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US-20260125650-A1 - METHODS AND COMPOSITIONS FOR GENERATING HEPATOCYTES

US20260125650A1US 20260125650 A1US20260125650 A1US 20260125650A1US-20260125650-A1

Abstract

Methods for generating hepatocytes from induced pluripotent stem cells (iPSCs) are provided using chemically-defined culture media in a four-stage culture protocol. Culture media and isolated cell populations are also provided.

Inventors

  • Michael A. Bukys
  • Xiaoguang FANG
  • Faruk SENTURK

Assignees

  • TRAILHEAD BIOSYSTEMS INC.

Dates

Publication Date
20260507
Application Date
20251003

Claims (14)

  1. 1 . A method of generating a population of hepatocytes from induced pluripotent stem cells (iPSCs) comprising: (a) culturing iPSCs in a culture medium comprising a glycogen synthase kinase 3 beta (GSK3B) inhibitor for 12-120 hours to obtain a first population of endodermal cells; (b) culturing the first population of endodermal cells from step (a) in a culture medium lacking a GSK33 inhibitor for 12-120 hours to obtain a second population of endodermal cells; (c) culturing the second population of endodermal cells from step (b) in a culture medium comprising a histone deacetylase (HDAC) inhibitor, a retinoic acid (RA) agonist, and a fibroblast growth factor receptor (FGFR; e.g., FGFR1) agonist for 24-240 hours to obtain a population of midgut cells; (d) culturing the population of midgut cells in a culture medium comprising a glucocorticoid, a cAMP response element-binding protein (CREB) agonist, an antioxidant, a Wnt antagonist, a transforming growth factor beta (TGF-β) antagonist, and a hepatocyte growth factor (HGF) agonist for at least 48 hours to obtain a population of hepatoblasts; and (e) culturing the population of hepatoblasts in a culture medium comprising an HDAC inhibitor, a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, an HGF agonist, a Notch antagonist, a bone morphogenetic protein (BMP) antagonist, and a hepatocyte function enhancer for at least 48 hours to obtain a population of hepatocytes.
  2. 2 . The method of claim 1 , wherein: (a) the GSK36 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, CHIR98023, 3 F8, A 1070722, AR-A014418, BIO, BIO-acetoxime, 6-BIO, Indirubin-3′-oxime, Alsterpaullone, 1-Azakenpaullone, Cazpaullone, Kenpaullone, Aloisine A, SB 216763, SB 415286, SB41528, SAR502250, TC-G 24, TWS119, LY2090314, AT7519, KY19382, AZD1080, AZD2858, Hymenialdisine, Debromohymenialdisine, Dibromocantherelline, Meridianine A, NSC 693868, IM-12, IMID1, IMID2, VP2.51, VP2.54, BIP-135, JGK-263, MMBO, TCS2002, PF-367, BRD0705, BRD3731, AF3581, TDZD-8, NP00111, Tideglusib (NP031112), NP031115, L803, L803-mts, L807-mts, HMK-32, Palinurin, Tricantin, Manzamine A, BTO, VP0.7, VP1.14, VP1.16, VP3.15, VP3.35, SC100, 6j, LCQFGS01, LCQFGS02, 4-3, and 4-4; (b) the HDAC inhibitor in each of steps (c) and (e) is independently selected from the group consisting of Vorinostat, Sodium Butyrate, Belinostat, Dacinostat, Droxinostat, Domatinostat, Entinostat, Ricolinostat, Resminostat, Abexinostat, Givinostat, Ivaltinostat, Panobinostat, Pracinostat, Quisinostat, Mocetinostat, Nanatinostat, Nexturastat A, Tefinostat, Tucidinostat, Fimepinostat, Citarinostat, Zabadinostat, GSK3117391, CUDC-101, AR-42, M344, Scriptaid, Sulforaphane, SR-4370, MC1568, CAY10603, CAY10683, RG2833, RGFP966, Cpd60, BRD3308, Tasquinimod, BML-210, LMK-235, BRD73954, PCI-34051, TMP195, TMP269, NKL22, TH34, SIS17, WT-161, ACY-738, BG45, CBHA, Pyroxamide, NCH-51, NCH-31, KD 5170, TCS HDAC6 20b, NSC 3852, NSC 69603, NSC 86371, NSC 305819, MS-27-275, Trapoxin A, Trapoxin B, Romidepsin, Apicidin, Trichostatin A, ACY-775, Tubastatin A, Tubacin, SKLB-23bb, HPOB, Curcumin, UF010, tc-H 106, Splitomicin, Raddeanin A, Depudecin, Tacedinaline, Isoguanosine, Parthenolide, Tinostamustine, Sodium Phenylbutyrate, Valproic Acid, Butyric acid, Phenylbutyric acid, 4-Phenylbutyric Acid, Divalproex Sodium, Sinapinic Acid, Suberohydroxamic Acid, Biphenyl-4-sulfonyl chloride, Thiophene Benzamide, Nicotinamide, Dihydrocoumarin, Naphthopyranone, and 2-Hydroxynaphthaldehyde; (c) the RA agonist is selected from the group consisting of TTNPB, ATRA, 9-cis-Retinoic Acid, Adapalene, Tretinoin, WYC-209, DC271, Acitretin, Arotinoid, AGN190168, AGN205327, LGD1550, Ch55, AM580 (CD336), CD2081, BMS 753, Tamibarotene, AGN194078, AGN195183, AGN193836, CD2314, CD2019, CD666, C286, BMS 641, AC-55649, AC261066, KCL-286, CD1530, CD437, CD2325, BMS 189961, BMS 270394, BMS 961, Trifarotene, and Palovarotene; (d) the FGFR (e.g., FGFR1) agonist is selected from the group consisting of FGF2, SUN11602, FGF1, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, and FGF23; (e) the glucocorticoid in steps (d) and (e) is independently selected from the group consisting of Hydrocortisone 21-hemisuccinate, Dexamethasone, Dexamethasone Acetate, Hydrocortisone, Cortisone, Prednisone, Prednisone Acetate, Meprednisone, Prednisolone, Methylprednisolone, Methylprednisolone Acetate, Fluprednisolone, Betamethasone, Paramethasone, Triamcinolone, Deflazacort, Fludrocortisone, Fludrocortisone Acetate, Deoxycorticosterone Acetate, Aldosterone, Beclometasone, Budesonide, Mometasone Furoate, Fluocinolone, Flunisolide, Fluorometholone, Fluticasone, Dagrocorat, Dagrocorat Hydrochloride, Mapracorat, Fosdagrocorat, GSK9027, GSK866, AZD2906, GW-870086, BAY 1003803, ZK 216348, LEO 134310, and RU28362; (f) the CREB agonist is selected from the group consisting of Forskolin, cAMP, Dibutyryl cAMP, 8-Br-CAMP, cAMPS-Sp, and CW 008; (g) the antioxidant in steps (d) and (e) is independently selected from the group consisting of N-Acetyl-L-Cysteine, Ascorbic Acid, Sodium Ascorbate, Glutathione, Ebselen, a-tocopherol, β-tocopherol, o-tocopherol, y-tocopherol, Lipoic Acid, Uric Acid, and Ubiquinol; (h) the Wnt antagonist in each of steps (d) and (e) is independently selected from the group consisting of IWR-1-endo, C59, XAV939, WIKI4, JW55, JW74, NVP-TNKS656, LZZ-02, TC-E 5001, IWP2, IWP4, WNT974, CGX1321, ETC-159, RXC004, GNF-6231, WIF-1, Ipafricept, DKK1, BMD4503-2, Salinomycin, NSC 668036, FJ9, 3289-8625, LF3, CCT036477, CCT251545, MSAB, KY1220, KY02111, FH535, Triptonide, KYA1797K, iCRT3, iCRT5, iCRT14, PNU-74654, PKF118-310, Cardionogen 1, ICG-001, JW67, NLS-StAx-h, PRI-724, GNE-781, Capmatinib, NCB-0846, TAK715, Nitazoxanide, Vantictumab, OTSA-101, and Fz7-21; (i) the TGF-β antagonist in each of steps (d) and (e) is independently selected from the group consisting of SB 431542, A 83-01, GW788388, SB505124, SB525334, TP0427736, RepSox, SD-208, Galunisertib, IN-1130, Ki 26894, LY2109761, LY2157299, LY550410, PF-03446962, TEW-7197, AP12009, AP11014, AP15012, ISTH0036, Fresolimumab, Lerdelimumab, GC1008, 2G7, 1 D11, CAT-192, LY2382770, and LY3022859; (j) the HGF agonist in each of steps (d) and (e) is independently selected from the group consisting of HGF, Dihexa, NK1, NK2, Fosgonimeton, and Terevalefim; (k) the Notch antagonist is selected from the group consisting of DAPT, GSI-XX, BMS 299897, BMS 433796, BMS 906024, BMS 986115, Compound E, Compound W, Compound 18, DBZ, DFK-167, L-685458 LY 3039478, LY 411575, LY 450139, LY 900009, MK-0752, MRK 003, MRK 560, PF 3084014, PF 3084014 Hydrobromide, Z-IL-CHO, Avagacestat, Begacestat, JLK6, AL101, RO 4929097, FLI-06, Thapsigargin, CAD204520, Tangeretin, Bruceine D, 15D11, Enoticumab, Demcizumab, ABT-165, Navicixizumab, Marimastat, ZLDI-8, IMR-1, IMR-1A, CB-103, RIN1, Brontictuzumab, Tarextumab, and PF 06650808; (l) the BMP antagonist is selected from the group consisting of LDN193189, Dorsomorphin, DMH-1, DMH-2, ML 347, LDN212854, LDN214117, K02288, Follistatin, Follistatin-like 1, Noggin, Chordin, Ventroptin, Twisted Gastrulation, Dan, Cerberus, PRDC, Dante, Caronte, Gremlin, and Sclerostin; (m) the hepatocyte function enhancer is FH1 or FPH1.
  3. 3 . The method of claim 2 , wherein: (a) the GSK3β inhibitor is CHIR99021; (b) the HDAC inhibitor in step (c) is Vorinostat and/or the HDAC inhibitor in step (e) is Sodium Butyrate; (c) the RA agonist is TTNPB; (d) the FGFR (e.g., FGFR1) agonist is FGF2; (e) the glucocorticoid in step (d) and/or step (e) comprises Hydrocortisone 21-hemisuccinate and Dexamethasone; (f) the CREB agonist is Forskolin; (g) the antioxidant in step (d) comprises N-Acetyl-L-Cysteine, Ascorbic Acid, and Sodium Ascorbate and/or the antioxidant in step (e) is Sodium Ascorbate; (h) the Wnt antagonist in step (d) is IWR-1-endo and/or the Wnt antagonist in step (e) comprises IWR-1-endo and C59; (i) the TGF-β antagonist in step (d) and/or step (e) is SB431542; (j) the HGF agonist in step (d) comprises HGF and Dihexa and/or the HGF agonist in step (e) is Dihexa; (k) the Notch antagonist is DAPT; (l) the BMP antagonist is LDN193189; (m) the hepatocyte function enhancer is FH1.
  4. 4 . The method of claim 3 , wherein: (a) CHIR 99021 is present in the culture medium at a concentration of 100 nM-10 μM: (b) Vorinostat is present in the culture medium at a concentration of 10 nM-5 μM and/or Sodium Butyrate is present in the culture medium at a concentration of 10 nM-5 μM; (c) TTNPB is present in the culture medium at a concentration of 5 nM-2 μM; (d) FGF 2 is present in the culture medium at a concentration of 500 pg/ml-500 ng/ml; (e) Hydrocortisone 21-hemisuccinate is present in the culture medium at a concentration of 10 nM-100 μM and/or Dexamethasone is present in the culture medium at a concentration of 500 pM-5 μM; (f) Forskolin is present in the culture medium at a concentration of 20 nM-2 mM; (g) (i) N-Acetyl-L-Cysteine is present in the culture medium at a concentration of 500 nM-2 M, and/or (ii) Ascorbic Acid is present in the culture medium at a concentration of 250 pg/ml-25 mg/ml, and/or (iii) Sodium Ascorbate is present in the culture medium at a concentration of 200 pg/ml-20 mg/ml, and/or (iv) Sodium Ascorbate is present in the culture medium at a concentration of 200 pg/ml-20 mg/ml; (h) IWR-1-endo is present in the culture medium at a concentration of 500 pM-10 μM and/or C59 is present in the culture medium at a concentration of 5 nM-100 μM; (i) SB 431542 is present in the culture medium at a concentration of 10 nM-200 μM; (j) HGF is present in the culture medium at a concentration of 100 pg/ml-10 μg/ml and/or Dihexa is present in the culture medium at a concentration of 500 pg/ml-1 μg/ml; (k) DAPT is present in the culture medium at a concentration of 10 nM-100 μM; (l) LDN 193189 is present in the culture medium at a concentration of 500 pM-2.5 μM; (m) FH 1 is present in the culture medium at a concentration of 10 nM-2.5 mM.
  5. 5 .- 53 . (canceled)
  6. 54 . A culture medium for obtaining: (a) endodermal cells, wherein the culture medium comprises a GSK3β inhibitor; (b) midgut cells, wherein the culture medium comprises an HDAC inhibitor, an RA agonist, and an FGFR (e.g., FGFR1) agonist; (c) hepatoblasts, wherein the culture medium comprises a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, and an HGF agonist; (d) hepatocytes, wherein the culture medium comprises an HDAC inhibitor, a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, an HGF agonist, a Notch antagonist, a BMP antagonist, and a hepatocyte function enhancer.
  7. 55 .- 57 . (canceled)
  8. 58 . An isolated cell culture comprising: (a) endodermal cells in a culture medium comprising a GSK3β inhibitor; (b) midgut cells in a culture medium comprising an HDAC inhibitor, an RA agonist, and an FGFR (e.g., FGFR1) agonist; (c) hepatoblasts in a culture medium comprising a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, and an HGF agonist; or (d) 50 million to 50 billion hepatocytes in a culture medium comprising an HDAC inhibitor, a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, an HGF agonist, a Notch antagonist, a BMP antagonist, and a hepatocyte function enhancer.
  9. 59 .-61. (canceled)
  10. 62 . Endodermal cells generated by steps (a) and (b) of the method of claim 1 .
  11. 63 . Midgut cells generated by steps (a)-(c) of the method of claim 1 .
  12. 64 . Hepatoblasts generated by steps (a)-(d) the method of claim 1 .
  13. 65 . Hepatocytes generated by the method of claim 1 .
  14. 66 . An isolated cell culture comprising 50 million to 50 billion engineered hepatocytes generated by the method of claim 1 .

Description

BACKGROUND OF THE INVENTION Hepatocytes are cells of the main parenchymal tissue of the liver that make up the majority of the liver's mass. Hepatocytes have several physiological roles and can be used for research, drug development, and tissue engineering of liver tissue. Hepatocytes can be obtained from induced pluripotent stem cells (IPSCs). Early approaches for obtaining hepatocytes from iPSCs involve screening of individual components that drive the differentiation, which results in a process optimization that does not consider the synergistic effects among the components. There is a need for efficient and robust methods and compositions for controlled differentiation of stem cells towards a hepatocyte fate, which takes into account the combinatorial effects of the added components. SUMMARY OF THE INVENTION This disclosure provides methods of generating hepatocytes from induced pluripotent stem cells (iPSCs), culture media, and other compositions for use in such methods. Hepatocytes can be obtained from iPSCs in a five-step, four-stage, 24-day protocol that sequentially generates endodermal cells, midgut cells, hepatoblasts, and hepatocytes. The methods use chemically defined culture media that allow for generation of endodermal cells within two days of culture, midgut cells within seven days of culture, hepatoblasts within 17 days of culture, and hepatocytes within 24 days of culture. The defined culture media used to obtain the different types of cells comprise small molecule agents that either agonize or antagonize particular signaling pathway activity in the pluripotent stem cells such that differentiation along the hepatocyte lineage is promoted, leading to cellular maturation and expression of hepatocyte-associated biomarkers, including but not limited to DLK1, GPC3, CYP3A7 and FXR, CAR, PXR, ASS, ARG1, FGL1, ORM1, CYP2C9, and CYP3A4. The methods of the disclosure use culture media for differentiation that comprise different components than those of earlier protocols. The methods of the disclosure also have the advantage that the use of small molecule agents in the culture media allows for precise control of the culture components and the time needed for differentiation to hepatocytes is significantly shortened compared to prior art protocols, due to the synergistic effects of the small molecule agents in the culture media. Accordingly, in one aspect, the disclosure pertains to a method of generating a population of hepatocytes. The method includes the steps of: (a) culturing iPSCs in a culture medium comprising a glycogen synthase kinase 3 beta (GSK3β) inhibitor for 12-120 hours to obtain a first population of endodermal cells;(b) culturing the first population of endodermal cells from step (a) in a culture medium lacking a GSK3ß inhibitor for 12-120 hours to obtain a second population of endodermal cells;(c) culturing the second population of endodermal cells from step (b) in a culture medium comprising a histone deacetylase (HDAC) inhibitor, a retinoic acid (RA) agonist, and a fibroblast growth factor receptor (FGFR; e.g., FGFR1) agonist for 24-240 hours to obtain a population of midgut cells;(d) culturing the population of midgut cells in a culture medium comprising a glucocorticoid, a cAMP response element-binding protein (CREB) agonist, an antioxidant, a Wnt antagonist, a transforming growth factor beta (TGF-β) antagonist, and a hepatocyte growth factor (HGF) agonist for at least 48 hours to obtain a population of hepatoblasts; and(e) culturing the population of hepatoblasts in a culture medium comprising an HDAC inhibitor, a glucocorticoid, a CREB agonist, an antioxidant, a Wnt antagonist, a TGF-β antagonist, an HGF agonist, a Notch antagonist, a bone morphogenetic protein (BMP) antagonist, and a hepatocyte function enhancer for at least 48 hours to obtain a population of hepatocytes. In some embodiments, the GSK36 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, CHIR98023, 3F8, A 1070722, AR-A014418, BIO, BIO-acetoxime, 6-BIO, Indirubin-3′-oxime, Alsterpaullone, 1-Azakenpaullone, Cazpaullone, Kenpaullone, Aloisine A, SB 216763, SB 415286,SB41528, SAR502250, TC-G 24, TWS119, LY2090314, AT7519, KY19382, AZD1080, AZD2858, Hymenialdisine, Debromohymenialdisine, Dibromocantherelline, Meridianine A, NSC 693868, IM-12,IMID1, IMID2, VP2.51, VP2.54, BIP-135, JGK-263, MMBO, TCS2002, PF-367, BRD0705, BRD3731, AF3581, TDZD-8, NP00111, Tideglusib (NP031112), NP031115, L803, L803-mts, L807-mts, HMK-32, Palinurin, Tricantin, Manzamine A, BTO, VP0.7, VP1.14, VP1.16, VP3.15, VP3.35, SC100, 6j, LCQFGS01, LCQFGS02, 4-3, and 4-4. In some embodiments, the GSK36 inhibitor is CHIR99021. In some embodiments, CHIR99021 is present in the culture medium at a concentration of 100 nM-10 μM. In some embodiments, the HDAC inhibitor in each of steps (c) and (e) is independently selected from the group consisting of Vorinostat, Sodium Butyrate, Belinostat, Dacinostat, Droxinostat, Domatinostat, En