Processes for clonal growth of hepatic progenitor cells

Abstract

A method of propagating mammalian endodermally derived progenitors such as hepatic progenitors, their progeny, or mixtures thereof is developed which includes culturing mammalian progenitors, their progeny, or mixtures thereof on a layer of embryonic mammalian feeder cells in a culture medium. The culture medium can be supplemented with one or more hormones and other growth agents. These hormones and other growth agents can include insulin, dexamethasone, transferrin, nicotinamide, serum albumin, β-mercaptoethanol, free fatty acid, glutamine, CuSO 4 , and H 2 SeO 3 . The culture medium can also include antibiotics. Importantly, the culture medium does not include serum. The invention includes means of inducing the differentiation of the progenitors to their adult fates such as the differentiation of hepatic progenitor cells to hepatocytes or biliary cells by adding, or excluding epidermal growth factor, respectively. The method of producing mammalian progenitors is useful in that the progenitors can be used subsequently in one or more of the following processes: identification of growth and differentiation factors, toxicological studies, drug development, antimicrobial studies, or the preparation of an extracorporeal organ such as a bioartificial liver.

1 . A method of identifying hepatic growth factors comprising providing hepatic progenitors, their progeny, or mixtures thereof, and a sample suspected of comprising at least one hepatic growth factor, combining the progenitors, their progeny, or mixtures thereof, and the sample, and observing stimulation of growth of the hepatic progenitors. 
   
   
       2 . A method of identifying hepatic differentiation factors comprising providing hepatic progenitors, their progeny, or mixtures thereof, and a sample suspected of comprising at least one hepatic differentiation factor, combining the progenitors, their progeny, or mixtures thereof, and the sample, and observing differentiation of the hepatic progenitors. 
   
   
       3 . A method of identifying a hepatic toxin comprising providing hepatic progenitors, their progeny, or mixtures thereof, and a sample suspected of comprising at least one hepatic toxin, combining the progenitors, their progeny, or mixtures thereof, and the sample, and observing the death of the hepatic progenitors. 
   
   
       4 . A method of developing a drug comprising providing hepatic progenitors, their progeny, or mixtures thereof, and a drug suspected of a capacity to affect hepatic progenitor, metabolisms and combining the progenitors, their progeny, or mixtures thereof, and the sample, and observing a change in the metabolism of the hepatic progenitors. 
   
   
       5 . A method of identifying novel antimicrobials comprising providing hepatic progenitors, their progeny, or mixtures thereof, and at least one agent suspected of having an antimicrobial effect, combining the progenitors, their progeny, or mixtures thereof, and the agent, and observing changes in growth of the hepatic progenitors. 
   
   
       6 . A method of preparing an extracorporeal liver comprising providing hepatic progenitors, their progeny, or mixtures thereof and culturing the progenitors, their progeny, or mixtures thereof in a bioreactor until a sufficient population is obtained effective to serve as an extracorporeal liver. 
   
   
       7 . A method of propagating hepatic progenitors, their progeny, or mixtures thereof comprising:
 (a) providing at least one hepatic progenitor and   (b) culturing the hepatic progenitor in a culture medium comprising at least one feeder cell biosynthetic product.   
   
   
       8 . The method of  claim 7  in which the culture medium further comprises glucocorticoid, insulin, epidermal growth factor, nicotinamide, or combinations. 
   
   
       9 . The method of  claim 7  in which the feeder cell biosynthetic product comprises a peptide, a glycopeptide, a lipopeptide, a lipid, a glycolipid, a carbohydrate, a protein, a glycoprotein, a lipoprotein, or combinations thereof, in which the biosynthetic product enhances the proliferation or differentiation of the hepatic progenitor. 
   
   
       10 . The method of  claim 7  in which the feeder cell biosynthetic product comprises insulin-like growth factors, interleukin-6 family growth factors, hepatocyte growth factor, fibroblast growth factor, extracellular matrix, or combinations thereof, in which the biosynthetic product enhances the proliferation or differentiation of the hepatic progenitor. 
   
   
       11 . The method of  claim 7  in which the at least one hepatic progenitor comprises at least one cell from human, non-human primate, pig, dog, rabbit, rat, or mouse. 
   
   
       12 . The method of  claim 7  which further comprises cloning the at least one hepatic progenitor. 
   
   
       13 . The method of  claim 7  further comprising adding a growth factor. 
   
   
       14 . The method of  claim 13  further comprising adding a differentiation factor to produce hepatocytes, biliary cells, or combinations thereof.