Isolation of bona fide pancreatic progenitor cells

Abstract

The present invention relates to a method for isolating bona fide pancreatic progenitor cells and to cell populations enriched for bona fide pancreatic progenitor cells.

1 . A method for isolating a population enriched for bona fide pancreatic progenitor cell, said method comprising the steps of:
 i) providing a cell population comprising at least one bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and   ii) exposing said cell population to:
 a) a first ligand which binds to a first marker specific for PDX1− cells and selecting the cells that do not bind to said first ligand from said cell population, thereby enriching the cell population for PDX1+ cells; 
 and/or 
 b) a second ligand which binds to a second marker specific for PDX1+ cells and selecting the cells that bind to said second ligand from the cells that do not bind to said second ligand, thereby enriching the cell population for PDX1+ cells; 
 and/or 
 c) a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, thereby enriching the cell population for PDX1+ NKX6-1+ cells; 
   thereby obtaining a cell population enriched for bona fide pancreatic progenitor cells.   
     
     
         2 . The method according to  claim 1 , wherein at least one of the first, second and third ligands is an antibody or fragment thereof. 
     
     
         3 . The method according to any one of the preceding claims, wherein the antibody is a monoclonal or polyclonal antibody. 
     
     
         4 . The method according to any one of the preceding claims, wherein at least one of the first, second and third ligand binds to a cell surface marker of the bona fide pancreatic progenitor cell. 
     
     
         5 . The method according to any one of the preceding claims, wherein at least one of the first, second and third ligand is conjugated to a label. 
     
     
         6 . The method according to any one of the preceding claims, wherein the expression of at least one of the first, second and third marker is detected by flow cytometry. 
     
     
         7 . The method according to any one of the preceding claims, wherein the cells are removed or selected by flow cytometry. 
     
     
         8 . The method according to any one of the preceding claims, wherein the first ligand is an antibody or fragment thereof directed against CD49d. 
     
     
         9 . The method according to any one of the preceding claims, wherein the second ligand is an antibody or fragment thereof directed against a target selected from the group consisting of: FOLR1, CDH1/ECAD, F3/CD142, PDX1, FOXA2, EPCAM, HES1 and GATA4. 
     
     
         10 . The method according to any one of the preceding claims, wherein the second ligand is an antibody or fragment thereof directed against FOLR1. 
     
     
         11 . The method according to any one of the preceding claims, wherein the third ligand is an antibody or fragment thereof directed against a target selected from the group consisting of: GP2, SCN9A, MPZ, NAALADL2, KCNIP1, CALB1, SOX9, NKX6-2 and NKX6-1. 
     
     
         12 . The method according to any one of the preceding claims, wherein the third ligand is an antibody or fragment thereof directed against GP2. 
     
     
         13 . The method according to any one of the preceding claims, wherein the first ligand is an antibody or fragment thereof directed against CD49d and the third ligand is an antibody directed against GP2. 
     
     
         14 . The method according to any one of the preceding claims, wherein the first ligand is an antibody or fragment thereof directed against CD49d, the second ligand is an antibody or fragment thereof directed against FOLR1 and the third ligand is an antibody or fragment thereof directed against GP2. 
     
     
         15 . The method according to any one of the preceding claims, said method comprising the steps of:
 i) providing a cell population comprising at least one bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and   ii) exposing said cell population to a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, thereby enriching the cell population for PDX1+ NKX6-1+ cells, wherein the third marker is GP2;
 thereby obtaining a cell population enriched for bona fide pancreatic progenitor cells. 
   
     
     
         16 . The method according to any one of the preceding claims, said method comprising the steps of:
 i) providing a cell population comprising at least one bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and   ii) exposing said cell population to:
 a) a second ligand which binds to a second marker specific for PDX1+ cells and selecting the cells that bind to said second ligand from the cells that do not bind to said second ligand, thereby enriching the cell population for PDX1+ cells, wherein the second marker is FOLR1; 
 and 
 b) a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, thereby enriching the cell population for PDX1+ NKX6-1+ cells, wherein the third marker is GP2; 
   thereby obtaining a cell population enriched for bona fide pancreatic progenitor cells.   
     
     
         17 . The method according to any one of the preceding claims, wherein the bona fide pancreatic progenitor cells are derived from cells capable of differentiation such as human pluripotent stem cells. 
     
     
         18 . The method according to any one of the preceding claims, wherein the cells capable of differentiation are selected from the group consisting of human iPS cells (hIPSCs), human ES cells (hESCs) and naive human stem cells (NhSCs). 
     
     
         19 . The method according to any one of the preceding claims, wherein the cells capable of differentiation are derived from cells isolated from an individual. 
     
     
         20 . The method according to any one of the preceding claims, wherein at least one cell of the cell population enriched for bona fide pancreatic progenitor cells has the capability to differentiate further. 
     
     
         21 . The method according to any one of the preceding claims, wherein at least one cell of the cell population enriched for bona fide pancreatic progenitor cells has the capability to differentiate further into pancreatic hormone-producing cells. 
     
     
         22 . The method according to any one of the preceding claims, wherein at least one of the pancreatic hormone-producing cells is an insulin-producing cell and/or is responsive to glucose. 
     
     
         23 . The method according to any one of the preceding claims, wherein at least one cell of the cell population enriched for bona fide pancreatic progenitor cells can produce insulin-producing islet cells. 
     
     
         24 . The method according to any one of the preceding claims, wherein CDKN1a and/or CDKN2a is inactivated in the cell population provided in step i). 
     
     
         25 . The method according to  claim 24 , wherein CDKN1a and/or CDKN2a is inactivated by knock-down, deletion, silencing or repression. 
     
     
         26 . The method according to any one of  claims 24  to  25 , wherein the starting cell population is a pancreatic progenitor population expressing PDX1. 
     
     
         27 . The method according to any one of  claims 24  to  26 , wherein inactivation of CDKN1a and/or CDKN2a results in an increase in the proportion of cells entering replicating stage compared to the proportion of cells entering replicating stage when CDKN1a and/or CDKN2a is not inactivated. 
     
     
         28 . The method according to  claim 27 , wherein the increase is at least 1.5-fold, such as at least 2.0-fold, such as at least 2.5-fold, such as at least 3.0-fold, such as at least 3.5-fold. 
     
     
         29 . A method for producing a cell population enriched for bona fide pancreatic progenitor cells, said enriched cell population comprising at least 70% bona fide pancreatic progenitor cells, such as at least 75% bona fide pancreatic progenitor cells, such as at least 80% bona fide pancreatic progenitor cells, such as at least 85% bona fide pancreatic progenitor cells, such as at least 90% bona fide pancreatic progenitor cells. 
     
     
         30 . The method of  claim 29 , said method comprising the steps of:
 i) providing a cell population comprising a bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and   ii) exposing said cell population to:
 a) a first ligand which binds to a first marker specific for PDX1− cells and selecting the cells that do not bind to said first ligand from said cell population, thereby enriching the cell population for PDX1+ cells; 
 and/or 
 b) a second ligand which binds to a second marker specific for PDX1+ cells and selecting the cells that bind to said second ligand from the cells that do not bind to said second ligand, thereby enriching the cell population for PDX1+ cells; 
 and/or 
 c) a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, thereby enriching the cell population for PDX1+ NKX6-1+ cells. 
   
     
     
         31 . The method of any one of  claims 29  to  30 , said method comprising the steps of:
 i) providing a cell population comprising a bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and 
 ii) exposing said cell population to a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, wherein the third marker is GP2, thereby enriching the cell population for PDX1+ NKX6-1+ cells. 
 
     
     
         32 . The method of any one of  claims 29  to  31 , said method comprising the steps of:
 i) providing a cell population comprising a bona fide pancreatic progenitor cell, wherein the bona fide pancreatic progenitor cell expresses PDX1 and NKX6-1; and 
 ii) exposing said cell population to:
 a) a second ligand which binds to a second marker specific for PDX1+ cells and selecting the cells that bind to said second ligand from the cells that do not bind to said second ligand, thereby enriching the cell population for PDX1+ cells, wherein the second marker is FOLR1; 
 and 
 b) a third ligand which binds to a third marker specific for PDX1+ NKX6-1+ cells and selecting the cells that bind to said third ligand from the cells that do not bind to said third ligand, wherein the third marker is GP2, 
 
 
       thereby enriching the cell population for PDX1+ NKX6-1+ cells. 
     
     
         33 . The method of  claim 29  or  30 , wherein the first ligand is an antibody or fragment thereof directed against CD49d, the second ligand is an antibody or fragment thereof directed against FOLR1 and the third ligand is an antibody or fragment thereof directed against GP2. 
     
     
         34 . The method of any one of  claims 29  to  31 , wherein CDKN1a and/or CDKN2a is inactivated in the cell population provided in step i). 
     
     
         35 . A cell population comprising at least 50% bona fide pancreatic progenitor cells, such as at least 75% bona fide pancreatic progenitor cells, such as at least 80% bona fide pancreatic progenitor cells, such as at least 85% bona fide pancreatic progenitor cells, such as at least 90% bona fide pancreatic progenitor cells. 
     
     
         36 . A cell population comprising bona fide pancreatic progenitor cells, obtainable by the method of any one of  claims 1  to  23 . 
     
     
         37 . The cell population according to  claim 36 , wherein said cell population comprises at least 50% bona fide pancreatic progenitor cells, such as at least 75% bona fide pancreatic progenitor cells, such as at least 80% bona fide pancreatic progenitor cells, such as at least 85% bona fide pancreatic progenitor cells, such as at least 90% bona fide pancreatic progenitor cells. 
     
     
         38 . A cell population comprising bona fide pancreatic progenitor cells, obtainable by the method of any one of  claims 1  to  23  for use in a method of treatment. 
     
     
         39 . A cell population comprising bona fide pancreatic progenitor cells, obtainable by the method of any one of  claims 1  to  23  for use in the treatment of a metabolic disorder in an individual in need thereof. 
     
     
         40 . The cell population for the uses according to any one of  claim 38  or  39 , wherein said cell population comprises at least 50% bona fide pancreatic progenitor cells, such as at least 75% bona fide pancreatic progenitor cells, such as at least 80% bona fide pancreatic progenitor cells, such as at least 85% bona fide pancreatic progenitor cells, such as at least 90% bona fide pancreatic progenitor cells. 
     
     
         41 . A cell population enriched for bona fide pancreatic progenitor cells according to any one of  claims 35  to  40  for use in the treatment of a metabolic disorder in an individual in need thereof. 
     
     
         42 . The cell population for the use of  claim 41 , wherein the metabolic disorder is diabetes mellitus, such as insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, malnutrition-related diabetes mellitus, type 1 diabetes, type 2 diabetes or unspecified diabetes mellitus. 
     
     
         43 . A method of treatment of a metabolic disorder in an individual in need thereof, wherein the method comprises a step of providing a cell population according to any one of  claims 35  to  40 . 
     
     
         44 . The method according to  claim 43 , said method further comprising a step of transplanting at least part of said cell population into said individual. 
     
     
         45 . The method according to any one of  claims 43  to  44 , said method further comprising a step of inducing differentiation of at least part of cell population into insulin-producing cells and optionally a step of isolating said insulin-producing cells. 
     
     
         46 . The method according to any one of  claims 43  to  45 , wherein the step of inducing differentiation of at least part of cell population into insulin-producing β-cells comprises the step of incubating at least part of said cell population in the presence of a Yap1 inhibitor, thereby obtaining a cell population enriched for insulin-producing β-cells. 
     
     
         47 . The method according to  claim 46 , wherein the Yap1 inhibitor is verteporfin. 
     
     
         48 . The method according to any of  claims 43  to  47 , wherein the insulin-producing β-cells have increased expression of at least one of Insm-1, Isl-1, MafA and MafB compared to cells that have been incubated in the absence of the Yap1 inhibitor. 
     
     
         49 . The method according to any one of  claims 43  to  48 , wherein the insulin-producing β-cells have increased insulin area compared to the insulin area in cells obtained when the cell population is incubated in the absence of Yap1 inhibitor. 
     
     
         50 . The method according to any one of  claims 43  to  47 , said method further comprising a step of transplanting said insulin-producing cells into said individual.