@article{8c138642b6084b04b133bb12fe639525,
title = "Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts",
abstract = "During early mammalian embryonic development, trophoblast cells play an essential role in establishing cell–cell interactions at the maternal–fetal interface to ensure a successful pregnancy. In a recent study, we showed that human fibroblasts can be reprogrammed into induced trophoblast stem (iTS) cells by transcription factor-mediated nuclear reprogramming using the Yamanaka factors OCT4, KLF4, SOX2 and c-MYC (OKSM) and a selection of TS cell culture conditions. The derivation of TS cells from human blastocysts or first-trimester placenta can be limited by difficulties in obtaining adequate material as well as ethical implications. By contrast, the described approach allows the generation of iTS cells from the adult cells of individuals with diverse genetic backgrounds, which are readily accessible to many laboratories around the world. Here we describe a step-by-step protocol for the generation and establishment of human iTS cells directly from dermal fibroblasts using a non-integrative reprogramming method. The protocol consists of four main sections: (1) recovery of cryopreserved human dermal fibroblasts, (2) somatic cell reprogramming, (3) passaging of reprogramming intermediates and (4) derivation of iTS cell cultures followed by routine maintenance of iTS cells. These iTS cell lines can be established in 2–3 weeks and cultured long term over 50 passages. We also discuss several characterization methods that can be performed to validate the iTS cells derived using this approach. Our protocol allows researchers to generate patient-specific iTS cells to interrogate the trophoblast and placenta biology as well as their interactions with embryonic cells in health and diseases.",
author = "Tan, {Jia Ping} and Xiaodong Liu and Polo, {Jose M.}",
note = "Funding Information: The authors thank the staff at Monash Flowcore Facility for providing high-quality cell sorting service and technical input. The authors acknowledge the use of the services and facilities of Micromon, Monash Micro Imaging and Monash Histology Platforms at Monash University. We also thank J. Hatwell-Humble and S. Nilsson for assistance with the mouse work. The schematics were created with BioRender.com. This work was supported by National Health and Medical Research Council (NHMRC) project grants APP1104560 and APP2004774 to J.M.P.; a Silvia and Charles Viertel Senior Medical Research Fellowship; and an ARC Future Fellowship FT180100674. J.P.T. was supported by a Monash International Tuition Scholarship and Research Training Program Scholarship. X.L. was supported by the Monash International Postgraduate Research Scholarship, a Monash Graduate Scholarship and the Carmela and Carmelo Ridolfo Prize in Stem Cell Research. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. Funding Information: The authors thank the staff at Monash Flowcore Facility for providing high-quality cell sorting service and technical input. The authors acknowledge the use of the services and facilities of Micromon, Monash Micro Imaging and Monash Histology Platforms at Monash University. We also thank J. Hatwell-Humble and S. Nilsson for assistance with the mouse work. The schematics were created with BioRender.com. This work was supported by National Health and Medical Research Council (NHMRC) project grants APP1104560 and APP2004774 to J.M.P.; a Silvia and Charles Viertel Senior Medical Research Fellowship; and an ARC Future Fellowship FT180100674. J.P.T. was supported by a Monash International Tuition Scholarship and Research Training Program Scholarship. X.L. was supported by the Monash International Postgraduate Research Scholarship, a Monash Graduate Scholarship and the Carmela and Carmelo Ridolfo Prize in Stem Cell Research. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. Publisher Copyright: {\textcopyright} 2022, Springer Nature Limited.",
year = "2022",
month = dec,
doi = "10.1038/s41596-022-00742-2",
language = "English",
volume = "17",
pages = "2739--2759",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "12",
}
