A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator of human naïve pluripotency and totipotency

Abstract

Regulation of totipotency and naïve pluripotency is crucial for early human embryo development. However, the mechanisms of naïve pluripotency and totipotency regulation in humans, especially the signaling pathways involved in these processes, remain largely unknown. Here, using the conversion of human extended pluripotent stem cells (hEPSCs) to naïve pluripotent stem cells as a model, we performed a CRISPR/Cas9-based kinome knockout screen to analyze the effect of disrupting 763 kinases in regulating human naïve pluripotency. Further validation using small molecules revealed that the inhibition of ErbB family kinases promoted the transition of hEPSCs to human naïve pluripotent stem cells. More importantly, chemical inhibition of the ErbB family also promoted induction of totipotent signatures in human pluripotent cells under different culture conditions. Our findings provide new mechanistic insights into the regulation of naïve pluripotency and totipotency in humans.

Keywords

totipotency / naïve pluripotency / hEPSCs / CRISPR screens / kinome

References

[1] Baker CL, Pera MF. Capturing totipotent stem cells. Cell Stem Cell 2018;22:25–34.

[2] Molè MA, Weberling A, Zernicka-Goetz M. Comparative analysis of human and mouse development: from zygote to pre-gastrulation. Curr Top Dev Biol 2020;136:113–38.

[3] Guo G, Stirparo GG, Strawbridge SE et al. Human naive epiblast cells possess unrestricted lineage potential. Cell Stem Cell 2021;28:1040–1056.e6.

[4] Zou Z, Zhang C, Wang Q et al. Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation. Science 2022;378:abo7923.

[5] De Iaco A, Planet E, Coluccio A et al. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat Genet 2017;49:941–5.

[6] Whiddon JL, Langford AT, Wong CJ et al. Conservation and innovation in the DUX4-family gene network. Nat Genet 2017;49:935–40.

[7] Martello G, Smith A. The nature of embryonic stem cells. Annu Rev Cell Dev Biol 2014;30:647–75.

[8] Li M, Belmonte JCI. Deconstructing the pluripotency gene regulatory network. Nat Cell Biol 2018;20:382–92.

[9] Kinoshita M, Barber M, Mansfield W et al. Capture of mouse and human stem cells with features of formative pluripotency. Cell Stem Cell 2021;28:453–71.e8.

[10] Yu L, Wei Y, Sun HX et al. Derivation of intermediate pluripotent stem cells amenable to primordial germ cell specification. Cell Stem Cell 2021;28:550–567.e12.

[11] Nichols J, Smith A. Naive and primed pluripotent states. Cell Stem Cell 2009;4:487–92.

[12] Takashima Y, Guo G, Loos R et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 2014;158:1254–69.

[13] Theunissen TW, Powell BE, Wang H et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 2014;15:471–87.

[14] Theunissen TW, Friedli M, He Y et al. Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 2016;19:502–15.

[15] Io S, Kabata M, Iemura Y et al. Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell 2021;28:1023–1039.e13.

[16] Yang Y, Liu B, Xu J et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 2017;169:243–257. e25.

[17] Liu B, Chen S, Xu Y et al. Chemically defined and xeno-free culture condition for human extended pluripotent stem cells. Nat Commun 2021;12:3017.

[18] Sozen B, Jorgensen V, Weatherbee BAT et al. Reconstructing aspects of human embryogenesis with pluripotent stem cells. Nat Commun 2021;12:5550.

[19] Taubenschmid-Stowers J, Rostovskaya M, Santos F et al. 8C-like cells capture the human zygotic genome activation program in vitro. Cell Stem Cell 2022;29:449–459.e6.

[20] Mazid MA, Ward C, Luo Z et al. Rolling back human pluripotent stem cells to an eight-cell embryo-like stage. Nature 2022;605:315–24.

[21] Weinberger L, Ayyash M, Novershtern N et al. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol 2016;17:155–69.

[22] Johnson SA, Hunter TK. Methods for deciphering the kinome. Nat Methods 2005;2:17–25.

[23] Ihry RJ, Worringer KA, Salick MR et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med 2018;24:939–46.

[24] Doench JG, Fusi N, Sullender M et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 2016;34:184–91.

[25] Bredenkamp N, Yang J, Clarke J et al. Wnt inhibition facilitates RNA-mediated reprogramming of human somatic cells to naive pluripotency. Stem Cell Rep 2019;13:1083–98.

[26] Bredenkamp N, Stirparo GG, Nichols J et al. The cell-surface marker sushi containing domain 2 facilitates establishment of human naive pluripotent stem cells. Stem Cell Rep 2019;12:1212–22.

[27] Li W, Xu H, Xiao T et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 2014;15:554.

[28] Hsu PD, Scott DA, Weinstein JA et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013;31:827–32.

[29] Szklarczyk D, Kirsch R, Koutrouli M et al. The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res 2023;51:D638–D646.28.

[30] Lea RA, McCarthy A, Boeing S et al. KLF17 promotes human naïve pluripotency but is not required for its establishment. Development 2021;148:dev199378.

[31] Wang SH, Hao J, Zhang C et al. KLF17 promotes human naive pluripotency through repressing MAPK3 and ZIC2. Sci China Life Sci 2022;65:1985–97.

[32] Roskoski R. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res 2014;79:34–74.

[33] Yan L, Yang M, Guo H et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 2013;20:1131–9.

[34] Xiong Z, Xu K, Lin Z et al. Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development. Nat Cell Biol 2022;24:968–80.

[35] Yu X, Liang S, Chen M et al. Recapitulating early human development with 8C-like cells. Cell Rep 2022;39:110994.

[36] Xue Z, Huang K, Cai C et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 2013;500:593–7.

[37] Collier AJ, Bendall A, Fabian C et al. Genome-wide screening identifies Polycomb repressive complex 13 as an essential regulator of human naïve pluripotent cell reprogramming. Sci Adv 2022;8:eabk0013.

[38] Doench JG. Am I ready for CRISPR? A user’s guide to genetic screens. Nat Rev Genet 2018;19:67–80.

[39] Bayerl J, Ayyash M, Shani T et al. Principles of signaling pathway modulation for enhancing human naive pluripotency induction. Cell Stem Cell 2021;28:1549–1565.e12.

[40] Tebbutt N, Pedersen MW, Johns TG. Targeting the ERBB family in cancer: couples therapy. Nat Rev Cancer 2013;13:663–73.

[41] Che M, Chaturvedi A, Munro SA et al. Opposing transcriptional programs of KLF5 and AR emerge during therapy for advanced prostate cancer. Nat Commun 2021;12:6377.

[42] Park JM, Wu T, Cyr AR et al. The role of Tcfap2c in tumorigenesis and cancer growth in an activated Neu model of mammary carcinogenesis. Oncogene 2015;34:6105–14.

[43] Kinisu M, Choi YJ, Cattoglio C et al. Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes. Cell Rep 2021;37:109982.

[44] Zhu M, Cornwall-Scoones J, Wang P et al. Developmental clock and mechanism of de novo polarization of the mouse embryo. Science 2020;370:eabd2703.

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2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.