Visualization of h3k9me3 in embryoid bodies using genetically encoded fluorescent sensor MPP8-Green

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Abstract

Epigenetic histone modifications play a key role in the differentiation of stem cells into various cell types. The ability of induced pluripotent stem cells (iPSCs) to differentiate is assessed using the embryoid body formation method, which is widely used and prevalent in iPSC research. In this study, we utilized a stable line of iPSCs with a genetically encoded sensor MPP8-Green to visualize the histone modification H3K9me3 during embryoid body formation. We identified two groups of cells based on the distribution of H3K9me3 in the formed embryoid bodies, using the MPP8-Green sensor. This study demonstrates that the MPP8-Green sensor can be used to track the dynamics of H3K9me3 during spontaneous differentiation and embryoid body formation. Using the sensor, we identified two groups of cells with different distributions of H3K9me3 and showed the potential application of such genetically encoded tools to reveal differences in patterns of epigenetic modifications during the spontaneous differentiation of iPSCs.

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About the authors

A. I. Stepanov

Skolkovo Institute of Science and Technology; Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Author for correspondence.
Email: gurskayanadya@gmail.com
Russian Federation, Moscow; Moscow

E. B. Zhigmitova

Pirogov Russian National Research Medical University

Email: gurskayanadya@gmail.com
Russian Federation, Moscow

E. B. Dashinimaev

Pirogov Russian National Research Medical University

Email: gurskayanadya@gmail.com
Russian Federation, Moscow

A. A. Galiakberova

Pirogov Russian National Research Medical University

Email: gurskayanadya@gmail.com
Russian Federation, Moscow

L. V. Putlyaeva

Skolkovo Institute of Science and Technology; Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gurskayanadya@gmail.com
Russian Federation, Moscow; Moscow

K. A. Lukyanov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry

Email: gurskayanadya@gmail.com
Russian Federation, Moscow

N. G. Gurskaya

Skolkovo Institute of Science and Technology; Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Pirogov Russian National Research Medical University

Email: gurskayanadya@gmail.com
Russian Federation, Moscow; Moscow; Moscow

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2. Fig. 1 Fluorescence microscopy of immunostained differentiated cells demonstrating markers of endoderm (a), mesoderm (b) and ectoderm (c); (a) – α-fetoprotein (AFP) – green fluorescence channel, desmin – red channel; (b) – smooth muscle actin (αSMA) – green channel, Pax6 – red channel; (c) – nestin – green channel, FOXA2 – red channel.

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3. Fig. 2. (a) – Fluorescence microscopy of MPP8-Green in formed embryoid bodies; (b) – fluorescence microscopy of two different groups of differentiated cells: cells with epigenetic “dots” on top, cells with diffuse distribution of the sensor on the bottom.

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