The Dual Effect of Lithium Chloride on the Efficiency of Generating Mouse-Induced Pluripotent Stem Cells

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Abstract

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) using certain factors. The low efficiency of the reprogramming, as well as the heterogeneity of iPSCs, limits the potential application for iPSCs in cell therapy. Here, we show that lithium chloride (LiCl), a known activator of the Wnt signaling pathway, reduces or enhances the efficiency of iPSC generation from mouse embryonic fibroblasts (MEFs) depending on the timing of its addition during the reprogramming. Our results not only demonstrate a method to improve the efficiency of iPSC formation by LiCL, but also indicate its dual role in this process.

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About the authors

A. V. Kuznetsov

Institute of Cytology RAS

Author for correspondence.
Email: atsimokha@incras.ru
Russian Federation, St. Petersburg

E. V. Skvortsova

Institute of Cytology RAS

Email: atsimokha@incras.ru
Russian Federation, St. Petersburg

A. N. Tomilin

Institute of Cytology RAS

Email: atsimokha@incras.ru
Russian Federation, St. Petersburg

A. S. Tsimokha

Institute of Cytology RAS

Email: atsimokha@incras.ru
Russian Federation, St. Petersburg

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Supplementary files

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2. Fig. 1. The efficiency of MEF reprogramming in iPSC is the same for 2iL and iCD1 environments. a is a diagram of the reprogramming process of MEF in iPSC; MEF was infected with viruses and on the next day (D0), OKSM expression was activated using doxycycline (DOX). After 2 days (D2), the reprogramming MEFs were dispersed onto the cell feeder layer and a day later (D3), the serum medium was changed to serum-free. b — The diagram shows the number of Nanog-positive iPSC colonies formed by the 15th day of reprogramming (D15) in the well of a 6-well tablet. The data is presented as averages and standard deviations of SD (n = 3). The lower panel shows representative images of entire wells of a 6-well tablet containing Nanog-positive iPSC colonies (red), and the color of DAPI nuclei (blue). b — Micrographs of iPSC colonies in transmitted light, Nanog-positive (red), obtained in 2iL (left) and iCD1 (right) media. Coloring of DAPI cores (blue). Scale ruler: 1000 microns.

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3. Fig. 2. The presence of LiCl from the 3rd day of MEF reprogramming reduces the efficiency of iPSC clone generation in the 2iL environment. a is a diagram of the experiment on reprogramming MEF in iPSC in the 2iL environment. The expression of OKSM was activated by doxycycline (DOX). Vitamin C (VitC) at a concentration of 50 micrograms/ml and (or) 5 mM LiCl was added starting from the 3rd day of reprogramming (D3) and until the end of the experiment (D15). b — The diagram shows the percentage of Nanog-positive clones on the 15th day of reprogramming. The data are presented in the form of average values and standard deviation (n = 3), ** — significant differences compared to the 2iL medium (p < 0.01).

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4. 3. The presence of LiCl reduces the efficiency of generation of iPSC clones from MEF in a medium with a serum substitute (KSR). a is a scheme of an experiment on reprogramming MEF in iPSC in a medium with a serum substitute (KSR). The expression of OKSM was activated by doxycycline (DOX). 10 mM LiCl was added between day 3 (D3) and day 8 (D8) of reprogramming. The serum medium was changed to KSR medium from the 6th day of reprogramming (D6). b - The diagram shows the percentage of Nanog—positive clones on the 15th day of reprogramming. The data is presented as averages ± standard deviation (n = 3). Asterisks indicate significant differences between the groups (*p < 0.05; **p < 0.01).

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5. Fig. 4. The presence of LiCl MEF from the beginning of reprogramming reduces the efficiency of generation of iPSC clones in the iCD1* environment, but increases from the 6th day of reprogramming. a is a diagram of an experiment on reprogramming MEF in iPSC in the iCD1* environment. The expression of OKSM was activated by doxycycline (DOX). 5 mM LiCl was added at different reprogramming periods. The serum to serum-free medium was changed on the 3rd day of reprogramming (D3). b — The diagram shows the percentage of Nanog-positive clones on the 15th day of reprogramming. The data is presented as averages and standard deviations (n = 3). Asterisks indicate significant differences compared to the iCD1* environment (*p < 0.05, **p < 0.01).

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