells were cultured with insulin (1 IU insulin/ml) defined media for 24 hours and later replaced with media without insulin medium. The cells were checked for the production of Insulin for 10 days (Table 1). Insulin estimation in invitro culture The insulin was estimated in medium and in the lysed cells. The cultured cells were trypsinised and washed twice with Hanks medium and the cells were lysed by using 0.2% Triton x 100 and spinned at 2000rpm for 5 min the supernatant is collected for the estimation of insulin production by ELISA as per the manfacturer's protocol. In Gr-I cells were cultured with insulin ( 1 IU insulin/ml ) defined media for 24 hours and later replaced with media without insulin medium. In Gr-II cells were cultured in DMEMwithout any growth harmone or any other supplements. The cells were checked for the production insulin. The results showed production of 49uIU/ml, whereas the control, which were not challenged with insulin have shown the production 7uIU/ml. Cultures Groups Gr-I : only Insulin + DMEM Gr-II without any growth harmone or any supplements The cultures maintained for 10 days and insulin levels were followed. Electron Microscopy of the differentiated Scanning cells Cultured cells were fixed in 2.5% gluteraldehyde and viewed under the Scanning Electron Microscope as shown in figure no. 7. Immuno-cytochemical staining for Insulin TD cells The cultured cells were fixed in 3:1 acetone methanol mixture for 15 min at room temp. The slides were washed twice with cold PBS and then incubated with monospecific mouse anti human insulin ( Zymed ) for 2 hours. After incubation the slides were washed with cold PBS and the nonspecific binding is blocked by incubating the slides with 1 % BSA for 30 min. Then the slides were washed twice with cold PBS and incubated with FITC conjugated goat anti mouse immunoglobin-G (Sigma aldrich ) for 1 hour in dark. Again wash the slides with cold PBS then the cells are observed under Leitz flourescent microscope as shown in figure No. 8. Development of diabetic animal model. All the animals received intraperitoneal injection (40 jig /gm) body wt. Transplantation of Cultured hepatic progenitors Approximately three million differentiated pancreatic progenitors were injected intrahepatically by opening the abdomen under anesthesia in aseptic condition after 24 hours of Streptozotocin injection. Animals were immunosuppressed by cyclosporin injection lmg/ kg body weight Blood glucose level was monitored daily for 10 days. Transplantation study Seven rats (wistar) were taken in study. Animals were divided in three groups; Gr-I : Only normal saline Gr-II: Streptozotocin +TD cells Gr-III: only Streptozotocin RESULTS t\-• All the animals in group III died by 5 day. • Gr-I animals maintained normal glucose level in the range of 97-102 mg/dl. • In group-II, glucose levels started falling following transplantation of TD cells intrahepatically as shown in figure no. 9. The blood glucose levels come to the normal within 7 to 10 days. 13. Aterman., "The Stem Cells of Liver-a Selective Review", J Cancer Research Clinical Oncol.", vol. 118, pp. 87-115 (1992). 14. Enat et. al., "Hepatocyte Proliferation in Vitro: Its Dependence on the use of Serum- Free Hormonally Defined Medium: and Substrata of Extracellular Matrix", "Proc. Natl. Acad. Of Sci. U.S.A.", vol. 81, pp. 1411-1415 (1984). 15. Enjoji et. al., " Establishment of a Facultative Human Hepatic Stem Cell Line with a Dual Differentiating Potentiality", "Gastroenterology 110", vol. 110 (4 suppl.), A1187 (1996). 16. Fulvvyler et. al. "Production of Uniform Microspheres", "Review of Scientific Instruments', vol.44 (No.2), pp. 204-206 (1973). 17. Lineages", "American J. of Pathology", vol. 144 (No.5), pp. 849-854 (1994). 18. Johe et. al., "Single Factors Direct the Differentiation of Stem Cells from the Fetal and Adult Central Nervous System", "Genes & Development", vol. 10 pp.3129-3140 (1996). 19. Reid. "Defining Hormone and Matrix Requirements for Differentiated Epithelia", "Methods in Molecular Biology", The Humana Press, Inc., vol.5 pp. 237-276 (1990). 20. Rutenberg et. al., "Histochemical and Ultrastructural Demonstration of Glutamyl Transpeptidase Activity", "Journal of Histochemical and Cytochemistry", vol. 17, pp. 517-526(1969). 21.Sigal et. al., "Characterization and Enrichment of Fetal Rat Hepatoblasts by Imunoabsorption ("Panning") and Fluorescene-activated Cell Sorting", Hepatology", vol.19 (No.4), pp. 39-47 (1995). 22. Gupta et. al., "Permananet Engraftment and Function of Hepatocytes Delivered to the Liver, Implications for Gene Therapy and Liver Repopulation", "Hepatology", vol. 14 (No.l), pp. 144-149(1991). 23. Koch, et. al., "Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes", "Journal of Cell Sciences", vol. 99, pp. 121-130 (1991). 24. Moran et. al., "Hepatoid Yolk Sac Tumors of the Mediastinum: LAMBDA. Clinocopathologic and Immunohistochemical Study of Four Cases", The American Journal of Surgical Pathology, vol.21 (No.10), pp.1210-1214, 1997. 25. Yamamasu, et. al., "Role of Glutathione Metabolism and Apoptosis in the Regression if Live Hemopoiesis", "Free Radical Biology & Medicine", vol. 23 (No.l), pp.100-109 (1997). 26. Ruck et. al., "Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6", Histopathology", vol.31, pp.324-329 (1997). 27. Smith et. al., "Hepatic stem cells in the human liver", "Correspondence", vol.29, pp. 589-594(1996). 28. Fisher, M., Neuronal Influence on Glial Enzyme Expression: Evidence from Mutant Mouse Cerebella," PNAS, vol.81, 4414-4418 (1984). 29. Langer, R., et. al., "Tissue Engineering, "Science, vol.260, 920-926 (1993). 30. Sarraf, C, et. al., "cell Behavior in the Acetylaminofluorene-Treated Regenerating Rat Liver", American Journal of Pathology, vol. 145, No.5, 1114-11126(1994). 31. Teitelman, G., "Precursor Cells of Mouse Endocrine Pancreas Coexpress Insulin, Glucagon and the Neuronal Proteins Tyrosine Hydoxylase and Neuropeptide Y, but not Pancreatic Polypeptide, "Development, vol.118, 1031-1039 (1993). 32. Dabeva M, et. al., "Models for Hepatic Progenitor Cell Activation, "Proceedings of the Society for Experimental Biology and Medicine, vol. 204, No.3, 242-252 (1993). 33. Brill et. al., "Hepatic Progenitor Populations in Embryonic, Neonatal, and Adult Liver sup.l (43662)". Hepatic Proc. Soc. Exp. Biol. Med. (U.S.), 204(3): 270-279, 1993. 34. Lemmer et al., "Isolation and Culture of Hepatic Stem cells from Human Foetal Liver: A Preliminary Report", Hepatology, 1-56 Iasl Abstracts 23(1): 157P, 1996. 35. Travis, "The Search for Liver Stem Cells Picks Up", Science 259:1829, 1993. 36. M. Alison, "Liver stem cells: a two compartment system", Current Opinion in Cell Biol., 10:710-715, 1998. 37. Alison et al., "Wound healing in the liver with particular reference to stem cells", Phil. Trans. R. Soc. Lond. B, 353:877-894, 1998. 38. Le mire et. al., "Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine", Am. J. of Path., 139:535-552, 1991. 39. Tian et al., "The oval shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and pyruvate kinase isoenzyme expression", Histochem. Cell. Biol., 107: 243-250, 1997. 40. Agelli et. al., "Survival in Culture of Putative Liver Stem Cells is Independent from Known Epithelial Cell Growth Factors", "Clinical Research", vol.39 (No.2), pp. 167A (1991). 41. Aterman., "The Stem Cells of Liver-a Selective Review", J Cancer Research Clinical Oncol.", vol. 118, pp. 87-115 (1992). 42. Enat et. al., "Hepatocyte Proliferation in Vitro: Its Dependence on the use of Serum-Free Hormonally Defined Medium: and Substrata of Extracellular Matrix", "Proc. Natl. Acad. Of Sci. U.S.A.", vol. 81, pp. 1411-1415 (1984). 43. Enjoji et. al., " Establishment of a Facultative Human Hepatic Stem Cell Line with a Dual Differentiating Potentiality", "Gastroenterology 110", vol. 110 (4 suppl.), Al 187 (1996). 44. Fulvvyler et. al. "Production of Uniform Microspheres", "Review of Scientific Instruments', vol.44 (No.2), pp. 204-206 (1973). 45. Lineages", "American J. of Pathology", vol. 144 (No.5), pp. 849-854 (1994). 46. Johe et. al., "Single Factors Direct the Differentiation of Stem Cells from the Fetal and Adult Central Nervous System", "Genes & Development", vol. 10 pp.3129-3140 (1996). 47. Reid, "Defining Hormone and Matrix Requirements for Differentiated Epithelia", "Methods in Molecular Biology", The Humana Press, Inc., vol.5 pp. 237-276 (1990). 48. Rutenberg et. al., "Histochemical and Ultrastructural Demonstration of Glutamyl Transpeptidase Activity", "Journal of Histochemical and Cytochemistry", vol. 17, pp. 517-526(1969). 49. Sigal et. al., "Characterization and Enrichment of Fetal Rat Hepatoblasts by Imunoabsorption ("Panning") and Fluorescene-activated Cell Sorting", Hepatology", vol.19 (No.4), pp. 39-47 (1995). 50. Gupta et. al., "Permananet Engraftment and Function of Hepatocytes Delivered to the Liver, Implications for Gene Therapy and Liver Repopulation", "Hepatology", vol. 14 (No.l), pp. 144-149(1991). 51. Koch, et. al, "Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes", "Journal of Cell Sciences", vol. 99, pp. 121-130 (1991). 52. Moran et. al., "Hepatoid Yolk Sac Tumors of the Mediastinum: LAMBDA. Clinocopathologic and Immunohistochemical Study of Four Cases", The American Journal of Surgical Pathology, vol.21 (No.10), pp.1210-1214, 1997. 53. Yamamasu, et. al., "Role of Glutathione Metabolism and Apoptosis in the Regression if Live Hemopoiesis", "Free Radical Biology & Medicine", vol. 23 (No.l), pp. 100-109(1997). 54. Ruck et. al., "Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6", Histopathology", vol.31, pp.324-329 (1997). 55. Smith et. al., "Hepatic stem cells in the human liver", "Correspondence", vol.29, pp. 589-594(1996). 56. Fisher, M., Neuronal Influence on Glial Enzyme Expression: Evidence from Mutant Mouse Cerebella," PNAS, vol.81, 4414-4418 (1984). 57. Langer, R., et. al., "Tissue Engineering, "Science, vol.260, 920-926 (1993). 58. Sarraf, C, et. al., "cell Behavior in the Acetylaminofluorene-Treated Regenerating Rat Liver", American Journal of Pathology, vol. 145, No.5, 1114-11126(1994). 59. Teitelman, G., "Precursor Cells of Mouse Endocrine Pancreas Coexpress Insulin, Glucagon and the Neuronal Proteins Tyrosine Hydoxylase and Neuropeptide Y, but not Pancreatic Polypeptide, "Development, vol.118, 1031-1039 (1993). 60. Dabeva M, et. al., "Models for Hepatic Progenitor Cell Activation, "Proceedings of the Society for Experimental Biology and Medicine, vol. 204, No.3, 242-252 (1993). 61. Brill et. al., "Hepatic Progenitor Populations in Embryonic, Neonatal, and Adult Liver sup.l (43662)". Hepatic Proc. Soc. Exp. Biol. Med. (U.S.), 204(3): 270-279, 1993. 62. Lemmer et al., "Isolation and Culture of Hepatic Stem cells from Human Foetal Liver: A Preliminary Report", Hepatology, 1-56 Iasl Abstracts 23(1): 157P, 1996. 63. Travis, "The Search for Liver Stem Cells Picks Up", Science 259:1829, 1993. 64. M. Alison, "Liver stem cells: a two compartment system", Current Opinion in Cell Biol., 10:710-715, 1998. 65. Alison et al., "Wound healing in the liver with particular reference to stem cells", Phil. Trans. R. Soc. Lond. B, 353:877-894, 1998. 66. Lemire et. al, "Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine", Am. J. of Path., 139:535-552, 1991. 67. Tian et al., "The oval shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and pyruvate kinase isoenzyme expression", Histochem. Cell. Biol., 107: 243-250, 1997. 68. Gebhardt, Pharmac. Ther., Vol. 53, pp. 275-354 (1990). 69. Brill et al., Liver Biology and Pathobiology, Arias et al., 3d eds, Raven Press, 70. Sigal et al., Amer. J. Physiol., Vol. 263, pp. G139-G148 (1993)). 71. Li, Heather Hatch, Kim Ahrens , Janet G. Cornelius , Bryon E Petersen (2002) Proc. Natl . Acad . Sci .USA 99, 8078 -8083. 72. Dabeva, M.D., Hwang, S.G., Vasa, S.R ., Hurston, E., Novikoff, P.M ., Hixson D.C., Gupta ,S & Shafritz D.A.(1997)Proc. Natl . Acad. Sci.USA94, 7356- 7361 73. Petersen ,BE., Goff J.P., Greenberger, J.S & Michalopoulos, G.K(1998) Hepatology 27, 433 -445 . 74. Petersen, BE., Zajac, V .F & Michalopoulos, G.K (1998) Hepatology 27, 1030-1038. 75. Malouf, N.N ., Coleman ,W.B, Grisham, J.W., Lininger, R .A ., Madden ,VJ Sproul , M.& Anderson, P.A(2001) Am . J.Pathol. 158, 1929 -1935. 76. Lijun yang , shivvu Li , Heather Hatch , Kimaherens , Janet g Cornelius, Bryom E Petersen (2002) Proc. Natl . Acad. Sci.USA 99, 8078 -8083. 77. David tosh , Chia- Ning Shen And Jonathan M .W .slack (2002) Hepatology , 36 534 -543 Claims 1. A method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps of: (a) obtaining pure hepatocytes from aborted fetuses, (b) suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 8-12% preferably 10% Fetal calf serum, (c) centrifuging the suspended cells at rate ranging between 2200-2500 rpm at about 8-12 °C, (d) obtaining 5 fractions at increasing pump flow rate of about 16 to 40 ml/minute with increasing rates of 16-20; 24-26; 28-30; 32-34; and 35-40 ml/minute respectively, (e) obtaining 6th blow-out fraction with rotor stopped, (f) centrifuging each fraction in cold at about 475-525 X g to obtain pellet, (g) re-suspending the pellets in the DMEM, and (h) obtaining liver stem cells in fraction no. 5. 2. A method as claimed in claim 1, wherein fetuses is from animals including humans. 3. A method as claimed in claim 1, wherein the centrifugation is by counterflow centrifugal elutriation system. 4. An in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions, said method comprising steps of: a. culturing liver stem cells in a culture comprising Dulbecco's Minimum Essential Media/HAM'SF-12, fungizone of concentration ranging between 20-30 ng/ml, penicillin of concentration ranging between 55-65 units/ml, streptomycin of concentration ranging between 550-650 j-ig/ml, and Fetal calf serum of concentration ranging between 7-13 %. b. maintaining the culture at about 37°C in water saturated atmosphere of about 3-7 % C02 preferably 5% and about 93-97 % preferably 95 air in a CO2 incubator, and c. obtaining transdifferentiated pancreatic cells. 5. The method as claimed in claim 4, wherein the fungizone is of concentration of about 23-27 \|.ig/ml preferably 25 (ig/ml. 6. The method as claimed in claim 4, wherein the penicillin is of concentration of about 57-63 units/ml preferably about 60 units/.ml. 7. The method as claimed in claim 4, wherein the streptomycin is of concentration of about 575-625 \|ig/ml preferably about 600 jxg/ml. 8. The method as claimed in claim 4, wherein the fetal calf serum is of concentration of about 8-12% preferably 10%. 9. A method of treating Type I diabetes in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells as claimed in claim 4 intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized blood glucose levels. 10. A method as claimed in claim 9, wherein the blood glucose levels comes to normal in about 7 days. 11. A method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture. 12. A method as claimed in claim 11, wherein injecting the hepatic stem cells twice or more. 13. A method as claimed in claim 11, wherein the transplanted liver attains normal architecture is about 25 days. 14. A method as claimed in claim 11, wherein the said method helps repopulation of the liver cells. 15. A method as claimed in claim 11, wherein the said method is used for treating Acute liver failures including fulminant hepatic failure. 16. A method as claimed in claim 11, wherein the success rate in management of acute liver failure is about 65-70%. 17. A method of isolating liver stem cells from the fetuses of gestation period ranging

Full Text

A METHOD OF ISOLATING PROGENITORS LIVER CELLS FROM FETAL LIVER CELLS AND THEIR TRANS-DIFFERENTIATION THEREOF
Field of the present invention
The present invention relates to a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks. Also, it relates to an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions and it use in treatment of diabetes Type I and acute liver failure.
Background and prior art references of the present invention
The term " Hepatic progenitors" refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitors having ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiated daughter cells. As used here, the term "progenitor cell" is also intended to encompass a cell which is sometimes referred to in the art as "stem cell". In the preferred embodiment, the term "progenitor cell" refers to a generalized mother cell whose descendents (progeny) specialize, often in different directions, by differentiation.
The term " isolated hepatic progenitors" in the present invention refers to the single or cluster of the hepatic progenitors.
Stem cells and early progenitors have long been known to exist in rapidly proliferating adult tissues such as bone marrow, gut and epidermis, but have only recently been thought to exist in quiescent tissues such as adult liver, an organ characterized by a long cellular life span. The ability of stem cells to self-replicate and produce daughter cells with multiple fates distinguishes them from committed progenitors. In contrast, committed progenitors produce daughter cells with only one fate in terms of cell type, and these cells undergo a gradual maturation process wherein differentiated functions appear in a lineage-position-dependent process.
In adult organisms, stem cells in somatic tissues produce a lineage of daughter cells that undergo a unidirectional, terminal differentiation process. In all well-characterized ineage systems, such as hematopoiesis, gut and epidermis, stem cells have been identified by empirical assays in which the stem cells were shown to be capable of producing the full

range of descendants. To date, no molecular markers are known which uniquely identify stem cells as a general class of cells, and no molecular mechanisms are known which result in the conversion of cells from self-replication and pluripotency to a commitment to differentiation and a single fate.
The structural and functional units of the hepatic parenchyma is the acinus, which is organized like a wheel around two distinct vascular beds. Six sets of portal triads, each with a portal venule, a hepatic arteriole and a bile duct, form the periphery, and the central vein forms the hub. The parenchyma, which comprises the "spokes" of the wheel, consists of plates of cells lined on both sides by the fenestrated sinusoidal endothelium. Blood flows from the portal venules and hepatic arterioles at the portal triads, through sinusoids which align plates of parenchyma, to the terminal hepatic venules, the central vein. Hepatocytes display marked morphologic, biochemical and functional heterogeneity based on their acinar location (see Gebhardt, Pharmac. Ther., Vol. 53, pp. 275-354 (1990)).
Comparatively, periportal parenchymal cells are small in size, midacinar cells are intermediate in size and pericentral cells are largest in size. There are acinar-position-dependent variations in the morphology of mitochondria, endoplasmic reticulum and glycogen granules. Of critical importance is that the diploid parenchymal cells and those with greatest growth potential are located periportally. In parallel, tissue-specific gene expression is acinar-position-dependent leading to the hypothesis that the expression of genes is maturation-dependent (see Sigal et al., Amer. J. Physiol., Vol. 263, pp. G139-
G148(1993)).
It is currently believed that the liver is a stem cell and lineage system which has several
parallels to the gut, skin and hemopoietic systems (see Sigal et al., Amer. J. Physiol., Vol.
263, pp. G139-G148 (1993); Sigal et al. In Extracellular Matrix, Zern and Reed, eds,
Marcel Dekker, NY., pp. 507-537 (1993); and Brill et al., Liver Biology and
Pathobiology, Arias et al., 3d eds, Raven Press, NY (1994 in press)). As such, it is
expected that there are progenitor cell populations in the livers of all or most ages of
animals.
A lineage model of the liver would clarify why researches have been unable to grow
adult, mature liver cells in culture for more than a few rounds of division, have observed

only a few divisions of mature, adult liver cells when injected in vivo into liver or into ectopic sites, and have had limited success in establishing artificial livers with adult liver cells. These impasses are of considerable concern in the use of isolated liver cells for liver transplantation, artificial livers, gene therapy and other therapeutic and commercial uses.
The success of the above-listed procedures requires the use of hepatic progenitor cells which are found in a high proportion of liver cells in early embryonic livers and in small numbers located periportally in adult livers. Because it is desirable to isolate such hepatic progenitors , a need has arisen to develop a method of successfully isolating said hepatic progenitos (liver stem cells).
Diabetes affects 16 million people in the U.S and is caused by the abnormal metabolism of insulin. Normally insulin is produced and secreted by the cellular structures called the islets of langerhans in the pancreas. Diabetes mellitus will frequently reach the stage of life threatening and severely disabling complications which cannot be kept under control by insulin alone. Since last two decades cell transplants programmes have been initiated by many centers around the world which has shown encouraging results. But, the shortage of the cell supply is the main hurdle for the success of cell based therapy. The knowledge of stem cell biology has revolutionized the area of cell biology. Stem cell transplantation appears to be the sole therapy available for the treatment of type I diabetes. Stem cells have two important characteristics that distinguish them from matured cells. Firstly, they can renew themselves for long periods through cell division. Secondly, is that under certain physiologic or experimental conditions, they can be induced to become other cell type. Other than the embryonic stem cells organ specific stem cells (adult stem cell) have shown much more plasticity than originally thought. Stem cells isolated from one tissue can differentiate into hematopoietic lineages. Similarly, bone marrow derived stem cells can differentiate into several non hemeatopoietic cell type including skeletal muscle, microglia, astroglia and hepatocytes. During embryogenesis, both liver and ventral pancreas appears to arise from the same cell population located with in the embryonic endoderm. Recently much attention has focused on the apparent plasticity of adult stem cells, especially the capability of such

cells to transdifferentiate into cells of other organs. The well studied transdifferentiation mechanism is between liver to pancreas and vise versa.
In various experimental conditions transdifferentaition of pancreas into liver cells has been reported. In hamsters, hepatocytes like ceils can be induced in a regenerating model based on methionine deficiency and the treatment with pancreatic carcinogen N-nitroso-bis (2-oxopropyl) amine. Hepatocytes can be produced in the rat pancreas either from feeding peroxisomal proliferators or in transgenic mice overexpressing keratinocyte growth factor in pancreas. Pancreatic epithelial cells transplanted into liver can also transdifferentiate to hepatocytes. The most reproducible method for inducing hepatocytes in the pancreas copper depletion of the diet in rats. Bartles et al. have shown that these pancreatic hepatocytes exhibit many of the characteristics of the normal hepatocytes; for example, at the level of electron microscope level, they form bile canaliculi between opposing cells, and the hepatocytes also express numerous liver specific proteins including albumin, dipeptidylpeptidase, and alpha 2 macroglobulim . Similarly, transdifferentiation of hepatic cells to pancreatic cells is well reported in literature. Yang et al. (2002) demonstrated that rat hepatic stem cells can transdifferentiate into functional pancreatic endocrine harmone producing cells after culture in high glucose concentration environment, insulin production was confirmed by immunocytochemistry and western blot analysis. These transdifferentiated cells, when transplanted in streptozotocin induced diabetic animal model. The animals received 200 islet -like clusters exhibited of similarly, Tosh et al. (2002) demonstrated the transdifferentiation of pancreatic cells to hepatocytes in cultures induced with dexamethasone. Further, these cells demonstrated all liver specific enzymes. Isolation of pancreatic progenitors from fetal source is of great clinical potential, because of higher proliferation rate and less imunogenecity.
In present invention, isolated hepatic progenitors from fetal liver and were transdifferentiated to pancreatic cells. Tansdifferentiated cells produced insulin, an important cellular function pancreatic cell type. The efficacy of these TD cells have shown to normalize the glucose levels of streptozotocin induced diabetic animal model. The methods of the invention have been developed with embryonic livers in which there are significant numbers of pluripotent liver cells (liver stem cells) and committed

progenitors (cells with a single fate to become either hepatocytes or bile duct cells). The
present of separation isolated hepatic progenitors is on the basis of size and density. This
is achieved by using centrifugation elutration system. The fraction with smallest size is
separated from other subpopulation of cells. This method gives homogenous population
of hepatic progenitors in a fraction. This method does not use antibody bound mediated
separation.
Objects of the present invention
The main object of the present invention is to develop a method of isolating mammalian
including human hepatic progenitors, also called liver stem cells.
Another object of the present invention is to develop a method to proliferate liver stem
cells in vitro culture.
Yet another object of the present invention is to develop a method of differentiating liver
stem cells in vitro culture condition.
Still another object of the present invention is to develop a method of transdifferentiating
liver stem cells into pancreatic progenitor cells.
Still another object of this invention to develop a method of treating insulin dependent
diabetes.
Still another object of this invention to develop a method of treating acute liver failure,
including fulminant hepatic failure.
Summary of the present invention
The present invention relates to a method of isolating liver stem cells from the fetuses of
gestation period ranging between 14-20 weeks, said method comprising steps suspending
the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 10%
Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last
fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method
of trans-differentiating liver stem cells into pancreatic cell under specific culture
conditions; in addition, a method of treating diabetes Type I in a subject, said method
comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells
intrahepatically in the subject, and obtaining the subject with increased levels of insulin,
and normalized glucose levels; further, a method of liver transplantation in a subject
using liver stem cells, said method comprising steps of injecting about 10 million

mammalian including Human tetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture. Detailed description of the present invention
The present invention relates to a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 10% Fetal calf serum, centrifuging the suspended cells, obtaining liver stem cells as a last fraction in the pump flow rate ranging between 35-40 ml/minute; also, an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions; in addition, a method of treating diabetes Type I in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized glucose levels; further, a method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian including human fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture.
In an embodiment of the present invention, wherein a method of isolating liver stem cells from the fetuses of gestation period ranging between 14-20 weeks, said method comprising steps of:
• obtaining pure hepatocytes from aborted fetuses of gestation period of about 14-20 weeks,
• suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 8-12 % Fetal calf serum,
• centrifuging the suspended cells at about 2200-2700 rpm preferably 2500 rpm at about 8-12°C, preferably at about 10°C,
• obtaining 5 fractions at increasing pump flow rate of about 16 to 40 ml/minute, with step wise increase of
a. about 16-20 ml/minutes, preferably 18 ml/minute;
b. about 24-26 ml/minute, preferably 25 ml/minute;
c. about 28-30 ml/minute, preferably 29 ml/minute;
d. about 32-34 ml/minute, preferably 33 ml/minute;

e. about 35-40 ml/minute; preferably 37 ml/minute;
• obtaining 6th blow-out fraction (residue)with rotor stopped,
• centrifuging each fraction in cold at about 475-525 X g preferably 500 X g to obtain pellet,
• re-suspending the pellets in the DMEM, and
• obtaining liver stem cells in fraction no. 5.
In another embodiment of the present invention, wherein fetuses is from animals including humans.
In yet another embodiment of the present invention, wherein the centrifugation is by using counterflow centrifugal elutriation system.
In still another embodiment of the present invention, wherein an in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions, said method comprising steps of:
culturing liver stem cells in a culture comprising Dulbecco's Minimum Essential Media/HAM'SF-12, fungizone of concentration ranging between 20-30 |ag/ml, penicillin of concentration ranging between 55-65 units/.ml, streptomycin of concentration ranging between 550-650 jxg/ml, and Fetal calf serum of concentration ranging between 7-13 %.
• maintaining the culture at about 37°C in water saturated atmosphere of about 3-7 % preferably about 5% C02 and about 93-97% preferably about 95% air in a C02 incubator, and
• obtaining transdifferentiated pancreatic cells.
In still another embodiment of the present invention, wherein the fungizone is of
concentration of about 23-27 |ig/ml preferably about 25 (ig/ml.
In still another embodiment of the present invention, wherein the penicillin is of
concentration of about 57-63 units/ml preferably about 60 units/.ml.
In still another embodiment of the present invention, wherein the streptomycin is of
concentration of about between 575-625 |ig/ml preferably 600 ng/ml.
In still another embodiment of the present invention, wherein the fetal calf serum is of
concentration of about 8-12% preferably about 10%.
In still another embodiment of the present invention, wherein a method of treating Type I
diabetes in a subject, said method comprising steps of transplanting transdifferentiated

(TD) pancreatic progenitors cells intrahepatically in the subject, and obtaining the subject
with increased levels of insulin, and normalized glucose levels.
In still another embodiment of the present invention, wherein the glucose levels comes to
normal in about 7 days.
In still another embodiment of the present invention, wherein a method of liver
transplantation in a subject using liver stem cells, said method comprising steps of
injecting about 10 million mammalian including human fetal hepatic stem cells
intrahepatically into the subject, and obtaining transplanted liver of normal architecture.
In still another embodiment of the present invention, wherein injecting the hepatic stem
cells twice or more.
In still another embodiment of the present invention, wherein the transplanted liver
attains normal architecture is about 25 days.
In still another embodiment of the present invention, wherein the said method helps
repopulation of the liver cells.
In still another embodiment of the present invention, wherein the said method is used for
treating Acute liver failures including fulminant hepatic failure.
In still another embodiment of the present invention, wherein the success rate in acute
liver failure is about 65-70%.
Brief description of the accompanying drawings
Fig 1 shows Scanning Electron Microscopy (SEM) figure of the single isolated
Human Hepatic Progenitor Cell. The size of the isolated hepatic progenitor is around 3
micron.
Fig 2 shows SEM figure of cluster of isolated human hepatic progenitors
Fig 3 shows SEM figure of the differentiated proliferative biliary cell. The biliary is a
important cell of hepatic lineage. These are characterized by specific perturbations. These
proliferating cells arrange themselves either in a well defined tubular architecture or
alternatively in an irregular, disorganized pattern sprouting into the parenchyma.
Fig 4 shows SEM figure of the differentiated hepatocyte, round shape with smooth
surface.

Fig 5 shows SEM figure showing cluster of cells, Majority 'of the cells differentiated to
hepatocyte (approximately 70%) with lesser number forming to biliary cell
(approximately 30%).
Fig 6 shows SEM figure also showing single differentiated pancreatic islet cell,
characterized by the production of Insulin in the culture supernatant.
Fig 7 shows Trans-differentiation of hepatic progenitors into pancreatic cells.
Fig 8 shows Immuno cytochemical staining for insulin trans differentiated cells.
Fig 9 shows decrease in the blood glucose level following TD cell transplantation
In still another embodiment of the present invention, wherein this invention relates to
methods for isolating hepatic progenitors (liver stem cells). The hepatic progenitors are
committed progenitors for either hepatocytes or bile duct cells. The isolated hepatic
progenitors of the invention may be used to treat liver of liver failure. In addition, the
isolated hepatic progenitors of the invention may be used for research, therapeutic and
commercial purposes which require the use of populations of functional liver cells.
Unlike mature liver cells, the hepatic progenitors of the invention generate daughter cells
that can mature through the liver lineage and offer the entire range of liver functions,
many of which are lineage-position specific. Further, the hepatic progenitors of the
invention have a greater capacity for proliferation and long-term viability than do mature
liver cells. As a result, the hepatic progenitors of the invention are better for research,
therapeutic and commercial uses than mature liver cells.
In still another embodiment of the present invention, wherein this invention relates to
method of isolating hepatic progenitors from developing fetal liver and
transdifferentaiting (TD) to pancreatic cells invitro culture. The TD cells produce
pancreatic specific harmone (insulin). This invention provides a method for the treatment
of diabetes type-I by transplantating the cells in intrahepatically.
Isolation of hepatocytes by in situ liver perfusion
In the present method aborted fetuses with the gestation period of 13-24 weeks were
taken. The time interval between fetal death (invitro or in vivo) and the commencement
of liver perfusion is less than two hours. The fetuses are weighed and its crown crump
length is measured in the lateral position.

The chest and the abdomen of the fetuses are prepared with antiseptics and the procedure is carried out under strict aseptic conditions. The fetus is draped in supine position exposing the chest and the upper abdomen. A thin polythene catheter of 22-24 guage is introduced into umbilical cord vein and passed down for about 3-5 cms. The cord is then ligated with the catheter in situ. 2 ml of normal saline (containing one drop of 1 in 5000 heparin) is gassed continuously with 100% oxygen and warmed to 37 C. Initially 10-30ml is perfused until the skin of the epigastrum is stetched.
The chest is opened transversely from the 9th costochondrial joint of one side to the other. Longitudinal incisions are taken on both sides along the costochondral junction to reach the level of manubrium sterni. This flap containing the sternum is now reflected towards the head. The pericardium is cut open with scissors and extended to both sides to expose the heart completely which is turned towards the left so the inferior vena cava can be seen entering the right atricum. A second plastic catheter is passed into the vena cava downwards to reach the tip of the hepatic vein and a ligature applied to the inferior vena cava with the catheter in situ. The inferior vena cava proximal to catheter is divided. The perfusate from the liver now flows out through this outlet catheter.
The liver is irrigated continuously with the buffer until the outflow fluid becomes pale to clear. Simultaneously, the abdomen is opened using an upper paramedian incision with care taken not to injure the liver capsule beneath. The abdominal inferior vena cava is clamped. The incision is extended along the subcostal margin towards the flanks on both the sides which gives full exposure of the liver. The initial rate of perfusion is increased from 20-30 ml./min. to 30-50 ml./min. The outflow fluid becomes clear on perfusion with 300-500 ml.
The perfusion is stopped and the liver is gently pressed to express as much irrigating fluid as possible. The inferior vena cava catheter is clamped with artery forceps and collagenase solution (0.050%) in Hank's buffer is perfused until the liver is swollen. The cord catheter is clamped and the enzyme solution kept in the liver for 10 minutes then the umbilical vein at its entrance into the liver is clamped and cut. The thoracic inferior vena cava is clamped near the diaphragm and cut and the liver is separated from all its attachments and removed.

The liver is placed in a sterlized Petri-dish in a laminar flow chamber. The liver capsule is removed and tissue is freed of connective and vascular tissue using a scalpel blace. More collaeenase is added to the freed tissue and incubated at 37°C with constant stirring for 20 minutes so that the cell dispersion is complete. It is then filtered serially through nylon meshes of 250, 100 and 40 micron size to remove the vascular and connective tissue.
The cells in suspension are washed with chilled Hank's washing medium containing calcium and magnesium salts" and kept for gravity sedimentation taking the following precautions (a) the pH of the washing solution is kept at 7.4 (b) the Hank's medium is always previously gassed with pure 100% oxygen before being added to the cell suspension. The supernatant containing non-hepatocytic cells is pipetted out. The viable hepatocytes form a light brown colour sediment at the bottom of the flask. The cell suspension is repeatedly washed (3-6 times) with the Hank's medium till a pure hepatocyte suspension is obtained. Medium 199 is then added to the cell suspension which is stored at 4°C.
Isolation of hepatic progenitors (liver stem cells)
The liver cell suspension obtained by collagenase digestion were separated according size and density using centrifugal elutration system. The equipment used was Beckman JE-6 elutration rotor with standard Beckman separator chamber(Beckman Instruments, Palo Alto California). The isolated cells were suspended in Dulbecco's Minimum Essential Medium with 10% Fetal Calf Serum was used as elutration medium and the sample was run at 2500rpm at IOC. Approximately 9ml of the cell suspension were injected into the mixing chamber five fraction of 100ml each were collected at increasing pump flow rate 24 ml /minute. Sixth blow out fraction was collected with the rotor stopped. The eutriated cellls from each fraction were centrifuged at 500xg 10 in cold high speed centrifuge and the pellets were resuspended in Dulbecco's Minmum Essential Medium. The ceils were fixed in 2.5% gluteraldehyde and processed for Scanning Electron Microscopy as shown in Figure 1. Please find below details of the composition of the buffers used in the experimentations.
HANKS BUFFER
Sodium Chloride

Potassium Chloride
Sodium Phosphate Dibasic (anhydrous)
Potassium Phosphate Dibasic (anhydrous), and optionally D-GIucose, and Calcium
Chloride2H20
Dulbecco, s Minimum Essential Medium
Components
Calcium Chloride anhydrous
Cupric Sulphate .5H20
Feme Nitrare .9H20
Ferrous Sulphate.7 H20
Magnesium Chloride
Magnesium Sulphate (anhydrous)
Potassium Chloride
Sodium Chloride
Sodium Phosphate Dibasic (anhydrous)
Sodium Phosphate Monobasic (anhydrous)
L- Alanine
L- Arsinine.HCL
L- Asparagine
L-Aspartic Acid
L-Cystine.HCL.H20
L-Cystine.2HCL
L-Glutamic Acid
L-GIutamine
Glycine
L-Histidine . HCL.H20
L-Isoleucine
L- Leucine
L- Lysine .HCL
L-methionine
L- Phenylalanine

L- proline L- Serine L-Threonine L- Tryptophan
L-Tyrosine.2Na2H20
L- Valine
D-Biotin
Choline Chloride
Foilic Acid
Myo- inositol
Niacinamide
D-Pantothenic Acid (hemicalcium)
Pyridoxal .HCL
Pyridoxin .HCL
Riboflavin
Thiamine.HCL
Vitamin B-12
D-Glucose
HEPES
Hypoxanthin
Lionic Acid
Phneol Red.Na
Putrescine.HCL
Pyruvicacid. Na
Thiocitic Ac
Characterization isolated mammalian including human hepatic progenitors (liver
stem cells)
Characterizing stem cells or their progeny in human liver has of course been more
difficult. A number of surface determinants are shared between haematpoietic derived
progenitors and hepatic progenitors (liver stem cells), including c-kit, CD34, and Thy-1
in rodents and c-kit and CD34 in humans.

In the present invention the isolated progenitors were found positive for CD34. Scanning Electron Microscopy (SEM) of the isolated hepatic progenitors .
The elutriated sample were fixed 2.5% gluteraldehyde and viewed under the Scanning Electron Microscope, the cells were viewed as small oval shaped cells. The size was found to be less than 3 micron as shown in figure No. 1.
In the present invention the isolated progenitors CD34 was used as one of the marker to identify hepatic progenitors. The cells were analysed in Flow Cytometer (FACS) using commercially available CD34 (Becton Dickinson USA).
In the present invention the cells were put in invitro culture and the AFP production was assessed by ELISA. Further, AFP secretion decreased from 5th day and disappeared by 10th day. Albumin production was observed from 3rd day of culture and continued. Morphological studies were carried by SEM after isolation and subsequent to a culture. The homogenous population of the 5 fraction of centrifugal elutriated sample was viewed under the Scanning Electron Microscope, the cells were viewed as small oval shaped within size range of 3-5 jaM as shown in figure Nos 1 and 2. Characterising stem cells or their progeny in mammalian including human liver has of course been more difficult. A number of surface determinants are shared between haematpoietic derived progenitors and hepatic progenitors (liver stem cells), including c-kit, CD34, and Thy-1 in rodents and c-kit and CD34 in humans. Alfafeto production (AFP): A liver specific marker
AFP is a liver specific marker and is also marker of proliferation. AFP also has been postulated as a transient marker of liver stem cells. In the present invention the cells were put in invitro culture and the AFP production was assessed by Enzyme Liked Immuno Sorbent Assay (ELISA) The isolated hepatic progenitors continued to produce high level of AFP in vitro culture condition (Figure 1 ) GammaGlutamy Transpeptidase: A proliferative marker
Gammaglutamyl transpeptidase is a marker of proliferation. In the present invention GGT has been used as one of the marker to assess the proliferative property of the isolated human hepatic progenitors. The isolated hepatic progenitors have shown to have high

level of GGT levels in the isolated human hepatic progenitors during invitro culture
condition (Figure 2)
Differentiation of hepatic progenitors in invitro culture
Invitro culture
The hepatic progenitors (liver stem cells), may be bipotential or multipotential. They can
differentiate into hepatocyte or cholangiocyte or they may produce other type of cell like
pancreatic cell in a specified culture environment as shown in figure nos. 3 and 4.
In the present invention majority of the hepatic progenitors differentiated to hepatocyte,
with lesser number differentiating to cholangiocytes as shown in figure nos. 5 and 7.
Clinical application of isolated hepatic progenitors
Intrahepatic transplantation of Hepatic progenitors in fulminant hepatic failure
animal model
Hepatocyte transplantation is an attractive therapy for most liver diseases. Our earlier
experience showed that the Intraperitoneal transplantation of human fetal hepatocytes in
fulminant hepatic failure has reversed the disease condition of >50% of the hepatocyte
transplantated cases (Habeebullah et al. Transplantation 1994).
However, the success requires injection of large number of cells (-20 billion cells) and
matured cells have limited growth potential in vivo. Further, the introduction of
substantial numbers of large matured hepatocytes with a diameter 20-25 micron is very
complicated.
Intrahepatic transplantation of hepatic progenitors has several advantages over other
ectopic sites
1 Transplanting cells into the unique hepatic architecture allows interaction with other
hepatocytes and nonparenchymal cells;
2. Proximity to hepatocyte-specific growth and differentiation factors creates an environment particularly conducive to hepatocyte engraftment.
3. Locally released mitogens and portal-born hepatotrophic factors can increase transplanted cell numbers.
4.The liver may be an immunological privileged site.
5, Only in the liver would hepatocytes be able to secrete bile into the biliary tree.

In the present invention, differentiated cells were transplanted intrahepatically for the treatment of acute liver failure animal model.
• Massive acute necrosis of liver cells leads to FHF a condition which is
characterized by a sudden onset of progressive jaundice, decrease in size of the
liver, hepatic encephalopathy within eight weeks of acute illness.
• The mortality rate ranges from 50-90 % primarirly because of associated cerebral edema, sepsis and multiorgan failure.
• The major problem in FHF is the loss of hepatocytes rtather than a deficiency of proliferation of surviving hepatocytes Therefore salvaging the injured liver in FHF requires liver repopulation.

• Orthotopic Liver Transplantation is the alternative sources for FHF.
• Only 10 % of patients with acute liver failure receive OLT.
• Different approaches to treat acute Hover failure include cross circulation, plasma exchange, hemoperfusion, hemodialysis, hemofilteration and exvivo isolated liver perfusions without any significant improvement of survival.
• The potential successful option might be hepatocyte transplantation.
• If temporary metabolic support is provided for a short period the damaged liver
could regenerate. If sufficient amounts of viable liver cells can successfully
transplanted to the ALF patient a bridge- to - time can be created to obtain wither
sufficient regenaration of the host liver or to stabilize the patient expecting OLT
in the near future.
Acute Liver Failure animal model (rat)
Acute liver failure injury animal model (rat) was developed injecting single dose of 875
mg/Kg body wt intraperitoneal^.
Animals were divided into three groups
Group I Only saline
Group II D-Galactosamine only
Group III Cell transplantation* after 24 hours of D-Gal induction Site of transplantation: Intrahepatic, by multiple injections at three points. Dosage: 10 X 10A6 million were transplanted intrahepatically after 24 hours of D-Gal induction.

Results
1. Survival following transplantation of Hepatic progenitors in acute liver
failure animal model
• None of the animals died in group -I,
• In group-II, 63% survival was observed, which received the cells intrahepatically,
• All the animals died in Group-II, which did not receive cell therapy following liver
• Injury
• Histopathology of the liver following transplantation in D-Gal injured liver was taken which shows marked improvement in the pathology of the liver. By 25th day liver regained normal architecture as shown in figures 4, 5, and 6.
Conclusion
• Liver provides necessary conditions for survival differentiation and proliferation for the transplanted.
• >60% (around 70%) survival in Gr-III shows that transplanted cells provides necessary support to the failing liver.
Transdifferentiation of hepatic progenitors in specified culture condition as shown
in figure no. 6
Culture Condition:
The isolated hepatic progenitors were cultured in Dulbecco's Minimum Essential
Media/HAM'SF-12 (Sigma, USA) was supplemented with fungizone (25 jig/ml),
pencillin (60units/.ml), streptomycin (600 |J.g/ml) (Hi Media Laboratories, India ) with
10% Fetal calf serum. The culture condition were maintained at 37C in water saturated
atmosphere of 5%C02 and 95% air in a C02 incubator.
In cells were cultured with insulin (1 IU insulin/ml) defined media for 24 hours and later
replaced with media without insulin medium. The cells were checked for the production
of Insulin for 10 days (Table 1).

Insulin estimation in invitro culture
The insulin was estimated in medium and in the lysed cells. The cultured cells were trypsinised and washed twice with Hanks medium and the cells were lysed by using 0.2% Triton x 100 and spinned at 2000rpm for 5 min the supernatant is collected for the estimation of insulin production by ELISA as per the manfacturer's protocol. In Gr-I cells were cultured with insulin ( 1 IU insulin/ml ) defined media for 24 hours and later replaced with media without insulin medium. In Gr-II cells were cultured in DMEMwithout any growth harmone or any other supplements. The cells were checked for the production insulin. The results showed production of 49uIU/ml, whereas the control, which were not challenged with insulin have shown the production 7uIU/ml.
Cultures Groups
Gr-I : only Insulin + DMEM
Gr-II without any growth harmone or any supplements
The cultures maintained for 10 days and insulin levels were followed.

Electron Microscopy of the differentiated Scanning cells
Cultured cells were fixed in 2.5% gluteraldehyde and viewed under the Scanning Electron Microscope as shown in figure no. 7. Immuno-cytochemical staining for Insulin TD cells
The cultured cells were fixed in 3:1 acetone methanol mixture for 15 min at room temp.
The slides were washed twice with cold PBS and then incubated with monospecific
mouse anti human insulin ( Zymed ) for 2 hours. After incubation the slides were washed
with cold PBS and the nonspecific binding is blocked by incubating the slides with 1 %
BSA for 30 min. Then the slides were washed twice with cold PBS and incubated with
FITC conjugated goat anti mouse immunoglobin-G (Sigma aldrich ) for 1 hour in dark.
Again wash the slides with cold PBS then the cells are observed under Leitz flourescent
microscope as shown in figure No. 8.
Development of diabetic animal model.
All the animals received intraperitoneal injection (40 jig /gm) body wt.
Transplantation of Cultured hepatic progenitors
Approximately three million differentiated pancreatic progenitors were injected
intrahepatically by opening the abdomen under anesthesia in aseptic condition after 24
hours of Streptozotocin injection. Animals were immunosuppressed by cyclosporin
injection lmg/ kg body weight Blood glucose level was monitored daily for 10 days.
Transplantation study
Seven rats (wistar) were taken in study. Animals were divided in three groups;
Gr-I : Only normal saline
Gr-II: Streptozotocin +TD cells
Gr-III: only Streptozotocin
RESULTS
t\-• All the animals in group III died by 5 day.
• Gr-I animals maintained normal glucose level in the range of 97-102 mg/dl.
• In group-II, glucose levels started falling following transplantation of TD cells intrahepatically as shown in figure no. 9. The blood glucose levels come to the normal within 7 to 10 days.

13. Aterman., "The Stem Cells of Liver-a Selective Review", J Cancer Research Clinical
Oncol.", vol. 118, pp. 87-115 (1992).
14. Enat et. al., "Hepatocyte Proliferation in Vitro: Its Dependence on the use of Serum-
Free Hormonally Defined Medium: and Substrata of Extracellular Matrix", "Proc.
Natl. Acad. Of Sci. U.S.A.", vol. 81, pp. 1411-1415 (1984).
15. Enjoji et. al., " Establishment of a Facultative Human Hepatic Stem Cell Line with a
Dual Differentiating Potentiality", "Gastroenterology 110", vol. 110 (4 suppl.), A1187
(1996).
16. Fulvvyler et. al. "Production of Uniform Microspheres", "Review of Scientific Instruments', vol.44 (No.2), pp. 204-206 (1973).
17. Lineages", "American J. of Pathology", vol. 144 (No.5), pp. 849-854 (1994).
18. Johe et. al., "Single Factors Direct the Differentiation of Stem Cells from the Fetal and Adult Central Nervous System", "Genes & Development", vol. 10 pp.3129-3140 (1996).
19. Reid. "Defining Hormone and Matrix Requirements for Differentiated Epithelia",
"Methods in Molecular Biology", The Humana Press, Inc., vol.5 pp. 237-276 (1990).

20. Rutenberg et. al., "Histochemical and Ultrastructural Demonstration of Glutamyl
Transpeptidase Activity", "Journal of Histochemical and Cytochemistry", vol. 17, pp.
517-526(1969). 21.Sigal et. al., "Characterization and Enrichment of Fetal Rat Hepatoblasts by
Imunoabsorption ("Panning") and Fluorescene-activated Cell Sorting", Hepatology",
vol.19 (No.4), pp. 39-47 (1995).
22. Gupta et. al., "Permananet Engraftment and Function of Hepatocytes Delivered to the Liver, Implications for Gene Therapy and Liver Repopulation", "Hepatology", vol. 14 (No.l), pp. 144-149(1991).
23. Koch, et. al., "Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes", "Journal of Cell Sciences", vol. 99, pp. 121-130 (1991).
24. Moran et. al., "Hepatoid Yolk Sac Tumors of the Mediastinum: LAMBDA. Clinocopathologic and Immunohistochemical Study of Four Cases", The American Journal of Surgical Pathology, vol.21 (No.10), pp.1210-1214, 1997.
25. Yamamasu, et. al., "Role of Glutathione Metabolism and Apoptosis in the Regression if Live Hemopoiesis", "Free Radical Biology & Medicine", vol. 23 (No.l), pp.100-109 (1997).
26. Ruck et. al., "Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6", Histopathology", vol.31, pp.324-329 (1997).
27. Smith et. al., "Hepatic stem cells in the human liver", "Correspondence", vol.29, pp. 589-594(1996).
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34. Lemmer et al., "Isolation and Culture of Hepatic Stem cells from Human Foetal Liver: A Preliminary Report", Hepatology, 1-56 Iasl Abstracts 23(1): 157P, 1996.
35. Travis, "The Search for Liver Stem Cells Picks Up", Science 259:1829, 1993.
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37. Alison et al., "Wound healing in the liver with particular reference to stem cells", Phil. Trans. R. Soc. Lond. B, 353:877-894, 1998.
38. Le mire et. al., "Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine", Am. J. of Path., 139:535-552, 1991.
39. Tian et al., "The oval shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and pyruvate kinase isoenzyme expression", Histochem. Cell. Biol., 107: 243-250, 1997.
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42. Enat et. al., "Hepatocyte Proliferation in Vitro: Its Dependence on the use of Serum-Free Hormonally Defined Medium: and Substrata of Extracellular Matrix", "Proc. Natl. Acad. Of Sci. U.S.A.", vol. 81, pp. 1411-1415 (1984).
43. Enjoji et. al., " Establishment of a Facultative Human Hepatic Stem Cell Line with a Dual Differentiating Potentiality", "Gastroenterology 110", vol. 110 (4 suppl.), Al 187 (1996).
44. Fulvvyler et. al. "Production of Uniform Microspheres", "Review of Scientific Instruments', vol.44 (No.2), pp. 204-206 (1973).
45. Lineages", "American J. of Pathology", vol. 144 (No.5), pp. 849-854 (1994).

46. Johe et. al., "Single Factors Direct the Differentiation of Stem Cells from the Fetal and Adult Central Nervous System", "Genes & Development", vol. 10 pp.3129-3140 (1996).
47. Reid, "Defining Hormone and Matrix Requirements for Differentiated Epithelia", "Methods in Molecular Biology", The Humana Press, Inc., vol.5 pp. 237-276 (1990).
48. Rutenberg et. al., "Histochemical and Ultrastructural Demonstration of Glutamyl Transpeptidase Activity", "Journal of Histochemical and Cytochemistry", vol. 17, pp. 517-526(1969).
49. Sigal et. al., "Characterization and Enrichment of Fetal Rat Hepatoblasts by Imunoabsorption ("Panning") and Fluorescene-activated Cell Sorting", Hepatology", vol.19 (No.4), pp. 39-47 (1995).
50. Gupta et. al., "Permananet Engraftment and Function of Hepatocytes Delivered to the Liver, Implications for Gene Therapy and Liver Repopulation", "Hepatology", vol. 14 (No.l), pp. 144-149(1991).
51. Koch, et. al, "Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes", "Journal of Cell Sciences", vol. 99, pp. 121-130 (1991).
52. Moran et. al., "Hepatoid Yolk Sac Tumors of the Mediastinum: LAMBDA. Clinocopathologic and Immunohistochemical Study of Four Cases", The American Journal of Surgical Pathology, vol.21 (No.10), pp.1210-1214, 1997.
53. Yamamasu, et. al., "Role of Glutathione Metabolism and Apoptosis in the Regression if Live Hemopoiesis", "Free Radical Biology & Medicine", vol. 23 (No.l), pp. 100-109(1997).
54. Ruck et. al., "Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6", Histopathology", vol.31, pp.324-329 (1997).
55. Smith et. al., "Hepatic stem cells in the human liver", "Correspondence", vol.29, pp. 589-594(1996).
56. Fisher, M., Neuronal Influence on Glial Enzyme Expression: Evidence from Mutant Mouse Cerebella," PNAS, vol.81, 4414-4418 (1984).
57. Langer, R., et. al., "Tissue Engineering, "Science, vol.260, 920-926 (1993).

58. Sarraf, C, et. al., "cell Behavior in the Acetylaminofluorene-Treated Regenerating Rat Liver", American Journal of Pathology, vol. 145, No.5, 1114-11126(1994).
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60. Dabeva M, et. al., "Models for Hepatic Progenitor Cell Activation, "Proceedings of the Society for Experimental Biology and Medicine, vol. 204, No.3, 242-252 (1993).
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62. Lemmer et al., "Isolation and Culture of Hepatic Stem cells from Human Foetal Liver: A Preliminary Report", Hepatology, 1-56 Iasl Abstracts 23(1): 157P, 1996.
63. Travis, "The Search for Liver Stem Cells Picks Up", Science 259:1829, 1993.
64. M. Alison, "Liver stem cells: a two compartment system", Current Opinion in Cell Biol., 10:710-715, 1998.
65. Alison et al., "Wound healing in the liver with particular reference to stem cells", Phil. Trans. R. Soc. Lond. B, 353:877-894, 1998.
66. Lemire et. al, "Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine", Am. J. of Path., 139:535-552, 1991.
67. Tian et al., "The oval shaped cell as a candidate for a liver stem cell in embryonic, neonatal and precancerous liver: identification based on morphology and pyruvate kinase isoenzyme expression", Histochem. Cell. Biol., 107: 243-250, 1997.
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Claims
1. A method of isolating liver stem cells from the fetuses of gestation period ranging
between 14-20 weeks, said method comprising steps of:
(a) obtaining pure hepatocytes from aborted fetuses,
(b) suspending the hepatocytes in Dulbecco's Minimum Essential Medium (DMEM) with about 8-12% preferably 10% Fetal calf serum,
(c) centrifuging the suspended cells at rate ranging between 2200-2500 rpm at about 8-12 °C,
(d) obtaining 5 fractions at increasing pump flow rate of about 16 to 40 ml/minute with increasing rates of 16-20; 24-26; 28-30; 32-34; and 35-40 ml/minute respectively,
(e) obtaining 6th blow-out fraction with rotor stopped,
(f) centrifuging each fraction in cold at about 475-525 X g to obtain pellet,
(g) re-suspending the pellets in the DMEM, and
(h) obtaining liver stem cells in fraction no. 5.
2. A method as claimed in claim 1, wherein fetuses is from animals including humans.
3. A method as claimed in claim 1, wherein the centrifugation is by counterflow centrifugal elutriation system.
4. An in vitro method of trans-differentiating liver stem cells into pancreatic cell under specific culture conditions, said method comprising steps of:
a. culturing liver stem cells in a culture comprising Dulbecco's Minimum
Essential Media/HAM'SF-12, fungizone of concentration ranging between
20-30 ng/ml, penicillin of concentration ranging between 55-65 units/ml,
streptomycin of concentration ranging between 550-650 j-ig/ml, and Fetal
calf serum of concentration ranging between 7-13 %.
b. maintaining the culture at about 37°C in water saturated atmosphere of
about 3-7 % C02 preferably 5% and about 93-97 % preferably 95 air in a
CO2 incubator, and
c. obtaining transdifferentiated pancreatic cells.
5. The method as claimed in claim 4, wherein the fungizone is of concentration of about
23-27 |.ig/ml preferably 25 (ig/ml.

6. The method as claimed in claim 4, wherein the penicillin is of concentration of about 57-63 units/ml preferably about 60 units/.ml.
7. The method as claimed in claim 4, wherein the streptomycin is of concentration of about 575-625 |ig/ml preferably about 600 jxg/ml.
8. The method as claimed in claim 4, wherein the fetal calf serum is of concentration of about 8-12% preferably 10%.
9. A method of treating Type I diabetes in a subject, said method comprising steps of transplanting transdifferentiated (TD) pancreatic progenitors cells as claimed in claim 4 intrahepatically in the subject, and obtaining the subject with increased levels of insulin, and normalized blood glucose levels.
10. A method as claimed in claim 9, wherein the blood glucose levels comes to normal in about 7 days.
11. A method of liver transplantation in a subject using liver stem cells, said method comprising steps of injecting about 10 million mammalian fetal hepatic stem cells intrahepatically into the subject, and obtaining transplanted liver of normal architecture.
12. A method as claimed in claim 11, wherein injecting the hepatic stem cells twice or more.
13. A method as claimed in claim 11, wherein the transplanted liver attains normal architecture is about 25 days.
14. A method as claimed in claim 11, wherein the said method helps repopulation of the liver cells.
15. A method as claimed in claim 11, wherein the said method is used for treating Acute liver failures including fulminant hepatic failure.
16. A method as claimed in claim 11, wherein the success rate in management of acute liver failure is about 65-70%.

17. A method of isolating liver stem cells from the fetuses of gestation period ranging

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Patent Number

207460

Indian Patent Application Number

395/MAS/2002

PG Journal Number

44/2007

Publication Date

02-Nov-2007

Grant Date

13-Jun-2007

Date of Filing

23-May-2002

Name of Patentee

CENTRE FOR LIVER RESEARCH AND DIAGNOSTICS

Applicant Address

DECCAN COLLEGE OF MEDICAL SCIENCES, KANCHAN BAGH, HYDERABAD 500 058.

Inventors:

#	Inventor's Name	Inventor's Address
1	ALEEM AHMED KHAN	CENTRE FOR LIVER RESEARCH AND DIAGNOSTICS DECCAN COLLEGE OF MEDICAL SCIENCES, KANCHAN BAGH, HYDERABAD 500 058.
2	PARVEEN(ETC)	CENTRE FOR LIVER RESEARCH AND DIAGNOSTICS DECCAN COLLEGE OF MEDICAL SCIENCES, KANCHAN BAGH, HYDERABAD 500 058.

PCT International Classification Number

A61K35/407

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA