Differentiation Method

Abstract

The invention relates to differentiation methods for progenitor cells, e.g. mammalian epithelial stem cells, differentiation media for use in said methods, organoids and cells obtainable by said methods and uses, including therapeutic uses, thereof.

Claims

1.-40. (canceled)

41. An organoid obtainable or obtained by a method comprising culturing progenitor cells in a differentiation medium comprising a basal medium, one or more receptor tyrosine kinase ligands, a Notch inhibitor, a glucocorticoid, a TGF-beta inhibitor, one or more Wnt inhibitors, and a Wnt agonist, wherein the Wnt agonist is a GSK 3 inhibitor, and wherein the one or more Wnt inhibitors comprises an inhibitor that acts downstream of the β catenin destruction complex.

42. The organoid of claim 41, wherein the differentiation medium further comprises one or more additional Wnt inhibitors selected from: (1) an inhibitor of Wnt secretion, (2) a competitive or non-competitive inhibitor of the interaction between Wnt or Rspondin and the Wnt receptor complex, (3) an inhibitor that promotes the degradation of components of the Wnt receptor complex, (4) an inhibitor of Dishevelled family proteins, (5) an activator that promotes destruction complex activity, (6) an inhibitor of the deoligomerisation of the destruction complex, and/or (7) an inhibitor of β-catenin target gene expression.

43. The organoid of claim 42, wherein the inhibitor of Wnt secretion is a Porc inhibitor selected from IWP-2, LGK974, and/or IWP-1.

44. The organoid of claim 41, wherein the GSK-3 inhibitor is selected from CHIR99201, 6-BIO, Dibromocantharelline, Hymenialdesine, Indirubins, Meridianins, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR-A014418, AZD-1080, Alsterpaullone, Cazpaullone, Kenpaullone, Aloisines, Manzamine A, Palinurine, Tricantine, TDZD-8, NP00111, NP031115, Tideglusib, HMK-32, and L803-mts.

45. The organoid of claim 41, wherein the inhibitor that acts downstream of the β catenin destruction complex is an inhibitor of β catenin target gene expression, optionally wherein the inhibitor of β-catenin target gene expression is selected from iCRT3, CGP049090, PKF118310, PKF115 584, ZTM000990, PNU 74654, BC21, iCRT5, iCRT14, and FH535.

46. The organoid of claim 41, wherein the differentiation medium further comprises an AP 1 stimulant, optionally wherein the AP 1 stimulant is a muscarinic acetylcholine receptor agonist, optionally selected from acetylcholine, bethanechol, carbachol, oxotremorine or pilocarpine.

47. The organoid of claim 41, wherein: (a) the one or more receptor tyrosine kinase ligands comprises one or more, or all, of a ligand for RTK class I (EGF receptor family or ErbB family), a ligand for RTK class IV (FGF receptor family), and a ligand for RTK class VI (HGF receptor family), optionally wherein the one or more receptor tyrosine kinase ligands are selected from epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF); (b) the Notch inhibitor is a gamma secretase inhibitor, optionally DAPT or dibenzazepine (DBZ) or benzodiazepine (BZ), or LY-411575; (c) the glucocorticoid is selected from: dexamethasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, beclomethasone, and fludrocortisone acetate; and/or (d) the TGF beta inhibitor is an inhibitor of ALK4, ALK5 or ALK7, optionally selected from: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208, and SJN 2511.

48. The organoid of claim 41, wherein the differentiation medium comprises EGF, FGF19, and HGF as receptor tyrosine kinase ligands, DAPT as a notch inhibitor, IWP2 and iCRT3 as Wnt inhibitor, dexamethasone as glucocorticoid, CHIR99021 as GSK-3 inhibitor, and carbachol as AP-1 inhibitor.

49. The organoid of claim 41, wherein: (a) the differentiation medium further comprises a BMP pathway activator, optionally selected from one or more of BMP7, BMP4, and BMP2; (b) the differentiation medium further comprises gastrin; and/or (c) the differentiation medium further comprises one or more components selected from: B27, B27 without retinoic acid, and N2.

50. The organoid of claim 41, wherein the organoid is in contact with an extracellular matrix, optionally wherein the organoid is embedded in a non-mesenchymal extracellular matrix.

51. The organoid of claim 41, wherein the progenitor cells are: (a) epithelial cells; (b) mammalian progenitor cells, optionally human progenitor cells; and/or (c) comprised in an organoid, optionally an expansion organoid.

52. The organoid of claim 51, wherein the epithelial cells are from the liver, pancreas, intestine, stomach, prostate, lung, breast, ovary, salivary gland, hair follicle, skin, oesophagus, bladder, ear, or thyroid, optionally from the liver or pancreas.

53. The organoid of claim 41, wherein the organoid is a liver organoid and is: (a) derived from a human, and in which at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the cells express hepatocyte markers; or (b) derived from a mouse, and in which at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the cells express hepatocyte markers.

54. The organoid of claim 41, wherein the organoid is a liver organoid and the mRNA expression level of Cyp3A4 is: (a) at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the expression level found in normal human hepatocytes; and/or (b) at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1.0 relative to GAPDH expression.

55. The organoid of claim 41, wherein the organoid does not comprise non-epithelial cells.

56. The organoid of claim 41, wherein: (a) the organoid has a cystic structure with a central lumen, optionally wherein the central lumen is surrounded by an epithelial monolayer; (b) one or more of the following cell types is absent in the organoid: liver macrophages, Kupffer cells, hepatic stellate cells, endothelial cells, smooth muscle cells, and fibroblasts; (c) the organoid does not comprise blood vessels or formed bile ducts; and/or (d) hepatocytes are integrated in an epithelial monolayer.

57. The organoid of claim 41, wherein the method further comprises an expansion step in which the progenitor cells are first cultured in an expansion medium before subsequently culturing in the differentiation medium.

58. The organoid of claim 57, wherein the expansion medium comprises a basal medium, EGF, FGF10, HGF, a Wnt agonist, Nicotinamide, a TGF-beta inhibitor, forskolin, and gastrin.

59. A method for obtaining a differentiated organoid, comprising: (a) culturing progenitor cells in the presence of an expansion medium to obtain an expanded population of progenitor cells or an expansion organoid; and (b) culturing the expanded population of progenitor cells or expansion organoid in a differentiation medium to obtain a differentiated organoid, wherein the differentiation medium comprises a basal medium, one or more receptor tyrosine kinase ligands, a Notch inhibitor, a glucocorticoid, a TGF-beta inhibitor, one or more Wnt inhibitors, and a Wnt agonist, wherein the Wnt agonist is a GSK 3 inhibitor, and wherein the one or more Wnt inhibitors comprises an inhibitor that acts downstream of the R catenin destruction complex.

60. A composition comprising the organoid of claim 41 and a differentiation medium.

61. The composition of claim 60, wherein the differentiation medium comprises a basal medium, one or more receptor tyrosine kinase ligands, a Notch inhibitor, a glucocorticoid, a TGF-beta inhibitor, one or more Wnt inhibitors, and a Wnt agonist, wherein the Wnt agonist is a GSK 3 inhibitor, and wherein the one or more Wnt inhibitors comprises an inhibitor that acts downstream of the β catenin destruction complex.

62. The composition of claim 61, wherein the differentiation medium further comprises one or more additional Wnt inhibitors is selected from: (1) an inhibitor of Wnt secretion, (2) a competitive or non-competitive inhibitor of the interaction between Wnt or Rspondin and the Wnt receptor complex, (3) an inhibitor that promotes the degradation of components of the Wnt receptor complex, (4) an inhibitor of Dishevelled family proteins, (5) an activator that promotes destruction complex activity, (6) an inhibitor of the deoligomerisation of the destruction complex, and/or (7) an inhibitor of β-catenin target gene expression.

63. The composition of claim 62, wherein the inhibitor of Wnt secretion is a Porc inhibitor selected from IWP-2, LGK974, and/or IWP-1.

64. The composition of claim 61, wherein the GSK-3 inhibitor is selected from CHIR99201, 6-BIO, Dibromocantharelline, Hymenialdesine, Indirubins, Meridianins, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR-A014418, AZD-1080, Alsterpaullone, Cazpaullone, Kenpaullone, Aloisines, Manzamine A, Palinurine, Tricantine, TDZD-8, NP00111, NP031115, Tideglusib, HMK-32, and L803-mts.

65. The composition of claim 61, wherein the inhibitor that acts downstream of the β catenin destruction complex is an inhibitor of β catenin target gene expression, optionally wherein the inhibitor of β-catenin target gene expression is selected from iCRT3, CGP049090, PKF118310, PKF115 584, ZTM000990, PNU 74654, BC21, iCRT5, iCRT14, and FH535.

66. The composition of claim 61, wherein the differentiation medium further comprises an AP 1 stimulant, optionally wherein the AP 1 stimulant is a muscarinic acetylcholine receptor agonist, optionally selected from acetylcholine, bethanechol, carbachol, oxotremorine, or pilocarpine.

67. The composition of claim 61, wherein: (a) the one or more receptor tyrosine kinase ligands comprises one or more, or all, of a ligand for RTK class I (EGF receptor family or ErbB family), a ligand for RTK class IV (FGF receptor family), and a ligand for RTK class VI (HGF receptor family), optionally wherein the one or more receptor tyrosine kinase ligands are selected from epidermal growth factor (EGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF); (b) the Notch inhibitor is a gamma secretase inhibitor, optionally DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), or LY-411575; (c) the glucocorticoid is selected from: dexamethasone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, beclomethasone, and fludrocortisone acetate; and/or (d) the TGF beta inhibitor is an inhibitor of ALK4, ALK5 or ALK7, optionally selected from: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208, and SJN 2511.

68. The composition according to claim 61, wherein the differentiation medium comprises EGF, FGF19 and HGF as receptor tyrosine kinase ligands, DAPT as a notch inhibitor, IWP2 and iCRT3 as Wnt inhibitor, dexamethasone as glucocorticoid, CHIR99021 as GSK-3 inhibitor, and carbachol as AP-1 inhibitor.

69. The composition according to claim 61, wherein: (a) the differentiation medium further comprises a BMP pathway activator, optionally selected from one or more of BMP7, BMP4, and BMP2; (b) the differentiation medium further comprises gastrin; and/or (c) the differentiation medium further comprises one or more components selected from: B27, B27 without retinoic acid, and N2.

Description

DESCRIPTION OF THE DRAWINGS

[0532] FIGS. 1A-1B. FIG. 1A: Improved method for expanding and differentiating a population of liver epithelial stem cells. The liver epithelial stem cells are grown for five days in EM+BMP7 medium (EM+BMP7 comprises Gibco Advanced DMEM/F12 as a basal medium, to which is added: N2, B27 without retinoic acid, EGF, HGF, TGF-beta inhibitor, nicotinamide, Rspondin, forskolin, gastrin, N-acetylcysteine, FGF10 and BMP7). After this pre-treatment, the organoid culture is disrupted, split and transferred to DM+ medium. The previous method for expanding and differentiating a population of liver epithelial stem cells involved liver epithelial stem cells being grown for five days in EM+BMP7 medium and then, without disruption or splitting, transferred to DM medium. FIG. 1B: Components of DM and DM+ media. DM+ medium comprises Gibco Advanced DMEM/F12 as a basal medium, to which is added: 10 mM HEPES, 1×B27-VitA, 1×N2, 1.25 mM N-Acetylcysteine, 50 ng/ml hEGF, 10 nM gastrin, 25 ng/ml HGF, 0.5 μM A83.01, 25 ng/ml BMP7, 100 ng/ml FGF19, 10 μM DAPT, 3 μM dexamethasone, 3 μM IWP-2, 3 μM Chir, 50 μM iCRT3 and 100 μM carbachol. DM medium comprises Gibco Advanced DMEM/F12 as a basal medium, to which is added: 10 mM HEPES, 1×B27-VitA, 1×N2, 1.25 mM N-Acetylcysteine, 50 ng/ml hEGF, 10 nM gastrin, 25 ng/ml HGF, 0.5 μM A83.01, 25 ng/ml BMP7, 100 ng/ml FGF19, 10 μM DAPT and 3 μM dexamethasone.

[0533] FIGS. 2A-2G. FIG. 2A List of factors that did not show positive effects on hepatocyte differentiation. FIG. 2B-2F qRT-PCR analysis of organoids in expansion medium (EM), after 11 days in differentiation medium (DM) or after 11 days in DM plus a modification. FIG. 2B The addition of the AP-1 stimulant carbachol to DM medium improves the expression of albumin and Cyp3A4, which are hepatocyte cell markers. FIG. 2C The addition of Wnt to DM medium decreases the expression of albumin and Cyp3A4. FIG. 2D The addition of an inhibitor of Wnt secretion, IWP-2, to DM medium enhances the expression of albumin and Cyp3A4. FIG. 2E The addition of CHIR99021 (a GSK3beta inhibitor that strongly increases Wnt signalling by interfering with the activity of the beta-catenin destruction complex) to DM medium increases the expression of albumin and Cyp3A4. The addition of CHIR99021 and iCRT3 (a small molecule that is capable of blocking the binding of beta-catenin to TCF4, and so capable of blocking Wnt signalling downstream of the beta-catenin destruction complex) leads to a significant increase in albumin and Cyp3A4 expression, while reducing the expression of CK19 (a biliary cell marker). FIG. 2F Disrupting and splitting the organoid culture after five days of EM+BMP7 pre-treatment and changing directly to DM medium improves both albumin and Cyp3A4 levels. FIG. 2G Human liver organoids after 11 days differentiation under standard conditions (standard DM) or using the new splitting technique (split DM). Splitting of the organoid culture results in decreased organoid size, which may contribute to enhanced differentiation. The complete removal of all expansion medium growth factors during the splitting procedure may also promote differentiation.

[0534] FIGS. 3A-3H. qRT-PCT analysis of primary hepatocytes or organoids in expansion medium (EM) or after 11 days differentiation following either the standard differentiation protocol (DM) or the improved procedure (DM+), which combines the DM+ medium with the splitting procedure. Expression levels of indicated genes are depicted relative to GAPDH. Each point represents an independent experiment. DM+ medium combined with the splitting procedure leads to a substantial improvement in expression of hepatocyte markers such as FIG. 3A albumin, FIG. 3B HNFa, FIG. 3C serpinA1 and FIG. 3D ASPGR. Most notably, however, the expression of cytochromes, such as FIG. 3E Cyp1A2 and FIG. 3F Cyp3A4 is strongly increased. The expression of Cyp3A4 is on average 50× higher in comparison to the DM protocol (without splitting) and reaches the level of primary hepatocytes. The expression of biliary markers, such as FIG. 3G CK7 and FIG. 3H CK19, is clearly reduced in the new DM+ protocol, which corroborates the increased differentiation to the hepatocyte lineage.

[0535] FIGS. 4A-4D. Improved hepatocyte functions in human liver organoids after DM+ differentiation. FIG. 4A Immunofluorescence staining for albumin after 11 days of differentiation following the standard (DM) or improved (DM+) protocol. Three views of exemplary organoids obtained using each protocol are provided: albumin, DAPI and brightfield. The DM+ protocol significantly increases the number of cell that differentiate to the hepatocyte fate. FIG. 4B Measurement of Cyp3A4 activity (P450-GLO™ assay) of HepG2, primary hepatocytes and human liver organoids in expansion medium (EM), standard differentiation medium (DM) or following the improved differentiation procedure (DM+) at day 11. The Cyp3A4 activity of cells differentiated using the DM+ protocol is at the level of primary hepatocytes and surpasses the levels achieved by HepG2 cells or the old DM condition by far. FIG. 4C Albumin secretion by HepG2, primary hepatocytes and human liver organoids in EM, DM or following the improved differentiation procedure (DM+) at day 11. FIG. 4D Conversion of Midazolam (a drug) to 1-OH-Midazolam by HepG2, human liver organoids in EM, DM or following the improved differentiation procedure (DM+) at day 11. Improved drug processing activity is seen in cells differentiated using the DM+ protocol. FIGS. 4B-4D Each point represents an independent donor.

EXAMPLES

Example 1

[0536] We significantly improved our ability to differentiate human liver organoids from bipotential stem cells to the hepatocyte state. Building on the previously established (old) differentiation protocol (DM) we developed a new improved procedure (DM+) that combines the treatment with 4 additional differentiation factors with a changed splitting protocol for the differentiation process (FIG. 1A and FIG. 1B). The components in the previous and improved differentiation media are summarised in the tables below. The DM+ protocol results in increased expression of all tested hepatocyte markers and reduces biliary fate commitment during the differentiation process. Most notably, DM+ leads to a significant improvement in the expression and activity of cytochromes (e.g. Cyp3A4 at the level of primary hepatocytes), which are the key enzymes of drug metabolism in hepatocytes. Thus, DM+ is a major step forward to adapt human liver organoids for commercial applications such as toxicology assays and drug development.

[0537] The human liver organoids were obtained from human liver stem cells cultured in an expansion medium (e.g. see table below) in accordance with methods previously described in WO2012/014076, WO2012/168930 and WO2015/173425. To improve human liver organoid differentiation, we tested a plethora of different factors with potentially beneficial effects. However, the majority of these did not contribute to differentiation improvement (see FIG. 2A). As human liver organoids have reduced expression of components of the AP-1 complex in comparison to primary hepatocytes (data not shown), we tested the AP-1 stimulant carbachol in addition to our standard differentiation medium and saw slight improvements in expression of Albumin and Cyp3A4 (FIG. 2B). Addition of WNT to the differentiation medium had the opposite effect and decreased expression of Albumin (FIG. 2C). Consequently, we tested an inhibitor of WNT secretion (IWP-2) to block endogenous WNT signaling and observed an increase in both Albumin and Cyp3A4 expression. In the same set of experiments we also tested CHIR, an inhibitor of GSK3beta, which strongly increases WNT signaling by interfering with activity of the beta-catenin destruction complex. Contrary to expectations, addition of CHIR99021 did not block differentiation (as stimulation by WNT did), but we observed a small increase in both Albumin and Cyp3A4 expression. This led us to the conclusion, that CHIR99021 stimulation has dual effects with a differentiation promoting and a differentiation inhibiting component. From the previous experiment we knew that WNT signaling would be the inhibitory component. Therefore, we looked into the possibility to combine CHIR99021 stimulation with a block of WNT signaling downstream of the destruction complex to cancel out the inhibitory component. Indeed, combination of CHIR99021 with iCRT3, a small molecule capable of blocking the binding of beta-catenin to TCF4, led to a significant increase in Albumin and Cyp3A4 expression, while reducing the expression of the biliary marker CK19 (FIG. 2E). In addition to medium additives we also looked into technical optimization of the differentiation process. We found that disrupting and splitting the organoid culture after 5 days of EM+BMP7 pretreatment and changing directly to differentiation medium improved both Albumin and Cyp3A4 levels (FIG. 2F). This is likely to be a combined effect of decreased organoid size (FIG. 2G) and the complete removal of all expansion medium growth factors during the splitting procedure.

[0538] We tested, whether the differentiation improvement by Carbachol, IWP-2, CHIR99021+iCRT3 and the new splitting procedure were additive and indeed this turned out to be the case. Thus, we defined our new differentiation protocol DM+ as a combination of the above (FIG. 1A and FIG. 1). DM+ leads to an even higher improvement in expression of hepatocyte markers such as Albumin (FIG. 3A), HNFa (FIG. 3B), SerpinA1 (FIG. 3C) and ASGPR (FIG. 3D). Most notably, however, the expression of Cytochromes such as Cyp1A2 and Cyp3A4 is strongly increased (FIG. 3E and FIG. 3F). The expression of Cyp3A4 is on average 50×higher in comparison to the old DM protocol and reaches the level of primary hepatocytes. The expression of biliary marker such as CK7 and CK19 is clearly reduced in the new DM+ protocol (FIG. 3G and FIG. 3H), which corroborates the increased differentiation to the hepatocyte lineage.

[0539] Generally, the DM+ protocol significantly increases the number of cells that differentiate to the hepatocyte fate (FIG. 4A). This is also evident in functional assays such as a Cyp3A4 activity assay and Albumin secretion (FIG. 4B and FIG. 4C). Cyp3A4 activity of cells differentiated according to the DM+ protocol is at the level of primary hepatocytes and surpasses levels achieved by HepG2 cells or the old DM condition by far. This is also reflected by improved drug processing activity, such as the processing of Midazolam to 1-OH-Midazolam (FIG. 4D). In summary, DM+ is an important step forward in differentiation of human liver organoids to the hepatocyte fate and improves their performance in various drug metabolism and toxicology assays. Beyond these applications, the improved differentiation profile may also indicate a better performance in disease modeling and transplantation.

TABLE-US-00005 Improved differentiation medium (DM+) Concentration Component Gibco Advanced DMEM/F12 10 mM HEPES 1 x B27 wo VitA 1 x N2 1.25 mM n-Acetylcysteine 50 ng/ml hEGF 10 nM Gastrin 25 ng/ml HGF 0.5 uM A83.01 25 ng/ml BMP7 100 ng/ml FGF19 10 uM DAPT 3 uM Dexamethasone 3 uM IWP-2 3 uM Chir 50 uM iCRT3 100 uM Carbachol

TABLE-US-00006 Old differentiation medium (DM) Concentration Component Gibco Advanced DMEM/F12 10 mM HEPES 1 x B27 wo VitA 1 x N2 1.25 mM n-Acetylcysteine 50 ng/ml hEGF 10 nM Gastrin 25 ng/ml HGF 0.5 uM A83.01 25 ng/ml BMP7 100 ng/ml FGF19 10 uM DAPT 3 uM Dexamethasone

TABLE-US-00007 Expansion medium (EM) Concentration Component Gibco Advanced DMEM/F12 10 mM HEPES 1 x B27 wo VitA 1 x N2 1.25 mM n-Acetylcysteine 50 ng/ml hEGF 10 nM Gastrin 25 ng/ml HGF 10 % Rspondin conditioned medium 0.01 M Nicotinamide 100 ng/ml FGF10 5 uM A83.01 10 uM FSK