Objective To use direct adherent method to induce human embryonic stem cells （hESCs） to become cardiomyocytes in vitro and examine their differentiation rate. Methods Undifferentiated hESCs were seeded onto Matrigel-coated plates at a density of 1×105 cells/cm2 and cultured in MEF-conditioned medium （MEF-CM） with 8 ng/ml basic fibroblast growth factor （bFGF） for 6 days. Then MEF-CM was replaced with RPMI 1640/B27 medium supplemented with 100 ng/ml human recombinant activin A for 24 hours in hESCs culture，followed by supplementation of 10 ng/ml human recombinant bone morphogenetic protein 4 （BMP4） for 4 days in hESCs culture. The medium was then replaced with RPMI 1640/B27 medium without supplementary cytokines，and hESCs were refed every 2-3 days for 2-3 additional weeks. Self-beating cardiomyocytes and the beating frequency were observed under the microscope，and the percentage of colonies showing beating cardiomyocytes was calculated. Cardiac troponin T （cTnT），a specific marker of cardiomyocytes，was examined by immunofluorescence. Spontaneous action potentials of cardiomyocytes were measured with patch clamp technique.Apoptotic rate of cardiomyocytes was detected with apoptosis-hoechst staining kit after beating cardiomyocytes were culturedunder hypoxia for 24 hours. Results A large number of spontaneous beating cardiomyocytes were observed 13 days after induction. The average time to show beating cardiomyocytes was 13.0±1.1 days after induction，the percentage of colonies showing beating cardiomyocytes was 66.7%，and the beating frequency of cardiomyocytes was 63.0±7.0 times/minutes. Beating cardiomyocytes were cTnT-positive. Spontaneous action potentials were detected in beating cardiomyocytes.Apoptotic rate of cardiomyocytes was 8.0%±0.5% after beating cardiomyocytes were cultured under hypoxia for 24 hours. Conclusion It’s the first time to use direct adherent method to induce hESCs to become cardiomyocytes in vitro in China with the differentiation rate of 66.7% and differentiation time of 13 days.
Objective To establ ish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-l ike cells using an adherent culture system with single-step induction. Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days, hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days. Then the induction medium was replaced by hepatocyte induction medium (HIM) supplemented with fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) for another 6 days. Finally, the cells were treated with HIM adding hepatocyte growth factor (HGF) and Oncostatin M (OSM) for 5-7 days. The characteristics of differentiated cells were determined by morphology, immunofluorescence staining, RT-PCR, and Periodicacid- Schiff (PAS) test. Results Differentiated cells treated with Activin A, FGF-1, BMP-4, HGF, and OSM sequentially were morphologically larger and became spherical, oval or polygon. Some cells had 2 or 3 nuclei, suggesting that the cells have a hepatocyte-l ike morphology. Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead (FOX)A2 after induction by Activin A. Then they turned to be α fetoprotein (AFP) and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4. Finally, the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection. The differentiated cells at various stages also expressed at early (SOX17, FOXA2, and GATA-4),middle (AFP, albumin, and cytokeratin 18), and mature (alcohol dehydrogenase 1C and Cytochrome P4501B1) stage hepatic genes, respectively. Conclusion Using a simple-step induction method and by suppl ied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-l ike cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.
Objective To study the differentially expressed genes (DEG) during the differentiation of human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) into pericytes and endothelial cells, and to identify key molecules and signaling pathways that may regulate this differentiation process. MethodshiPSC and hESC were selected and expanded using mTeSR medium. A "two-step method" was used to induce the differentiation of hiPSC and hESC into pericytes and endothelial cells. Pericytes were identified using immunofluorescence staining, while endothelial cells were isolated and identified using flow cytometry. Total RNA samples were extracted on days 0, 4, 7, and 10 of differentiation and consistently significant DEGs were screened. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis were performed on the screened DEGs. ResultsBoth hiPSCs and hESCs successfully differentiated into pericytes and endothelial cells under induction conditions. Transcriptome sequencing results showed that with the extension of differentiation time, the DEGs in hiPSCs and hESCs were significantly upregulated or downregulated, following a generally consistent trend. During the differentiation process, marker genes for pericytes and endothelial cells were significantly upregulated. A total of 491 persistent DEGs were detected in both hiPSC and hESC, with 164 unique to hiPSCs and 335 to hESCs, while 8 DEGs were co-expressed in both cell lines. Among these, SLC30A3, LCK, TNFRSF8, PRDM14, and GLB1L3 showed sustained downregulation, whereas CLEC18C, CLEC18B, and F2RL2 exhibited sustained upregulation. GO enrichment analysis revealed that DEGs with sustained upregulation were primarily enriched in terms related to neurogenesis, differentiation, and developmental proteins, while DEGs with sustained downregulation were enriched in terms related to membrane structure and phospholipid metabolic processes. KEGG pathway analysis showed that upregulated genes were primarily enriched in cancer-related pathways, pluripotency regulatory pathways, the Wnt signaling pathway, and the Hippo signaling pathway, whereas downregulated genes were predominantly enriched in metabolism-related pathways. ConclusionsDuring the differentiation of hiPSC and hESC into pericytes and endothelial cells, 8 DEGs exhibit sustained specific expression changes. These changes may promote pericyte and endothelial cell differentiation by activating the Wnt and Hippo pathways, inhibiting metabolic pathways, releasing the maintenance of stem cell pluripotency, affecting the cell cycle, and inhibiting cell proliferation.