Increased mesodermal and mesendodermal populations by BMP4 treatment facilitates human iPSC line differentiation into a cardiac lineage.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have attracted attention as a novel tool for drug safety screening and several differentiation protocols of hiPSC lines into cardiomyocytes have been reported; the standardization of these protocols will expand their applications for safety assessments such as "clinical safety trial-on-dish". Bone morphogenetic protein 4 (BMP4) is an important factor in promoting mesoderm differentiation and BMP4 treatment has been used at the early stage of cardiac differentiation into different hiPSCs. In the present study, we evaluated the effects of BMP4 treatment at the early stage of cardiac differentiation. We performed gene expression profiling of the germ layer during mesoderm differentiation of hiPSCs derived from three different donors. The expression of T (a mesoderm marker) and GATA6 (an endoderm marker) increased and that of PAX6 (a neuroectoderm marker) decreased in pooled embryoid bodies (EBs) after BMP4 treatment. Single-cell gene expression analysis revealed that mesodermal and mesendodermal populations increased in EBs derived from 253G1. Finally, BMP4 treatment increased mesodermal and mesendodermal populations compared with that without BMP4 in two other hiPSC lines, confirming the reproducibility of multiple hiPSC lines. Thus, our results suggest that BMP4 treatment increases mesodermal and mesendodermal populations at the early stage of cardiac differentiation in different hiPSC lines.

Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform activity in response to convulsant drugs. Our study indicates the possibility that co-culture system of hiPSC-derived neurons with hiPSC-derived astrocytes could be useful in the risk assessment of drug-induced seizure.

Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes.

To predict drug-induced serious adverse events (SAE) in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs) of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs) as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox)-induced QT prolongation. Different Mox-induced field potential duration (FPD) prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66) in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

BACKGROUND: Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. RESULTS: In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3ß inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis. CONCLUSIONS: Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.

Involvement of ER stress in dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 missense mutations shown by iPSC-derived oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.

A gene expression-based screening system for compounds influencing differentiation of mouse embryonic stem cells.

Low molecular weight compounds have been shown to be useful for controlling stem cells, and various high-throughput screening systems have been developed for identifying compounds that regulate the differentiation of stem cells. However, the effects of such compounds on stem cell differentiation are usually evaluated by assessing a single parameter, which is insufficient for proper monitoring of the cellular status. In this study, to classify a number of compounds, the authors established a gene expression-based screening system using mouse embryonic stem (ES) cells that monitored multiple parameters. ES cells were differentiated into three germ layers by embryoid body formation and then treated with the test compounds. Next, cellular changes were assessed by analyzing the expression of multiple genes with the multiplex quantitative reverse transcriptase polymerase chain reaction. By screening a library of pharmacologically active compounds with this system, the authors were able to classify 52 compounds that influenced the gene expression profile of ES cells. They also found that some compounds identified by screening could enhance osteogenic or adipogenic differentiation of human mesenchymal stem cells. These results indicate that the screening system is effective for identifying compounds involved in regulating the differentiation of both ES cells and adult stem cells.

Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells.

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic ß-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3ß inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-ß (TGF-ß) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-ß inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.

The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline.

Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice.

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.

RNA interference during spermatogenesis in mice.

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.