COMPOSITIONS AND METHODS FOR ENHANCING THE PLURIPOTENCY OF STEM CELLS

• US 20190330288A1
• Filed: 05/07/2019
• Published: 10/31/2019
• Est. Priority Date: 03/23/2011
• Status: Active Grant

• Current Assignee: Elixirgen, LLC
• Original Assignee: Elixirgen, LLC
• Inventors: KO, Minoru S.H., AMANO, Tomokazu
• Granted Patent: US 11,186,621 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Described herein is the finding that increasing the frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into a 4N blastocyst at an unexpectedly high success rate. The studies disclosed herein indicate that ES cells acquire higher potency by going through the transient Zscan4 activation state more frequently than the regular state. Particularly disclosed herein is the finding that the constitutive presence of Zscan4-ERT2, even in the absence of its usual activator tamoxifen, can increase the frequency of endogenous Zscan4 activation in ES cells, resulting in the increase of developmental potency of the ES cells. Accordingly, provided herein are Zscan4-ERT2 fusion proteins and nucleic acid molecules and vectors encoding Zscan4-ERT2 fusion proteins. Further provided are methods of prolonging and/or enhancing stem cell pluripotency using the disclosed Zscan4-ERT2 nucleic acid molecules and fusion proteins.

Claims

1. :

A recombinant polypeptide selected from a Zscan4-ERT2 fusion protein and a Zscan4-Δ

C protein.

2. :

The recombinant polypeptide of claim 1, wherein the Zscan4 or the Zscan4-Δ

C protein comprises mouse Zscan4c, mouse Zscan4d, mouse Zscan4f or human ZSCAN4, or a functional fragment or variant thereof.

3. :

The recombinant polypeptide of claim 1, comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO;

22 or SEQ ID NO;

23.

4. :

The recombinant polypeptide of claim 1, wherein the Zscan4-Δ

C protein comprises mouse Zscan4c, mouse Zscan4d, mouse Zscan4f or human ZSCAN4 comprising a deletion of at least one zinc finger domain.

5. :

An isolated cell comprising the recombinant polypeptide of claim 1.

6. :

The isolated cell of claim 5, which is a stem cell.

7. :

The isolated cell of claim 6, which is an ES cell or an iPS cell.

8. :

The isolated cell of claim 6, wherein the stem cell is a mouse, rat, human or non-human primate stem cell.

9. :

A composition comprising the recombinant polypeptide of claim 1 and a pharmaceutically acceptable carrier.

10. :

A method of enhancing or prolonging the pluripotency of a stem cell or a stem cell population, comprising contacting the stem cell or stem cell population with a nucleic acid encoding the recombinant polypeptide of claim 1.

11. :

A method of increasing the frequency of Zscan4-positive cells in a stem cell population, comprising contacting the stem cell population with the nucleic acid molecule encoding the recombinant polypeptide of claim 1.

12. :

A method of promoting genome stability or increasing telomere length, or both, in a stem cell or a stem cell population, comprising contacting the stem cell or stem cell population with the nucleic acid molecule encoding the recombinant polypeptide of claim 1.

13. :

The method of claim 10, wherein the stem cell is an ES cell or an iPS cell.

14. :

The method of claim 10, wherein the stem cell is a mouse, rat, human or non-human primate stem cell.

15. :

A method for increasing the developmental potency of a stem cell or a stem cell population, comprising transiently overexpressing Zscan4.

16. :

The method of claim 15, wherein the overexpressed Zscan4 is mouse Zscan4c.

17. :

A method of enhancing or prolonging the pluripotency of a stem cell or a stem cell population, comprising contacting the stem cell or stem cell population with an isolated nucleic acid molecule encoding a Zscan4 protein or a vector comprising a nucleic acid molecule encoding a Zscan4 protein.

18. :

The method of claim 17 wherein the vector further comprises an inducible promoter.