Methods of differentiation to neuronal cells and kits therefor

Patent metadata

Field	Value
Publication number	US-11566221-B2
Application number	US-201716344497-A
Country	US
Kind code	B2
Filing date	Oct 20, 2017
Priority date	Oct 24, 2016
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

Embodiments herein provide methods of differentiating neural stem cells to neuronal cells while concomitantly retarding neural stem cell proliferation. Resultant cultures demonstrate reduced clumping of cells, increased purity of neuronal cells and accelerated electrophysiology as compared to control methods.

First claim

What is claimed is:

1. A method for accelerating differentiation of at least one neural stem cell to at least one neuronal cell and concomitantly retarding neural stem cell proliferation, comprising:
culturing the at least one neural stem cell in a differentiation medium for a time and under conditions to form the at least one neuronal cell,
wherein the differentiation medium comprises a serum-free neural stem cell culture medium, and a serum-free supplement comprising at least one gamma secretase inhibitor wherein a differentiation signal of the at least one neuronal cell is greater by at least 100% at Day 7 of differentiation as compared to the differentiation signal of at least one neural stem cell at Day 7 of culturing in the differentiation medium lacking the at least one gamma secretase inhibitor.

2. The method of claim 1 , wherein the at least one neural stem cell is derived from an induced pluripotent stem cell.

3. The method of claim 1 , wherein the at least one neural stem cell is derived from an embryonic stem cell.

4. The method of claim 1 wherein the at least one neural stem cell is a SOX1 positive neural stem cell and the at least one neuronal cell is a MAP2 positive neuronal cell.

5. The method of claim 1 , wherein the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, MK-0752, a salt thereof, and a combination thereof.

6. The method of claim 5 , wherein the serum-free supplement of the differentiation medium comprises a gamma secretase inhibitor selected from the group consisting of Compound E, YO-01027, LY411575, a salt thereof, and a combination thereof.

7. The method of claim 5 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.1 micromolar to 40 micromolar.

8. The method of claim 6 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.2 micromolar to 10 micromolar.

9. The method of claim 6 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of 0.2 micromolar to 2.0 micromolar.

10. The method of claim 1 wherein the at least one neuronal cell is maintained in culture for at least a period of five weeks.

11. The method of claim 1 , wherein the gamma secretase inhibitor is present in the differentiation medium at a concentration of up to 2.0 micromolar.

12. The method of claim 1 , wherein the gamma secretase inhibitor is other than N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT).

Assignees

• Life Technologies Corp

Inventors

• Shin Soojung
• Yan Yiping
• Beacham Daniel

Classifications

• C12N5/0619 Primary

  Neurons · CPC title

• C12N5/0606

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N2506/08

  from cells of the nervous system · CPC title

• C12N5/0037

  Serum-free medium, which may still contain naturally-sourced components · CPC title

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title