Retinoic Acid Enhanced Human Stem Cell Derived Blood Brain Barrier Model

• US 20140127800A1
• Filed: 03/11/2013
• Published: 05/08/2014
• Est. Priority Date: 11/08/2012
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Shusta, Eric V., Palecek, Sean P., Azarin, Samira M., Lippmann, Ethan S.
• Granted Patent: US 10,590,393 B2
• Time in Patent Office: Days
• US Class Current: 435/347
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2502/08 cells of the nervous system
• CPC Class Codes: C12N 2502/088 neural stem cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

In one embodiment, the present invention is a method of creating a fully-human blood-brain barrier (BBB) model, comprising the steps of (a) obtaining a mixture of neural cells and brain microvascular endothelial cells (BMECs), wherein the neural cells and BMECs that comprise the mixture were produced from the differentiation of human pluripotent stem cells (hPSCs); (b) purifying BMECs from the mixture of neural cells and BMECs of step (a); and (c) co-culturing the purified BMECs with a cell type selected from the group consisting of pericytes, astrocytes and differentiated neural progenitor cells (NPCs), wherein a blood brain barrier model is created.

Claims

1. A method of creating a fully-human blood-brain barrier (BBB) model, comprising the steps of:

(a) obtaining a mixture of neural cells and brain microvascular endothelial cells (BMECs), wherein the neural cells and BMECs that comprise the mixture were produced from the differentiation of human pluripotent stem cells (hPSCs);

(b) purifying BMECs from the mixture of neural cells and BMECs of step(a); and

(c) co-culturing the purified BMECs with a cell type selected from the group consisting of pericytes, astrocytes and differentiated neural progenitor cells (NPCs), wherein a blood brain barrier model is created.

2. The method of claim 1 wherein the cell types of step(c) are human cells.

3. The method of claim 1 wherein the hPSCs are human embryonic stem cells (hESCs)

4. The method of claim 1 wherein the hPSCs are induced pluripotent stem cells (iPSCs).

5. A fully-human blood-brain barrier (BBB) model created by the method of claim 1.

6. A method of creating a retinoic acid (RA)-enhanced or RA-like compound-enhanced mammalian blood-brain barrier (BBB) model, comprising the steps of:

(a) obtaining a mixture of neural cells and brain microvascular endothelial cells (BMECs) in the presence of RA or RA-like compound, wherein the mixture of neural cells and BMECs was produced from the differentiation of human pluripotent stem cells (hPSCs);

(b) purifying BMECs from the mixture of neural cells and BMECs; and

(c) co-culturing the purified BMECs with a cell type selected from the group consisting of astrocytes, pericytes and differentiated neural progenitor cells (NPCs), wherein a BBB model is created.

7. A mammalian blood-brain barrier (BBB) model created by the method of creating a retinoic acid (RA)-enhanced or RA-like compound-enhanced mammalian blood-brain barrier (BBB) model, the method comprising the steps of:

a) obtaining a mixture of neural cells and brain microvascular endothelial cells (BMECs) in the presence of RA or RA-like compound, wherein the mixture of neural cells and BMECs was produced from the differentiation of human pluripotent stem cells (hPSCs);

b) purifying BMECs from the mixture of neural cells and BMECs; and

c) co-culturing the purified BMECs with a cell type selected from the group consisting of astrocytes, pericytes and differentiated neural progenitor cells (NPCs), wherein a BBB model is created.

8. A method of creating a retinoic acid (RA)-enhanced or RA-like compound-enhanced fully-human blood-brain barrier (BBB) model in an optimized endothelial cell medium (OECM) wherein the OECM does not contain basic fibroblast growth factor (bFGF), comprising the steps of:

(a) supplying a mixture of neural cells and brain microvascular endothelial cells (BMECs) in the presence of RA or RA-like compound, wherein the mixture of neural cells and BMECs was produced from the differentiation of human pluripotent stem cells (hPSCs);

(b) purifying BMECs from the mixture of neural cells and BMECs; and

(c) co-culturing the purified BMECs with a cell type selected from the group consisting of astrocytes, pericytes and differentiated NPCs in OECM.

9. The method of claim 8 wherein the cell types of step(c) are human cells.

10. The method of claim 8 wherein OECM contains at least 1% platelet-poor plasma-derived serum (PDS).

11. The method of claim 8 wherein step(c) comprises human pericytes co-cultured with BMECs within 30 minutes after the purification of BMECs.

12. The method of claim 8 wherein the mixture of human pericytes and BMECs is further co-cultured with differentiated hNPCs.

13. The method of claim 11 wherein the TEER of the confluent monolayer formed from the co-cultured BMECs and human pericytes within 30 minutes after the purification of BMECs, is greater than 4000 Ohm×

cm².

14. The method of claim 12 wherein the TEER of the confluent monolayer formed from the co-cultured BMECs, pericytes, and differentiated hNPCs, is greater than 5000 Ohm×

cm².

15. A mammalian blood-brain barrier (BBB) model created by the method of creating a retinoic acid (RA)-enhanced or RA-like compound-enhanced fully-human blood-brain barrier (BBB) model in an optimized endothelial cell medium (OECM) wherein the OECM does not contain basic fibroblast growth factor (bFGF), the method comprising the steps of:

a) supplying a mixture of neural cells and brain microvascular endothelial cells (BMECs) in the presence of RA or RA-like compound, wherein the mixture of neural cells and BMECs was produced from the differentiation of human pluripotent stem cells (hPSCs);

b) purifying BMECs from the mixture of neural cells and BMECs; and

c) co-culturing the purified BMECs with a cell type selected from the group consisting of astrocytes, pericytes and differentiated NPCs in OECM.

16. A retinoic acid (RA) or RA-like compound-enhanced mammalian blood-brain barrier (BBB) model in optimized endothelial cell medium (OECM) expressing a TEER greater than 4000 Ohm×

cm², wherein the OECM does not contain basic fibroblast growth factor (bFGF), comprising;

(a) brain microvascular endothelial cells (BMECs) wherein BMECs have been purified from a mixture of neural cells and BMECs, wherein the mixture of neural cells and BMECs was produced from the differentiation of human pluripotent stem cells (hPSCs) in the presence of RA or RA-like compound; and

(b) a cell type selected from the group consisting of astrocytes, pericytes and differentiated neural progenitor cells (NPCs), wherein the cell type was co-cultured with the purified BMECs in OECM such that the purified BMECs form a monolayer wherein the cells were confluent and the TEER of the confluent monolayer may be measured at greater than 4000 Ohm×

cm².

17. The RA or RA-like compound-enhanced mammalian BBB model in OECM according to claim 16 wherein the cell types of step(b) are human cells.

18. The RA or RA-like compound-enhanced mammalian BBB model in OECM according to claim 16 wherein step(b) comprises human pericytes co-cultured with the BMECs within 30 minutes after the purification of BMECs, wherein the TEER of the confluent monolayer formed from the co-cultured BMECs and human pericytes is greater than 4000 Ohm×

cm².

19. The RA or RA-like compound-enhanced mammalian BBB model in OECM according to claim 16 wherein the mixture of human pericytes and BMECs is further co-cultured with differentiated hNPCs and wherein the TEER of the confluent monolayer formed from the co-cultured BMECs, human pericytes, and differentiated hNPCs, is greater than 5000 Ohm×

cm².

20. The RA or RA-like compound-enhanced mammalian BBB model in OECM according to claim 16 wherein OECM contains at least 1% platelet-poor plasma-derived serum (PDS).