Functional decline and loss of the retinal pigment epithelium (RPE) cause retinal diseases. Clinical studies using human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived RPE cells have shown the safety and potential efficacy of hESC/iPSC-RPE cell transplantation. However, the production of RPE cells remains somewhat problematic. hESCs/iPSCs co-cultured with mouse feeder cells carry the risk of xeno-transmitted infections and immune reactions. Moreover, increasing the rate of cell division to ensure the quantity and purity of cells with low differentiation efficiency elevates the risk of gene mutations and chromosomal abnormalities. Here, we show that the transient inhibition of the FGF/MAPK signaling pathway during the hiPSC maintenance period markedly promotes RPE differentiation efficiency under feeder-free culture conditions. Blockage of FGF/MAPK signal induces neural differentiation and generates RPE cells without subsequent inhibition of Wnt and Nodal signals, which is known to be effective for retinal specification. We also found that additional inhibition of the PKC or BMP signaling pathway together with FGF/MAPK signal inhibition further elevates RPE differentiation efficiency. Our study will be helpful for producing clinical-grade RPE cells and will facilitate the development of therapies using hESC/hiPSC-RPE cells.

Some chemicals are harmful in to light-exposed tissues such as skin and eyes. The 3T3 Neutral Red Uptake Phototoxicity Test has been validated and adopted by the Organization of Economic and Community Development (OECD) as a method of evaluating chemical phototoxicity using mouse 3T3 fibroblasts. However, the high rate of false positive results associated with this test eventually led to increased laboratory animal usage. Although the eye is vulnerable to light damage because of constant exposure to environmental radiation, few approaches are available to predict ocular phototoxicity in humans. Here, we propose a tier one test that identifies the potential ocular phototoxicity of chemical substances. Using a three-dimensional culture technique, human embryonic stem cells (hESCs) were differentiated to retinal pigment epithelial cell (RPE) precursors. The precursors after prolonged treatment with FBS formed a uniform hexagonal lattice of cells with well-developed tight junctions and time-dependent elevation of melanin content and RPE maturation marker levels. Hierarchical clustering of gene transcripts revealed that hESC-derived RPEs were very similar to tissue-derived adult RPEs. Interestingly, there were a high percentage of chemicals eliciting a positive response in 3T3 cells and negative in hESC-derived RPEs under the experimental conditions used in the phototoxicity test. The response to treatment of hESC-derived RPEs with these negative chemicals became positive at a higher dose of UVA irradiation; however, the biological responses to these chemicals differed between the two cells. Taken together, we conclude that hESC-derived RPEs are novel tool for future toxicological and mechanistic studies of ocular phototoxicity in humans.

The widely used antiepileptic drug valproic acid (VPA) is known to exhibit teratogenicity in the form of a failure of the neural tube in humans. Embryonic stem cells (ESCs) are reported to be a promising cell source for evaluating chemical teratogenicity, because they are capable of reproducing embryonic developmental model and enable reduction in the number of experimental animals used. We previously investigated 22 genes for which expressions are altered by teratogens, specifically focusing on neural differentiation of mouse ESCs. In the present study, expressions of the investigated genes were evaluated by quantitative real-time PCR and compared during differentiation of human ESCs into neurons with or without VPA. Under the conditions, almost all gene expressions significantly changed in VPA-containing culture. Specifically, in neural development-related genes such as DCX, ARX, MAP2, and NNAT, more than 2-fold expression was observed. The findings suggest that the genes focused on in this study may help to elucidate the teratogenic effects of VPA and might be a useful tool to analyze embryotoxic potential of chemicals in humans.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galβ O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

In this report, we demonstrate that an optic cup structure can form by self-organization in human ESC culture. The human ESC-derived optic cup is much larger than the mouse ESC-derived one, presumably reflecting the species differences. The neural retina in human ESC culture is thick and spontaneously curves in an apically convex manner, which is not seen in mouse ESC culture. In addition, human ESC-derived neural retina grows into multilayered tissue containing both rods and cones, whereas cone differentiation is rare in mouse ESC culture. The accumulation of photoreceptors in human ESC culture can be greatly accelerated by Notch inhibition. In addition, we show that an optimized vitrification method enables en bloc cryopreservation of stratified neural retina of human origin. This storage method at an intermediate step during the time-consuming differentiation process provides a versatile solution for quality control in large-scale preparation of clinical-grade retinal tissues.

The embryonic stem cell test (EST) is a validated in vitro alternative test for prediction of embryotoxicity with inhibition of cardiomyocyte differentiation under the microscope as beating areas at day 10 as an endpoint. However, improvements are necessary for regulatory acceptance and application to high-throughput screening. We have previously reported that heart and neural crest derivatives expressed transcript 1 (Hand1), a transcription factor essential for mammalian heart development, and cardiomyopathy associated 1 (Cmya1), an intercalated disk protein implicated in cardiac morphogenesis, are quantitative and objective molecular endpoints for predicting embryotoxicity, detected at day 6 when mouse embryonic stem (ES) cells differentiate into cardiomyocytes. In established stable transgenic ES cells with Hand1 or Cmya1 promoters upstream of luciferase reporter gene, changes in each gene expression were found to be coincident with those in luciferase activities during cardiomyocyte differentiation, suggesting that monitoring might be possible by chemiluminescent determination. In our novel EST, differentiation toxicity and cytotoxicity of test chemicals were here analyzed using ES cells and 3T3 fibroblasts by this approach in 96-microwell plates. Extensive investigations were performed to explore predictive power and validity by comparing a set of 24 well-known test chemicals. The novel EST offers high predictability and accuracy with a reduced test duration and manpower compared with the original EST protocol, thus providing a new rapid and sensitive in vitro method for screening embryotoxicants.

Embryonic stem cells (ES cells), pluripotent cells derived from the inner cell mass of blastocysts, differentiate in vitro into a variety of cell types representing all three germ layers. They therefore constitute one of the most promising in vitro tools for developmental toxicology. To assess the developmental toxicity of chemicals using ES cells easily, identification of effective marker genes is a high priority. We report here altered gene expression during ES cell differentiation into myocardiac and neural cells on treatment with some embryotoxic and non-embryotoxic chemicals. Decreases in several undifferentiated markers such as Oct3/4 and Nanog, and elevated expression of genes associated with heart development or the central nervous system, respectively, were found on microarray analysis. Under differentiation of ES cells into myocardic cells, 107 genes were substantially up-regulated. Decrease in the expression of 13 genes of these (Hand1, Pim2, Tbx20, Myl4, Myl7, Hbb-bh1, Hba-a1, Col1a2, Hba-x, Cmya1, Pitx2, Smyd1 and Adam19) was observed specifically by embryotoxic chemicals. Of the 107 genes up-regulated under differentiation into neurons, 22 genes (Map2, Cpe, Marcks, Ptbp2, Sox11, Tubb2b, Vim, Arx, Emx2, Pax6, Basp1, Ddr1, Ndn, Sfrp, Ttc3, Ubqln2, Six3, Dcx, L1cam, Reln, Wnt1 and Nnat) showed reduced expression specifically by embryotoxic chemicals. Almost all gene sets identified in this study are known to be indispensable for differentiation and development of heart and brain tissues, and thus may serve in early detection or prediction of embryotoxicity of chemicals in vitro.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax+/Six3+/Vax1+) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax+ progenitors generate Otp+/Brn2+ neuronal precursors (characteristic of rostral–dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral–ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax+ progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral–dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.

Over the last decade, much progress has been made toward an understanding of the mechanism of regulation of neural differentiation. In this article, following a brief overview of neural induction research, I would like to discuss the potential contribution of basic embryological research to the progress of human therapeutic development in the present and future, focusing on the medical application of in vitro differentiation of neural tissues. This kind of linkage between basic and medical research will probably be strengthened even more by the recent emergence of human induced pluripotent stem cells. Human pluripotent stem cells are powerful tools for bridging the gap from our accumulated knowledge of embryology to regenerative medicine, as well as to a wide spectrum of medical and pharmaceutical research and development. In this commentary, I describe these issues with a particular emphasis on the contributions made by Japanese scientists.