Immortal Unipotent Porcine PICM-19H and PICM-19B Stem Cell Lines

Abstract

Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent ARS-PICM-19 pig liver stem cell line. The unipotent porcine stem cell line PICM-19H differentiates exclusively into hepatocytes and can be induced to express CYP450 enzymes. The growth rate and cell density in culture, morphological features, and hepatocyte detoxification functions, i.e., inducible CYP450 activity, ammonia clearance, and urea production of the PICM-19H cells were evaluated for their application in artificial liver devices. PICM-19H cells contain numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles and display inducible CYP450 activity, clear ammonia, and produce urea in a glutamine-free medium. The data indicate that both cell lines, either together or alone, may be useful as the cellular substrate for an artificial liver device. The results demonstrate the potential for the use of PICM-19H cells in drug biotransformation and toxicity testing.

1 - 3 . (canceled) 
     
     
         4 . An isolated, immortal, unipotent porcine stem cell line wherein cells of said cell line are capable of differentiating exclusively into functional bile duct cells expressing cholangiocyte functions. 
     
     
         5 . The stem cell line of  claim 4  wherein said cells exhibit all the identifying characteristics of PICM-19B cells and said characteristics are that the cells are basolaterally polarized cells exhibiting basal membrane fluid transport, high GGT activity and a lack of serum protein production. 
     
     
         6 . The stem cell line of  claim 26  wherein the stem cell line is the immortal, unipotent PICM-19B stem cell line deposited as ATCC PTA-9173. 
     
     
         7 . The cell line according to  claim 4  wherein a culture of said cells further comprises feeder cells. 
     
     
         8 . A composition comprising cells of the cell line according to  claim 4 , said cells being attached to microbeads. 
     
     
         9 . A composition comprising cells of the cell line according to  claim 4 , said cells being encapsulated in alginate microcapsules. 
     
     
         10 . A composition comprising cells of the cell line according to or  claim 4 , said cells being attached to and between single hollow fibers. 
     
     
         11 . (canceled) 
     
     
         12 . A bioartificial liver device comprising cells of an isolated, immortal, unipotent porcine stem cell line wherein cells of said cell line are capable of differentiating exclusively into functional bile duct cells expressing cholangiocyte functions, and a support for said cells. 
     
     
         13 . A bioartificial liver device comprising cells of an isolated, immortal, unipotent porcine stem cell line wherein cells of said cell line are capable of differentiating exclusively into hepatocytes, cells of an isolated, immortal, unipotent porcine stem cell line wherein cells of said cell line are capable of differentiating exclusively into functional bile duct cells expressing cholangiocyte functions, and a support for said cells. 
     
     
         14 . The bioartificial liver device of any one of  claims 12  and  13 , wherein said support comprises a collection of single hollow fibers, microbeads, or alginate microcapsules. 
     
     
         15 . (canceled) 
     
     
         16 . The method of using the cell lines of any one of  claims 4 - 6  as a bioartificial liver device, wherein said cells are capable of functioning as functional bile duct cells expressing cholangiocyte functions, for a patient in need of functioning cholangiocytes. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The method of using a composition comprising cells of the cell lines of any one of  claims 4 - 6 , said cells being attached to microbeads, attached to inorganic or organic porous supports, attached to single hollow-fibers or encapsulated in hydrogel-based supports to treat body fluids, such as whole blood, blood plasma, and peritoneal fluids as a bioartificial liver device, wherein said cells are capable of functioning as functional be duct cells expressing cholangiocyte functions, for a patient in need of functioning cholangiocytes. 
     
     
         20 - 24 . (canceled) 
     
     
         25 . A screening assay kit comprising PICM-19H cells, PICM-19B cells or both. 
     
     
         26 . An isolated, immortal, unipotent porcine stem cell line wherein cells of said cell line are capable of differentiating exclusively into functional bile duct cells expressing cholangiocyte functions exhibiting all the identifying characteristics of PICM-19 Bile duct (PICM-19B) cells and said characteristics are that the cells are basolaterally polarized cells exhibiting basal membrane fluid transport, high GGT cholangiocyte-like activity observed as a recognized functional marker of bile duct (cholangiocyte) cells, and a lack of serum protein production. 
     
     
         27 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174 as a bioartificial liver device, wherein said cells are capable of functioning as hepatocytes, for a patient in need of functioning hepatocytes. 
     
     
         28 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stern cell line deposited as ATCC PTA-9174 together with the cell lines of any one of  claims 4 - 6  as bioartificial liver device, wherein said cells are capable of functioning as hepatocytes and as functional bile duct cells expressing cholangiocyte functions, for a patient in need of functioning hepatocytes and cholangiocytes. 
     
     
         29 . The method of using a composition comprising cells of the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174, said cells being attached to microbeads, attached to inorganic or organic porous supports, attached to single hollow-fibers or encapsulated in hydrogel-based supports to treat body fluids, such as whole blood, blood plasma, and peritoneal fluids as a bioartificial liver device, wherein said cells are capable of functioning as hepatocytes, for a patient in need of functioning hepatocytes. 
     
     
         30 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174, together with the cell lines of any one of  claims 4 - 6 , said cells being attached to microbeads, attached to inorganic or organic porous supports, attached to single hollow-fibers or encapsulated in hydrogel-based supports to treat body fluids, such as whole blood, blood plasma, and peritoneal fluids as a bioartificial liver device, wherein said cells are capable of functioning as hepatocytes and as functional bile duct cells expressing cholangiocyte functions, for a patient in need of functioning hepatocytes and cholangiocytes. 
     
     
         31 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell line of  claim 4  to screen for compounds or new chemical entities which inhibit or promote an enzyme activity involved in the metabolism of xenobiotics in the liver. 
     
     
         32 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell line of  claim 4  to screen for compounds or new chemical entities which results in cytotoxicity due to the metabolism of xenobiotics and/or endogenous substrates in the liver. 
     
     
         33 . The method of using the immortal, unipotent P1CM-1Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell line of  claim 4  to screen for compounds or new chemical entities which results in hepatotoxicity due to metabolism of xenobiotics and/or endogenous substrates in the liver. 
     
     
         34 . The method of using the immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell line of  claim 4  to screen for compounds or new chemical entities which results in hepatic dysfunction due to metabolism of xenobiotics and/or endogenous substrates in the liver.