CHIR99021 and Brdu Are Critical in Chicken IPSC Reprogramming Via Small-Molecule Screening

Overview

Journal Genes (Basel)

Publisher MDPI

Date 2024 Sep 28

PMID 39336797

Authors

Kai Jin

Jing Zhou

Gaoyuan Wu

Zeyu Li

XiLin Zhu

Youchen Liang

Tingting Li

Guohong Chen

Qisheng Zuo

Yingjie Niu

Jiuzhou Song

Wei Han

Affiliations

Soon will be listed here.

Abstract

Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 10 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms.

References

Xu Y, Shi Y, Ding S . A chemical approach to stem-cell biology and regenerative medicine. Nature. 2008; 453(7193):338-44. DOI: 10.1038/nature07042. View

Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

Naujok O, Diekmann U, Lenzen S . The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling. Stem Cell Rev Rep. 2014; 10(4):480-93. DOI: 10.1007/s12015-014-9509-0. View

Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K . Induced pluripotent stem cells generated without viral integration. Science. 2008; 322(5903):945-9. PMC: 3987909. DOI: 10.1126/science.1162494. View

Kim Y, Park Y, Lim J, Jung H, Han J . Technical note: Induction of pluripotent stem cell-like cells from chicken feather follicle cells. J Anim Sci. 2017; 95(8):3479-3486. DOI: 10.2527/jas.2017.1418. View

Warren L, Manos P, Ahfeldt T, Loh Y, Li H, Lau F . Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010; 7(5):618-30. PMC: 3656821. DOI: 10.1016/j.stem.2010.08.012. View

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-72. DOI: 10.1016/j.cell.2007.11.019. View

Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S . A more efficient method to generate integration-free human iPS cells. Nat Methods. 2011; 8(5):409-12. DOI: 10.1038/nmeth.1591. View

Chen G, Guo Y, Li C, Li S, Wan X . Small Molecules that Promote Self-Renewal of Stem Cells and Somatic Cell Reprogramming. Stem Cell Rev Rep. 2020; 16(3):511-523. DOI: 10.1007/s12015-020-09965-w. View

10.

Gore A, Li Z, Fung H, Young J, Agarwal S, Antosiewicz-Bourget J . Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011; 471(7336):63-7. PMC: 3074107. DOI: 10.1038/nature09805. View

11.

Yu J, Vodyanik M, Smuga-Otto K, Antosiewicz-Bourget J, Frane J, Tian S . Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858):1917-20. DOI: 10.1126/science.1151526. View

12.

Okita K, Ichisaka T, Yamanaka S . Generation of germline-competent induced pluripotent stem cells. Nature. 2007; 448(7151):313-7. DOI: 10.1038/nature05934. View

13.

Li W, Ding S . Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming. Trends Pharmacol Sci. 2009; 31(1):36-45. DOI: 10.1016/j.tips.2009.10.002. View

14.

Han X, Han J, Ding F, Cao S, Lim S, Dai Y . Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells. Cell Res. 2011; 21(10):1509-12. PMC: 3193448. DOI: 10.1038/cr.2011.125. View

15.

Liu H, Zhu F, Yong J, Zhang P, Hou P, Li H . Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell. 2008; 3(6):587-90. DOI: 10.1016/j.stem.2008.10.014. View

16.

Hawley R . Does retroviral insertional mutagenesis play a role in the generation of induced pluripotent stem cells?. Mol Ther. 2008; 16(8):1354-5. PMC: 2572214. DOI: 10.1038/mt.2008.142. View

17.

Ji J, Ng S, Sharma V, Neculai D, Hussein S, Sam M . Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells. Stem Cells. 2011; 30(3):435-40. DOI: 10.1002/stem.1011. View

18.

Cao S, Yu S, Li D, Ye J, Yang X, Li C . Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency. Cell Stem Cell. 2018; 22(4):529-542.e5. DOI: 10.1016/j.stem.2018.03.005. View

19.

Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin I . Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009; 324(5928):797-801. PMC: 2758053. DOI: 10.1126/science.1172482. View

20.

Schugar R, Robbins P, Deasy B . Small molecules in stem cell self-renewal and differentiation. Gene Ther. 2007; 15(2):126-35. DOI: 10.1038/sj.gt.3303062. View