Mika Suga

Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of ‘hPSC colony morphology’, permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

Protein Kinase C-Induced Early Growth Response Protein-1 Binding to SNAIL Promoter in Epithelial–Mesenchymal Transition of Human Embryonic Stem Cells

Epithelial-mesenchymal transition (EMT) has been thought to occur during early embryogenesis, and also the differentiation process of human embryonic stem (hES) cells. Spontaneous differentiation is sometimes observed at the peripheral of the hES cell colonies in conventional culture conditions, indicating that EMT occurs in hES cell culture. However, the triggering mechanism of EMT is not yet fully understood. The balance between self-renewal and differentiation of human pluripotent stem (hPS) cells is controlled by various signal pathways, including the fibroblast growth factor (FGF)-2. However, FGF-2 has a complex role for self-renewal of hES cells. FGF-2 activates phosphatidylinositol-3 kinase/AKT, mitogen-activated protein kinase/extracellular signal-regulated kinase-1/2 kinase, and also protein kinase C (PKC). Here, we showed that a PKC rapidly induced an early growth response protein-1 (EGR-1) in hES cells, which was followed by upregulation of EMT-related genes. Before the induction of EMT-related genes, EGR-1 was translocated into the nucleus, and then bound directly to the promoter region of SNAIL, which is a master regulator of EMT. SNAIL expression was attenuated by knockdown of EGR-1, but upregulated by ectopic expression of EGR-1. EGR-1 as the downstream signal of PKC might play a key role in EMT initiation during early differentiation of hES cells. This study would lead to a more robust understanding of the mechanisms underlying the balance between self-renewal and initiation of differentiation in hPS cells.

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis

Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50–100 cells for passaging, cell counting is time‐consuming. In the present study, using a time‐lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase‐contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time‐lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase‐contrast images. This method provided real‐time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC‐based therapy.

Synergistic effects of FGF-2 and Activin A on early neural differentiation of human pluripotent stem cells.

Neural differentiation is an important target of human embryonic stem cells, which provide a source for cell-based therapy, developmental biology, and pharmaceutical research. Previous studies revealed that inhibition of the bone morphogenetic protein is required for neural induction from human embryonic stem cells. On the contrary, the functions of fibroblast growth factors and Activin/Nodal signaling are controversial. Fibroblast growth factor-2 and Activin/Nodal pathways exert divergent influences on human embryonic stem cell concerning the maintenance of both pluripotency and cellular differentiation. We hypothesized that the combination of fibroblast growth factor-2 and Activin A at various concentrations synergistically exerts diverse effects on cell differentiation. To determine the effects of fibroblast growth factor-2 and Activin A on cellular differentiation into neural lineages, we examined the expression of neural differentiation markers in human embryonic stem cells treated with fibroblast growth factor-2 and/or Activin A at various concentrations in a growth factor-defined serum-free medium in short-term culture. In this study, we provide evidence that fibroblast growth factor-2 and Activin A synergistically regulated the initiation of human embryonic stem cell differentiation into neural cell lineages even though human embryonic stem cells autonomously differentiate into neural cell lineages.

A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency

Mitochondrial oxidative phosphorylation is a major source of cellular ATP. Its usage as an energy source varies, not only according to the extracellular environment, but also during development and differentiation, as indicated by the reported changes in the flux ratio of glycolysis to oxidative phosphorylation during embryonic stem (ES) cell differentiation. The fluorescent probe JC‐1 allows visualization of changes in the mitochondrial membrane potential produced by oxidative phosphorylation. Strong JC‐1 signals were localized in the differentiated cells located at the edge of H9 ES colonies that expressed vimentin, an early differentiation maker. The JC‐1 signals were further intensified when individual adjacent colonies were in contact with each other. Time‐lapse analyses revealed that JC‐1‐labeled H9 cells under an overconfluent condition were highly differentiated after subculture, suggesting that monitoring oxidative phosphorylation in live cells might facilitate the prediction of induced pluripotent stem cells, as well as ES cells, that are destined to lose their undifferentiated potency.

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells

Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75(NTR) or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells

Abstract Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However, the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study, we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line, H9, which is known to differentiate into hepatocytes, and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs, hPSC-derived hepatoblast-like differentiated cells, and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus, our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines.

Imaging-cytometry revealed spatial heterogeneities of marker expression in undifferentiated human pluripotent stem cells

Human pluripotent stem cells (hPSCs) provide a good model system for studying human development and are expected as a source for both cell-based medical and pharmaceutical research application. However, stable maintenance of undifferentiated hPSCs is yet challenging, and thus routine characterization is required. Flow-cytometry is one of the popular quantitative characterization tools for hPSCs, but it has drawback of spatial information loss of the cells in the culture. Here, we have applied a two-dimensional imaging cytometry that examines undifferentiated state of hPSCs to analyze localization and morphological information of immunopositive cells in the culture. The whole images of cells in a culture vessel were acquired and analyzed by an image analyzer, IN Cell Analyzer 2000, and determined staining intensity of the cells with their positional information. We have compared the expression of five hPSC-markers in four hPSC lines using the two-dimensional imaging cytometry and flow cytometry. The results showed that immunopositive ratios analyzed by the imaging cytometry had good correlation with those by the flow cytometry. Furthermore, the imaging cytometry revealed spatially heterogenic expression of hPSC-markers in undifferentiated hPSCs. Imaging cytometry is capable of reflecting minute aberrance without losing spatial and morphological information of the cells. It would be a powerful, useful, and time-efficient tool for characterizing hPSC colonies.

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions

ABSTRACT Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug‐induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)‐derived hepatocyte‐like cells in vitro are expected as a tool for predicting drug‐induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte‐like cells, however its differentiation process is time‐consuming, labor‐intensive, cost‐intensive, and unstable. In order to solve this problem, expansion culture for hPSC‐derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self‐renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC‐derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC‐derived hepatic progenitor cells, we tried to develop serum‐free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC‐derived hepatic progenitor cells which could differentiate into hepatocyte‐like cells through hepatoblast‐like cells. We have confirmed that the hepatocyte‐like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC‐derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self‐renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.