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Dual-specificity Tyrosine Phosphorylation-regulated Kinase Inhibitor ID-8 Promotes Human Somatic Cell Reprogramming by Activating PDK4 Expression

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Abstract

Background

Human induced pluripotent stem cells (hiPSCs) hold great potentials in disease modeling, drug screening and cell therapy. However, efficiency and costs of hiPSCs preparation still need to be improved.

Methods

We screened the compounds that target signaling pathways, epigenetic modifications or metabolic-process regulation to replace the growth factors. After small molecule treatment, TRA-1-60, which is a cell surface antigen expressed by human embryonic stem cells (hESCs), staining was performed to quantify the efficiency of somatic cell reprogramming. Next, small molecule cocktail-induced ESCs or iPSCs were examined with pluripotent markers expression. Finally, Genome-wide gene expression profile was analyzed by RNA-seq to illustrate the mechanism of human somatic cell reprogramming.

Result

Here, we found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of pyruvate dehydrogenase kinase 4 (PDK4) and activation of glycolysis. Furthermore, we identified a novel growth-factor-free hiPSC generation system using small molecules ID-8 (I) and TGFβ signal pathway agonist Kartogenin (K). Importantly, we developed IK medium combined with low-dose bFGF to support the long-term expansion of human pluripotent stem cells. IK-iPSCs showed pluripotency and normal karyotype.

Conclusions

Our studies may provide a novel growth-factor-free culture system to facilitate the generation of hiPSCs for multiple applications in regenerative medicine.

Graphical abstract

In Brief

Xu et at. found that a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor ID-8 robustly enhanced human somatic cell reprogramming by upregulation of PDK4 and activation of glycolysis. Furthermore, we established a novel growth-factor-free hiPSC generation system using small molecules ID-8/Kartogenin (IK). IK medium combined with Low-dose bFGF (IKB medium) supported the long-term expansion of human pluripotent stem cells.

Highlights

ID-8 Enhanced Reprogramming of Human Fibroblasts and Astrocytes

Establishment of the Growth-factor-free Reprogramming System Using Small Molecule Compounds IK

IKB Medium Maintained the Long-term Expansion of Human Pluripotent Stem Cells

ID-8 Promoted Human Somatic Cell Reprogramming by Activating PDK4 Expression

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Availability of Data and Materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

hiPSCs:

Human induced pluripotent stem cells

IKB:

ID-8, Kartogenin and Low-dose bFGF

E8:

Essential 8

E6:

E8 without bFGF and TGFβ

I:

ID-8

K:

Kartogenin

C:

CHIR99021

AK:

1-Azakenpaullone

T:

Tacrolimus

IK-iPSCs:

iPSC cultured in E6 with ID-8 and Kartogenin

TZDs:

Thiazolidinediones

DCA:

Sodium dichloroacetate

2-DG:

2-Deoxyglucose

PDK4:

Pyruvate dehydrogenase kinase 4

Go:

Go6983

SP:

SP600125

Dora:

Doramapimod

DGs:

Differential genes

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Acknowledgements

We would like to thank School of Medicine, Xiamen University and MOE Frontiers Center for Brain Science, Fudan University for providing platform for experiments of cell culture, staining and images.

Funding

This work was supported by National Natural Science Foundation of China (grants 32070956 to Zhicheng Shao), Fundamental Research Funds for Central Universities from Xiamen Universities (grants 20720180040 to Zhicheng Shao), Natural Science Foundation of Shanghai (grants 20ZR1405200 to Zhicheng Shao), Natural Science Foundation of Fujian Province of China (grants 2018J01055 to Zhicheng Shao), MOE Frontiers Center for Brain Science fund and starting fund from Fudan Universities.

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Authors and Affiliations

  1. Fujian Provincial Key Laboratory of Neurodegenerative, Disease and Aging Research, Institute of Neuroscience, School of Medicine, Xiamen University, Xiamen, 361005, Fujian, China

    Jinhong Xu, Shi Fang, Yongheng Huang & Zhicheng Shao

  2. Institute for Translational Brain Research, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, 200032, China

    Jinhong Xu, Shi Fang, Naweng Wang, Yongheng Huang, Qi Fan, Jingyi Shi, Huihui Liu & Zhicheng Shao

  3. Key Laboratory of Biomedical Engineering of Fujian Province, Research Center of Biomedical Engineering of Xiamen, Department of Biomaterials, College of Materials, Xiamen University, Xiamen, 361005, People’s Republic of China

    Bo Li

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  1. Jinhong Xu
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  2. Shi Fang
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  4. Bo Li
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Contributions

Z.S and J.X. designed the experiments. J.X., S.F., N.W., B.L., Y.H., Q.F., J.S.and H.L. performed fibroblast and astrocyte reprogramming, screening of small molecules for promoting reprogramming efficiency and maintaining human pluripotency stem cells. Z.S and J.X. did RNA-seq analysis. Z.S., J.X. and N.W. wrote the manuscript and data interpretation. Z.S. supported this study financially.

Corresponding author

Correspondence to Zhicheng Shao.

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This article belongs to the Topical Collection: Special Issue on Stem Cell Technology and Skin Disorders (Dermatology): from Stem Cell Biology to Clinical Application

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Xu, J., Fang, S., Wang, N. et al. Dual-specificity Tyrosine Phosphorylation-regulated Kinase Inhibitor ID-8 Promotes Human Somatic Cell Reprogramming by Activating PDK4 Expression. Stem Cell Rev and Rep 18, 2074–2087 (2022). https://doi.org/10.1007/s12015-021-10294-9

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