MANUFACTURE OF VASCULAR SMOOTH MUSCLE CELLS AND THE USE

• US 20150218523A1
• Filed: 02/05/2015
• Published: 08/06/2015
• Est. Priority Date: 02/05/2014
• Status: Abandoned Application

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Cheung, Christine
• Application Number:
  US14/614,889
• Publication Number:
  US 20150218523A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 5/0691 Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels

Abstract

A method for preparing brain-specific vascular smooth muscle cells comprising the step of: (a) contacting a population of stem cells with a composition comprising a bone morphogenetic protein (BMP) antagonist, a fibroblast growth factor (FGF) and an activin or nodal inhibitor to produce a population of neural crest cells.

Claims

1. A method for preparing brain-specific vascular smooth muscle cells, the method comprising the step of:

(a) contacting a population of stem cells with a composition comprising a bone morphogenetic protein (BMP) antagonist, a fibroblast growth factor (FGF) and an activin or nodal inhibitor to produce a population of neural crest cells.

2. The method according to claim 1, wherein the composition in step (a) does not comprise any one of the following which is selected from the group consisting glycogen synthase kinase (GSK) inhibitor, a Wnt protein, and a composition that is serum-free.

3. The method according to claim 1, further comprising the step of:

(b) isolating a population of CD57+ and/or CD271+ neural crest progenitor cells from the population of neural crest cells produced in step (a).

4. The method according to claim 1, further comprising the step of:

(c) contacting the population of neural crest progenitor cells with a composition comprising platelet-derived growth factor (PDGF) and transforming growth factor (TGF) to produce said brain-specific vascular smooth muscle cells.

5. The method according to claim 1, wherein said population of stem cells in step (a) are human pluripotent stem cells.

6. The method according to claim 5, wherein said human pluripotent stem cells are selected from the group consisting of human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs).

7. The method according to claim 6, wherein said human pluripotent stem cells are derived from stem cell lines and/or via stem cell preparation methods that do not involve destruction of human embryos.

8. The method according to claim 1, wherein the contacting of the population of stem cells with the composition in step (a) is carried out in a monolayer of said population of stem cells.

9. The method according to claim 1, wherein the composition in step (a) comprises:

(i) a BMP antagonist selected from the group consisting of noggin, an inhibitor of the transcriptional activity of the BMP type I receptors ALK2 and/or ALK3, chordin, and LDN193189; and

/or

(ii) a FGF which is selected from the group consisting of a basic fibroblast growth factor (bFGF), FGF-17, FGF-5, FGF-16, FGF-6, FGF-20, FGF-12, FGF-4, FGF-10, FGF-21, FGF-8a, FGF-23, FGF-9, FGF-19, FGF-22, and FGF-3; and

/or

(iii) an activin or nodal inhibitor selected from the group consisting of SB431542, LY2157299, SB525334, SB505124 and LY2109761.

10. The method according to claim 1, wherein the composition in step (a) comprises:

(i) a BMP antagonist present in a concentration selected from the group consisting of about 100 ng/ml to about 500 mg/ml, about 100 ng/ml, about 200 ng/ml, about 300 ng/ml, about 400 ng/ml, and about 500 ng/ml; and

/or

(ii) a FGF present in a concentration selected from the group consisting of about 5 ng/ml to about 20 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml and about 20 ng/ml; and

/or

(iii) an activin or nodal inhibitor present in a concentration selected from the group consisting of about 5 μ

M to about 20 μ

M, about 5 μ

M, about 6 μ

M, about 7 μ

M, about 8 μ

M, about 9 μ

M, about 10 μ

M, about 11 μ

M, about 12 μ

M, about 13 μ

M, about 14 μ

M, about 15 μ

M, about 16 μ

M, about 17 μ

M, about 18 μ

M, about 19 μ

M, and about 20 μ

M.

11. The method according to claim 1, wherein the composition in step (a) comprises:

(i) a BMP antagonist which is noggin present in a concentration of about 200 ng/ml; and

/or

(ii) a FGF which is bFGF present in a concentration of about 12 ng/ml; and

/or

(iii) an activin or nodal inhibitor which is SB431542 present in a concentration of about 10 μ

M.

12. The method according to claim 1, wherein the contacting in step (a) is for a duration selected from the group consisting of about 8 days to about 18 days, about 10 days to about 16 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days and about 18 days.

13. The method according to claim 3, wherein the population of CD57+ and/or CD271+ neural crest progenitor cells are isolated in step (b) using Fluorescence Assisted Cell Sorting (FACS) or magnetic assisted cell sorting (MACs).

14. The method according claim 4, wherein the composition in step (c) comprises:

(i) a PDGF which is platelet-derived growth factor BB (PDGF-BB); and

/or

(ii) a TGF which is transforming growth factor-beta 1 (TGF-β

1).

15. The method according claim 4, wherein the composition in step (c) comprises:

(i) a PDGF present in a concentration selected from the group consisting of about 5 ng/ml to about 20 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, and about 20 ng/ml; and

/or

(ii) a TGF present in a concentration selected from the group consisting of about 1 ng/ml to about 5 ng/ml, about 1 ng/ml, about 1.5 ng/ml, about 1.6 ng/ml, about 1.7 ng/ml, about 1.8 ng/ml, about 1.9 ng/ml, about 2 ng/ml, about 2.1 ng/ml, about 2.2 ng/ml, about 2.3 ng/ml, about 2.4 ng/ml, about 2.5 ng/ml, about 3 ng/ml, about 4 ng/ml and about 5 ng/ml.

16. The method according to claim 4, wherein the contacting in step (c) is for a duration selected from the group consisting of about 9 days to about 20 days, about 10 days to about 16 days, about 10 days to about 14 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days and about 20 days.

17. A method for inducing a disease phenotype associated with abnormal amyloid-beta (Aβ

) protein uptake and clearance, the method comprising the step of exposing the brain-specific vascular SMCs prepared according to the method of claim 1 to hypoxic condition for a length of time sufficient to induce said disease phenotype.

18. The method according to claim 17, wherein the hypoxic condition comprises a condition selected from the group consisting of less than about 5% of oxygen, less than about 4% of oxygen, less than about 3% of oxygen, less than about 2% of oxygen, and less than about 1% of oxygen.

19. The method according to claim 17, where the length of time sufficient to induce said disease phenotype is selected from the group consisting of at least about 48 hr, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, about 2 weeks or more, about 3 weeks or more, about 4 weeks or more, about 2 months or more, about 3 months or more, about 4 months or more, and about 5 months or more.

20. The method according to claim 17, wherein the Aβ

protein is selected from the group consisting of Aβ

40 and Aβ

42.

21. The method according to claim 17, wherein said disease phenotype is selected from the group consisting of aging, neurological disorders, and cerebrovascular disorders.

22. A method for simulating or modeling a disorder associated with abnormal amyloid-beta (Aβ

) protein uptake and clearance, comprising the method according to claim 17.

23. A composition comprising a BMP antagonist which is noggin present in a concentration of about 200 ng/ml, a FGF which is bFGF present in a concentration of about 12 ng/ml, an activin or nodal inhibitor which is SB431542 present in a concentration of about 10 μ

M.

24. The composition according to claim 23, wherein the composition does not comprise any one of the following which is selected from the group consisting of a glycogen synthase kinase (GSK) inhibitor, a Wnt protein, and a serum-free composition.