Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Rapid process to differentiate iPSCs into iEPCs. ( A ) Schematic representation of the protocol for the differentiation of hiPSCs to iEPCs. ( B ) The cell morphology from day 1 to day 4 during differentiation. Scale bar, 50 μm. ( C ) The cells were treated with different concentrations of VEGF (0 ng/ml, 25 ng/ml, 50 ng/ml, or 100 ng/ml) from days 2–3. Representative FACS plots for iPSC-derived cells on day 4 are shown. ( D ) Changes in the gene expression of OCT4, NANOG, T, SCL, KDR, PECAM1, CD34 and CDH5 during differentiation. The results are shown as the mean ± SD of 3 independent experiments. ( E ) Immunofluorescence staining for CD31, CD34, and VE-CADHERIN on day 4. Nuclei were counterstained with DAPI. Scale bar, 50 μm. *** p < 0.001 and **** p < 0.0001.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Gene Expression, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Generation of iLSECs from iEPCs. ( A ) Schematic of the differentiation from hiPSC-derived EPCs into LSECs. ( B ) Expression levels of LSEC gene markers in CD34 sorted and no-sorted cells at D8. The results are shown as the mean ± SD of 3 independent experiments. **p < 0.01, ***p < 0.001. ( C ) FACS analysis of differentiated LSECs. Representative plots are shown. ( D ) Gene expression of STAB2, LYVE1, FCGR2B, and CD36 in hiPSC-derived LSEC-like cells cultured in differentiation medium A, B and C under hypoxic (5% O2) or normoxic conditions (21% O2). The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns not significant. ( E ) Percentages of LYVE1 + cells at D4 and D8 (n = 5). The results are shown as representative plots for purified D8 cells. On D4, 0.92 ± 0.57% of the cells were LYVE1 + cells, and on D8, 76.94 ± 5.16% were LYVE1 + cells.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Derivative Assay, Expressing, Gene Expression, Cell Culture, Purification

Journal: Scientific Reports

Article Title: A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells

doi: 10.1038/s41598-024-64195-1

Figure Lengend Snippet: Evaluation of iLSECs. ( A ) The morphology of iLSEC-like cells after CD34 sorting. Scale bar, 100 μm. ( B ) qPCR analysis of canonical LSEC gene markers in iLSEC-like cells and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. ** p < 0.01, *** p < 0.001 and **** p < 0.0001. ( C ) qRT‒PCR analysis of novel LSEC-specific marker genes (screened by ScRNA-seq analyses) in iLSECs and HUVECs. The results are shown as the mean ± SD of 3 independent experiments. * p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001. ( D ) Immunostaining of LYVE-1 (green) in the iLSECs revealed irregular round or oval-shaped perforations. The nuclei were counterstained with DAPI. Scale bar, 50 μm. E Fluorescence microscopy analysis of Dil-AcLDL and CyTM5-conjugated IgG uptake. hiPSCs, purified iLSEC-like cells and HUVECs were cultured in medium supplemented with DiI-ac-LDL or CyTM5-conjugated IgG. Scale bar, 100 μm.

Article Snippet: EPCs were purified based on CD34 expression using mouse anti-human CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi, 130046702).

Techniques: Marker, Immunostaining, Fluorescence, Microscopy, Purification, Cell Culture