Isolation and characterization of ventricular-like cells derived from NKX2-5eGFP/w and MLC2vmCherry/w double knock-in human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5eGFP/w and MLC2vmCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5eGFP/w and MLC2vmCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources.

OpenTein: a database of digital whole-slide images of stem cell-derived teratomas.

Human stem cells are promising sources for regenerative therapy. To ensure safety of future therapeutic applications, the differentiation potency of stem cells has to be tested and be widely opened to the public. The potency is generally assessed by teratoma formation comprising differentiated cells from all three germ layers, and the teratomas can be inspected through high-quality digital images. The teratoma assay, however, lacks consistency in transplantation protocols and even in interpretation, which needs community-based efforts for improving the assay quality. Here, we have developed a novel database OpenTein (Open Teratoma Investigation, http://opentein.hgc.jp/) to archive and freely distribute high-resolution whole-slide images and relevant records. OpenTein has been designed as a searchable, zoomable and annotatable web-based repository system. We have deposited 468 images of teratomas derived by our transplantation of human stem cells, and users can freely access and process such digital teratoma images. Approximately, the current version of OpenTein responds within 11.2 min for processing 2.03 gigapixel teratoma images. Our system offers valuable tools and resources in the new era of stem cell biology.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

The role of NF-κB signaling in the maintenance of pluripotency of human induced pluripotent stem cells.

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.

Wnt signaling orchestration with a small molecule DYRK inhibitor provides long-term xeno-free human pluripotent cell expansion.

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-ß (TGFß) signaling pathways, and none provide for replacement of FGF/TGFß ligands with chemical compounds. The Wnt/ß-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/ß-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/ß-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFß activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells.

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or, alternatively, induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects, we focused on SNON (SKIL), a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast, enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus, our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment.

Dynamic link between histone H3 acetylation and an increase in the functional characteristics of human ESC/iPSC-derived cardiomyocytes.

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes in vitro, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA) recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.

Comparative study of transplantation of hepatocytes at various differentiation stages into mice with lethal liver damage.

Hepatocyte transplantation utilizing induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) has been expected to provide an alternative to liver transplantation. However, it remains uncertain precisely which cell type is the best suited for cell transplantation. In particular, it is unclear whether mature hepatocytes, which have sufficient liver function, or immature hepatic progenitor cells, which have a higher proliferative capacity, will provide a better outcome. The main objective of this study was to investigate the therapeutic efficacy of the transplantation of hepatocytes at various differentiation stages. We utilized transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. ESC-derived endodermal cells, fetal hepatocytes, and adult hepatocytes were transplanted into these mice with experimentally induced lethal acute liver injury caused by DT administration. The transplanted cells were marked by enhanced green fluorescent protein. We evaluated their effects on survival. At 35 days after transplantation, the survival rate of the adult hepatocyte-transplanted group (8/20, 40%) was significantly improved in comparison to that of the sham-operated group (2/25, 8%), the fetal hepatocyte-transplanted group (1/20, 5%), and the ESC-derived endodermal cell-transplanted group (0/21, 0%). The adult hepatocytes proliferated in the recipient livers and replaced a large part of their parenchyma. The transplantation of adult hepatocytes for acute liver failure significantly improved the survival rate in comparison to that of transplantation of immature cells, thus suggesting that ESCs and iPSCs should be differentiated into mature hepatocytes before cell transplantation for acute liver failure.

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease.

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)-derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC-derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinson's disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42-day culture with brain-derived neurotrophic factor/glial cell line-derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain.

Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

Alpha-fetoprotein-producing pancreatic cancer cells possess cancer stem cell characteristics.

We aimed to demonstrate the existence of cancer stem cells in human pancreatic cancer, and to clarify that they are alpha-fetoprotein (AFP) producing cells. Six cell lines derived from human pancreatic cancers were examined, and AsPC-1 and PANC-1 were noted to express AFP. Single cell culture assays and xenotransplantation revealed that the AFP-producing cells had the capacity for self-renewal and differentiation, and that these cells were tumorigenic. Furthermore, they were resistant to anti-cancer agents. The ABCA12 transporter was expressed in the AFP-producing cells at a level more than twice as high as that in the non-AFP-producing cells. The AFP-producing cells were shown to be putative pancreatic cancer stem cells. Furthermore, the expression of ABCA12 appears to be associated with drug resistance.

Simple and highly efficient method for production of endothelial cells from human embryonic stem cells.

Endothelial cells derived from human embryonic stem cells (hESC-ECs) hold much promise as a valuable tool for basic vascular research and for medical application such as cell transplantation or regenerative medicine. Here we have developed an efficient approach for the production of hESC-ECs. Using a differentiation method consisting of a stepwise combination of treatment with glycogen synthase kinase-3ß (GSK-3ß) inhibitor and culturing in vascular endothelial growth factor (VEGF)-supplemented medium, hESC-ECs are induced in 5 days with about 20% efficiency. These cells express vascular endothelial cadherin (VE-cadherin), VEGF receptor-2 (VEGFR-2), CD34, and platelet endothelial cell adhesion molecule-1 (PECAM-1). These hESC-ECs can then be isolated with 95% purity using a magnetic sorting system, and expanded to more than 100-fold within a month. The hESC-ECs thus produced exhibit the endothelial morphological characteristics and specific functions such as capillary tube formation and acetylated low-density lipoprotein uptake. We propose that our methodology is useful for efficient and large-scale production of hESC-ECs.

Neutrophil differentiation from human-induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells are of potential value not only for regenerative medicine, but also for disease investigation. The present study describes the development of a neutrophil differentiation system from human iPS cells (hiPSCs) and the analysis of neutrophil function and differentiation. The culture system used consisted of the transfer of hiPSCs onto OP9 cells and their culture with vascular endothelial growth factor (VEGF). After 10 days, TRA 1-85(+) CD34(+) VEGF receptor-2 (VEGFR-2)(high) cells were sorted and co-cultured with OP9 cells in the presence of hematopoietic cytokines for 30 days. Floating cells were collected and subjected to morphological and functional analysis. These hiPSC-derived neutrophils were similar to peripheral blood mature neutrophils in morphology, contained functional neutrophil specific granules, and were equipped with the basic functions such as phagocytosis, superoxide production, and chemotaxis. In the process of differentiation, myeloid cells appeared sequentially from immature myeloblasts to mature segmented neutrophils. Expression patterns of surface antigen, transcription factors, and granule proteins during differentiation were also similar to those of granulopoiesis in normal bone marrow. In conclusion, differentiation of mature neutrophils from hiPSCs was successfully induced in a similar process to normal granulopoiesis using an OP9 co-culture system. This system may be applied to elucidate the pathogenesis of various hematological diseases that affect neutrophils.

Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.

Human embryonic stem cells (hESCs), unlike mouse ones (mESCs), are vulnerable to apoptosis upon dissociation. Here, we show that the apoptosis, which is of a nonanoikis type, is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr, a unique Rho-GEF family factor containing a functional Rac-GAP domain, is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation, but not in Abr-depleted hESCs or mESCs. Furthermore, artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus, the Abr-dependent "Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs.

Alpha-fetoprotein producing cells act as cancer progenitor cells in human cholangiocarcinoma.

We aimed to demonstrate that alpha-fetoprotein (AFP)-producing cells in cholangiocarcinomas possessed cancer stem cell (CSC)-like properties. AFP enhancer/promoter-driven EGFP gene was transfected into human cholangiocarcinoma cell lines. One cell line, RBE, expressed both AFP and EGFP. Clonal analyses revealed that one EGFP-positive cell generated both EGFP-positive and EGFP-negative cell fractions. However, one EGFP-negative cell never produced EGFP-positive cells. The EGFP-positive cells had a greater tumorigenic potential. Only the EGFP-positive cells expressed Notch1. AFP and Notch1 expression was observed in clinical intrahepatic cholangiocarcinomas. The AFP-producing cells were suggested to be CSCs. The Notch pathway might play an important role in maintaining the CSC characteristics.

Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO.

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me(2)SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9+/-7.7%, compared to 0.4+/-0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me(2)SO-free, chemically-defined medium.

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.

Establishment of a cell line derived from a mouse fetal liver that has the characteristic to promote the hepatic maturation of mouse embryonic stem cells by a coculture method.

Stromal cells residing in murine fetal livers have the ability to promote the hepatic maturation of murine embryonic stem cells (ESCs) and hepatic progenitor cells (HPCs) 3848 in vitro. These stromal cells were isolated as the CD49f(+/-)CD45(-)Thy1(+)gp38(+) cell fraction. The present study established a murine fetal liver stromal cell line that induced hepatic maturation in mouse ESCs and HPCs. A transgene containing a temperature-sensitive SV40 large T antigen was transfected into the primary fetal liver stromal cells. These immortalized cells, which were named as the gp38-positive and Thy1-positive murine liver stromal (MLSgt) cells, induced both mouse ESCs and HPCs to differentiate into mature hepatocyte-like cells using a coculture method. Since MLSgt is not a cloned cell line, one clone, MLSgt20, was selected as a line with the characteristic to induce hepatic differentiation, which was comparable to its parental stromal cells. The ESC-derived endoderm cells cocultured with the MLSgt20 cells expressed mature hepatocyte-specific gene markers, including glucose-6-phosphatase, tyrosine aminotransferase, tryptophan 2,3-dioxgenase, and cytochrome P450 (CYP1a1, Cyp1b1, Cyp1a2, and Cyp3a11). In addition, these cells also exhibited hepatic functions, such as glycogen storage and ammonia metabolism. Transmission electron microscopy showed that the cocultured ESCs expressed the morphologic features of mature hepatocytes. In conclusion, a cell line was established that has the characteristic to promote the hepatic maturation of mouse ESCs and HPCs by a coculture method.

Adipogenic differentiation of human induced pluripotent stem cells: comparison with that of human embryonic stem cells.

Induced pluripotent stem (iPS) cells were recently established from human fibroblasts. In the present study we investigated the adipogenic differentiation properties of four human iPS cell lines and compared them with those of two human embryonic stem (ES) cell lines. After 12 days of embryoid body formation and an additional 10 days of differentiation on Poly-l-ornithine and fibronectin- coated dishes with adipogenic differentiation medium, human iPS cells exhibited lipid accumulation and transcription of adipogenesis-related molecules such as C/EBPalpha, PPARgamma2, leptin and aP2. These results demonstrate that human iPS cells have an adipogenic potential comparable to human ES cells.