{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T05:15:32Z","timestamp":1785906932950,"version":"3.56.0"},"reference-count":30,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T00:00:00Z","timestamp":1748563200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical College of Georgia-Augusta University and by the National Institutes of Health","award":["DK127861"],"award-info":[{"award-number":["DK127861"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Definitive endoderm (DE) differentiation leads to the development of the major internal organs including the liver, intestines, pancreas, gall bladder, prostate, bladder, thyroid, and lungs. The two primary methods utilized for in vitro differentiation of induced pluripotent stem cells (iPSCs) into DE cells are the growth factor (GF) and the small molecule (SM) approaches. The GSK-3 inhibitor (CHIR99021) is a key factor for the SM approach. Activin A and Wnt3a are utilized in the GF approach. In this study, both the GF and SM protocols were compared to each other. The results show that both the GF and SM protocol produce DE with a similar morphological phenotype, gene and protein expression, and a similar level of homogeneity and functionality. However, on both the gene expression and proteomic level, there is a divergence between the two protocols during hepatic specification. Proteomic analysis shows that hepatoblasts from the GF protocol have significantly differentially expressed proteins (DEPs) involved in liver metabolic pathways compared to the SM protocol. Well-validated DE differentiation protocols are needed to fully unlock the clinical potential of iPSCs. In the first step of generating DE-derived tissue, either protocol can be utilized. However, for hepatic specification, the GF protocol is more effective.<\/jats:p>","DOI":"10.3390\/cells14110815","type":"journal-article","created":{"date-parts":[[2025,6,2]],"date-time":"2025-06-02T12:27:09Z","timestamp":1748867229000},"page":"815","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Efficient Generation of Induced Pluripotent Stem Cell-Derived Definitive Endoderm Cells with Growth Factors and Small Molecules"],"prefix":"10.3390","volume":"14","author":[{"given":"Faizal Z.","family":"Asumda","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"},{"name":"Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shadia","family":"Alzoubi","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5969-9392","authenticated-orcid":false,"given":"Kiyasha","family":"Padarath","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina","family":"John","sequence":"additional","affiliation":[{"name":"Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimya","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8283-2403","authenticated-orcid":false,"given":"Ravindra","family":"Kolhe","sequence":"additional","affiliation":[{"name":"Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3826-9489","authenticated-orcid":false,"given":"Ashis Kumar","family":"Mondal","sequence":"additional","affiliation":[{"name":"Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tae Jin","family":"Lee","sequence":"additional","affiliation":[{"name":"Center for Biotechnology and Genomics, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7720-7048","authenticated-orcid":false,"given":"Wenbo","family":"Zhi","sequence":"additional","affiliation":[{"name":"Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9812-7573","authenticated-orcid":false,"given":"Robert C.","family":"Huebert","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan P.","family":"Staff","sequence":"additional","affiliation":[{"name":"Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lindsey A.","family":"Kirkeby","sequence":"additional","affiliation":[{"name":"Center for Regenerative Biotherapeutics, Mayo Clinic, Rochester, MN 55905, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"dev150920","DOI":"10.1242\/dev.150920","article-title":"The endoderm: A divergent cell lineage with many commonalities","volume":"146","author":"Nowotschin","year":"2019","journal-title":"Development"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1146\/annurev.cellbio.15.1.393","article-title":"Vertebrate endoderm development","volume":"15","author":"Wells","year":"1999","journal-title":"Annu. 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