Generation of glucose-responsive beta cells

Abstract

The present invention relates to a method for generating glucose-responsive beta cells.

1 . A method of generating beta cells, comprising the steps of providing a starting cell population comprising at least one cell capable of differentiation; wherein the cell capable of differentiation is a pluripotent stem cell or a pancreatic progenitor cell expressing PDX1 and NKX6.1, wherein:
 a. If the cell capable of differentiation is a pluripotent stem cell, the method comprises the steps of:   i) Incubating said cell population in RPMI medium comprising Activin A and a glycogen synthase kinase (GSK3) inhibitor for a duration, thereby differentiating at least part of the cell population into definitive endoderm cells;   ii) Incubating the cell population of i) in RPMI medium comprising B27−insulin, for a duration, thereby further differentiating the cell population into definitive endoderm cells;   iii) Incubating the cell population of ii) in DMEM/F12 medium comprising B27+insulin and retinoic acid, for a duration, thereby differentiating at least part of the cell population into gut tube cells;   iv) incubating the cell population of iii) in DMEM/F12 medium comprising B27+insulin and human FGF2, and optionally human Noggin, for a duration, thereby differentiating at least part of the cell population into posterior foregut cells;   v) Incubating the cell population of iv) in DMEM/F12 medium comprising B27+insulin, ((2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam) (TPB), and human Noggin for a duration, thereby differentiating at least part of the cell population into early pancreatic progenitor cells; and   vi) Incubating the cell population of v) in DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, and human Noggin for a duration, thereby differentiating at least part of the cell population into mature pancreatic progenitor cells; and   vii) Further incubating the cell population of vi) for an additional duration, thereby differentiating at least part of the cell population into beta cells;   or   b. If the cell capable of differentiation is a pancreatic progenitor cell, the method comprises the steps of:   viii) Incubating the starting cell population in DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, human Noggin and Rock inhibitor for a duration; and   ix) Incubating the cell population obtained in step viii) in DMEM/F12 medium comprising B27+insulin, Forskolin, Alk5 inhibitor, Nicotinamide, human Noggin without Rock inhibitor for a duration.   
     
     
         2 . The method according to  claim 1 , wherein the incubation of step i) is for a duration of one day. 
     
     
         3 . The method according to any one of the preceding claims, wherein the incubation of step ii) is for a duration of between 3 and 6 days. 
     
     
         4 . The method according to any one of the preceding claims, wherein the incubation of step iii) is for a duration of between 3 and 6 days. 
     
     
         5 . The method according to any one of the preceding claims, wherein the incubation of step iv) is for a duration of between 3 and 6 days. 
     
     
         6 . The method according to any one of the preceding claims, wherein the incubation of step v) is for a duration of between 3 and 6 days. 
     
     
         7 . The method according to any one of the preceding claims, wherein the incubation of step vi) is for a duration of between 3 and 6 days. 
     
     
         8 . The method according to any one of the preceding claims, wherein the incubation of step vii) is for a duration of between 7 and 23 days. 
     
     
         9 . The method according to any one of the preceding claims, wherein the incubation of step viii) is for a duration of between 1 and 2 days. 
     
     
         10 . The method according to any one of the preceding claims, wherein the incubation of step ix) is for a duration of between 7 and 14 days. 
     
     
         11 . The method according to any one of the preceding claims, wherein the RPMI medium of step i) comprises between 1 and 500 ng/mL Activin A, such as between 10 and 400 ng/mL Activin A, such as between 25 and 300 ng/mL Activin A, such as between 50 and 200 ng/mL Activin A, such as between 75 and 150 ng/mL Activin A. 
     
     
         12 . The method according to  claim 2 , wherein the RPMI medium of step i) comprises 100 ng/mL Activin A. 
     
     
         13 . The method according to any one of the preceding claims, wherein the RPMI medium of step i) comprises between 1 and 100 μM of a glycogen synthase kinase (GSK3) inhibitor such as CHIR, preferably CHIR99021, such as between 1 and 75 μM of a GSK3 inhibitor, such as between 1 and 50 μM of a GSK3 inhibitor, such as between 1 and 25 μM of a GSK3 inhibitor, such as between 1 and 10 μM of a GSK3 inhibitor, such as between 1 and 5 μM of a GSK3 inhibitor, such as 3 μM GSK3 inhibitor, for example 3 μM CHIR, preferably 3 μM CHIR99021. 
     
     
         14 . The method according to any one of the preceding claims, wherein the RPMI medium of step ii) comprises 1× B27−insulin. 
     
     
         15 . The method according to any one of the preceding claims, wherein the incubation of step ii) is for a duration of 3 days, 4 days, 5 days or 6 days, preferably 4 days. 
     
     
         16 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step iii) comprises 1× B27+insulin. 
     
     
         17 . The method according to anyone of the preceding claims, wherein the DMEM/F12 medium of step iii) comprises between 0.5 and 10 μM retinoic acid, such as between 0.75 and 7.5 μM retinoic acid, such as between 1.0 and 5 μM retinoic acid, such as between 1.5 and 2.5 μM retinoic acid, such as 2 μM retinoic acid. 
     
     
         18 . The method according to any one of the preceding claims, wherein the incubation of step iii) is for a duration of 3 days, 4 days, 5 days or 6 days, preferably 3 days. 
     
     
         19 . The method according to any one of the preceding claims, wherein the cell population obtained in step iii) is washed prior to step iv). 
     
     
         20 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step iv) comprises 1× B27+insulin. 
     
     
         21 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step iv) comprises between 10 and 200 ng/mL human FGF2, such as between 20 and 175 ng/mL human FGF2, such as between 30 and 150 ng/mL human FGF2, such as between 40 and 125 ng/mL human FGF2, such as between 50 and 100 ng/mL human FGF2, such as between 55 and 90 ng/mL human FGF2, such as between 60 and 80 ng/mL human FGF2, such as between 60 and 70 ng/mL human FGF2, such as 64 ng/mL human FGF2. 
     
     
         22 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step iv) comprises between 10 and 500 ng/mL human noggin, such as between 15 and 400 ng/mL human noggin, such as between 20 and 300 ng/mL human noggin, such as between 30 and 200 ng/mL human noggin, such as between 40 and 100 ng/mL human noggin, such as between 45 and 75 ng/mL human noggin, such as 50 ng/mL human noggin. 
     
     
         23 . The method according to any one of the preceding claims, wherein the incubation of step iv) is for a duration of 3 days, 4 days, 5 days or 6 days, preferably 3 days. 
     
     
         24 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step v) comprises 1× B27+insulin. 
     
     
         25 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step v) comprises between 0.1 and 10 μM TPB, such as between 0.2 and 7.5 μM TPB, such as between 0.3 and 5 μM TPB, such as between 0.4 and 2.5 μM TPB, such as between 0.4 and 1 μM TPB, such as 0.5 μM TPB. 
     
     
         26 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step v) comprises between 10 and 500 ng/mL human noggin, such as between 25 and 400 ng/mL human noggin, such as between 50 and 300 ng/mL human noggin, such as between 75 and 200 ng/mL human noggin, such as between 75 and 150 ng/mL human noggin, such as 100 ng/mL human noggin. 
     
     
         27 . The method according to any one of the preceding claims, wherein the incubation of step v) is for a duration of 3 days, 4 days, 5 days or 6 days, preferably 3 days. 
     
     
         28 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of steps vi), vii), viii) and/or ix) comprises 1× B27+insulin. 
     
     
         29 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of steps vi), vii), viii) or ix) comprises between 1 and 500 μM Forskolin, such as between 2 and 400 μM Forskolin, such as between 3 and 300 μM Forskolin, such as between 4 and 200 μM Forskolin, such as between 5 and 100 μM Forskolin, such as between 6 and 75 μM Forskolin, such as between 7 and 50 μM Forskolin, such as between 8 and 25 μM Forskolin, such as between 9 and 15 μM Forskolin, such as 10 μM Forskolin. 
     
     
         30 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of steps vi), vii), viii) or ix) comprises between 1 and 100 μM Alk5 inhibitor, such as between 1.5 and 75 μM Alk5 inhibitor, such as between 2 and 50 μM Alk5 inhibitor, such as between 3 and 40 μM Alk5 inhibitor, such as between 3.5 and 30 μM Alk5 inhibitor, such as between 4.0 and 20 μM Alk5 inhibitor, such as between 4.5 and 10 μM Alk5 inhibitor, such as 4.5 μM Alk5 inhibitor. 
     
     
         31 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step vi), vii), viii) or ix) comprises between 1 and 100 mM Nicotinamide, such as between 2 and 90 mM Nicotinamide, such as between 3 and 80 mM Nicotinamide, such as between 4 and 70 mM Nicotinamide, such as between 5 and 60 mM Nicotinamide, such as between 6 and 50 mM Nicotinamide, such as between 7 and 40 mM Nicotinamide, such as between 8 and 30 mM Nicotinamide, such as between 9 and 20 mM Nicotinamide, such as 10 mM Nicotinamide. 
     
     
         32 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of steps vi), vii), viii) or ix) comprises between 10 and 500 ng/mL human noggin, such as between 25 and 400 ng/mL human noggin, such as between 50 and 300 ng/mL human noggin, such as between 75 and 200 ng/mL human noggin, such as between 75 and 150 ng/mL human noggin, such as 100 ng/mL human noggin. 
     
     
         33 . The method according to any one of the preceding claims, wherein the DMEM/F12 medium of step ix) comprises between 1 and 100 μM Rock inhibitor, such as between 2.5 and 75 μM Rock inhibitor, such as between 5 and 50 μM Rock inhibitor, such as between 7.5 and 25 μM Rock inhibitor, such as between 10 and 15 μM Rock inhibitor. 
     
     
         34 . The method according to any one of the preceding claims, wherein the incubation of step vi) is for a duration of 3 days, 4 days, 5 days or 6 days, preferably 3 days. 
     
     
         35 . The method according to any one of the preceding claims, wherein the incubation of step vii) is for a duration of 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days or 23 days. 
     
     
         36 . The method according to any one of the preceding claims, wherein the incubation of step ix) is for a duration of 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. 
     
     
         37 . The method according to any one of the preceding claims, wherein the medium is replaced by fresh medium on a daily basis in steps i) to v). 
     
     
         38 . The method according to any one of the preceding claims, wherein the medium is replaced by fresh medium every second day in steps vi), vii), viii) and ix). 
     
     
         39 . The method according to any one of the preceding claims, wherein the medium of step i) does not comprise bovine serum albumin (BSA). 
     
     
         40 . The method according to any one of the preceding claims, wherein the medium of step ii) does not comprise bovine serum albumin (BSA). 
     
     
         41 . The method according to any one of the preceding claims, wherein the medium of step iii) does not comprise bovine serum albumin (BSA). 
     
     
         42 . The method according to any one of the preceding claims, wherein the medium of step iv) does not comprise bovine serum albumin (BSA). 
     
     
         43 . The method according to any one of the preceding claims, wherein the medium of step v) does not comprise bovine serum albumin (BSA). 
     
     
         44 . The method according to any one of the preceding claims, wherein the medium of step vi) does not comprise bovine serum albumin (BSA). 
     
     
         45 . The method according to any one of the preceding claims, wherein the medium of step vii) does not comprise bovine serum albumin (BSA). 
     
     
         46 . The method according to any one of the preceding claims, wherein the starting cell population is a population of pluripotent stem cells. 
     
     
         47 . The method according to  claim 46 , wherein the population of pluripotent stem cells is a population of induced pluripotent stem cells, such as human induced pluripotent stem cells. 
     
     
         48 . The method according to  claim 46 , wherein the population of pluripotent stem cells is a population of embryonic stem cells, such as human embryonic stem cells. 
     
     
         49 . The method according to any one of the preceding claims, wherein the starting cell population is a population of naïve stem cells, such as human naïve stem cells. 
     
     
         50 . The method according to any one of the preceding claims, wherein the starting cell population is a population of somatic cells. 
     
     
         51 . The method according to any one of the preceding claims, wherein the starting cell population is a population of pancreatic progenitor cells. 
     
     
         52 . The method according to  claim 51 , wherein the method further comprises a step of enriching the starting cell population for cells expressing PDX1 and NKX6.1 between steps v) and vi) and/or between steps vi) and vii). 
     
     
         53 . The method according to  claim 52 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises exposing the cell population to 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. 
     
     
         54 . The method according to any one of  claims 52  to  53 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises exposing the cell population to 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. 
     
     
         55 . The method according to any one of  claims 52  to  54 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises exposing the 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. 
     
     
         56 . The method according to any one of  claims 52  to  55 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises the steps of,
 in any order:
 a) exposing the cell population to 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; and 
 b) exposing the cell population to 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. 
 
 
     
     
         57 . The method according to any one of  claims 52  to  56 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises the steps of, in any order:
 a) exposing the cell population to 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; and 
 b) exposing the 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. 
 
     
     
         58 . The method according to any one of  claims 52  to  57 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises the steps of, in any order:
 a) exposing the cell population to 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 
 b) exposing the 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. 
 
     
     
         59 . The method according to any one of  claims 52  to  58 , wherein the step of enriching the cell population for cells expressing PDX1 and NKX6.1 comprises the steps of, in any order:
 a) exposing the cell population to 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; and 
 b) exposing the cell population to 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 
 c) exposing the 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. 
 
     
     
         60 . The method according to any one of  claims 52  to  59 , wherein at least one of the first, second and third ligands is an antibody or fragment thereof. 
     
     
         61 . The method according to any one of  claims 52  to  60 , wherein the antibody is a monoclonal or polyclonal antibody. 
     
     
         62 . The method according to any one of  claims 52  to  61 , wherein at least one of the first, second and third ligands binds to a cell surface marker of the pancreatic progenitor cell. 
     
     
         63 . The method according to any one of  claims 52  to  62 , wherein at least one of the first, second and third ligands is conjugated to a label. 
     
     
         64 . The method according to any one of  claims 52  to  63 , wherein the expression of at least one of the first, second and third marker is detected by flow cytometry. 
     
     
         65 . The method according to any one of  claims 52  to  64 , wherein the cells are removed or selected by flow cytometry. 
     
     
         66 . The method according to any one of  claims 52  to  65 , wherein the first ligand is an antibody or fragment thereof directed against CD49d. 
     
     
         67 . The method according to any one of  claims 52  to  66 , 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. 
     
     
         68 . The method according to any one of  claims 52  to  67 , wherein the second ligand is an antibody or fragment thereof directed against FOLR1. 
     
     
         69 . The method according to any one of  claims 52  to  68 , 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. 
     
     
         70 . The method according to any one of  claims 52  to  69 , 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 and CALB1. 
     
     
         71 . The method according to any one of  claims 52  to  70 , wherein the third ligand is an antibody or fragment thereof directed against GP2. 
     
     
         72 . The method according to any one of  claims 52  to  71 , wherein the first ligand is an antibody or fragment thereof directed against CD49d and the third ligand is an antibody directed against GP2. 
     
     
         73 . The method according to any one of  claims 52  to  72 , 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. 
     
     
         74 . The method according to any one of the preceding claims, wherein if the starting population is a pluripotent stem cell population, the method further comprises the step of enriching the cell population obtained in step v) and/or in step vi) for cells expressing PDX1 and NKX6.1 as defined in any one of  claims 53  to  74 . 
     
     
         75 . The method according to any one of the preceding claims, wherein the starting cell population is derived from cells isolated from an individual. 
     
     
         76 . The method according to any one of the preceding claims, wherein at least one cell of the cell population obtained in any of steps i) to iix) has the capability to differentiate further. 
     
     
         77 . The method according to any one of the preceding claims, wherein at least one cell of the cell population obtained in any of steps i) to ix) has the capability to differentiate further into pancreatic hormone-producing cells. 
     
     
         78 . The method according to any one of the preceding claims, wherein the cell population obtained in step vii) or ix) is capable of producing insulin. 
     
     
         79 . The method according to any one of the preceding claims, wherein the cell population obtained in step vii) or ix) is glucose-responsive. 
     
     
         80 . The method according to any one of the preceding claims, wherein the cell population obtained in step vii) or ix) can produce insulin-producing islet cells. 
     
     
         81 . A population of cells obtainable by the method according to any one of the preceding claims, for treatment of a metabolic disorder in an individual in need thereof. 
     
     
         82 . The population according to  claim 81 , wherein the cells are beta cells. 
     
     
         83 . A method of treatment of a metabolic disorder in an individual in need thereof, wherein the method comprises a step of providing a population of beta cells obtainable by the method according to any one of  claims 1  to  80  and transplanting said population of beta cells into said individual. 
     
     
         84 . The method according to  claim 83 , 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.