Fig. 10

1 The process of differentiating human iPSCs, nuclear transfer embryonic stem cells (nt-ESCs), and in vitro fertilization embryonic stem cells (IVF-ESCs) into cardiac cells. Panel (A) presents an overview of the experimental design used in the study. Panel (B) illustrates a monolayer cardiac differentiation protocol facilitated by small molecules. Panel (C) shows the sarcomere structures of pluripotent stem cell-derived cardiac cells (PSC-CMs) and rat adult cardiac cells, stained for cardiac troponin T (green), α-actinin (red), and the nuclei counterstained with DAPI (blue). The scale bars represent 25 μm, and the magnification is 600 × . Panel (D) quantifies the efficiency of cardiac differentiation by calculating the percentage of cells positive for TNNT2 (cardiac troponin T) using flow cytometry. Panel (E) compares the expression of TNNT2 in iPSC-derived cardiac cells (iPSC-CMs), nt-ESC-derived cardiac cells (nt-ESC-CMs), and IVF-ESC-derived cardiac cells (IVF-ESC-CMs). Panels (F–H) depict the heterogeneity of cardiac cells derived from different types of pluripotent stem cells using single-cell quantitative polymerase chain reaction (qPCR) analysis. Blue, red, and green colors represent iPSC-CMs, nt-ESC-CMs, and IVF-ESC-CMs, respectively. Heavy and light colors indicate two different cell lines within each category. Each row represents a single cell, while each column represents a single gene. The color key applies to panels F–H. Statistical analysis using one-way ANOVA was performed, and the error bars represent the standard error of the mean (SEM). 2 The generation of endothelial cells (ECs) from various types of pluripotent stem cells (PSCs) using different methods. In (A), a protocol involving small molecules is shown for inducing endothelial differentiation. The effectiveness of the differentiation process is evaluated in (B) by determining the percentage of CD31 + cells at day 12 of differentiation. Representative immunofluorescence staining of PSC-derived ECs using CD31 and CD144 antibodies is presented in (C), with the nuclei counterstained using DAPI. (D) compares the maintenance of endothelial characteristics among induced pluripotent stem cell-derived ECs (iPSC-ECs), nuclear transfer embryonic stem cell-derived ECs (nt-ESC–ECs), and in vitro fertilization-derived ECs (IVF-ESC-ECs) by measuring the percentage of CD144 + cells. No significant differences were observed among these cell types. The expression levels of EC-specific marker genes, PECAM1 (E), CDH5 (F), and NOS3 (G), were similar in iPSC-ECs, nt-ESC–ECs, and IVF-ESC-ECs. The production of nitric oxide by PSC-ECs and human umbilical vein endothelial cells (HUVECs) is shown in (H), while (I) presents the average number of branches in tubes formed by PSC-ECs. 3 The results obtained from analyzing the global gene-expression profiles of pluripotent stem cells (PSCs), PSC-derived cardiomyocytes (PSC-CMs), and PSC-derived endothelial cells (PSC-ECs) using RNA sequencing (RNA-seq). A) The differentially expressed genes (DEGs) between PSCs, PSC-CMs, and PSC-ECs were clustered using unsupervised hierarchical clustering (with a statistical significance threshold of q < 0.1). B) PSC-ECs were grouped together based on the specific reprogramming approaches used to generate the iPSCs (i12C, i12J), non-transgenic embryonic stem cells (nt-ESCs) (NT1, NT2), and in vitro fertilization-derived embryonic stem cells (IVF-ESCs) (ESO7, ESO8) (with a statistical significance threshold of q < 0.1). C) The number of DEGs identified in PSCs, PSC-CMs, and PSC-ECs due to the different reprogramming approaches is shown, with overlapping regions indicating the number of consistent DEGs shared among the different cell types. D) Gene ontology (GO) analysis was performed to identify enriched functional terms for the DEGs between iPSC-CMs, nt-ESC-CMs, and IVF-ESC-CMs (with a statistical significance threshold of P < 0.05). E) GO terms associated with the DEGs in ECs derived from iPSCs, nt-ESCs, and IVF-ESCs were identified using GO analysis (with a statistical significance threshold of P < 0.05). 4 The results obtained from analyzing the global DNA methylome of pluripotent stem cells (PSCs), PSC-derived cardiomyocytes (PSC-CMs), and PSC-derived endothelial cells (PSC-ECs) using a technique called RRBS-seq. A) This part shows the percentages of different types of methylated cytosines (mCG, mCHG, and mCHH) among all observed 5-methylcytosines in PSCs, PSC-CMs, and PSC-ECs.B) An unsupervised hierarchical clustering analysis is performed based on the global CpG methylation levels of PSCs, PSC-CMs, and PSC-ECs. The clustering groups include iPSCs, non-transgenic embryonic stem cells (nt-ESCs), in vitro fertilization-derived ESCs (IVF-ESCs), and their respective differentiated cells (CMs and ECs). The height of the cluster trees represents the similarity or dissimilarity between different objects and groups. C) The number of differentially methylated cytosines (DMCs) identified through pairwise comparisons is depicted in this section. The identified DMCs have a statistical significance (q < 0.01) and a methylation difference of at least 25%. D) Another unsupervised hierarchical clustering analysis is conducted, but this time for differentially methylated regions (DMRs) in CpG islands (CGIs) across the genome in PSCs, PSC-CMs, and PSC-ECs. The DMRs shown here have a statistical significance (q < 0.01) and amount to a total of 3,452. E) Lastly, an unsupervised hierarchical clustering analysis is presented for 2,324 DMRs located in CGI shores (regions adjacent to CGIs) in PSCs, PSC-CMs, and PSC-ECs. These DMRs also have a statistical significance (q < 0.01). 5 The identification of consistent differentially methylated regions (DMRs) in undifferentiated pluripotent stem cells (PSCs) and fully differentiated cells. In panel A, 42 consistent DMRs within CpG islands (CGIs) were found in both PSCs and differentiated cells. Panel B shows 40 consistent DMRs located in CGI shores, which were either hypermethylated or hypomethylated in in vitro fertilization (IVF) samples. Panel C provides the numbers of IVF-specific hypermethylated, IVF-specific hypomethylated, and inter-individual DMRs persistently present in PSCs, PSC-derived cardiomyocytes (PSC-CMs), and PSC-derived endothelial cells (PSC-ECs). The consistent DMRs specific to iPSCs were not found in CGI shores. Panels D and E represent IVF-specific consistent CGI-DMRs identified in undifferentiated PSCs and differentiated cells. Panels F and G demonstrate that the methylation levels of iPSC-specific consistent CGI-DMRs in iPSCs were higher compared to those in non-transgenic embryonic stem cells (nt-ESCs) and IVF-ESCs. Panel H shows the results of Spearman's correlation analysis, indicating a significant correlation between consistent promoter DMRs and the mRNA abundance of the associated genes (P < 2.2e − 16). 6 The results of a study examining the toxic effects of doxorubicin on cardiomyocytes (CMs) derived from iPSCs, non-transgenic embryonic stem cells (nt-ESCs), and in vitro fertilization-derived ESCs (IVF-ESCs). Panel (A) shows the dose-dependent impact of doxorubicin on the viability of PSC-CMs. The viability was measured using a Prestoblue cell viability assay, and the results indicate that as the dose of doxorubicin increases, the viability of PSC-CMs decreases. The values were normalized to the viability at 0 μM doxorubicin. Panel (B) displays the effect of doxorubicin treatment on the production of ATP in PSC-CMs. ATP production was measured using a CellTiter-Glo assay, and the data suggests that doxorubicin treatment negatively affects ATP production in PSC-CMs. Panel (C) demonstrates the assessment of cellular apoptosis in PSC-CMs after doxorubicin treatment. A luminescent Caspase 3/7 assay was used to measure apoptosis, and the results indicate that doxorubicin treatment leads to increased cellular apoptosis in PSC-CMs. Panel (D) reveals that the viability of PSC-CMs is not significantly affected after 24 h of doxorubicin treatment. Panel (E) presents the detection of whole-cell reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2), in PSC-CMs after different doses of doxorubicin treatment for 24 h. The data suggests that doxorubicin administration leads to an increase in ROS levels in PSC-CMs. Panel (F) shows the acute influence of doxorubicin treatment on the mitochondrial glutathione (GSH) concentration in PSC-CMs. The GSH concentration was measured using a GSH-Glo Glutathione kit, and the results indicate that doxorubicin treatment has an impact on the mitochondrial GSH concentration in PSC-CMs. Reprinted from [547] with permission from the PNAS