Methods for enhancing the pluripotency of stem cells
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.
1. An in vitro method of prolonging pluripotency, increasing frequency of Zscan4-positive cells and/or increasing telomere length of a mouse embryonic stem (ES) cell, the method comprising:
a) transfecting an expression vector comprising a nucleic acid encoding a Zscan4-ΔC protein consisting of the amino acid sequence as set forth in SEQ ID NO:25 into the mouse ES cell; and
b) culturing the transfected mouse ES cell in culture medium such that the Zscan4-ΔC protein is expressed in the mouse ES cell.
2. An in vitro method of prolonging pluripotency, increasing frequency of Zscan4-positive cells and/or increasing telomere length of a mouse embryonic stem (ES) cell, the method comprising:
a) transfecting an expression vector comprising a nucleic acid encoding a Zscan4-ERT2 fusion protein consisting of the amino acid sequence as set forth in SEQ ID NO:22 or SEQ ID NO:23 into the mouse ES cell; and
b) culturing the transfected mouse ES cell in culture medium lacking tamoxifen such that the Zscan4-ERT fusion protein is expressed in the mouse ES cell.
3. The method of claim 2 , wherein the Zscan4-ERT2 fusion protein consists of the amino acid sequence of SEQ ID NO:23.
4. The method of claim 2 , wherein the Zscan4-ERT2 fusion protein consists of the amino acid sequence of SEQ ID NO:22.