Additional file 1: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Daf1-positive cells are negative for Nanog expression. Mouse Nanog-iPS cells, in which GFP expression is driven by Nanog promoter [45], are differentiated into DE. Cxcr4+/E-cadherin + cells were sorted and analyzed for Daf1 and Nanog-GFP expression. Daf1-positive cells are negative for Nanog expression. (TIF 312 kb

Additional file 3: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Primer sequences used for RT-PCR analysis. Primer sequences used for detection of gene expression in Fig. 1, 2. (DOCX 15 kb

Additional file 2: of Late stage definitive endodermal differentiation can be defined by Daf1 expression

Flow cytometric analyses of the cell cycle. Histograms of flow cytometric analyses of Daf1-DE and Daf1 + DE (n = 5) are shown. Cell cycle was analyzed by measuring DNA quantities using DyeCycle. (TIF 314 kb

Label-Free Evaluation of Maturation and Hepatotoxicity of Human iPSC-Derived Hepatocytes Using Hyperspectral Raman Imaging

To promote the clinical application of human induced pluripotent stem cell (hiPSC)-derived hepatocytes, a method capable of monitoring regenerative processes and assessing differentiation efficiency without harming or modifying these cells is important. Raman microscopy provides a powerful tool for this as it enables label-free identification of intracellular biomolecules in live samples. Here, we used label-free Raman microscopy to assess hiPSC differentiation into hepatocyte lineage based on the intracellular chemical content. We contrasted these data with similar phenotypes from the HepaRG and from commercially available hiPSC-derived hepatocytes (iCell hepatocytes). We detected hepatic cytochromes, lipids, and glycogen in hiPSC-derived hepatocyte-like cells (HLCs) but not biliary-like cells (BLCs), indicating intrinsic differences in biomolecular content between these phenotypes. The data show significant glycogen and lipid accumulation as early as the definitive endoderm transition. Additionally, we explored the use of Raman imaging as a hepatotoxicity assay for the HepaRG and iCell hepatocytes, with data displaying a dose-dependent reduction of glycogen accumulation in response to acetaminophen. These findings show that the nondestructive and high-content nature of Raman imaging provides a promising tool for both quality control of hiPSC-derived hepatocytes and hepatotoxicity screening