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However, traditional methods of engineering CARs into human tissue\u2010derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large\u2010scale and cryopreserved mesothelin (MSLN) CAR\u2010NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR\u2010expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR\u2010NK cells via an efficient organoid induction system. The MSLN CAR\u2010NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR\u2010NK cells show increased secretion of IFN\u2010\u03b3 and TNF\u2010\u03b1, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR\u2010NK cells in liquid nitrogen using a clinical\u2010grade freezing medium (CS10) for more than 6\u2009months to mimic an off\u2010the\u2010shelf CAR\u2010NK cell product. The thawed MSLN CAR\u2010NK cells immediately recovered after 48\u201372\u2010h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR\u2010NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour\u2010bearing mice. Our study provides insights into the clinical translation of hESC\u2010derived MSLN CAR\u2010NK cells as a promising off\u2010the\u2010shelf cell product.<\/jats:p>","DOI":"10.1111\/cpr.13727","type":"journal-article","created":{"date-parts":[[2024,8,13]],"date-time":"2024-08-13T08:25:20Z","timestamp":1723537520000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Mesothelin <scp>CAR<\/scp>\u2010engineered <scp>NK<\/scp> cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals"],"prefix":"10.1111","volume":"57","author":[{"given":"Yanhong","family":"Liu","sequence":"first","affiliation":[{"name":"Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology Institute of Zoology, Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Institute for Stem Cell and Regenerative Medicine  Beijing 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