Human pancreatic beta cell lines for diagnostic of diabetes

Abstract

The present invention relates to a method for preparing commercial scale quantities of human functional Betacells and to the establishment of cell lines. It also relates to a method of diagnosis using Beta cell tumors or cells derived thereof. The method comprises sub-transplantation procedure to enrich the graft in proliferating Betacells, allowing to generate human Betacell lines. Such lines express little amount of insulin and have a gene expression profile that resembles to adult Betacells. In addition, the human Betacell lines are able to normalize glycemia of diabetic mice when transplanted, demonstrating their insulin secretion capabilities.

1 .- 30 . (canceled) 
     
     
         31 . An isolated human transduced beta cell capable of producing insulin and PDX-1, wherein the transduced human beta cell comprises one or more lentiviral vector comprising an insulin promoter controlling the expression of at least one immortalization gene, wherein the at least one immortalization gene is inserted within two Lox P or FRT sites allowing reversible or conditional immortalization, and wherein the transduced human beta cell issued from immature pancreatic cells. 
     
     
         32 . The isolated human transduced beta cell of  claim 31 , wherein the immortalization gene is SV40 Large T antigen and/or hTERT. 
     
     
         33 . The isolated human transduced beta cell of  claim 31 , wherein the immature pancreatic cells are fetal pancreas cells or stem cells that have done a first differentiation in endodermic cells. 
     
     
         34 . The isolated human transduced beta cell of  claim 31 , wherein the transduced human beta cell forms insulinomas and restores normoglycemia in diabetic SCID mice after transplantation. 
     
     
         35 . The isolated human transduced beta cell of  claim 31 , wherein the transduced human beta cell further expresses at least one gene encoding a protein selected from the group consisting of transcription factor MafA, proconvertase Pcsk1, glucose transporter Glut2, potassium channel subunit Kcnj1 or Abcc8, zinc transporter Znt8. 
     
     
         36 . The isolated human transduced beta cell of  claim 31 , wherein the Lox P sites are targeted by Cre recombinase or the FRT sites are targeted by FLP recombinase. 
     
     
         37 . The isolated human transduced beta cell of  claim 31 , wherein SV40 Large T antigen gene and hTert gene are expressed by the same lentiviral vector or by two different lentiviral vectors. 
     
     
         38 . The isolated human transduced beta cell of  claim 31 , wherein the insulin promoter of the one or more lentiviral vector controlling the expression of SV40 Large T antigen gene and/or hTert gene further controls the expression of an antibiotic resistance gene. 
     
     
         39 . The isolated human transduced beta cell of  claim 38 , wherein the antibiotic resistance gene is selected from the group consisting of hygromycin resistance gene, neomycin resistance genes, tetracyclin resistance gene, ampicillin resistance gene, kanamycin resistance gene, phleomycin resistance gene, bleomycin resistance gene, geneticin resistance gene, carbenicillin resistance gene, chloramphenicol resistance gene, puromycin resistance gene and blasticidin-S-deaminase gene. 
     
     
         40 . The isolated human transduced cell of  claim 38 , wherein the SV40 LargeT antigen, hTert and antibiotic resistance genes are expressed. 
     
     
         41 . The isolated human transduced beta cell of  claim 31 , wherein the one or more lentiviral vector comprising an insulin promoter controlling the expression of SV40 Large T antigen gene and/or hTert gene further comprises one or more negative selection marker gene. 
     
     
         42 . The isolated human transduced beta cell of  claim 31 , further comprising a vector or a plasmid comprising a DNA sequence coding for a Cre recombinase or a FLP recombinase. 
     
     
         43 . The isolated human transduced beta cell of  claim 31 , wherein the cell is cultured and amplified on matrigel and fibronectin coated plates in a culture medium containing glucose, BSA fraction V fatty acid free, nicotinamide, 2-betamercapoethanol, antibiotics, human transferrin and sodium selenite so as to obtain a human transduced beta cell line, a human transduced beta cell population or a human transduced beta cell bank. 
     
     
         44 . The isolated human transduced beta cell of  claim 42 , such that expression of Cre recombinase or FLP recombinase leads to the removal of the SV40 Large T antigen and/or hTert genes, so as to obtain a de-immortalized human transduced beta cell comprising:
 (i) one or more lentiviral vector comprising an insulin promoter and one Lox P or FRT site and,   (ii) a vector or a plasmid comprising a DNA sequence coding for a Cre recombinase or a FLP recombinase.   
     
     
         45 . A pharmaceutical composition comprising an effective amount of the isolated human transduced beta cell of  claim 31  and at least one pharmaceutically acceptable excipient. 
     
     
         46 . The pharmaceutical of  claim 45 , wherein the transduced beta cell is encapsulated. 
     
     
         47 . A method for deleting SV40 Large T antigen and/or hTert genes in isolated human transduced beta cell, comprising a step of cultivating the isolated human transduced beta cell according to  claim 31  under conditions such that following the expression of Cre recombinase the SV40 Large T antigen and hTert genes are removed.