Fig. 11

1 The process of deriving iPSCs with characteristics similar to naïve mouse embryonic stem cells (mESCs). Panel A shows the overall strategy and representative images of C1 cultures and a subcloned cell line called C1.2 at various reprogramming stages. The passage number (p) is indicated. Additionally, images of NOD mESCs (mouse ESCs) and C1.2 hiPSCs (human iPSCs derived from C1) after withdrawal of doxycycline (DOX) are presented. Panel B demonstrates the maintenance of the C1 hiPSC line in a conventional growth condition for human embryonic stem cells (hESCs) supplemented with basic fibroblast growth factor (bFGF) and serum. The C1 line is transferred to a medium called N2B27 PD/CH/LIF + DOX, and emerging colonies are subcloned. A representative clone called C1.10 hiPSC is shown. Panel C explores the signaling dependence of pluripotent cell lines. Pluripotent cells are divided equally and plated on feeders in different growth media that are typically used for maintaining these cell lines. After 36 h, the wells are treated with specific inhibitors or growth factors. After 6 days, the wells are fixed and stained for a pluripotency marker called Nanog to determine the relative percentage of pluripotent colonies. Colony formation is normalized to an internal control growth medium without inhibitors. Panel D focuses on the reprogramming process of the C1.2 hiPSC line. The cells are electroporated with mammalian expression vectors expressing specific reprogramming factors and subjected to puromycin selection. The cells are then passaged in a medium called PD/CH/LIF without DOX. The values indicate the relative percentage of SSEA4 + colonies obtained compared to control cells that were transfected with a polycistronic construct encoding Oct4, Klf4, and Sox2. Panel E investigates the screening of factors that enable the propagation of transgene-independent C1 hiPSCs, meaning these cells no longer require DOX for stabilization. The effects of removing individual factors from a pool of 13 small molecules or cytokines are examined on the survival and pluripotency maintenance of C1 hiPSCs. C1 cells are plated on feeders in N2B27 media with the indicated factors. The P values obtained using Student’s t-test indicate significant changes compared to cells grown in DOX/PD/CH/LIF conditions, which are defined as the control with 100% survival. 2 The characteristics of naïve human embryonic stem cell (hESC) lines. In panel A, a diagram outlines the process of reverting hESCs to generate naïve hESCs. Representative images of WIBR3 hESCs at different stages of the reversion process in the presence of PD (small molecule), CH (chemical), LIF (leukemia inhibitory factor), and FK (forskolin) are shown. The passage number (p) and magnifications of the captured images are indicated. Panel B presents the single-cell cloning efficiency of various pluripotent stem cell lines. This efficiency is determined by counting the number of wells containing colonies positive for Nanog (a pluripotency marker) after 7 days. Panel C displays the estimated cell doubling time. Plated cells were counted at 1, 4, and 7 days after plating in triplicates, and the increase in cell number was used to calculate the average doubling time. The error bars represent the standard deviation (SD), and the P values, determined using Student's t-test, indicate significant differences between the average values of hESC/hiPSC lines compared to the average values of naïve hESC/hiPSC lines. 3 The similarities in signaling and epigenetic characteristics between naïve human embryonic stem cells (hESCs) and mouse embryonic stem cells (mESCs). In panel A, the dependence of pluripotent cell lines on specific signaling pathways was assessed. After a 7-day period, the wells were fixed and stained to determine the percentage of colonies positive for pluripotency markers. Mouse stem cells were stained with SSEA1. The colony formation was normalized to a control growth medium without inhibitors, which was represented in the first left column. Normalized percentages below 5% were categorized as “sensitivity” to the presence of the supplemented inhibitor. Panel B shows the expression of early germ-cell markers through RT-PCR in the presence or absence of BMP4/7/8 cytokines. Lastly, in panel C, a representative analysis using fluorescence in situ hybridization (FISH) was conducted to examine the presence of XIST RNA (red) and Cot1 nuclear RNA (green). The Pri-WIBR3.2 cell line was analyzed after being passaged in conventional bFGF/serum-containing human ESC growth conditions. The numbers provided in the figure indicate the average percentage of XIST-positive nuclei counted. 4 The similarities in gene expression between naive human embryonic stem cells (hESCs) and naive human-hiPSCs with mouse embryonic stem cells (mESCs). (A) a bar chart comparing the expression levels of pluripotency and lineage-specific marker genes in hESCs and naive hESCs, with asterisks indicating genes that showed significant differences between the two groups of samples; (B) a fluorescence-activated cell sorting (FACS) analysis measuring the surface expression of human and mouse major histocompatibility complex (MHC) class I alleles, with a black graph representing the control isotype match; (C) a cross-species gene expression clustering depicting the grouping of mESCs and naive hESCs as distinct from mEpiSCs (mouse epiblast stem cells) and hESCs. The legend on the right explains that yellow and blue colors represent positive and negative correlations, respectively. The gene expression levels were clustered based on Spearman correlation and average linkage, with mouse samples labeled in purple and human samples labeled in brown. Reprinted from [628] with permission from the PNAS