Comparative Analysis of Commercial Kit versus In-House Protocol for Assessing Endodermal Differentiation Potential in Human Embryonic Stem Cells
نویسندگان
1 Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Basic and Population Based Studies in NCD, Reproductive Epidemiology Research Center, Royan Institute, ACECR, Tehran, Iran
3 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
4 Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
5 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
6 Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
7 Department of Developmental Biology, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, ACECR, Tehran, Iran
8 Department of Basic and Population Based Studies in NCD, Reproductive Epidemiology Research Center, Royan Institute, ACECR, Tehran, Iran
9 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
10 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
doi
10.22074/cellj.2025.2056939.1819چکیده
Objective: Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell lineages, making theminvaluable for cell-based therapies. However, the ability of individual hESC lines to generate the three embryonic germlayers varies. Selecting a cell line with a high propensity for definitive endoderm (DE) differentiation is crucial for producingpancreatic progenitors, hepatocytes, and other endodermal derivatives. Direct and spontaneous differentiation are twocommonly used methods to assess hESC differentiation potential; both are complex and time-consuming. This studyevaluates a commercially available differentiation kit as a simple and reproducible screening assay to predict the DEdifferentiation potential of various hESC lines.Materials and Methods: In this experimental study, three hESC lines obtained from the Royan Institute Stem Cell Bank(Tehran, Iran) were used. Pluripotency was confirmed by evaluating cell morphology, OCT4 protein expression, andalkaline phosphatase staining. Early-stage lineage differentiation was compared between the StemMACS™ Trilineagekit and a previously established DE protocol. Differentiation outcomes were assessed using gene and protein expressionanalyses, including flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and immunostaining.Results: The three hESC lines exhibited varying capacities for DE differentiation. Flow cytometry analysis of thesurface marker CXCR4 showed differentiation efficiencies of 70% for RH2, compared with 35% and 40% for RH5 andRH6, respectively. The superior efficiency of RH2 was consistent across both differentiation protocols, as evidenced byits strong CXCR4 expression using the in-house method.Conclusion: hESC lines exhibit variable capacities for differentiation. Commercial kit-based screening provides asimple and reliable method to identify suitable hESC lines for generating endoderm-derived cells.
