{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T05:34:28Z","timestamp":1763789668733,"version":"3.45.0"},"reference-count":35,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,20]],"date-time":"2025-11-20T00:00:00Z","timestamp":1763596800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science and ICT, Ministry of Trade, Industry and Energy, Ministry of Health &amp; Welfare, and Ministry of Food and Drug Safety","award":["RS-2023-KH140925"],"award-info":[{"award-number":["RS-2023-KH140925"]}]},{"name":"Ministry of Health &amp; Welfare","award":["RS-2025-02213795"],"award-info":[{"award-number":["RS-2025-02213795"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Platelet shortage poses a significant barrier to research and transfusion therapies because native megakaryocytes (MKs) are scarce in blood. To overcome this limitation, pluripotent stem cell\u2013derived MKs (PSC-MKs) offer a standardized, donor-independent platform for research and therapeutic development, including disease modeling and ex vivo platelet production. Here, we report a chemically defined, feeder-free protocol to generate MKs from human pluripotent stem cells (hPSCs). The protocol combines the small molecule MPL agonist Butyzamide, macrophage colony-stimulating factor (M-CSF), and three-dimensional (3D) suspension culture, achieving high efficiency and reproducibility. Butyzamide replaced recombinant thrombopoietin (TPO), yielding comparable CD41+\/CD42b+ populations and enhanced polyploidization. M-CSF accelerated nuclear lobulation and induced 4N MKs, while 3D culture increased yield, cell size, and substrate detachment. Multiple independent assays confirmed mature MK hallmarks, multi-nuclei, demarcation membranes, granules, and elevated mitochondrial respiration. Single-cell RNA sequencing outlined a continuous trajectory from early progenitors to functionally specialized MK subsets. This platform enables reliable MK supply for mechanistic studies and in vitro platelet production, advancing both basic research and therapeutic development.<\/jats:p>","DOI":"10.3390\/cells14221835","type":"journal-article","created":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T08:59:46Z","timestamp":1763715586000},"page":"1835","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Chemically Defined, Efficient Megakaryocyte Production from Human Pluripotent Stem Cells"],"prefix":"10.3390","volume":"14","author":[{"given":"Jae Eun","family":"Kim","sequence":"first","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yeonmi","family":"Lee","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"},{"name":"Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yonghee","family":"Kim","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0056-5189","authenticated-orcid":false,"given":"Sae-Byeok","family":"Hwang","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoo Bin","family":"Choi","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jongsuk","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juyeol","family":"Jung","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, Artificial Intelligence Omics Research Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jae-woo","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Yonsei University, College of Medicine, Seodaemoon-gu, Seoul 03722, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Je-Gun","family":"Joung","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeong-Jae","family":"Ko","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7240-7868","authenticated-orcid":false,"given":"Eunju","family":"Kang","sequence":"additional","affiliation":[{"name":"CHA R&D Institute, CHA Bundang Medical Center, Seongnam 13488, Republic of Korea"},{"name":"Department of Biomedical Science, College of Life Science, CHA University, Seongnam 13488, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2088","DOI":"10.1161\/ATVBAHA.123.318782","article-title":"Diversity of megakaryocytes","volume":"43","author":"Puhm","year":"2023","journal-title":"Arterioscler. 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