Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile.

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

The backs of our eyes are lined with retinal pigment epithelial cells (or RPE cells for short). These cells provide nutrition to surrounding cells and contain a pigment called melanin that absorbs excess light that might interfere with vision. By doing so, they support the cells that receive light to enable vision. However, with age, RPE cells can become damaged and less able to support other cells. This can lead to a disease called age-related macular degeneration, which can cause blindness. One potential way to treat this disease is to transplant healthy RPE cells into eyes that have lost them. These healthy cells can be grown in the laboratory from human pluripotent stem cells, which have the capacity to turn into various specialist cells. Stem cell-derived RPE cells growing in a dish contain varying amounts of melanin, resulting in some being darker than others. This raised the question of whether pigment levels affect the function of RPE cells. However, it was difficult to compare single cells containing various amounts of pigment as most previous studies only analyzed large numbers of RPE cells mixed together. Nakai-Futatsugi et al. overcame this hurdle using a technique called Automated Live imaging and cell Picking System (also known as ALPS). More than 2300 stem cell-derived RPE cells were photographed individually and the color of each cell was recorded. The gene expression of each cell was then measured to investigate whether certain genes being switched on or off affects pigment levels and cell function. Analysis did not find a consistent pattern of gene expression underlying the pigmentation of RPE cells. Even gene expression related to the production of melanin was only slightly linked to the color of the cells. These findings suggests that the RPE cell color fluctuates and is not primarily determined by which genes are switched on or off. Future experiments are required to determine whether the findings are the same for RPE cells grown naturally in the eyes and whether different pigment levels affect their capacity to protect the rest of the eye.

Zscan4 Is Activated after Telomere Shortening in Mouse Embryonic Stem Cells.

ZSCAN4 is a DNA-binding protein that functions for telomere elongation and genomic stability. In vivo, it is specifically expressed at the two-cell stage during mouse development. In vitro, it is transiently expressed in mouse embryonic stem cells (ESCs), only in 5% of the population at one time. Here we attempted to elucidate when, under what circumstances, Zscan4 is activated in ESCs. Using live cell imaging, we monitored the activity of Zscan4 together with the pluripotency marker Rex1. The lengths of the cell cycles in ESCs were diverse. Longer cell cycles were accompanied by shorter telomeres and higher activation of Zscan4. Since activation of Zscan4 is involved in telomere elongation, we speculate that the extended cell cycles accompanied by Zscan4 activation reflect the time for telomere recovery. Rex1 and Zscan4 did not show any correlation. Taken together, we propose that Zscan4 is activated to recover shortened telomeres during extended cell cycles, irrespective of the pluripotent status.

Epiblast and primitive endoderm differentiation: fragile specification ensures stable commitment.

Specification of the epiblast and primitive endoderm is one of the earliest differentiation steps during embryogenesis. In vitro tracking of pluripotency markers in ESCs suggests that epiblast specification may be plastic; however, live imaging of blastocysts, as detailed in a recent paper from Xenopoulos et al (2015), showed that, unlike in ESCs, fate commitment in vivo is largely irreversible.

LIF signal in mouse embryonic stem cells.

Since the establishment of mouse embryonic stem cells (mESCs) in the 1980s, a number of important notions on the self-renewal of pluripotent stem cells in vitro have been found. In serum containing conventional culture, an exogenous cytokine, leukemia inhibitory factor (LIF), is absolutely essential for the maintenance of pluripotency. In contrast, in serum-free culture with simultaneous inhibition of Map-kinase and Gsk3 (so called 2i-culture), LIF is no longer required. However, recent findings also suggest that LIF may have a role not covered by the 2i for the maintenance of naïve pluripotency. These suggest that LIF functions for the maintenance of naïve pluripotency in a context dependent manner. We summarize how LIF-signal pathway is converged to maintain the naïve state of pluripotency.

Transcription factor network in embryonic stem cells: heterogeneity under the stringency.

Leukemia inhibitory factor (LIF) signaling regulates transcription factors to maintain the self-renewability and pluripotency of embryonic stem (ES) cells. Recently, we have proposed a network model that consists of transcription factors such as, Klf4, Sox2, Tbx3, Nanog, and Oct3/4, which form a parallel pathway downstream from LIF signaling (Nature, 460, 2009, Niwa et al.). In this parallel pathway, the transcription factors maintain the pluripotency of ES cells through mutual balance with some degree of redundancy and compensation. While self-renewability and pluripotency are maintained well under such seemingly stringent regulation, studies of single cells revealed heterogeneity among individual ES cells. This heterogeneity may underlie the mechanism that allows ES cells to exit self-renewal and enter into differentiation to exert pluripotency. Here we focus on recent studies on the heterogeneity of ES cells and discuss their inherent metastability.