Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation.

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for corneal reconstruction.

Cells of renin lineage are adult pluripotent progenitors in experimental glomerular disease.

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFß receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFß-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells.

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

Empirical gradient threshold technique for automated segmentation across image modalities and cell lines.

New microscopy technologies are enabling image acquisition of terabyte-sized data sets consisting of hundreds of thousands of images. In order to retrieve and analyze the biological information in these large data sets, segmentation is needed to detect the regions containing cells or cell colonies. Our work with hundreds of large images (each 21,000×21,000 pixels) requires a segmentation method that: (1) yields high segmentation accuracy, (2) is applicable to multiple cell lines with various densities of cells and cell colonies, and several imaging modalities, (3) can process large data sets in a timely manner, (4) has a low memory footprint and (5) has a small number of user-set parameters that do not require adjustment during the segmentation of large image sets. None of the currently available segmentation methods meet all these requirements. Segmentation based on image gradient thresholding is fast and has a low memory footprint. However, existing techniques that automate the selection of the gradient image threshold do not work across image modalities, multiple cell lines, and a wide range of foreground/background densities (requirement 2) and all failed the requirement for robust parameters that do not require re-adjustment with time (requirement 5). We present a novel and empirically derived image gradient threshold selection method for separating foreground and background pixels in an image that meets all the requirements listed above. We quantify the difference between our approach and existing ones in terms of accuracy, execution speed, memory usage and number of adjustable parameters on a reference data set. This reference data set consists of 501 validation images with manually determined segmentations and image sizes ranging from 0.36 Megapixels to 850 Megapixels. It includes four different cell lines and two image modalities: phase contrast and fluorescent. Our new technique, called Empirical Gradient Threshold (EGT), is derived from this reference data set with a 10-fold cross-validation method. EGT segments cells or colonies with resulting Dice accuracy index measurements above 0.92 for all cross-validation data sets. EGT results has also been visually verified on a much larger data set that includes bright field and Differential Interference Contrast (DIC) images, 16 cell lines and 61 time-sequence data sets, for a total of 17,479 images. This method is implemented as an open-source plugin to ImageJ as well as a standalone executable that can be downloaded from the following link: https://isg.nist.gov/.

From pluripotent stem cells to multifunctional cordocytic phenotypes in the human brain: an ultrastructural study.

Light microscopy and transmission electron microscopy were used to investigate surgical cases in a variety of pathological conditions (thromboses, tumors, cerebrovascular malformations, Moyamoya disease) to identify and characterize different phenotypes belonging to a new interstitial cell recently described ultrastructurally in the brain and here named "cordocyte." Also, this work is an attempt to identify and characterize precursor/stem cells for cordocytic lineage in the perivascular areas, within perivascular nerves and pia mater (now considered a cordocytic-vascular tissue). Unexpected relationships and functions emerge from observations concerning these phenotypes, almost ubiquitous, but not yet fully studied in the brain.

Luminal particles within cellular microtubules.

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

iPS programmed without c-MYC yield proficient cardiogenesis for functional heart chimerism.

RATIONALE: Induced pluripotent stem cells (iPS) allow derivation of pluripotent progenitors from somatic sources. Originally, iPS were induced by a stemness-related gene set that included the c-MYC oncogene. OBJECTIVE: Here, we determined from embryo to adult the cardiogenic proficiency of iPS programmed without c-MYC, a cardiogenicity-associated transcription factor. METHODS AND RESULTS: Transgenic expression of 3 human stemness factors SOX2, OCT4, and KLF4 here reset murine fibroblasts to the pluripotent ground state. Transduction without c-MYC reversed cellular ultrastructure into a primitive archetype and induced stem cell markers generating 3-germ layers, all qualifiers of acquired pluripotency. Three-factor induced iPS (3F-iPS) clones reproducibly demonstrated cardiac differentiation properties characterized by vigorous beating activity of embryoid bodies and robust expression of cardiac Mef2c, alpha-actinin, connexin43, MLC2a, and troponin I. In vitro isolated iPS-derived cardiomyocytes demonstrated functional excitation-contraction coupling. Chimerism with 3F-iPS derived by morula-stage diploid aggregation was sustained during prenatal heart organogenesis and contributed in vivo to normal cardiac structure and overall performance in adult tumor-free offspring. CONCLUSIONS: Thus, 3F-iPS bioengineered without c-MYC achieve highest stringency criteria for bona fide cardiogenesis enabling reprogrammed fibroblasts to yield de novo heart tissue compatible with native counterpart throughout embryological development and into adulthood.

Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states.

Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have a reduced nuclear:cytoplasmic ratio, are more transcriptionally active, and contain abundant cellular organelles. This study also revealed cavitation and potential unfolding of the epiblast. As the ICM forms the epiblast, SSEA1 is lost and VIMENTIN is lost and re-expressed. The D6 blastocyst expressed high levels of STELLA, TERF1, and GDF3, and the epiblast expressed epithelial markers, MUC1 and E-CADHERIN, and the pluripotency markers, DNMT3B and CRIPTO.

Human pluripotent stem cell-derived eosinophils reveal potent cytotoxicity against solid tumors.

Eosinophils are attractive innate immune cells to use to potentiate T cell antitumor efficacy because they are capable of infiltrating tumors at early stages and modulating the tumor microenvironment. However, the limited number of functional eosinophils caused by the scarcity and short life of primary eosinophils in peripheral blood has greatly impeded the development of eosinophil-based immunotherapy. In this study, we established an efficient chemically defined protocol to generate a large quantity of functional eosinophils from human pluripotent stem cells (hPSCs) with nearly 100% purity expressing eosinophil peroxidase. These hPSC-derived eosinophils transcriptionally resembled their primary counterpart. Moreover, hPSC-derived eosinophils showed competent tumor killing capacity in established solid tumors. Furthermore, the combination of hPSC-derived eosinophils with CAR-T cells exhibited potential synergistic effects, inhibiting tumor growth and enhancing mouse survival. Our study opens up new avenues for the development of eosinophil-based immunotherapies to treat cancer.

Mice cloned from induced pluripotent stem cells (iPSCs).

Differentiated somatic cells of various species can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopically expressing a combination of several transcription factors that are highly enriched in embryonic stem cells (ESCs). The generation of iPSCs in large animals has raised the possibility of producing genetically modified large animals through the nuclear transplantation approach. However, it remains unknown whether iPSCs could be used for generating cloned animals through the nuclear transfer method. Here, we show the successful production of viable cloned mice from inducible iPSCs through the nuclear transfer approach, and the efficiency is similar to that of using ESCs derived via normal fertilization. Furthermore, the cloned mice are fertile and can produce second-generation offspring. These efforts strengthen the possibility of utilizing iPSCs to generate gene-modified large animals for pharmaceutical purposes in the future.

Identification and characterization of aortic valve mesenchymal progenitor cells with robust osteogenic calcification potential.

Advanced valvular lesions often contain ectopic mesenchymal tissues, which may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. The identity, frequency, and differentiation potential of the putative progenitor subpopulation are unknown. The objectives of this study were to determine whether valve interstitial cells (VICs) contain a subpopulation of multipotent mesenchymal progenitor cells, to measure the frequencies of the mesenchymal progenitors and osteoprogenitors, and to characterize the osteoprogenitor subpopulation because of its potential role in calcific aortic valve disease. The multilineage potential of freshly isolated and subcultured porcine aortic VICs was tested in vitro. Progenitor frequencies and self-renewal capacity were determined by limiting dilution and colony-forming unit assays. VICs were inducible to osteogenic, adipogenic, chondrogenic, and myofibrogenic lineages. Osteogenic differentiation was also observed in situ in sclerotic porcine leaflets. Primary VICs had strikingly high frequencies of mesenchymal progenitors (48.0 +/- 5.7%) and osteoprogenitors (44.1 +/- 12.0%). High frequencies were maintained for up to six population doublings, but decreased after nine population doublings to 28.2 +/- 9.9% and 5.8 +/- 1.3%, for mesenchymal progenitors and osteoprogenitors, respectively. We further identified the putative osteoprogenitor subpopulation as morphologically distinct cells that occur at high frequency, self-renew, and elaborate bone matrix from single cells. These findings demonstrate that the aortic valve is rich in a mesenchyma l progenitor cell population that has strong potential to contribute to valve calcification.

Cellular and molecular dissection of pluripotent adult somatic stem cells in planarians.

Freshwater planarians, Plathelminthes, have been an intriguing model animal of regeneration studies for more than 100 years. Their robust regenerative ability is one of asexual reproductive capacity, in which complete animals develop from tiny body fragments within a week. Pluripotent adult somatic stem cells, called neoblasts, assure this regenerative ability. Neoblasts give rise to not only all types of somatic cells, but also germline cells. During the last decade, several experimental techniques for the analysis of planarian neoblasts at the molecular level, such as in situ hybridization, RNAi and fluorescence activated cell sorting, have been established. Moreover, information about genes involved in maintenance and differentiation of neoblasts has been accumulated. One of the molecular features of neoblasts is the expression of many RNA regulators, which are involved in germline development in other animals, such as vasa and piwi family genes. In this review, we introduce physiological and molecular features of the neoblast, and discuss how germline genes regulate planarian neoblasts and what differences exist between neoblasts and germline cells.

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics.

BACKGROUND AIMS: Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS: Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS: We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS: The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.

Folliculo-stellate cells of the human pituitary as adult stem cells: examples of their neoplastic potential.

Two unique, formerly unrecorded sellar neoplasms were observed in two women of 60 and 63 years of age. One lesion consisted of small epithelial cells and the other was a large-cell oncocytic tumor, yet they had the same simple cytoplasmic organization with dominance of polyribosomes and a sprinkle of glycogen. Striking markers shared by the neoplasms: (1) network of typical pituitary follicles, and (2) unexpected similarity to fetal human pituitary tissue at different gestational ages of 6 and 10-12 weeks. The latter showed appreciable endocrine differentiation. The assumed parent cell is the folliculo-stellate cell as pluripotent adult stem cell.

Primary cilia-dependent signaling is involved in regulating mesenchymal stem cell proliferation and pluripotency maintenance.

Using a large-scale quantitative mesenchymal stem cells (MSCs) membrane proteomics analysis, we identified a large group of ciliary proteins in the MSCs membrane fraction, which prompted us to examine the cilia, intricate organelles that were originally discovered approximately 100 years ago. Here we characterize their primary structure and function in MSCs. We first characterized the primary cilia on undifferentiated human MSCs by immunostaining and verified these observation with scanning and 3D electronic microscopy. To investigate the function of the primary cilia of the MSCs we induced loss of function by means of siRNA knockdown (targeted to two known ciliary proteins: IFT172 and KIF3A). After either of these two proteins was knocked down by the respective siRNA, the MSCs showed fewer and shortened primary cilia. The MSCs proliferation assays showed increased cell proliferative activity under confluent conditions after the siRNA knockdown of IFT172 or KIF3A; among these MSCs, the proportion in S phase was increased in the IFT172 siRNA knockdown group. The expression of stem cell markers on the MSCs, namely, Oct4, Nanog and Sox2, were downregulated after the siRNA-induced knockdown of IFT172 or KIF3A, and the gene expression upregulation of ectoderm lineage markers was notable. Furthermore, manipulation of the primary cilia-dependent Shh pathway, using the Shh activator SAG (smoothened agonist), upregulated the gene expression of pluripotency markers, including Nanog and Oct4, and transcriptional target genes in the Shh pathway. This study confirms that MSCs have primary cilia and provides evidence that primary cilia-dependent signaling pathways play functional roles in MSCs proliferation and stemness maintenance.

Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells.

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin.

Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.