Vascularized brain organoids and methods of making and using them

Abstract

Disclosed herein is an organoid model of blood-brain barrier and the method for producing it from pluripotent stem cells.These organoids show the complex organization of cells, including neurons, endothelial cells, glial cells and pericytes, which is similar to the natural blood-brain barrier structure.These organoids can be used to study the function of blood-brain barrier and cerebrovascular disease.

Description

[Technical field]

[0001] Aspects of the present disclosure relate generally to organoid compositions that exhibit blood-brain barrier structures, and methods of making and using the same. [Background technology]

[0002] The blood-brain barrier (BBB) ​​is a very important biological structure because it selectively allows or prevents the passage of molecules and other substances from the blood to the central nervous system. This confers protection against pathogens and immune-related components (e.g., immune cells, antibodies, and cytokines) and protects the central nervous system from the effects of peripheral immune function. However, this also makes it difficult for pharmaceutical molecules to access the central nervous system, including the brain, limiting their therapeutic efficacy unless specifically designed to cross the BBB. Therefore, the BBB is an essential component that must be considered during drug development, and there is a great need for robust in vitro and / or in vivo models to study the BBB. Summary of the Invention

[0003] Disclosed herein is a method for generating vascularized brain organoids. In some embodiments, the method comprises contacting vascular organoids with brain organoids, and culturing the vascular organoids and brain organoids for a period of time until the vascular organoids and brain organoids fuse together and the blood vessels of the vascular organoids infiltrate into the brain organoids, thereby forming vascularized brain organoids. In some embodiments, the neurons of the brain organoids innervate the blood vessels of the vascular organoids that infiltrate into the organoids. In some embodiments, the vascularized brain organoids comprise a blood-brain barrier formed between the brain organoids and all or part of the blood vessels of the vascular organoids that infiltrate into the brain organoids. In some embodiments, the blood-brain barrier comprises endothelial cells connected by tight junctions, astrocytes, and pericytes.

[0004] Also disclosed herein are vascularized cerebral organoids generated according to the methods disclosed herein.

[0005] Also disclosed herein is the method for treating cerebrovascular disease or the disease associated with blood-brain barrier dysfunction in a subject that needs to do so.In some embodiments, the method comprises administering to subject any of the vascularized brain organoids disclosed herein, or part or fragment thereof.

[0006] Also disclosed herein is a screening method.In some embodiments, the method comprises: contacting any of the vascularized brain organoids disclosed herein or a part thereof with a candidate compound or composition; and evaluating the effect of the candidate compound or composition on the vascularized brain organoid or a part thereof.In some embodiments, the effect is the transport of the candidate compound or composition across the blood-brain barrier of the organoid or a part thereof.

[0007] Exemplary embodiments of the present disclosure are provided in the following numbered embodiments: 1. A method for generating vascularized cerebral organoids, comprising: Contacting a vascular organoid with a brain organoid; Culturing the vascular organoids and the cerebral organoids for a period of time until the vascular organoids and the cerebral organoids fuse together and blood vessels of the vascular organoids infiltrate the cerebral organoids; Neurons from the brain organoid innervate the blood vessels of the vascular organoid that have infiltrated into the organoid. This results in the formation of vascularized brain organoids, The vascularized cerebral organoid comprises a blood-brain barrier formed between the cerebral organoid and all or a portion of the blood vessels of the vascular organoid that have infiltrated into the cerebral organoid; A method, wherein the blood-brain barrier comprises endothelial cells connected by tight junctions, astrocytes, and pericytes.

[0008] 2. The method of embodiment 1, wherein the endothelial cells express CD31, GLUT-1 and PDGFR-β, the tight junctions contain claudin-5, the astrocytes express S100B, GFAP and AQP4, and the pericytes express PDGFR-β, αSMA and NG2.

[0009] 3. The method of embodiment 1 or 2, wherein the brain organoid is a forebrain organoid, a midbrain organoid, a hypothalamic organoid, a hippocampal organoid, a spinal cord organoid, or a striatal brain organoid.

[0010] 4. The method according to any one of embodiments 1 to 3, wherein the vascular organoids and cerebral organoids are contacted and / or cultured in a basement membrane matrix or a component thereof, optionally Matrigel.

[0011] 5. The method according to any one of embodiments 1 to 4, wherein the vascular organoids and cerebral organoids are cultured for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 days, or any number of days within a range defined by any two of the aforementioned days.

[0012] 6. The method according to any one of embodiments 1 to 5, wherein the vascular organoids and cerebral organoids are cultured with stirring, optionally with shaking, for at least part of the period.

[0013] 7. Vascular organoids and brain organoids 1) cultured without agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding days, and then 2) cultured with agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the preceding days; 7. The method of any one of embodiments 1 to 6, optionally wherein the agitation comprises shaking.

[0014] 8. The method according to any one of embodiments 1 to 7, wherein the vascular organoids and cerebral organoids are cultured in a medium that promotes neuronal and / or vascular growth.

[0015] 9. The method according to any one of embodiments 1 to 8, wherein the vascular organoids and cerebral organoids are cultured in a medium containing a growth factor that promotes neuronal growth and / or a growth factor that promotes vascular growth.

[0016] 10. The method of embodiment 9, wherein the growth factors that promote neuronal growth comprise cAMP pathway activators, ascorbic acid, BDNF, GDNF, or any combination thereof.

[0017] 11. The method of embodiment 9 or 10, wherein the growth factors that promote vascular growth comprise growth serum, a VEGF pathway activator, an FGF pathway activator, or any combination thereof.

[0018] 12. Culturing vascular organoids and cerebral organoids, a) culturing vascular and cerebral organoids without agitation for 4, 5, 6, 7, 8, 9, or 10 days, optionally for 7 days; b) culturing the organoids of step a) with agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days; The organoids of steps a) and b) are cultured in a medium containing a growth factor that promotes neuronal growth and / or a growth factor that promotes vascular growth; Optionally, the agitation comprises shaking; Optionally, the growth factors that promote neuronal growth include cAMP pathway activators, ascorbic acid, BDNF, GDNF, or any combination thereof; Optionally, the growth factors that promote vascular growth include growth serum, a VEGF pathway activator, an FGF pathway activator, or any combination thereof; Optionally, the method according to any one of embodiments 1 to 11, wherein the vascular organoid and the cerebral organoid are cultured in a basement membrane matrix or a component thereof, optionally Matrigel.

[0019] 13. The method of any one of embodiments 10 to 12, wherein the cAMP pathway activator is cAMP.

[0020] 14. The method of any one of embodiments 10-13, wherein the cAMP pathway activator is provided at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 50 μM.

[0021] 15. Ascorbic acid is at or about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μM 15. The method of any one of embodiments 10-14, wherein the compound is provided at a concentration of 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 200 μM.

[0022] 16. The method of any one of embodiments 10-15, wherein BDNF is provided at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 20 ng / mL.

[0023] 17. The method of any one of embodiments 10-16, wherein GDNF is provided at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 20 ng / mL.

[0024] 18. The method of any one of embodiments 10 to 17, wherein the growth serum is fetal bovine serum (FBS).

[0025] 19. The method of any one of embodiments 10-18, wherein the growth serum is provided at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 15%.

[0026] 20. The method of any one of embodiments 10 to 19, wherein the VEGF pathway activator is VEGF.

[0027] 21. The method of any one of embodiments 10-19, wherein the VEGF pathway activator is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 100 ng / mL.

[0028] 22. The method of any one of embodiments 10 to 21, wherein the FGF pathway activator is FGF2.

[0029] 23. The method of any one of embodiments 10-22, wherein the FGF pathway activator is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 100 ng / mL.

[0030] 24. The method according to any one of embodiments 1 to 23, wherein the vascular and / or cerebral organoids are derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

[0031] 25. Vascular organoids contacting the angiogenic sprouts with a Wnt pathway activator, an FGF pathway activator, a VEGF pathway activator, and optionally a growth serum for a first period of time; The method according to any one of embodiments 1 to 24, wherein the vascular organoids are generated according to the method.

[0032] 26. The method of embodiment 25, wherein the first period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or any number of days in a range defined by any two of the aforesaid numbers of days, optionally 5 days or at least 5 days.

[0033] 27. Angiogenic sprouts a) contacting the pluripotent stem cells with a Wnt pathway activator and a BMP pathway activator for a second period of time to form vascular lineage cells; b) contacting the vascular lineage cells with a VEGF pathway activator and a second cAMP pathway activator for a third period of time; 27. The method according to embodiment 25 or 26, produced according to the method, thereby forming angiogenic sprouts.

[0034] 28. The method of embodiment 27, wherein the second period of time is 1, 2, 3, 4, or 5 days, or any number of days within a range defined by any two of the foregoing numbers of days, optionally 3 days.

[0035] 29. The method of embodiment 27 or 28, wherein the third period of time is 1, 2, 3, or 4 days, or any number of days within a range defined by any two of the preceding numbers of days, optionally 2 days.

[0036] 30. The method of any one of embodiments 25 to 29, wherein the BMP pathway activator is BMP4.

[0037] 31. The method of any one of embodiments 25 to 30, wherein the BMP pathway activator is provided at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 30 ng / mL.

[0038] 32. The method of any one of embodiments 25 to 31, wherein the Wnt pathway activator is CHIR99021.

[0039] 33. The method of any one of embodiments 25-32, wherein the Wnt pathway activator is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 4 μM, or optionally at or about 12 μM.

[0040] 34. The method of any one of embodiments 25-33, wherein the second cAMP pathway activator is forskolin.

[0041] 35. The method of any one of embodiments 25-34, wherein the second cAMP pathway activator is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 2 μM.

[0042] 36. The method according to any one of embodiments 25 to 35, wherein the vascular organoids are distinct from vascular organoids generated without contacting the angiogenic sprouts with a Wnt pathway activator by increasing the expression of blood-brain barrier specific endothelial markers, optionally GLUT-1 and ZO-1.

[0043] 37. The method according to any one of embodiments 1 to 36, wherein the cerebral organoid is contacted with LIF and growth serum to induce astrocyte formation in the cerebral organoid.

[0044] 38. Brain organoids a) contacting pluripotent stem cells with a BMP pathway inhibitor, a TGF-beta pathway inhibitor, and a Wnt pathway inhibitor for a first period of time to form neuroectodermal cells; b) contacting the neuroectodermal cells of step a) with a second TGF-beta pathway inhibitor and a Wnt pathway activator for a second period of time to form neuroepithelial cells; c) contacting the neuroepithelial cells of step b) with insulin for a third period of time to form brain tissue organoids; d) contacting the brain tissue organoids of step c) with GDNF, BDNF, ascorbic acid, and a cAMP pathway activator for a fourth period of time to form brain organoids; Optionally, the brain tissue organoid is further contacted with LIF and growth serum for a portion of a fourth period to induce astrocyte proliferation in the brain organoid, according to any one of the methods described in embodiments 1 to 37.

[0045] 39. The method of embodiment 38, wherein the first period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the foregoing numbers of days, optionally 7 days.

[0046] 40. The method of embodiment 38 or 39, wherein the second period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding numbers of days, optionally 7 days.

[0047] 41. The method of any one of embodiments 38-40, wherein the third period of time is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the aforesaid numbers of days, optionally 56 days.

[0048] 42. The method of any one of embodiments 38-41, wherein the fourth period of time is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the aforesaid numbers of days.

[0049] 43. The method of embodiment 42, wherein the portion of the fourth period is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 17, 18, 19, 20, or 21 days, or any number of days within a range defined by any two of the foregoing numbers of days, optionally 14 days; optionally, the portion of the fourth period is the beginning of the fourth period.

[0050] 44. The method of any one of embodiments 38 to 43, wherein the BMP pathway inhibitor is LDN193189.

[0051] 45. The method of any one of embodiments 38-44, wherein the BMP pathway inhibitor is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0052] 46. ​​The method of any one of embodiments 38-45, wherein the TGF-beta pathway inhibitor and the second TGF-beta pathway inhibitor are the same or different.

[0053] 47. The method of any one of embodiments 38 to 46, wherein the TGF-beta pathway inhibitor is A83-01.

[0054] 48. The method of any one of embodiments 38-47, wherein the TGF-beta pathway inhibitor is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 2 μM.

[0055] 49. The method of any one of embodiments 38-48, wherein the second TGF-beta pathway inhibitor is SB-431542.

[0056] 50. The method of any one of embodiments 38-49, wherein the second TGF-beta pathway inhibitor is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0057] 51. The method of any one of embodiments 38 to 50, wherein the Wnt pathway inhibitor is IWR-1.

[0058] 52. The method of any one of embodiments 38-51, wherein the Wnt pathway inhibitor is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 3 μM.

[0059] 53. The method of any one of embodiments 38 to 52, wherein the Wnt pathway activator is CHIR99021.

[0060] 54. The method of any one of embodiments 38-53, wherein the Wnt pathway activator is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0061] 55. The method of any one of embodiments 38-54, wherein insulin is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at a concentration of 2.5 μg / mL or about 2.5 μg / mL.

[0062] 56. The method of any one of embodiments 38 to 55, wherein LIF is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL, optionally at or about 10 mg / mL.

[0063] 57. The method of any one of embodiments 38 to 56, wherein the brain organoid comprises astrocytes expressing S100B, GFAP, and AQP4.

[0064] 58. The method according to any one of embodiments 1 to 57, wherein the vascular organoids and / or cerebral organoids are human.

[0065] 59. The method according to any one of embodiments 1 to 58, wherein the vascular organoids and / or cerebral organoids are derived from a subject, optionally a human subject.

[0066] 60. The method of embodiment 59, wherein the subject has a cerebrovascular disease or a disease associated with blood-brain barrier dysfunction, optionally wherein the cerebrovascular disease or a disease associated with blood-brain barrier dysfunction comprises cavernous vascular malformation, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0067] 61. A vascularized cerebral organoid produced by a method according to any one of embodiments 1 to 60.

[0068] 62. A method for treating cerebrovascular disease or a disease associated with blood-brain barrier dysfunction in a subject in need thereof, comprising administering to the subject a vascularized cerebral organoid, or a part or fragment thereof, as described in embodiment 61.

[0069] 63. A screening method comprising contacting the vascularized cerebral organoid or a portion thereof described in embodiment 61 with a candidate compound or composition, and evaluating the effect of the candidate compound or composition on the vascularized cerebral organoid or a portion thereof.

[0070] 64. The method of embodiment 63, wherein the effect comprises transport of the candidate compound or composition across the blood-brain barrier of the organoid or portion thereof.

[0071] 65. The method of embodiment 63 or 64, wherein the vascularized cerebral organoid is a model for cerebrovascular disease or a disease associated with blood-brain barrier dysfunction, and evaluating the effect of a candidate compound or composition on the vascularized organoid comprises evaluating the effect of a candidate compound or composition on cerebrovascular disease or a disease associated with blood-brain barrier dysfunction.

[0072] 66. The method according to any one of embodiments 63 to 65, wherein the vascularized cerebral organoids are generated from cells derived from a subject, and optionally, the cells derived from the subject are induced pluripotent stem cells.

[0073] 67. The method of embodiment 66, wherein the subject has or is susceptible to developing cerebrovascular disease or a disease associated with blood-brain barrier dysfunction.

[0074] Further exemplary embodiments of the present disclosure are provided in the following numbered embodiments: 1. A method for generating vascularized cerebral organoids, comprising: Contacting a vascular organoid with a brain organoid; Culturing the vascular organoids and the cerebral organoids for a period of time until the vascular organoids and the cerebral organoids fuse together and blood vessels of the vascular organoids infiltrate the cerebral organoids; Neurons from the brain organoid innervate the blood vessels of the vascular organoid that have infiltrated into the organoid. This results in the formation of vascularized brain organoids, The vascularized cerebral organoid comprises a blood-brain barrier formed between the cerebral organoid and all or a portion of the blood vessels of the vascular organoid that have infiltrated into the cerebral organoid; A method, wherein the blood-brain barrier comprises endothelial cells connected by tight junctions, astrocytes, and pericytes.

[0075] 2. The method of embodiment 1, wherein the endothelial cells express CD31, GLUT-1 and PDGFR-β, the tight junctions contain claudin-5, ZO-1 and cadherin 5, the astrocytes express S100B, GFAP and AQP4, and the pericytes express PDGFR-β, αSMA and NG2.

[0076] 3. The method of embodiment 1 or 2, wherein the endothelial cells form a continuous basement membrane and express collagen IV.

[0077] 4. The method according to any one of embodiments 1 to 3, wherein the vascularized brain organoid comprises cells selected from the group consisting of neural precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, cerebrovascular endothelial cells, vascular leptomeningeal cells, perivascular adipocytes, and tendon cells.

[0078] 5. The method according to any one of embodiments 1 to 3, wherein the vascularized brain organoid comprises cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells.

[0079] 6. The method of embodiment 4 or 5, wherein the cells of the vascular brain organoids are identified by cell type-specific gene expression markers.

[0080] 7. The method of any one of the preceding embodiments, wherein the blood vessels comprise capillaries.

[0081] 8. The method of embodiment 7, wherein the capillaries are covered by the endfeet of pericytes and astrocytes.

[0082] 9. The method according to any one of embodiments 1 to 8, wherein the brain organoid is a forebrain organoid, a midbrain organoid, a hypothalamic organoid, a hippocampal organoid, a spinal cord organoid, or a striatal brain organoid.

[0083] 10. The method according to any one of embodiments 1 to 9, wherein the vascular organoids and cerebral organoids are contacted and / or cultured in a basement membrane matrix or a component thereof, optionally Matrigel.

[0084] 11. Vascular and cerebral organoids are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 11. The method of any one of embodiments 1-10, wherein the cells are cultured for a period of 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 days, or any number of days within a range defined by any two of the aforesaid numbers of days.

[0085] 12. The method according to any one of embodiments 1 to 11, wherein the vascular organoids and cerebral organoids are cultured with stirring, optionally with shaking, for at least a portion of the time period.

[0086] 13. Vascular organoids and brain organoids 1) cultured without agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding days, and then 2) cultured with agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the preceding days; 13. The method of any one of embodiments 1 to 12, optionally wherein the agitation comprises shaking.

[0087] 14. The method according to any one of embodiments 1 to 13, wherein the vascular organoids and cerebral organoids are cultured in a medium that promotes neuronal and / or vascular growth.

[0088] 15. The method according to any one of embodiments 1 to 14, wherein the vascular organoids and cerebral organoids are cultured in a medium containing a growth factor that promotes neuronal growth and / or a growth factor that promotes vascular growth.

[0089] 16. The method of embodiment 15, wherein the growth factors that promote neuronal growth comprise cAMP pathway activators, ascorbic acid, BDNF, GDNF, or any combination thereof.

[0090] 17. The method of embodiment 15 or 16, wherein the growth factors that promote vascular growth comprise growth serum, a VEGF pathway activator, an FGF pathway activator, or any combination thereof.

[0091] 18. Culturing vascular organoids and cerebral organoids a) culturing vascular and cerebral organoids without agitation for 4, 5, 6, 7, 8, 9, or 10 days, optionally for 7 days; b) culturing the organoids of step a) with agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days; The organoids of steps a) and b) are cultured in a medium containing a growth factor that promotes neuronal growth and / or a growth factor that promotes vascular growth; Optionally, the agitation comprises shaking; Optionally, the growth factors that promote neuronal growth include cAMP pathway activators, ascorbic acid, BDNF, GDNF, or any combination thereof; Optionally, the growth factors that promote vascular growth include growth serum, a VEGF pathway activator, an FGF pathway activator, or any combination thereof; Optionally, the method according to any one of embodiments 1 to 17, wherein the vascular organoid and the cerebral organoid are cultured in a basement membrane matrix or a component thereof, optionally Matrigel.

[0092] 19. The method of any one of embodiments 16 to 18, wherein the cAMP pathway activator is cAMP.

[0093] 20. The method according to any one of embodiments 16 to 19, wherein the cAMP pathway activator is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 50 μM.

[0094] 21. Ascorbic acid is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μM or less. 21. The method of any one of embodiments 16-20, wherein the compound is provided at a concentration of 50, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 200 μM.

[0095] 22. The method of any one of embodiments 16-21, wherein BDNF is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 20 ng / mL.

[0096] 23. The method of any one of embodiments 16-22, wherein GDNF is provided at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 20 ng / mL.

[0097] 24. The method of any one of embodiments 17-23, wherein the growth serum is fetal bovine serum (FBS).

[0098] 25. The method of any one of embodiments 17-24, wherein the growth serum is provided at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 15%.

[0099] 26. The method of any one of embodiments 17 to 25, wherein the VEGF pathway activator is VEGF.

[0100] 27. The method of any one of embodiments 17-26, wherein the VEGF pathway activator is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 100 ng / mL.

[0101] 28. The method of any one of embodiments 17 to 27, wherein the FGF pathway activator is FGF2.

[0102] 29. The method of any one of embodiments 17-28, wherein the FGF pathway activator is provided at a concentration of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 100 ng / mL.

[0103] 30. The method according to any one of embodiments 1 to 29, wherein the vascular and / or cerebral organoids are derived from pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

[0104] 31. Vascular organoids contacting the angiogenic sprouts with an FGF pathway activator, a VEGF pathway activator, optionally a Wnt pathway activator, and optionally a growth serum for a first period of time; The method according to any one of embodiments 1 to 30, wherein the method is produced according to the method, thereby forming a vascular organoid.

[0105] 32. The method of embodiment 31, wherein the first period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or any number of days in a range defined by any two of the aforesaid numbers of days, optionally 5 days or at least 5 days.

[0106] 33. Angiogenic sprouts a) contacting the pluripotent stem cells with a Wnt pathway activator and a BMP pathway activator for a second period of time to form vascular lineage cells; b) contacting the vascular lineage cells with a VEGF pathway activator and a second cAMP pathway activator for a third period of time; 33. The method according to embodiment 31 or 32, produced according to the method, thereby forming angiogenic sprouts.

[0107] 34. The method of embodiment 33, wherein the second period of time is 1, 2, 3, 4, or 5 days, or any number of days within a range defined by any two of the foregoing numbers of days, optionally 3 days.

[0108] 35. The method of embodiment 33 or 34, wherein the third period of time is 1, 2, 3, or 4 days, or any number of days within a range defined by any two of the preceding days, optionally 2 days.

[0109] 36. The method of any one of embodiments 33 to 35, wherein the BMP pathway activator is BMP4.

[0110] 37. The method of any one of embodiments 33-36, wherein the BMP pathway activator is provided at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 30 ng / mL.

[0111] 38. The method of any one of embodiments 31 to 37, wherein the Wnt pathway activator is CHIR99021.

[0112] 39. The method of any one of embodiments 31-38, wherein the Wnt pathway activator is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 4 μM, or optionally at or about 12 μM.

[0113] 40. The method of any one of embodiments 33-39, wherein the second cAMP pathway activator is forskolin.

[0114] 41. The method of any one of embodiments 33-40, wherein the second cAMP pathway activator is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 2 μM.

[0115] 42. The method according to any one of embodiments 31 to 41, wherein the vascular organoid is different from the vascular organoid generated without contacting the angiogenic sprouts with a Wnt pathway activator by increasing the expression of blood-brain barrier specific endothelial markers, optionally GLUT-1 and ZO-1.

[0116] 43. The method according to any one of embodiments 1 to 42, wherein the cerebral organoid is contacted with LIF and growth serum to induce astrocyte formation in the cerebral organoid.

[0117] 44. Brain organoids a) contacting pluripotent stem cells with a BMP pathway inhibitor, a TGF-beta pathway inhibitor, and a Wnt pathway inhibitor for a first period of time to form neuroectodermal cells; b) contacting the neuroectodermal cells of step a) with a second TGF-beta pathway inhibitor and a Wnt pathway activator for a second period of time to form neuroepithelial cells; c) contacting the neuroepithelial cells of step b) with insulin for a third period of time to form brain tissue organoids; d) contacting the brain tissue organoids of step c) with GDNF, BDNF, ascorbic acid, and a cAMP pathway activator for a fourth period of time to form brain organoids; Optionally, the brain tissue organoid is further contacted with LIF and growth serum for a portion of a fourth period to induce astrocyte proliferation in the brain organoid, according to any one of the methods described in embodiments 1 to 43.

[0118] 45. The method of embodiment 44, wherein the first period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the foregoing numbers of days, optionally 7 days.

[0119] 46. ​​The method of embodiment 44 or 45, wherein the second period of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding numbers of days, optionally 7 days.

[0120] 47. The method of any one of embodiments 44-46, wherein the third period of time is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the aforesaid numbers of days, optionally 56 days.

[0121] 48. The method of any one of embodiments 44-47, wherein the fourth period of time is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the aforesaid numbers of days.

[0122] 49. The method of any one of embodiments 44-48, wherein the portion of the fourth period is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or any number of days within a range defined by any two of the preceding days, optionally 14 days, optionally wherein the portion of the fourth period is the start of the fourth period.

[0123] 50. The method of any one of embodiments 44 to 49, wherein the BMP pathway inhibitor is LDN-193189.

[0124] 51. The method of any one of embodiments 44 to 50, wherein the BMP pathway inhibitor is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0125] 52. The method of any one of embodiments 44-51, wherein the TGF-beta pathway inhibitor and the second TGF-beta pathway inhibitor are the same or different.

[0126] 53. The method of any one of embodiments 44 to 52, wherein the TGF-beta pathway inhibitor is A83-01.

[0127] 54. The method of any one of embodiments 44-53, wherein the TGF-beta pathway inhibitor is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 2 μM.

[0128] 55. The method of any one of embodiments 44-54, wherein the second TGF-beta pathway inhibitor is SB-431542.

[0129] 56. The method of any one of embodiments 44-55, wherein the second TGF-beta pathway inhibitor is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0130] 57. The method of any one of embodiments 44 to 56, wherein the Wnt pathway inhibitor is IWR-1.

[0131] 58. The method of any one of embodiments 44-57, wherein the Wnt pathway inhibitor is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 3 μM.

[0132] 59. The method of any one of embodiments 44 to 58, wherein the Wnt pathway activator is CHIR99021.

[0133] 60. The method of any one of embodiments 44-59, wherein the Wnt pathway activator is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at or about 1 μM.

[0134] 61. The method of any one of embodiments 44-60, wherein insulin is provided at a concentration of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or any concentration within a range defined by any two of the preceding concentrations, optionally at a concentration of 2.5 μg / mL or about 2.5 μg / mL.

[0135] 62. The method of any one of embodiments 44 to 61, wherein LIF is provided at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL, optionally at or about 10 mg / mL.

[0136] 63. The method of any one of embodiments 44 to 62, wherein the brain organoid comprises astrocytes expressing S100B, GFAP, and AQP4.

[0137] 64. The method according to any one of embodiments 1 to 63, wherein the vascular organoids and / or cerebral organoids are human.

[0138] 65. The method according to any one of embodiments 1 to 64, wherein the vascular organoids and / or cerebral organoids are derived from a subject, optionally a human subject.

[0139] 66. The method of embodiment 65, wherein the subject has a cerebrovascular disease or a disease associated with blood-brain barrier dysfunction, optionally wherein the cerebrovascular disease or a disease associated with blood-brain barrier dysfunction comprises cavernous vascular malformation, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0140] 67. A vascularized cerebral organoid produced by a method according to any one of embodiments 1 to 66.

[0141] 68. Vascularized brain organoids containing endothelial cells connected by tight junctions, astrocytes, and pericytes.

[0142] 69. The vascularized brain organoid of embodiment 68, wherein the endothelial cells express CD31, GLUT-1 and PDGFR-β, the tight junctions contain claudin-5, ZO-1 and cadherin-5, the astrocytes express S100B, GFAP and AQP4, and the pericytes express PDGFR-β, αSMA and NG2.

[0143] 70. The vascularized cerebral organoid of embodiment 68 or 69, wherein the endothelial cells form a continuous basement membrane and express collagen IV.

[0144] 71. A vascularized brain organoid according to any one of embodiments 68 to 70, comprising cells selected from the group consisting of neural precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, cerebrovascular endothelial cells, vascular leptomeningeal cells, perivascular adipocytes, and tendon cells.

[0145] 72. The vascularized brain organoid of any one of embodiments 68 to 71, comprising cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells.

[0146] 73. A vascularized cerebral organoid according to embodiment 71 or 72, wherein the cells are identified by cell type-specific gene expression markers.

[0147] 74. A vascularized cerebral organoid according to any one of embodiments 68 to 73, wherein the blood vessels comprise capillaries.

[0148] 75. A vascularized cerebral organoid according to any one of embodiments 68 to 74, wherein the capillaries are covered by the endfeet of pericytes and astrocytes.

[0149] 76. A method for treating a cerebrovascular disease or a disease associated with blood-brain barrier dysfunction in a subject in need thereof, comprising administering to the subject a vascularized cerebral organoid, or a part or fragment thereof, according to any one of embodiments 67 to 75.

[0150] 77. A screening method comprising contacting a vascularized cerebral organoid or a portion thereof described in any one of embodiments 67 to 75 with a candidate compound or composition, and evaluating the effect of the candidate compound or composition on the vascularized cerebral organoid or a portion thereof.

[0151] 78. The method of embodiment 77, wherein the effect comprises transport of the candidate compound or composition across the blood-brain barrier of the organoid or portion thereof.

[0152] 79. The method of embodiment 77 or 78, wherein the vascularized cerebral organoid is a model for cerebrovascular disease or a disease associated with blood-brain barrier dysfunction, and evaluating the effect of a candidate compound or composition on the vascularized organoid comprises evaluating the effect of a candidate compound or composition on cerebrovascular disease or a disease associated with blood-brain barrier dysfunction.

[0153] 80. The method of any one of embodiments 77 to 79, wherein the vascularized cerebral organoids are generated from cells derived from a subject, and optionally, the cells derived from the subject are induced pluripotent stem cells.

[0154] 81. The method of embodiment 80, wherein the subject has or is susceptible to developing a cerebrovascular disease or a disease associated with blood-brain barrier dysfunction. [Brief description of the drawings]

[0155] In addition to the features described herein, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, it being understood that these drawings illustrate embodiments and are not intended to be limiting in scope. [Figure 1A] FIG. 1 shows one embodiment of a schematic diagram for forming vascular organoids from pluripotent stem cells. [Figure 1B] According to the exemplary schematic diagram of Figure 1A, the bright field and fluorescent images of the vascular organoid differentiated from pluripotent stem cells are shown in one embodiment.The fluorescent images show cells expressing GFP, which early pluripotent stem cells are engineered to express. [Figure 1C] FIG. 1A shows an embodiment of a fluorescent image of vascular organoids differentiated from pluripotent stem cells according to the exemplary schematic diagram. The fluorescent image shows that the cells of the vascular organoids express GFP (which early pluripotent stem cells were engineered to express), platelet endothelial cell adhesion molecule (PECAM-1, CD31) and platelet-derived growth factor receptor beta (PDGFR-β), which are markers of early endothelial and pericyte progenitor cells. The fluorescent image in FIG. 1D shows tubular structures with a lumen, indicating the generation of 3D self-organizing vascular organoids. [Figure 1D]FIG. 1A shows an embodiment of a fluorescent image of vascular organoids differentiated from pluripotent stem cells according to the exemplary schematic diagram. The fluorescent image shows that the cells of the vascular organoids express GFP (which early pluripotent stem cells were engineered to express), platelet endothelial cell adhesion molecule (PECAM-1, CD31) and platelet-derived growth factor receptor beta (PDGFR-β), which are markers of early endothelial and pericyte progenitor cells. The fluorescent image in FIG. 1D shows tubular structures with a lumen, indicating the generation of 3D self-organizing vascular organoids. [Figure 2A] FIG. 1 shows one embodiment of a schematic diagram for generating dorsal forebrain organoids, which are considered a type of cortical organoid, from pluripotent stem cells. [Figure 2B] Figure 2A shows an embodiment of bright field and fluorescent images of forebrain organoids differentiated from pluripotent stem cells according to the exemplary schematic diagram. Fluorescent images show that cells of early stage (day 26) brain organoids express neural cell marker class III beta-tubulin (Tuj1) and neural stem cell marker SRY-Box transcription factor (Sox2). Cells of late stage (day 61) forebrain organoids express neural cell marker B cell lymphoma / leukemia 11B (BCL11B, Ctip1) and T-Box brain transcription factor 1 (Tbr1). [Figure 2C] 1 shows an embodiment of a fluorescent image of forebrain organoids differentiated from pluripotent stem cells and cultured to induce astrocyte formation. The fluorescent image shows that the forebrain organoids contain cells that are positive for the astrocyte markers S100 calcium binding protein B (S100B) and glial fibrillary acidic protein (GFAP). [Figure 2D]1 shows an embodiment of a fluorescent image of forebrain organoids differentiated from pluripotent stem cells and cultured to induce astrocyte formation. The fluorescent image shows that the forebrain organoids contain cells that are positive for the astrocyte markers S100 calcium binding protein B (S100B) and glial fibrillary acidic protein (GFAP). [Figure 3A] FIG. 1 shows one embodiment of a schematic diagram for fusing blood vessel organoid (VO) and forebrain organoid (FBO) in culture to form fused vascularized forebrain organoid (fvFBO). Alternative cortical organoids may be used instead of forebrain organoids. [Figure 3B] FIG. 1 shows one embodiment of a schematic diagram for generating vascularized cerebral organoids from human pluripotent stem cells (hPSCs). Cerebral organoids and vascular organoids are generated separately and then assembled to mimic neurovascular co-generation. [Figure 3C] FIG. 1 shows one embodiment of a schematic diagram of the vascularized brain organoid and its similarity to an in vivo human BBB-like structure with endothelial cells of the capillary walls connected via tight junctions, astrocytic endfeet and pericytic coverings, and neuronal innervation. [Figure 3D] Figure 1 shows an embodiment of an exemplary protocol for culturing vascular organoids and forebrain organoids together to form fused vascularized forebrain organoids (fvFBOs).Vascular organoids and forebrain organoids are directly contacted and fused in culture containing growth factors that promote both neural cell growth and blood vessel growth.Also shown is a fluorescent image showing fused vascularized forebrain organoids with endothelial cell infiltration into brain organoids.Alternative cortical organoids may be used instead of forebrain organoids. [Figure 3E]1 shows an embodiment of a fluorescent image showing the integration of vascular organoids and forebrain organoids in fused vascularized forebrain organoids after 14 and 21 days of culture.As the culture time progresses, endothelial cells (labeled with GFP) gradually infiltrate into the forebrain organoids and form a vascular network within the brain organoids. [Figure 3F] 1 shows an embodiment of a fluorescent image showing the occurrence of CD31-positive brain capillaries in forebrain organoids and the close association of GFAP-positive neural cells with brain capillaries. [Figure 3G] 1 shows one embodiment of a fluorescent image showing co-localization of claudin-5 and glucose transporter 1 (Glut-1) with endothelial cells, indicating an intact blood-brain barrier. [Figure 3H] 1 shows one embodiment of a fluorescent image showing co-localization of claudin-5 and glucose transporter 1 (Glut-1) with endothelial cells, indicating an intact blood-brain barrier. [Figure 3I] FIG. 1 shows an embodiment of an electron microscope image of a cross section of a fused vascularized pre-brain organoid showing the presence of brain capillary lumens, microvesicles (MVs), tight junctions (TJs), and adherens junctions (AJs). [Figure 3J] One embodiment of the fluorescent image of the vascularized brain organoid at day 21 immunostained for GFP, CD31 and collagen IV is shown. The fluorescent image shows that the brain endothelium (CD31) is a key structure that regulates angiogenesis and maintains the BBB defined by the molecular marker collagen IV, and is covered by a continuous basement membrane. [Figure 3K] FIG. 1 shows one embodiment of a fluorescent image showing the presence of GFAP-positive astrocytes in close association with GFP-expressing endothelial cells in fused vascularized forebrain organoids. [Figure 3L]FIG. 1 shows one embodiment of a fluorescent image showing the presence of GFAP-positive astrocytes in close association with GFP-expressing endothelial cells in fused vascularized forebrain organoids. [Figure 3M] FIG. 1 shows one embodiment of a fluorescent image showing the presence of AQP-4 positive astrocytes (importantly, also involved in blood-brain barrier function) in vascularized cerebral organoids. [Figure 3N] One embodiment of a fluorescent image shows the presence of newly formed capillaries covered by astrocyte processes and human pericytes (stained for PDGFR-β, FIG. 3N) in vascularized brain organoids, showing similarities to an in vivo human BBB-like structure with endothelial cells of the capillary walls connected via tight junctions, astrocyte endfeet, and pericyte covering. Also shown in FIG. 3O is the colocalization of cadherin 5 (CDH5) with endothelial cells, indicating the tight junctions of the blood-brain barrier. [Figure 3O] One embodiment of a fluorescent image shows the presence of newly formed capillaries covered by astrocyte processes and human pericytes (stained for PDGFR-β, FIG. 3N) in vascularized brain organoids, showing similarities to an in vivo human BBB-like structure with endothelial cells of the capillary walls connected via tight junctions, astrocyte endfeet, and pericyte covering. Also shown in FIG. 3O is the colocalization of cadherin 5 (CDH5) with endothelial cells, indicating the tight junctions of the blood-brain barrier. [Figure 4A] Figure 1 shows an embodiment of single-cell RNA sequencing of fused vascularized precerebral organoids.The data shows the presence of multiple cell types that compose the brain vascular network, including neurons, astrocytes, and endothelial cells. [Figure 4B] FIG. 1 shows an embodiment of single-cell RNA sequencing data showing the presence of multiple sub-clusters of endothelial cells, suggesting the presence of diverse cell populations in fused vascularized procerebral organoids. [Figure 4C]1 shows an embodiment of a heatmap showing the relative expression of cell markers in cell types identified in single-cell RNA sequencing of fused vascularized prebrain organoids: NPs1: neural precursors, Ast: astrocytes, PAst: proliferative astrocytes, GABAs: GABAergic neurons, GluNs: glutamatergic neurons: VLMCs: vascular leptomeningeal cells, brain VEs: brain endothelial cells, VEs2: vascular endothelial cells 2, PAs: perivascular adipocytes, TCs: tendon cells, PCs: proliferative cells. [Figure 4D] FIG. 1 shows one embodiment of a violin plot illustrating the expression of various neuronal and vascular markers in cell types identified in single-cell RNA sequencing of fused vascularized precerebral organoids. [Figure 4E] Figure 1 shows an embodiment of a map showing the relative abundance of potential interactions between protein receptors and ligands expressed by two different cell types identified in single-cell RNA sequencing of fused vascularized forebrain organoids. The data points shown here relate to vascular cell type to vascular cell type crosstalk. NPs1: neural precursor 1, NPs2: neural precursor 2, PPs: proliferative precursor, GABAs: GABAergic neurons, GluNs: glutamatergic neurons, VLMCs: vascular leptomeningeal cells, VEs1: vascular endothelial cells 1, VEs2: vascular endothelial cells 2, Pas: perivascular adipocytes, TCs: tendon cells, PCs: proliferative cells. [Figure 4F] Figure 1 shows an embodiment of a map showing the relative abundance of potential interactions between protein receptors and ligands expressed by two different cell types identified in single-cell RNA sequencing of fused vascularized pre-brain organoids.The data points shown here relate to crosstalk from neural cell types to vascular cell types. [Figure 4G] Figure 1 shows an embodiment of a map showing the relative abundance of potential interactions between protein receptors and ligands expressed by two different cell types identified in single-cell RNA sequencing of fused vascularized forebrain organoids.The data points shown here are related to crosstalk from neuronal cell type to neuronal cell type. [Figure 5A] 1 shows an embodiment of single-cell RNA sequencing data from vascularized brain organoids at day 30. The data shows the presence of multiple cell types that compose the complete neurovascular unit, including excitatory neurons, inhibitory neurons, neural progenitors (NPs), astrocytes, endothelial cells (ECs), pericytes, mesenchymal stem cells (MSCs), smooth muscle cells (SMCs), and fibroblasts. [Figure 5B] Figure 1 shows an embodiment of gene expression analysis of endothelial cells in vascularized brain organoid.Compare gene expression data from endothelial cells in vascularized brain organoid with gene expression data from organ-specific endothelial cells generated by Tabula Muris Consortium.Data shows that endothelial cells in vascularized brain organoid show the same gene expression pattern as brain microvascular endothelial cells (BMEC), but do not show the same gene expression pattern as other organ-specific endothelial cells, indicating the endothelial acquisition of brain-specific transcriptome signature in vascularized brain organoid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0156] The blood-brain barrier (BBB) ​​provides a critical boundary to limit exposure of the central nervous system from the rest of the body by regulating the transport of essential molecules such as oxygen, carbon dioxide, and nutrients, but preventing the passage of other molecules as well as larger biological entities such as cells and pathogens. The BBB is mediated by the formation of tight junctions between the endothelial cells that make up the blood vessels and capillaries in the brain. Additional nearby cells such as astrocytes and pericytes also support the endothelial cells to maintain the BBB. This highly organized structure, which forms the boundary between the vascular and neuronal parts of the model, is necessary for a model of the BBB to be representative of the native structure.

[0157] Described herein are human blood-brain barrier models that are generated by vascularizing human brain organoids. These blood-brain barrier models can be used to model and study cerebrovascular disorders. These vascularized brain organoids can be transplanted in vivo, such as into mouse cortex, to reconstitute active brain perfusion and integrate the organoids with living animals for advanced function and in vivo study of blood-brain barrier. These organoids can also serve as a powerful drug screening platform to evaluate drug delivery through the blood-brain barrier.

[0158] term In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0159] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs when read in light of this disclosure. For purposes of this disclosure, the following terms are described below.

[0160] The disclosure herein uses categorical language to describe numerous embodiments, and the disclosure also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is completely or partially excluded.

[0161] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0162] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 10% from the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0163] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the recited steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements may not be present. "Consisting essentially of" means the inclusion of any elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action set forth in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they have a substantial effect on the activity or action of the recited elements.

[0164] The terms "individual", "subject" or "patient" as used herein have their common and usual meaning as understood in the context of this specification, and refer to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as any other vertebrate or invertebrate. The term "mammal" is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, and the like.

[0165] The term "effective amount" or "effective dose" as used herein has its common and usual meaning as understood in the context of this specification and refers to that amount of the described composition or compound that produces an observable effect. The actual dosage level of the active ingredients in the active composition of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound that is effective to achieve a desired response for a particular subject and / or application. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the composition, the formulation, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Contemplated herein are the determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments.

[0166] The terms "function" and "functional" as used herein have their common and ordinary meaning as understood in the context of this specification and refer to biological, enzymatic, or therapeutic functions.

[0167] The term "inhibit" as used herein has its common and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be, about, at least, at least about, less than, or a percentage that is about, less than, or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount within a range defined by any two of the foregoing values. The term "delay" as used herein has its common and ordinary meaning as understood in light of the present specification and refers to a delay, postponement, or postponement of a biological event to a later time than would otherwise be expected. The delay may be 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about, at least about, less than, or a percentage less than about, or an amount within a range defined by any two of the preceding values. The terms inhibition and delay do not necessarily indicate 100% inhibition or delay. Partial inhibition or delay may be achieved.

[0168] As used herein, the term "isolated" has its common and ordinary meaning as understood in light of the present specification and refers to a substance and / or entity that is (1) separated from at least some of the components with which it is associated when originally produced (in nature and / or in an experimental environment) and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from equal to, about, at least about, less than, or about less than 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or substantially 100% (or ranges including and / or spanning the foregoing values) of other components with which they are originally associated. In some embodiments, an isolated agent is, about, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure, about, at least about, at least about, less than, or less than (or ranges including and / or spanning the aforementioned values). As used herein, an "isolated" material can be "pure" (e.g., substantially free of other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0169] As used herein, "in vivo" is given its ordinary and ordinary meaning as understood in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, as opposed to tissue extracts or dead organisms.

[0170] As used herein, "ex vivo" is given its ordinary and usual meaning as understood in light of the present specification and refers to the performance of the method outside the body with little change in natural conditions.

[0171] As used herein, "in vitro" is given its common and ordinary meaning as understood in the context of this specification and refers to the performance of methods outside biological conditions, for example in a petri dish or test tube.

[0172] The terms "nucleic acid" or "nucleic acid molecule" as used herein have their common and ordinary meaning as understood in the context of this specification and refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those naturally occurring in cells, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Furthermore, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications of the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotides" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid may be contained in a nucleic acid vector or construct (e.g., a plasmid, a virus, a retrovirus, a lentivirus, a bacteriophage, a cosmid, a fosmid, a phagemid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a human artificial chromosome (HAC)) that can be used for amplification and / or expression of the nucleic acid in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, an enhancer, a terminator, an inducer, a ribosome binding site, a translation initiation site, a start codon, a stop codon, a polyadenylation signal, an origin of replication, a cloning site, a multiple cloning site, a restriction enzyme site, an epitope, a reporter gene, a selection marker, an antibiotic selection marker, a targeting sequence, a peptide purification tag, or an accessory gene, or any combination thereof.

[0173] A nucleic acid or nucleic acid molecule can include one or more sequences encoding different peptides, polypeptides, or proteins, which can be adjacent in the same nucleic acid or nucleic acid molecule, or can be joined with extra nucleic acid, for example, between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, or any length within the range defined by any two of the aforementioned lengths. The term "downstream" as used herein with respect to a nucleic acid has its common and usual meaning as understood in the context of this specification and refers to the sequence behind the 3' end of the previous sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded. The term "upstream" as used herein with respect to a nucleic acid has its common and usual meaning as understood in the context of this specification and refers to the sequence ahead of the 5' end of the subsequent sequence on the strand containing the coding sequence (sense strand) if the nucleic acid is double-stranded.The term "grouping" as used herein with respect to nucleic acids has its ordinary and usual meaning as understood in the context of the present specification and refers to two or more sequences that occur either directly or in close proximity with, for example, extra nucleic acid between linkers, repeats, or restriction enzyme sites, or with any other sequence that is, about, at least, at least about, less than, or about less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, but generally not with sequences that are in between that encode a functional or catalytic polypeptide, protein, or protein domain.

[0174] The nucleic acid described herein comprises nucleobases. The primary, normal, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil. Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases or biotin-labeled bases.

[0175] The terms "peptide", "polypeptide" and "protein" as used herein have their common and usual meaning as understood in light of the present specification and refer to a polymer composed of amino acids linked by peptide bonds. Many functions of peptides, polypeptides and proteins are known in the art, including, but not limited to, enzymatic, structural, transport, defensive, hormonal or signal transduction. Peptides, polypeptides and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating the nucleic acid template, peptide, polypeptide and protein mutations such as substitution, deletion, truncation, addition, duplication or fusion of two or more peptides, polypeptides or proteins can be performed. These fusions of two or more peptides, polypeptides, or proteins can be adjacent in the same molecule or can be joined with extra amino acids between, for example, linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about, at least, at least about, less than, or about, less than, or any length within the range defined by any two of the aforementioned lengths. As used herein, the term "downstream" in reference to a polypeptide has its ordinary and usual meaning as understood in the context of this specification and refers to a sequence following the C-terminus of the preceding sequence. The term "upstream" as used herein with respect to a polypeptide has its ordinary and usual meaning as understood in the context of this specification and refers to the sequence preceding the N-terminus of the subsequent sequence.

[0176] The term "purity" of any given substance, compound, or material as used herein has its common and ordinary meaning as understood in light of the present specification, and refers to the actual abundance of the substance, compound, or material compared to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimal points therebetween. Purity may be affected by undesirable impurities, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious infectious agents. Purity can be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0177] The term "yield" of any given substance, compound, or material as used herein has its ordinary and usual meaning as understood in light of the present specification, and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be, about, at least, at least about, less than, or about less than 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected total amount, including all decimal points therebetween. Yield may be affected by the efficiency of a reaction or process, undesired side reactions, decomposition, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of production.

[0178] As used herein, "pharmacologically acceptable" has its ordinary and usual meaning as understood in the context of the present specification and refers to a carrier, excipient, and / or stabilizer that is non-toxic or has an acceptable level of toxicity to cells or mammals exposed at the dosages and concentrations used. As used herein, "pharmacologically acceptable", "diluent", "excipient", and / or "carrier" have their ordinary and usual meaning as understood in the context of the present specification and are intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those diluents, excipients, and / or carriers approved by federal, state, or other regulatory agencies or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, including humans and non-human mammals such as cats and dogs. The terms diluent, excipient, and / or "carrier" can refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous solutions of dextrose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.Physiologically acceptable carriers may also include one or more of the following: antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, carbohydrates such as amino acids, glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions can also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. The formulation should suit the mode of administration.

[0179] Cryoprotectants are cell composition additives to improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cryopreservation medium that includes other components such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum, FCS) to increase the survival rate of cells after thawing. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, about, at least about, less than, or equal to 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the foregoing numbers.

[0180] Additional excipients having desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugar, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of excipient may be found in the composition at, about, at least, at least about, less than, or equal to 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, or any weight percentage within a range defined by any two of the foregoing numbers.

[0181] The term "pharmaceutically acceptable salts" has its common and ordinary meaning as understood in the context of this specification and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such organic bases may include, but are not limited to, mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids, including glycine, arginine, and lysine; guanidine, N-methylglucosamine, N-methylglucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, trihydroxymethylaminoethane.

[0182] The appropriate formulation will vary depending on the route of administration selected. Techniques for the formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques for administering compounds exist in the art, including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, intravenous), intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injection. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0183] As used herein, "carrier" has its common and ordinary meaning as understood in light of the present specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or body organs.

[0184] As used herein, "diluent" has its common and usual meaning as understood in light of the present specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharma- ceutically necessary or desirable. For example, a diluent can be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It can also be a liquid for dissolving a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline, which mimics the composition of human blood.

[0185] The term "w / w%" or "weight / weight %" as used herein has its ordinary and usual meaning as understood in the context of this specification and refers to a percentage expressed in terms of the weight of a component or agent relative to the total weight of the composition, multiplied by 100. The term "v / v%" or "volume / volume %" as used herein has its ordinary and usual meaning as understood in the context of this specification and refers to a percentage expressed in terms of the liquid volume of a compound, substance, component or agent relative to the total liquid volume of the composition, multiplied by 100.

[0186] stem cells The term "totipotent stem cells" (also known as omnipotent stem cells) as used herein has its ordinary and usual meaning as understood in the context of this specification, and is a stem cell that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. These cells are produced from the fusion of an egg and a sperm cell. The cells produced by the first few divisions of a fertilized egg are also totipotent.

[0187] As used herein, the term "embryonic stem cells (ESCs)", commonly abbreviated as ES cells, has its common and ordinary meaning as understood in the context of this specification and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of this disclosure, the term "ESCs" may be used broadly to encompass embryonic germ cells.

[0188] The term "pluripotent stem cell (PSC)" as used herein has its common and ordinary meaning as understood in the context of this specification and includes any cell that can differentiate into almost any cell type of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the descendants of inner cell mass cells of a preimplantation blastocyst, or may be obtained by induction of non-pluripotent cells, e.g., adult somatic cells, by forcing the expression of certain genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0189] As used herein, the term "induced pluripotent stem cell (iPSC)" has its common and usual meaning as understood in the context of this specification, and is generally abbreviated as iPS cell, and refers to a type of pluripotent stem cell artificially derived from normally non-pluripotent cells, such as adult somatic cells, by inducing "forced" expression of certain genes. hiPSC refers to human iPSC. In some methods known in the art, iPSC can be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes can include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes can also improve the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, retroviral systems are used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, lentiviral systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (POU5F1), certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof. Other methods of generating induced pluripotent stem cells conventionally known in the art are also contemplated.

[0190] The term "progenitor cell" as used herein has its common and ordinary meaning as understood in light of the present specification and encompasses any cell that can be used in the methods described herein, during which one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, the progenitor cell is pluripotent or has the ability to become pluripotent. In some embodiments, the progenitor cell is subjected to treatment with an external factor (e.g., growth factor) to acquire pluripotency. In some embodiments, the progenitor cell can be a totipotent (or omnipotent) stem cell, a pluripotent stem cell (artificial or non-artificial), a multipotent stem cell, an oligopotent stem cell, and a unipotent stem cell. In some embodiments, the progenitor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, the progenitor cell can be a somatic cell that is subjected to treatment to confer pluripotency via genetic engineering or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), epiblast stem cells (EpiSCs), and induced pluripotent stem cells.

[0191] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "differentiation" or "directed differentiation" describes the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or modify the fate of the initial cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0192] The term "feeder cells" as used herein has its common and usual meaning as understood in light of the present specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth arrested by irradiation (e.g., gamma radiation), mitomycin-C treatment, electrical pulses, or mild chemical fixation (e.g., with formaldehyde or glutaraldehyde). However, feeder cells are not necessarily growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which may affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human forehead fibroblasts, human skin fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are not used during the expansion of target stem cells.

[0193] cell differentiation In some embodiments, known methods for generating downstream cell types from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, the pluripotent cells are derived from morulae. In some embodiments, the pluripotent stem cells are stem cells. Stem cells used in these methods include, but are not limited to, embryonic stem cells, or induced pluripotent stem cells. Embryonic stem cells can be derived from the inner cell mass of an embryo or the gonadal ridges of an embryo. Embryonic stem cells or germ cells can originate from a variety of animal species, including, but not limited to, various mammalian species, including humans.

[0194] In some embodiments, the pluripotent stem cells are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time period that is, is about, is at least, is at least about, is less than, or is less than about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours, or any time period within a range defined by any two of the aforementioned times, such as 6 hours to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the two or more small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0195] In some embodiments, the pluripotent stem cells are cultured in a growth medium that supports stem cell growth. In some embodiments, the pluripotent stem cells are cultured in a stem cell growth medium. In some embodiments, the stem cell growth medium is RPMI1640, DMEM, DMEM / F12, Advanced DMEM / F12. In some embodiments, the stem cell growth medium includes fetal bovine serum (FBS). In some embodiments, the stem cell growth medium comprises FBS at a concentration that is, is about, is at least about, is less than, or is less than 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the foregoing concentrations, e.g., 0%-20%, 0.2%-10%, 2%-5%, 0%-5%, or 2%-20%. In some embodiments, the stem cell growth medium does not contain xenogeneic components. In some embodiments, the growth medium comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0196] In some embodiments, the pluripotent stem cells are prepared from somatic cells. In some embodiments, the pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, the pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, the pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, the PBMCs are grown on a feeder cell substrate. In some embodiments, the PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, the PBMCs are grown on an irradiated MEF feeder cell substrate.

[0197] In some embodiments, the iPSCs are expanded in cell culture. In some embodiments, the iPSCs are expanded in Matrigel. In some embodiments, the iPSCs are expanded in cell culture medium containing a ROCK inhibitor (e.g., Y-27632).

[0198] In some aspects, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof are contacted with a Wnt pathway activator or a Wnt pathway inhibitor. In some embodiments, the Wnt pathway activator comprises a Wnt protein. In some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt pathway activator comprises Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt pathway activator comprises a GSK3 pathway inhibitor. In some embodiments, the Wnt pathway activator comprises CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD8, or TWS119, or any combination thereof. In some embodiments, the Wnt pathway inhibitor comprises IWR-1, C59, PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells are not treated with a Wnt pathway activator or Wnt pathway inhibitor. The Wnt pathway activators or Wnt pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0199] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof are contacted with an FGF pathway activator. In some embodiments, the FGF pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF pathway activator comprises one or more of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15 (FGF19, FGF15 / FGF19), FGF 16, FGF 17, FGF 18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with an FGF pathway activator. The FGF pathway activators provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0200] In some aspects, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof are contacted with a BMP pathway activator or a BMP pathway inhibitor. In some embodiments, the BMP pathway activator comprises a BMP protein. In some embodiments, the BMP protein is a recombinant BMP protein. In some embodiments, the BMP pathway activator comprises BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, IDE1, or IDE2, or any combination thereof. In some embodiments, the BMP pathway inhibitor comprises noggin, dorsomorphin, RepSox, LY364947, LDN-193189, SB-431542, or any combination thereof. In some embodiments, the cells are not treated with a BMP pathway activator or a BMP pathway inhibitor. The BMP pathway activators or BMP pathway inhibitors provided herein can be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0201] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with a VEGF pathway activator. In some embodiments, the VEGF pathway activator comprises one or more of VEGF or GS4012. In some embodiments, the cells are not treated with a VEGF pathway activator. The VEGF pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0202] In some aspects, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof are contacted with a TGF-beta (TGF-b) pathway activator or a TGF-b pathway inhibitor. In some embodiments, the TGF-b family includes bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Mullerian hormone, activin, and nodal pathways. In some embodiments, the TGF-b pathway activators include TGF-b 1, TGF-b 2, TGF-b 3, activin A, activin B, nodal, BMPs, IDE1, IDE2, or any combination thereof. In some embodiments, the TGF-b pathway inhibitors include A8301, RepSox, LY365947, SB-431542, or any combination thereof. In some embodiments, the cells are not treated with a TGF-b pathway activator or a TGF-b pathway inhibitor. The TGF-b pathway activators or TGF-b pathway inhibitors provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0203] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with a cAMP pathway activator. In some embodiments, the cAMP pathway activator comprises forskolin or cAMP. In some embodiments, the cells are not treated with a cAMP pathway activator. The cAMP pathway activators provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0204] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with ascorbic acid. In some embodiments, the cells are not treated with ascorbic acid. Ascorbic acid provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0205] In some embodiments, the pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with leukemia inhibitory factor (LIF). In some embodiments, the cells are not treated with LIF. The LIF provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0206] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with glial cell line-derived neurotrophic factor (GDNF). In some embodiments, the cells are not treated with GDNF. GDNF provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0207] In some embodiments, pluripotent stem cells, mesoderm, vascular lineage cells, ectoderm, neural lineage cells, or any combination thereof, are contacted with brain-derived neurotrophic factor (BDNF). In some embodiments, the cells are not treated with BDNF. BDNF provided herein may be used in combination with any of the other growth factors, pathway activators, or pathway inhibitors provided herein.

[0208] In some embodiments, the cells are contacted with any of the small molecule compounds, pathway activators, pathway inhibitors, or growth factors for a period of time that is, is about, is at least, is at least about, is less than, or is less than about, or any period of time within a range defined by any two of the preceding periods, for example, 1 hour to 300 hours, 24 hours to 120 hours, 48 ​​hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, two or more small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the two or more small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0209] In some embodiments, PSCs are differentiated into mesodermal cells. In some embodiments, PSCs are differentiated into vascular lineage cells. In some embodiments, PSCs are differentiated into vascular organoids. In some embodiments, PSCs are differentiated into ectodermal cells. In some embodiments, PSCs are differentiated into neural lineage cells. In some embodiments, PSCs are differentiated into cortical organoids.

[0210] In some embodiments, any of the cells disclosed herein can be cryopreserved for later use. In some embodiments, the cells are cryopreserved according to methods commonly known in the art.

[0211] How to Generate Vascular Organoids Exemplary methods for generating vascular (vascular) organoids from pluripotent stem cells can be found in Wimmer et al. Generation of blood vessel organoids from human pluripotent stem cells. Nature Protocols (2019) 14(11): 3082-3100 and Wimmer et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature (2019) 565(7740): 505-510, each of which is expressly incorporated herein by reference in its entirety. A schematic diagram of an improved method for generating vascular organoids from pluripotent stem cells is shown in FIG. 1A. The method may include the use of Wnt pathway activators, such as CHIR99021, during differentiation to generate endothelial cells, similar to those found in cerebral blood vessels.

[0212] Disclosed herein is a method for generating vascular organoid.In some embodiments, the method comprises contacting angiogenic sprouts with Wnt pathway activator, FGF pathway activator, VEGF pathway activator, and optionally growth serum for a first period, thereby forming vascular organoid.In some embodiments, the method comprises contacting angiogenic sprouts with FGF pathway activator, VEGF pathway activator, optionally Wnt pathway activator, and optionally growth serum for a first period, thereby forming vascular organoid.

[0213] In some embodiments, the angiogenic sprouts are derived from pluripotent stem cells, e.g., induced pluripotent stem cells. In some embodiments, the angiogenic sprouts are cultured in a basement membrane matrix. In some embodiments, the angiogenic sprouts are cultured in collagen I and / or matrigel. In some embodiments, the first period of time is, is about, is at least, is at least about, is less than, or is less than, or is any number of days within a range defined by any two of the preceding days, e.g., 1-30 days, 1-10 days, 5-20 days, 10-30 days, or 5-25 days. In some embodiments, the first period of time is 5 days or at least 5 days. In some embodiments, the angiogenic sprouts are generated according to a method comprising: a) contacting pluripotent stem cells with a Wnt pathway activator and a BMP pathway activator for a second period of time to form vascular lineage cells; and b) contacting the vascular lineage cells with a VEGF pathway activator and a second cAMP pathway activator for a third period of time, thereby forming angiogenic sprouts. In some embodiments, the vascular lineage cells are cultured in a basement membrane matrix. In some embodiments, the vascular lineage cells are cultured in collagen I and / or matrigel. In some embodiments, the second period of time is, is about, is at least, is at least about, is less than, or is less than about, 1, 2, 3, 4, or 5 days, or any number of days within a range defined by any two of the foregoing days, e.g., 1-5 days, 1-3 days, or 3-5 days. In some embodiments, the second period of time is 3 days.In some embodiments, the third period of time is, is about, is at least, is at least about, is less than, or is less than about 1, 2, 3, or 4 days, or any number of days within a range defined by any two of the foregoing numbers of days, e.g., 1-4 days, 1-2 days, or 2-4 days. In some embodiments, the third period of time is 2 days.

[0214] In some embodiments, the BMP pathway activator is provided at a concentration of, about, at least about, less than, or equal to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, e.g., 10-100 ng / mL, 10-30 ng / mL, 30-100 ng / mL, or 20-70 ng / mL. In some embodiments, the BMP pathway activator is provided at a concentration of 30 ng / mL or about 30 ng / mL. In some embodiments, the BMP pathway activator is BMP4.

[0215] In some embodiments, the Wnt pathway activator is provided at a concentration of, about, at least about, at most, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, or any concentration within a range defined by any two of the preceding concentrations, such as 1-20 μM, 1-12 μM, 4-12 μM, 4-20 μM, 2-6 μM, or 10-15 μM. In some embodiments, the Wnt pathway activator is provided at a concentration of 4 μM or about 4 μM. In some embodiments, the Wnt pathway activator is provided at a concentration of 12 μM or about 12 μM. In some embodiments, the Wnt pathway activator is CHIR99201.

[0216] In some embodiments, the second cAMP pathway activator is provided at a concentration that is, is about, is at least about, is at most, is at most, is at most, is at most, is at most, is at most, is at most, or is at most, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, e.g., 0.5-4 μM, 0.5-2 μM, 2-4 μM, or 1-3 μM. In some embodiments, the second cAMP pathway activator is provided at a concentration of 2 μM or about 2 μM. In some embodiments, the second cAMP pathway activator is forskolin.

[0217] In some embodiments, the growth serum is provided at a concentration that is, is about, is at least about, is less than, or is less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any concentration within a range defined by any two of the preceding concentrations, e.g., 0.5%-20%, 0.5%-5%, 1%-15%, 10-15%, 15-20%, or 12-18%. In some embodiments, the growth serum is provided at or about 15%. In some embodiments, the growth serum is provided at or about 1%. In some embodiments, the growth serum is (FBS).

[0218] In some embodiments, the VEGF pathway activator is provided at a concentration that is, is about, is at least about, is at most, is less than, or is less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the preceding concentrations, e.g., 10-150 ng / mL, 10-100 ng / mL, 100-150 ng / mL, or 80-120 ng / mL. In some embodiments, the VEGF pathway activator is provided at or about 100 ng / mL. In some embodiments, the VEGF pathway activator is VEGF.

[0219] In some embodiments, the FGF pathway activator is provided at a concentration that is, is about, is at least about, is at most, is less than, or is about less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-150 ng / mL, 10-100 ng / mL, 100-150 ng / mL, or 80-120 ng / mL. In some embodiments, the FGF pathway activator is provided at or about 100 ng / mL. In some embodiments, the FGF signaling pathway activator is FGF2.

[0220] In some embodiments, vascular organoids are different from the vascular organoids generated without contacting the cells of step b) with the Wnt pathway activator in step c) in that they have increased expression of blood-brain barrier specific endothelial markers.In some embodiments, blood-brain barrier specific endothelial markers include glucose transporter 1 (GLUT-1) and zonula occludens-1 (tight junction protein-1, ZO-1).In some embodiments, vascular organoids include endothelial cells that express CD31 and pericyte cells that express PDGFR-β, or their precursors.

[0221] How to Generate Brain Organoids Exemplary methods for generating brain organoids from pluripotent stem cells can be found in Qian et al. Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell (2016) 165(5): 1238-1254 and Qian et al. Generation of human brain region-specific organoids using a miniaturized spinning bioreactor. Nature Protocols (2018) 13(3): 565-580, each of which is expressly incorporated herein by reference in its entirety. A schematic diagram of an exemplary method for generating dorsal forebrain organoids, a type of cortical (brain) organoid, is provided in FIG. 2A. These methods can be adapted to generate alternative types of cortical organoids, such as midbrain, striatal brain, hypothalamic, hippocampal, or spinal cord organoids. The methods may involve the use of LIF and / or fetal bovine serum during differentiation to induce the formation of astrocytes in the brain organoids.

[0222] Disclosed herein is a method for generating brain organoid.In some embodiments, the method comprises: a) contacting pluripotent stem cells with BMP pathway inhibitor, TGF-beta pathway inhibitor and Wnt pathway inhibitor for a first period, forming neuroectodermal cells; b) contacting the neuroectodermal cells of step a) with a second TGF-beta pathway inhibitor and Wnt pathway activator for a second period, forming neuroepithelial cells; c) contacting the neuroepithelial cells of step b) with insulin for a third period, forming brain tissue organoid; and d) contacting the brain tissue organoid of step c) with GDNF, BDNF, ascorbic acid and cAMP pathway activator for a fourth period, forming brain organoid.In some embodiments, the brain tissue organoid is further contacted with LIF and growth serum for a part of the fourth period, to induce astrocyte proliferation in brain organoid. In some embodiments, the first period of time is, is about, is at least, is at least about, is less than, or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding days, such as 1-14 days, 1-7 days, 7-14 days, or 5-10 days. In some embodiments, the first period of time is 7 days. In some embodiments, the second period of time is, is about, is at least, is at least about, is less than, or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding days, such as 1-14 days, 1-7 days, 7-14 days, or 5-10 days. In some embodiments, the second period of time is 7 days.In some embodiments, the third period of time is, is about, is at least about, is less than, or is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days in a range defined by any two of the foregoing numbers of days, e.g., 20-70 days, 20-60 days, 40-70 days, or 40-60 days. In some embodiments, the third period is 56 days. In some embodiments, the fourth period is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 In some embodiments, the portion of the fourth period is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 17, 18, 19, 20, or 21 days, or is about, at least, at least about, at most, or is ... In some embodiments, the portion of the fourth time period is 14 days. In some embodiments, the portion of the fourth time period is the beginning of the fourth time period.In some embodiments, the brain organoid comprises cells that express Tuj1, Sox2, Ctip1, Tbr1, or any combination thereof.In some embodiments, the brain organoid comprises astrocytes that express S100B, GFAP, and AQP4.

[0223] In some embodiments, the BMP pathway inhibitor is provided at a concentration that is, is about, is at least about, is less than, or is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, such as 0.1-2 μM, 0.1-1 μM, 1-2 μM, or 0.5-1.5 μM. In some embodiments, the BMP pathway inhibitor is provided at a concentration of 1 μM or about 1 μM. In some embodiments, the BMP pathway inhibitor is LDN-193189.

[0224] In some embodiments, the TGF-beta pathway inhibitor and the second TGF-beta pathway inhibitor are the same or different. In some embodiments, the TGF-beta pathway inhibitor is provided at a concentration that is, is about, is at least, is at least about, is less than, or is less than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 μM, or any concentration within a range defined by any two of the preceding concentrations, such as 0.5-4 μM, 0.5-2 μM, 2-4 μM, or 1-3 μM. In some embodiments, the TGF-beta pathway inhibitor is provided at a concentration of 2 μM or about 2 μM. In some embodiments, the second TGF-beta pathway inhibitor is provided at a concentration that is, is about, is at least about, is less than, or is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 1-2 μM, or 0.5-1.5 μM. In some embodiments, the second TGF-beta pathway inhibitor is provided at a concentration of 1 μM or about 1 μM. In some embodiments, the TGF-beta pathway inhibitor and / or the second TGF-beta pathway inhibitor is A83-01. In some embodiments, the TGF-beta pathway inhibitor and / or the second TGF-beta pathway inhibitor is SB-431542.

[0225] In some embodiments, the Wnt pathway inhibitor is provided at a concentration that is, is about, is at least about, is at most, is at most, is at most, is at most, is at most, is at most, or is at most, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.5-5 μM, 0.5-3 μM, 3-5 μM, or 2-4 μM. In some embodiments, the Wnt pathway inhibitor is provided at a concentration of 3 μM or about 3 μM. In some embodiments, the Wnt pathway inhibitor is IWR-1.

[0226] In some embodiments, the Wnt pathway activator is provided at a concentration that is, is about, is at least about, is less than, or is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 μM, or any concentration within a range defined by any two of the preceding concentrations, e.g., 0.1-2 μM, 0.1-1 μM, 1-2 μM, or 0.5-1.5 μM. In some embodiments, the Wnt pathway activator is provided at a concentration of 1 μM or about 1 μM. In some embodiments, the Wnt pathway activator is CHIR99021.

[0227] In some embodiments, insulin is provided at a concentration that is, is about, is at least about, is less than, or is about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 μg / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 0.5-5 μg / mL, 0.5-2.5 μg / mL, 2.5-5 μg / mL, or 1-3 μg / mL. In some embodiments, insulin is provided at a concentration of 2.5 μg / mL or about 2 μg / mL.

[0228] In some embodiments, LIF is provided at a concentration that is, is about, is at least about, is less than, or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL, e.g., 1-20 mg / mL, 1-10 mg / mL, 10-20 mg / mL, or 5-15 mg / mL. In some embodiments, LIF is provided at a concentration of 10 mg / mL or about 10 mg / mL.

[0229] Vascularized brain organoids and methods for producing vascularized brain organoids A method for generating vascularized brain organoid is disclosed herein. In some embodiments, the method comprises contacting vascular organoid with brain organoid, and culturing the vascular organoid and brain organoid for a period of time until the vascular organoid and the brain organoid fuse together and the blood vessels of the vascular organoid infiltrate into the brain organoid, and the neurons of the brain organoid innervate the blood vessels of the vascular organoid that infiltrate into the organoid, thereby forming a vascularized brain organoid. In some embodiments, the vascularized brain organoid comprises a blood-brain barrier formed between the brain organoid and all or part of the blood vessels of the vascular organoid that infiltrate into the brain organoid. In some embodiments, the blood-brain barrier comprises endothelial cells connected by tight junctions, astrocytes, and pericytes. In some embodiments, the endothelial cells express CD31, GLUT-1, and PDGFR-β. In some embodiments, the tight junction comprises claudin-5. In some embodiments, the endothelial cells express CD31, GLUT-1, and PDGFR-β. In some embodiments, the tight junctions include claudin-5, ZO-1, and cadherin 5. In some embodiments, the astrocytes express S100B, GFAP, and AQP4. In some embodiments, the pericytes express PDGFR-β, α-smooth muscle actin (αSMA), and neural / glial antigen 2 (NG2). In some embodiments, the endothelial cells form a continuous basement membrane and express collagen IV. In some embodiments, the vascularized brain organoids include cells selected from the group consisting of neural precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, brain vascular endothelial cells, vascular leptomeningeal cells, perivascular adipocytes, and tendon cells.In some embodiments, the vascularized brain organoids comprise cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells. In some embodiments, the cells of the vascular brain organoids are identified by cell type specific gene expression markers. In some embodiments, the cells of the vascular brain organoids are identified by cell type specific gene expression markers. In some embodiments, the blood vessels comprise capillaries. In some embodiments, the capillaries are covered by the endfeet of pericytes and astrocytes. In some embodiments, the brain organoids are forebrain organoids, midbrain organoids, hypothalamic organoids, hippocampal organoids, spinal cord organoids, or striatal brain organoids. In some embodiments, the vascular organoids and the brain organoids are contacted and / or cultured in a basement membrane matrix or a component thereof, optionally Matrigel. In some embodiments, the vascular organoids and the brain organoids are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 101, 104, 105, 106, 107, 108, 109, 109, 109, 1 The organoids are cultured for a period of 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, about, at least, at least about, less than, or about less than 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the preceding days, such as 1-70 days, 1-50 days, 30-70 days, or 30-60 days. In some embodiments, the vascular organoids and cerebral organoids are cultured with agitation for at least a portion of the period. In some embodiments, the agitation comprises shaking.In some embodiments, vascular and cerebral organoids are cultured 1) without agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or any number of days within a range defined by any two of the preceding days, e.g., 1-14 days, 1-7 days, 7-14 days, or 5-10 days, and then 2) with agitation for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 9 The organoids may be cultured for 3, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days, or any number of days within a range defined by any two of the preceding days, such as 1-70 days, 1-50 days, 30-70 days, or 30-60 days. In some embodiments, the vascular organoids and cerebral organoids are cultured in a medium that promotes neuronal and / or vascular growth. In some embodiments, vascular organoids and brain organoids are cultured in a medium that includes growth factors that promote neuronal growth and / or growth factors that promote blood vessel growth. In some embodiments, the growth factors that promote neuronal growth include cAMP pathway activators, ascorbic acid, BDNF, GDNF, or any combination thereof. In some embodiments, the growth factors that promote blood vessel growth include growth serum, VEGF pathway activators, FGF pathway activators, or any combination thereof. In some embodiments, vascular organoids are generated according to the adaptation of the methods provided herein or methods generally known in the art. In some embodiments, brain organoids are generated according to the adaptation of the methods provided herein or methods generally known in the art.

[0230] In some embodiments, culturing vascular organoid and cerebral organoid comprises: a) culturing vascular organoid and cerebral organoid for 4, 5, 6, 7, 8, 9 or 10 days without stirring; and b) culturing the organoid of step a) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days with stirring.In some embodiments, the organoid of step a) and step b) is cultured in a medium that comprises growth factor that promotes neuronal growth and / or growth factor that promotes blood vessel growth. In some embodiments, the vascular organoid and the cerebral organoid are cultured in step a) for 7 days without stirring. In some embodiments, the organoid in step a) is cultured in step b) for at least 30 days with stirring. In some embodiments, the stirring comprises shaking. In some embodiments, the growth factor that promotes neuronal growth comprises cAMP pathway activator, ascorbic acid, BDNF, GDNF, or any combination thereof. In some embodiments, the growth factor that promotes blood vessel growth comprises FBS, VEGF, FGF2, or any combination thereof. In some embodiments, the vascular organoid and the cerebral organoid are cultured in basement membrane matrix or its components, for example, Matrigel.

[0231] In some embodiments of any of the methods disclosed herein, the cAMP pathway activator is provided at a concentration that is, is about, is at least about, is less than, or is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-150 μM, 10-50 μM, 50-150 μM, or 20-100 μM. In some embodiments, the cAMP pathway activator is provided at or about 50 μM. In some embodiments, the cAMP pathway activator is cAMP.

[0232] In some embodiments of any of the methods disclosed herein, ascorbic acid is provided at a concentration that is, is about, is at least about, is less than, or is about equal to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μM, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 50-300 μM, 50-200 μM, 200-300 μM, or 150-250 μM. In some embodiments, ascorbic acid is provided at or about 200 μM.

[0233] In some embodiments of any of the methods disclosed herein, BDNF is provided at a concentration that is, is about, is at least about, is less than, or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-30 ng / mL, 10-20 ng / mL, 20-30 ng / mL, or 15-25 ng / mL. In some embodiments, BDNF is provided at or about 20 ng / mL.

[0234] In some embodiments of any of the methods disclosed herein, GDNF is provided at a concentration that is, is about, is at least about, is less than, or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 1-30 ng / mL, 10-20 ng / mL, 20-30 ng / mL, or 15-25 ng / mL. In some embodiments, GDNF is provided at or about 20 ng / mL.

[0235] In some embodiments of any of the methods disclosed herein, the growth serum is provided at a concentration that is, is about, is at least about, is less than, or is about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any concentration within a range defined by any two of the preceding concentrations, e.g., 0.5%-20%, 0.5%-5%, 1%-15%, 10-15%, 15-20%, or 12-18%. In some embodiments, the growth serum is provided at or about 15%. In some embodiments, the growth serum is provided at or about 1%. In some embodiments, the growth medium is fetal bovine serum (FBS).

[0236] In some embodiments of any of the methods disclosed herein, the VEGF pathway activator is provided at a concentration that is, is about, is at least about, is less than, or is about equal to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-150 ng / mL, 10-100 ng / mL, 100-150 ng / mL, or 80-120 ng / mL. In some embodiments, the VEGF is provided at or about 100 ng / mL. In some embodiments, the VEGF pathway activator is VEGF.

[0237] In some embodiments of any of the methods disclosed herein, the FGF pathway activator is provided at a concentration that is, is about, is at least about, is less than, or is about equal to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 ng / mL, or any concentration within a range defined by any two of the foregoing concentrations, e.g., 10-150 ng / mL, 10-100 ng / mL, 100-150 ng / mL, or 80-120 ng / mL. In some embodiments, the FGF pathway activator is provided at or about 100 ng / mL. In some embodiments, the FGF signaling pathway activator is FGF2.

[0238] In some embodiments of any of the methods disclosed herein, vascular organoid and / or brain organoid are derived from pluripotent stem cells.In some embodiments, pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.In some embodiments, vascular organoid and / or brain organoid are human.In some embodiments, vascular organoid and / or brain organoid are derived from a subject, for example, a human subject.In some embodiments, the subject comprises cerebrovascular disease or disease related to blood-brain barrier dysfunction, for example, cavernous vascular malformation, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0239] Also disclosed herein is a vascularized brain organoid produced by any of the methods disclosed herein. In some embodiments, the vascularized brain organoid comprises endothelial cells connected with tight junctions, astrocytes, and pericytes. In some embodiments, the endothelial cells express CD31, GLUT-1, and PDGFR-β, the tight junctions include claudin-5, ZO-1, and cadherin 5, the astrocytes express S100B, GFAP, and AQP4, and the pericytes express PDGFR-β, αSMA, and NG2. In some embodiments, the endothelial cells form a continuous basement membrane and express collagen IV. In some embodiments, the vascularized brain organoid comprises cells selected from the group consisting of neural precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, cerebrovascular endothelial cells, vascular leptomeningeal cells, perivascular adipocytes, and tendon cells. In some embodiments, the vascularized brain organoid comprises cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells. In some embodiments, the cells are identified by cell type-specific gene expression markers. In some embodiments, the blood vessels comprise capillaries. In some embodiments, the capillaries are covered by the endfeet of pericytes and astrocytes.

[0240] How to use Also disclosed herein is the method for treating cerebrovascular disease or the disease associated with blood-brain barrier dysfunction in a subject that needs to do so.In some embodiments, the method comprises administering to subject any of the vascularized brain organoids disclosed herein, or part or fragment thereof.

[0241] Also disclosed herein is a method of screening. In some embodiments, the method comprises contacting any of the vascularized brain organoids disclosed herein, or a portion thereof, with a candidate compound or composition, and evaluating the effect of the candidate compound or composition on the vascularized brain organoid or a portion thereof. In some embodiments, the effect comprises transport of the candidate compound or composition across the blood-brain barrier of the organoid or a portion thereof. In some embodiments, the vascularized brain organoid is a model for cerebrovascular disease or a disease associated with blood-brain barrier dysfunction, and evaluating the effect of the candidate compound or composition on the vascularized organoid comprises evaluating the effect of the candidate compound or composition on cerebrovascular disease or a disease associated with blood-brain barrier dysfunction. In some embodiments, the vascularized brain organoid is generated from cells derived from a subject. In some embodiments, the cells derived from the subject are pluripotent stem cells. In some embodiments, the subject has or is susceptible to cerebrovascular disease or a disease associated with blood-brain barrier dysfunction. EXAMPLES

[0242] Example 1. Generation of vascular organoids Exemplary methods for generating vascular (vascular) organoids from pluripotent stem cells can be found in Wimmer et al. Generation of blood vessel organoids from human pluripotent stem cells. Nature Protocols (2019) 14(11): 3082-3100 and Wimmer et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature (2019) 565(7740): 505-510, each of which is expressly incorporated herein by reference in its entirety. A schematic diagram of the improved method for generating vascular organoids from pluripotent stem cells is shown in Figure 1A.

[0243] Preparation of feeder-free iPSC cultures: Matrigel was thawed on ice for up to 1 hour. 100 μL of thawed Matrigel was diluted in 6 mL of cold DMEM / F12 (for 6-well plates). Matrigel dilution was mixed thoroughly and used to coat 6-well plates with 1 mL of Matrigel mixture per well. Plates were placed in a 37°C incubator for up to 2 hours or overnight. iPSCs grown to 60-80% confluence were washed with approximately 2 mL of room temperature phosphate buffered saline (PBS). 1 mL of ReleSR dissociation reagent (StemCell Technologies) was added per well containing iPSCs, and after 1 minute, most of the ReleSR was aspirated, leaving only a portion covering the iPSCs. iPSCs were incubated in ReleSR reagent at room temperature for 6-8 minutes. The side of the iPSC plate was tapped to detach the iPSCs. One mL of mTeSR Plus medium (StemCell Technologies) was added per well to neutralize the ReleSR dissociation reagent. The plate was gently shaken to wash the iPSCs. The dissociated iPSCs were transferred to a 15 mL tube and dissociated into single cells by pipetting. The Matrigel mixture was aspirated from the coated 6-well plate and 2 mL of mTeSR Plus was added per well to the Matrigel plate. The iPSCs were added to the Matrigel-coated plate at the desired density (1:20 to 1:50 dilution, or 10,000 to 30,000 cells / well). The plate was briefly shaken and returned to the 37°C incubator. Growth medium was changed daily until the iPSCs reached approximately 80% confluence (5 to 7 days). These iPSCs can be used downstream for vascular organoid differentiation or passaged again for later use.

[0244] Preparation of vascular organoids: One day before starting the vascular organoid differentiation process (day -1), iPSCs were plated at 1.2 × 10 per plate in aggregation medium (KnockOut DMEM / F12, 99 μM β-mercaptoethanol, Knockout Serum Replacement, 1 × Glutamax, 1 × non-essential amino acids (NEAA), 1 × penicillin-streptomycin) supplemented with 50 μM Y-27632 (ROCK inhibitor). 6 Cells were seeded onto Aggrewell 400 (StemCell Technologies) 24-well plates to form uniform stem cell aggregates.

[0245] On day 1 (day 0), iPSCs were induced to differentiate into mesoderm by culturing the aggregates in N2B27 medium (50% DMEM / F12, 50% neurobasal medium, 99 μM β-mercaptoethanol, 1× Glutamax, 1× Penicillin-Streptomycin, 1× B27 supplement, 1× N2 supplement) supplemented with 12 μM CHIR99021 and 30 ng / mL BMP4.

[0246] After 4 days of mesodermal induction (on day 3), the differentiated mesodermal cells were induced to differentiate into the vascular lineage by culturing the mesodermal cells in N2B27 medium supplemented with 100 ng / mL VEGF and 2 μM forskolin.

[0247] Two days after vascular induction (day 5), the differentiated vascular lineage cells were further cultured to allow blood vessel formation to proceed.

[0248] For one 12-well plate, 5 mL of 2 mg / mL collagen I solution was prepared by mixing 300 μL 0.1 N NaOH, 450 μL ddH2O, 313 μL 10×DMEM, 63 μL HEPES, 49 μL 7.5% sodium bicarbonate, 31 μL Glutamax, 460 μL Ham's F-12, and 3.33 mL of 3 mg / mL collagen stock solution (PureCol; Advanced Biomatrix). The pH of this collagen I solution should be 7.4. A 4:1 collagen I solution to matrigel mixture was then prepared by mixing 4.5 mL of collagen I solution with 1.5 mL of growth factor reduced matrigel on ice.

[0249] 0.5 mL of the collagen I / matrigel mixture was used to coat the wells of a 12-well plate and incubated at 37° C. for 2 hours to allow the mixture to solidify. Vascular lineage cells were resuspended in another fresh batch of collagen I / matrigel mixture (unsolidified) and 0.5 mL of the cell suspension in collagen I / matrigel was used to seed each well of the collagen I / matrigel-coated plate. The plate was returned to a 37° C. incubator for 2 hours to allow the collagen I / matrigel mixture containing the vascular lineage cells to solidify. Complete StemPro-34 serum-free medium (Thermo Fisher) supplemented with 15% fetal bovine serum (FBS), 4 μM CHIR99021, 100 ng / mL VEGF, and 100 ng / mL FGF2 was then added to the vascular lineage cells.

[0250] Over the course of 5 days of culture in StemPro-34 medium containing 15% FBS, 4 μM CHIR99021, 100ng / mL VEGF and 100ng / mL FGF2, vascular lineage cells mature to form vascular organoids that contain vascular networks.These vascular organoids can be used for optional downstream studies, such as isolating blood vessels from organoids or transplanting organoids in vivo.

[0251] In FIG. 1B, stem cells engineered to express GFP were differentiated into vascular organoids according to the methods provided herein. The cells of the vascular organoids were organized in a vascular network. As shown in FIG. 1C-FIG. 1D, the cells of the vascular organoids expressed platelet endothelial cell adhesion molecule (PECAM-1, CD31) and platelet-derived growth factor receptor beta (PDGFR-β), which are markers of early endothelial cells. PDGFR-β is also expressed by pericyte progenitor cells that give rise to pericytes that are involved in the blood-brain barrier. In FIG. 1D, endothelial tube structures with a lumen were present within the vascular organoids.

[0252] Example 2: Generation of Cortical Organoids Exemplary methods for generating vascular organoids from pluripotent stem cells can be found in Qian et al. Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell (2016) 165(5): 1238-1254 and Qian et al. Generation of human brain region-specific organoids using a miniaturized spinning bioreactor. Nature Protocols (2018) 13(3): 565-580, each of which is expressly incorporated herein by reference in its entirety. A schematic diagram of an exemplary method for generating dorsal forebrain organoids, a type of cortical organoid, is provided in FIG. 2A. These methods can be adapted to generate alternative types of cortical organoids, such as midbrain, striatal brain, hypothalamic, hippocampal, and spinal cord organoids.

[0253] On day 1 (day 0), iPSCs were plated on Aggrewell 800 24-well plates (StemCell Technologies) at approximately 4 × 10 cells per Aggrewell in 2 mL of Essential 8 medium (Thermo Fisher) supplemented with 10 μM Y-27632 (ROCK inhibitor). 6The cells were seeded at a final cell density of 100×g. The Aggrewell was centrifuged at 100×g for 3 minutes at 4° C. to collect the stem cells at the bottom of the Aggrewell microwell. The Aggrewell was first washed with Anti-Adherence Rinsing solution (StemCell Technologies) and then with Essential 8 medium before adding the cells. The use of Aggrewell plates is optional, but the use of these plates helps achieve a higher final number of organoids with a more uniform size. Standard low-attachment plates may also be used, which results in larger organoids.

[0254] After 1 day of culture (day 1) (or after 2 days if the embryoid bodies were not large enough), the Aggrewell cultures were agitated with a pipette to resuspend the embryoid bodies (EBs) and the resuspension was transferred to a 15 mL tube. EBs were washed with fresh DMEM / F12, resuspended in H1 medium (77% DMEM / F12, 20% KnockOut Serum Replacement medium, 1x Glutamax, 1x NEAA, 1x β-mercaptoethanol, 1x penicillin-streptomycin supplemented with 1 μM LDN-193189, 2 μM A83-01, and 3 μM IWR-1) and plated into ultra-low attachment 6-well plates. EBs were allowed to settle, the medium was aspirated, and 3 mL of H1 medium supplemented with 10 μM Y-27632 was added to each well. Plates were incubated at 37°C with shaking (120 rpm) for 48 hours. After 48 hours, the medium was replaced with 3 mL of fresh H1 medium (without ROCK inhibitors). After another 24 hours (day 3 of culture), the medium was replaced again with fresh H1 medium (without ROCK inhibitors).

[0255] After 4 days of culture in H1 medium (day 4), the medium was replaced with H1 medium without IWR-1 and cultured at 37° C. with shaking (120 rpm) for an additional 2 days.

[0256] Two days later (day 7), the resulting neuroectodermal cells were transferred to a 1.5 mL tube and allowed to settle. The supernatant was removed and the EBs were washed twice with 1 mL of F2 medium (DMEM / F12, 1x N2, 1x Glutamax, 1x NEAA, 1x β-mercaptoethanol, 1x penicillin-streptomycin, supplemented with 1 μM SB-431542 and 1 μM CHIR99021). EBs (containing less than 60 EBs) resuspended in 67 μL of F12 medium were transferred to a new tube and combined with 100 μL of Matrigel. The EB / Matrigel mixture was added to the plate and allowed to solidify for 30 minutes in a 37° C. incubator. 3 mL of F2 medium was carefully added to the wells containing the EB / Matrigel droplets and returned to the 37° C. incubator without shaking for 48 hours. The medium was replaced with fresh F2 medium every 2 days (days 9, 11, 13) for a total of 6 days.

[0257] On day 14, the resulting neuroepithelial cells were detached from the Matrigel and resuspended in 1–3 mL of H3 medium (50% DMEM / F12, 50% neurobasal medium, 1x N2, 1x B27, 1x Glutamax, 1x NEAA, 1x β-mercaptoethanol, 1x penicillin-streptomycin, supplemented with 2.5 μg / mL insulin). The EB resuspension in H3 medium was added to a regular 6-well plate, and an additional 4.5 mL of H3 medium was added to each well. The cells were incubated at 37 °C with shaking (120 rpm).

[0258] Over a period of 54 days (days 16-70), replace the medium with fresh H3 medium every 2 days.

[0259] When the brain tissue organoids grow to be more than 1 mm in diameter (after day 70), replace the medium with 3 mL of F4 medium (neurobasal medium, 1x B27, 1x Glutamax, 1x NEAA, 1x β-mercaptoethanol, 1x penicillin-streptomycin supplemented with 0.05 mM cAMP, 0.2 mM ascorbic acid, 20 ng / mL BDNF, and 20 ng / mL GDNF) to differentiate the brain tissue into cortical forebrain organoids. Replace the medium with fresh F4 medium every 2 days.

[0260] For astrocyte induction, F4 medium used at any time point after day 70 is supplemented with 1% FBS (can be increased up to 15%) and 10 mg / mL leukemia inhibitory factor (LIF) for 2 weeks.

[0261] As shown in FIG. 2B, forebrain organoids generated according to the methods herein expressed the neuronal marker class III beta-tubulin (Tuj1) and the neural stem cell marker SRY-Box transcription factor (Sox2) as detected at day 26 of culture, and the neuronal markers B-cell lymphoma / leukemia 11B (BCL11B, Ctip1) and T-Box brain transcription factor 1 (Tbr1) as detected at day 61 of culture.

[0262] Figures 2C-D show images of forebrain organoids further cultured with 1% FBS and 10 mg / mL LIF for 2 weeks to induce astrocyte formation. The presence of astrocytes was confirmed by detection of the astrocyte markers S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4).

[0263] Example 3: Generation of fused vascularized cortical organoids Vascular organoid and cortical organoid are generated according to the method provided herein (e.g., in the examples) or according to the method generally known in the art.To generate the vascularized cortical organoid that models the intact blood-brain barrier, the cortical organoid that is cultured to contain astrocytes is used.The schematic diagram of the approach to form fused vascularized cortical organoid is given in Figure 3A, Figure 3B and Figure 3D.

[0264] A Matrigel mixture was prepared with 100 μL of Matrigel and 60 μL of ice-cold brain vascularization medium (a) StemPro-34 medium with 15% FBS, 100 ng / mL VEGF, and 100 ng / mL FGF2, and b) F4 medium (a 50% / 50% mixture of neurobasal medium, 1× B27, 1× Glutamax, 1× NEAA, 1× β-mercaptoethanol, 1× penicillin-streptomycin, supplemented with 0.05 mM cAMP, 0.2 mM ascorbic acid, 20 ng / mL BDNF, and 20 ng / mL GDNF). The volume of this preparation is sufficient to generate approximately 5 vascularized cortical organoids using 30 μL of each mixture.

[0265] A single vascular organoid and a single cortical organoid were placed in a 1.5 mL tube, and the medium in the tube that was carried away from the transfer of the organoids was removed. This process ensured that the two organoids were in direct contact with each other. 30 μL of the Matrigel / brain vascularization medium mixture was added to the organoids, and the tube was placed in a 37 °C incubator to allow the Matrigel mixture to solidify. If smaller sized organoids need to be sedimented, the tube can be gently centrifuged before the solidification of the Matrigel. Additional brain vascularization medium was added to the tube, and the tube was then incubated at 37 °C for 1 day. After 1 day, half of the liquid medium was replaced with fresh brain vascularization medium. The tube should be opened for approximately 30-60 minutes every day in a sterile hood to allow gas exchange. After another day, the Matrigel droplets were transferred to a low-attachment 6-well plate containing brain vascularization medium and cultured for another 3 days. After 3 days, the plates were incubated with slow shaking (100 rpm) for 3 days at 37° C. The plates were then incubated with faster shaking (120 rpm) at 37° C. After a total of 20 days from contacting the primary vascular and cortical organoids in Matrigel, the fused organoids were harvested for use.

[0266] In an alternative approach, a single vascular organoid and a single cortical organoid were placed on a sterile surface (e.g., a piece of sterile plastic). A drop of Matrigel / brain angiogenesis medium mixture was placed on the sterile surface, and the two organoids were manipulated to directly contact each other in the center of the Matrigel / brain angiogenesis medium mixture. The sterile surface holding the organoids was placed in a 37°C incubator to allow the Matrigel mixture to solidify. The solidified Matrigel drop containing the organoids was then transferred to an appropriate tissue culture plate containing brain angiogenesis medium and incubated at 37°C for 4 days. After 4 days, the plate was incubated at 37°C with slow shaking (100 rpm) for 3 days. The plate was then incubated at 37°C with faster shaking (120 rpm). After a total of 20 days from the initial vascular and cortical organoids being in contact in the Matrigel, the fused organoids were harvested for use.

[0267] As shown in Figure 3D and Figure 3E, direct contact between vascular and forebrain organoids resulted in their fusion and infiltration of endothelial cells from vascular organoids into forebrain organoids. The formation of brain capillaries in forebrain organoids was confirmed by detection of endothelial cell markers CD31 and cadherin 5 (CDH5) in the forebrain organoids, and neural cells were closely associated with endothelial cells. Figure 3K and Figure 3N show the presence of GFAP-positive astrocytes and PDGFR-β-positive pericyte progenitor cells, further cell types essentially involved in the blood-brain barrier. Importantly, Figure 3H shows the expression of claudin-5, which represents tight junctions that are important for blood-brain barrier function and has been observed to be absent or poorly present in previous models of the BBB.

[0268] Moreover, by day 21, the majority of endothelial cells expressed BBB-specific markers such as glucose transporter 1 (Glut-1) and tight junction proteins such as claudin-5 (Figure 3G-H) and (ZO-1 (not shown)), indicating that endothelial cells were differentiating towards a BBB-specific fate. Day 21 vascularized brain organoids immunostained for GFP, CD31, and collagen IV show that the brain endothelium (CD31), a key structure regulating angiogenesis and maintaining the BBB defined by the molecular marker collagen IV, was covered by a continuous basement membrane (Figure 3J). It is also noteworthy that the astrocyte processes aligned well with the endothelial tubes (Fig. 3K-L) and that human astrocytes extended their endfeet, labeled by AQP-4, to wrap around the abluminal capillary surface (Fig. 3M). The newly formed capillaries were not only covered by astrocyte processes but also by human pericytes (stained for PDGFR-β, Fig. 3N), indicating similarities to the in vivo human BBB-like structure with endothelial cells of the capillary wall connected via tight junctions, astrocyte endfeet and pericyte sheaths, and neuronal innervation (Fig. 3C and 3O).

[0269] Figure 3I shows an electron micrograph of fused provascularized brain organoids showing the presence of microvesicles (MVs), tight junctions (TJs) and adherens junctions (AJs) protruding into the lumen of brain capillary structures, demonstrating that human brain microvascular endothelial cells (BMECs) formed capillaries through tight junctions.

[0270] Example 4: Single-cell transcriptome profiling of fused vascularized cortical organoids The fused vascularized forebrain organoids were analyzed by single-cell RNA transcriptome sequencing. A total of 9342 cells were analyzed after quality control. Cells with mitochondrial gene ratios >10% and gene expression <200 were excluded. Clustering resolution was set to 0.5.

[0271] As shown in Figure 4A, the vascularized organoids contain multiple cell types representative of the blood-brain barrier, including neurons, astrocytes, and endothelial cells. Figure 4B shows the presence of subclusters within the endothelial cell cluster, suggesting that the organoids contain a diverse population of cell types. Figures 4C and 4D show the relative expression of various cell markers in the different cell types identified in single-cell RNA sequencing. Figures 4E-G show maps of interactions between different known protein receptors and ligands expressed by cells involved in 1) vascular cell type to vascular cell type, 2) neuronal cell type to vascular cell type, and 3) vascular cell type to neuronal cell type communication, respectively.

[0272] Example 5: Further single-cell transcriptome profiling of vascularized brain organoids Additional single-cell RNA sequencing (scRNA-seq) was performed on day 30 vascularized brain organoids derived from H9 embryonic stem cells using three replicate cultures to comprehensively decipher the cell populations present in the vascularized brain organoids at the transcriptome level. Sequencing data were aligned and quantified using Cell Ranger (10x Genomics) to obtain raw count data. The raw count data was normalized using the R package Seurat (version 4), and DoubletFinder was applied to remove doublet cells in the scRNA-seq data. After doublet cell removal, a total of 28,062 cells were extracted for further analysis (n=3 cultures). FindClusters was then applied to identify differentially expressed gene markers for each cell cluster. Figure 5A shows clusters of excitatory neurons, inhibitory neurons, neural precursors (NPs), astrocytes, endothelial cells (ECs), pericytes, mesenchymal stem cells (MSCs), smooth muscle cells (SMCs), and fibroblasts, identified by gene expression markers. The presence of these cell types suggests that vascularized brain organoids may resemble complete neurovascular units. Single-cell transcriptome analysis showed similar cell populations between biological replicas, indicating the reliability of the protocol. Gene expression data from endothelial cells in vascularized brain organoids was compared to gene expression data from organ-specific endothelial cells generated by the Tabula Muris Consortium. Figure 5B shows that endothelial cells in vascularized brain organoids displayed identical gene expression patterns to brain microvascular endothelial cells (BMECs), but not to other organ-specific endothelial cells, indicating endothelial acquisition of brain-specific transcriptome signatures in vascularized brain organoids.

[0273] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the present subject matter, as defined by the appended claims.

[0274] With respect to the use of substantially all plural and / or singular terms herein, those of skill in the art may translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, the various singular / plural permutations may be expressly set forth herein.

[0275] In general, it will be understood by those of ordinary skill in the art that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Those of ordinary skill in the art will further understand that where a specific number of introduced claim recitations are intended, such intent will be expressly set forth in the claims, and in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the appended claims below may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitations. However, the use of such phrases should not be interpreted to imply that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to an embodiment that includes only one such recitation, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. In addition, even if a specific number of introduced claim recitations is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the number recited (e.g., an explicit recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, where a convention similar to "such as at least one of A, B, and C" is used, such syntax generally is intended to mean what one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to "such as at least one of A, B, or C" is used, such syntax generally is intended to mean what one of ordinary skill in the art would understand this convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the detailed description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0276] In addition, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0277] As would be understood by one of ordinary skill in the art, for all purposes, including in terms of being written, all ranges disclosed herein encompass all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least 2, 3, 4, 5, 10, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As would be understood by one of ordinary skill in the art, all words such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the numbers recited and that may be subsequently broken down into subranges as discussed herein. Finally, as would be understood by one of ordinary skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, and so forth.

[0278] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those of ordinary skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0279] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or is intended to take precedence over such conflicting material.

[0280] References Blanchard et al.Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes.Nature Medicine(2020)26(6):952-963. Cakir et al.Engineering of human brain organoids with a functional vascular-like system.Nature Methods(2019)16(11):1169-1175. Chandrasekaran et al.Astrocyte Differentiation of Human Pluripotent Stem Cells: New Tools for Neurological Disorder Research.Front Cell.Neurosci.(2016)10:215. Daviaud et al.Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex.eNeuro(2018)5(6):ENEURO.0219-18. Ham et al.Blood vessel formation in cerebral organoids formed from human embryonic stem cells.Biochem Biophys.Res.Commun.(2020)521(1):84-90. Huang et al.Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.Cell Stem Cell(2021)28(9):1657-1670. Jacob et al.Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.Cell Stem Cell.(2020)27(6):937-950. Lippmann et al.Human Blood-Brain Barrier Endothelial Cells Derived from Pluripotent Stem Cells.Nat.Biotechnol.(2012)30(8):783-791. Mansour et al. An in vivo model of functional and vascularized human brain organoids Nature Biotechnology(2018)36:432-441. Perriot et al.Differentiation of functional astrocytes from human-induced pluripotent stem cells in chemically defined media.STAR Protoc.(2021)2(4):100902. Perriot et al.Human Induced Pluripotent Stem Cell-Derived Astrocytes Are Differentially Activated by Multiple Sclerosis-Associated Cytokines.Stem Cell Reports.(2018)11(5):1199-1210. Qian et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell(2016)165(5):1238-1254. Qian et al.Generation of human brain region-specific organoids using a miniaturized spinning bioreactor.Nature Protocols(2018)13(3):565-580. Shi et al.Vascularized human cortical organoids(vOrganoids)model cortical development in vivo.PLoS Biol.(2020)18(5):e3000705. Shin et al.Blood-Brain Barrier Dysfunction in a 3D In Vitro Model of Alzheimer's Disease.Adv.Sci.(Weinh).(2019)6(20):1900962. Song et al.Assembly of Human Stem Cell-Derived Cortical Spheroids and Vascular Spheroids to Model 3-D Brain-like Tissues.Scientific Reports 9:5977. TCW et al.An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.Stem Cell Reports.(2017)9(2):600-614. Wimmer et al.Generation of blood vessel organoids from human pluripotent stem cells.Nature Protocols(2019)14(11):3082-3100. Wimmer et al.Human blood vessel organoids as a model of diabetic vasculopathy.Nature(2019)565(7740):505-510.

Claims

1. A method for generating neovascular brain organoids, Bringing vascular organoids and brain organoids into contact, The process includes culturing the vascular organoid and the brain organoid for a predetermined period of time until they fuse together and the blood vessels of the vascular organoid infiltrate the brain organoid. The neurons of the brain organoid innervate the blood vessels of the vascular organoid that have infiltrated the organoid. This forms the angiogenic brain organoids, The angiogenic brain organoid includes the brain organoid and a blood-brain barrier formed between all or part of the blood vessels of the vascular organoid that has infiltrated the brain organoid. A method wherein the blood-brain barrier comprises endothelial cells linked by tight junctions, astrocytes, and pericytes.

2. The method according to claim 1, wherein the endothelial cells express CD31, GLUT-1 and PDGFR-β, the tight junctions contain claudin-5, ZO-1 and cadherin 5, the astrocytes express S100B, GFAP and AQP4, and the pericytes express PDGFR-β, αSMA and NG2.

3. The method according to claim 1 or 2, wherein the endothelial cells form a continuous basement membrane and express collagen IV.

4. The method according to claim 1, wherein the angiogenic brain organoid comprises cells selected from the group consisting of neuronal precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, cerebral vascular endothelial cells, vascular leucomeninge cells, perivascular adipocytes, and tendinocytes.

5. The method according to claim 1, wherein the angiogenic brain organoid comprises cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells.

6. The method according to claim 1, wherein the blood vessels include capillaries.

7. The method according to claim 6, wherein the capillaries are covered by pericytes and astrocyte terminals.

8. The method according to claim 1, wherein the brain organoid is a forebrain organoid, a midbrain organoid, a hypothalamic organoid, a hippocampal organoid, a spinal cord organoid, or a striatal brain organoid.

9. The method according to claim 1, wherein the vascular organoid and the brain organoid are in contact with and / or cultured in a basement membrane matrix or its components.

10. The method according to claim 1, wherein the vascular organoids and the brain organoids are cultured with stirring for at least a portion of the period.

11. The vascular organoid and the brain organoid are 1) The culture is incubated for at least one day without stirring, and then, 2) The culture is incubated with stirring for at least one day. The method according to claim 1, wherein the stirring optionally includes shaking.

12. The method according to claim 1, wherein the vascular organoids and the brain organoids are cultured in a medium that promotes neuronal growth and / or vascular growth.

13. The method according to claim 1, wherein the vascular organoids and the brain organoids are cultured in a culture medium containing growth factors that promote neuronal growth and / or growth factors that promote vascular growth.

14. The method according to claim 13, wherein the growth factor that promotes neuronal growth includes cAMP pathway activator, ascorbic acid, BDNF, GDPR, or any combination thereof.

15. The method according to claim 13 or 14, wherein the growth factor that promotes vascular growth includes growth serum, VEGF pathway activator, FGF pathway activator, or any combination thereof.

16. The method according to claim 14, wherein the cAMP pathway activator is cAMP.

17. The method according to claim 15, wherein the growth serum is fetal bovine serum (FBS).

18. The method according to claim 15, wherein the VEGF pathway activator is VEGF.

19. The method according to claim 15, wherein the FGF pathway activator is FGF2.

20. The method according to claim 1, wherein the vascular organoid and / or the brain organoid is derived from pluripotent stem cells, optionally embryonic stem cells, or induced pluripotent stem cells.

21. The aforementioned vascular organoid, The method involves exposing angiocytes to FGF pathway activator, VEGF pathway activator, optionally Wnt pathway activator, and optionally growth serum over a first period of time. The method according to claim 1, wherein the vascular organoid is formed according to the method described above.

22. The aforementioned angiocytes a) To form vascular cells by exposing pluripotent stem cells to Wnt pathway activator and BMP pathway activator over a second period, b) The vascular cells are brought into contact with a VEGF pathway activator and a second cAMP pathway activator over a third period of time, The method according to claim 21, wherein a neovascular bud is formed according to the method described above.

23. The method according to claim 22, wherein the BMP pathway activator is BMP4.

24. The method according to claim 21, wherein the Wnt pathway activator is CHIR99021.

25. The method according to claim 22, wherein the second cAMP pathway activator is forskolin.

26. The method according to claim 21, wherein the vascular organoid differs from vascular organoids generated without contacting the angiocytes with the Wnt pathway activator, by selectively increasing the expression of blood-brain barrier-specific endothelial markers, GLUT-1 and ZO-1.

27. The method according to claim 1, wherein the brain organoid is brought into contact with LIF and growth serum to induce astrocyte formation in the brain organoid.

28. The aforementioned brain organoid, a) To form neuroectoderm cells by exposing pluripotent stem cells to a BMP pathway inhibitor, a TGF-beta pathway inhibitor, and a Wnt pathway inhibitor over a first period of time, b) The neuroectoderm cells from step a) are brought into contact with a second TGF-beta pathway inhibitor and a Wnt pathway activator over a second period of time to form neuroepithelial cells. c) The neuroepithelial cells from step b) are brought into contact with insulin over a third period to form brain tissue organoids. d) The brain tissue organoid from step c) is brought into contact with GDNF, BDNF, ascorbic acid, and cAMP pathway activator over a fourth period of time to form the brain organoid, The method according to claim 1, wherein the brain tissue organoid is optionally further contacted with LIF and growth serum for part of a fourth period to induce astrocyte proliferation in the brain organoid.

29. The method according to claim 28, wherein the BMP pathway inhibitor is LDN-193189.

30. The method according to claim 28, wherein the TGF-beta pathway inhibitor and the second TGF-beta pathway inhibitor are the same or different.

31. The method according to claim 28, wherein the TGF-beta pathway inhibitor is A83-01.

32. The method according to claim 28, wherein the second TGF-beta pathway inhibitor is SB-431542.

33. The method according to claim 28, wherein the Wnt pathway inhibitor is IWR-1.

34. The method according to claim 28, wherein the Wnt pathway activator is CHIR99021.

35. The method according to claim 28, wherein the brain organoid comprises astrocytes expressing S100B, GFAP, and AQP4.

36. The method according to claim 1, wherein the vascular organoid and / or the brain organoid is human.

37. The method according to claim 1, wherein the vascular organoid and / or the brain organoid were, optionally, derived from a human subject.

38. The method according to claim 37, wherein the subject includes cerebrovascular diseases or diseases related to blood-brain barrier dysfunction, and optionally, the cerebrovascular diseases or diseases related to blood-brain barrier dysfunction include cavernous malformations, Alzheimer's disease, or amyotrophic lateral sclerosis.

39. Angiogenic brain organoid produced by the method described in claim 1.

40. Angiogenic brain organoids containing endothelial cells linked by tight junctions, astrocytes, and pericytes.

41. The angiogenic brain organoid according to claim 40, wherein the endothelial cells express CD31, GLUT-1 and PDGFR-β, the tight junctions contain claudin-5, ZO-1 and cadherin 5, the astrocytes express S100B, GFAP and AQP4, and the pericytes express PDGFR-β, αSMA and NG2.

42. The angiogenic brain organoid according to claim 40 or 41, wherein the endothelial cells form a continuous basement membrane and express collagen IV.

43. The angiogenic brain organoid according to claim 40, comprising cells selected from the group consisting of neuronal precursors, proliferative astrocytes, GABAergic neurons, glutamatergic neurons, proliferative cells, cerebral vascular endothelial cells, vascular leptomeningeal cells, perivascular adipocytes, and tendinocytes.

44. The angiogenic brain organoid according to claim 40, comprising cells selected from the group consisting of neural progenitor cells, GABAergic neurons, glutamatergic neurons, proliferative astrocytes, proliferative GABAergic neurons, mesenchymal stem cells, endothelial cells, pericytes, vascular smooth muscle cells, fibroblasts, and proliferative cells.

45. The neovascular brain organoid according to claim 40, wherein the blood vessels include capillaries.

46. The neovascular brain organoid according to claim 40, wherein the capillaries are covered by pericytes and astrocyte terminals.

47. The angiogenic brain organoid according to claim 39, for use in the treatment of cerebrovascular disease or diseases related to blood-brain barrier dysfunction.

48. A screening method comprising: contacting an angiogenic brain organoid or a part thereof described in claim 39 with a candidate compound or composition; and evaluating the effect of the candidate compound or composition on the angiogenic brain organoid or a part thereof.

49. The method according to claim 48, wherein the effect includes the transport of the candidate compound or composition across the blood-brain barrier of the organoid or a portion thereof.

50. The method according to claim 48, wherein the angiogenic brain organoid is a model for cerebrovascular disease or a disease related to blood-brain barrier dysfunction, and evaluating the effect of the candidate compound or composition on the angiogenic organoid is equivalent to evaluating the effect of the candidate compound or composition on cerebrovascular disease or a disease related to blood-brain barrier dysfunction.

51. The method according to claim 48, wherein the angiogenic brain organoid is generated from cells derived from the subject, and optionally, the cells derived from the subject are induced pluripotent stem cells.

52. The method according to claim 51, wherein the subject has or is prone to developing the cerebrovascular disease or the disease related to blood-brain barrier dysfunction.

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