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. 2024 Sep 20;5(3):103263.
doi: 10.1016/j.xpro.2024.103263. Epub 2024 Aug 10.

Protocol for generation and engineering of thyroid cell lineages using CRISPR-Cas9 editing to recapitulate thyroid cancer histotype progression

Vincenzo Davide Pantina  1 , Francesco Verona  1 , Alice Turdo  2 , Veronica Veschi  3 , Chiara Modica  1 , Melania Lo Iacono  2 , Miriam Gaggianesi  1 , Sebastiano Di Bella  1 , Matilde Todaro  4 , Simone Di Franco  5 , Giorgio Stassi  6
Affiliations

Protocol for generation and engineering of thyroid cell lineages using CRISPR-Cas9 editing to recapitulate thyroid cancer histotype progression

Vincenzo Davide Pantina et al. STAR Protoc. .

Abstract

Thyroid carcinoma represents the first malignancy among the endocrine organs. Investigating the cellular hierarchy and the mechanisms underlying the initiation of thyroid carcinoma is crucial in thyroid cancer research. Here, we present a protocol for deriving thyroid cell lineage from human embryonic stem cells. We also describe steps for engineering thyroid progenitor cells utilizing CRISPR-Cas9 technology, which can be used to perform in vivo studies, thus facilitating the development of representative thyroid tumorigenesis models. For complete details on the use and execution of this protocol, please refer to Veschi et al.1.

Keywords: CRISPR; Cancer; Cell Differentiation; Cell culture.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Differentiation model of human embryonic stem cells (hESC) in a complete thyroid lineage
Figure 2
Figure 2
Validation of hESC-derived differentiated progenies (A) Phase contrast microscopy of hESCs-derived stages of thyroid differentiation. Scale bars measure 20 μm. (B) mRNA levels of HHEX and FOXE1 in hESCs-derived specific stages of thyroid differentiation. Data represent mean ± SD of three independent experiments. (C) Heatmap representing flow cytometry analysis of specific markers in hESCs-derived various stages of thyroid differentiation lineages (D0, D6, D22, D30). Data are presented as mean ± SD from three separate experiments.
Figure 4
Figure 4
Schematic diagram of gene-edited OFP-positive cell sorting (A) Gating strategy for the sorting of OFP+ transduced TPC. (B) Post sorting evaluation of OFP+ TPC cell population.
Figure 3
Figure 3
Engineering of D22 TPCs via CRISPR/Cas9 technology
Figure 5
Figure 5
Orthotopic injection of engineered thyroid progenitor cells recapitulates thyroid cancer in vivo
Figure 6
Figure 6
Histopathological comparison of TPCs-derived xenografts and patient-derived thyroid tumors demonstrates that the orthotopic injection of engineered thyroid progenitor cells recapitulates thyroid cancer in vivo Scale bars represent 100 μm.
See this image and copyright information in PMC

References

    1. Veschi V., Turdo A., Modica C., Verona F., Di Franco S., Gaggianesi M., Tirrò E., Di Bella S., Iacono M.L., Pantina V.D., et al. Recapitulating thyroid cancer histotypes through engineering embryonic stem cells. Nat. Commun. 2023;14:1351. doi: 10.1038/s41467-023-36922-1. - DOI - PMC - PubMed
    1. Longmire T.A., Ikonomou L., Hawkins F., Christodoulou C., Cao Y., Jean J.C., Kwok L.W., Mou H., Rajagopal J., Shen S.S., et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012;10:398–411. doi: 10.1016/j.stem.2012.01.019. - DOI - PMC - PubMed
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    1. Li X., Krawetz R., Liu S., Meng G., Rancourt D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum. Reprod. 2009;24:580–589. doi: 10.1093/humrep/den404. - DOI - PubMed
    1. Liang X., Potter J., Kumar S., Zou Y., Quintanilla R., Sridharan M., Carte J., Chen W., Roark N., Ranganathan S., et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 2015;208:44–53. doi: 10.1016/j.jbiotec.2015.04.024. - DOI - PubMed

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