RATIONALE:Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical ...applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology.
OBJECTIVE:Here we performed droplet-based single-cell RNA-sequencing (scRNA-seq) of the human iPSCs following iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity.
METHODS AND RESULTS:Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of nitric oxide. Non-endothelial cell populations resulting from the differentiation protocol were identified, which included immature and atrial-like cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with four major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes respectively.
CONCLUSIONS:Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of non-endothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.
Virulence of human cytomegalovirus (HCMV) clinical isolates correlates with carriage of a 15 kb segment in the UL/b′ region of the viral genome, which is absent from attenuated strains. The ...mechanisms by which this segment contributes to HCMV virulence remain obscure. We observed that intergenic RNA sequences within the 15 kb segment function as a microRNA (miRNA) decay element (miRDE) and direct the selective, sequence-specific turnover of mature miR-17 and miR-20a encoded within the host miR-17-92 cluster. Unlike canonical miRNA-mRNA interactions, the miRNA-miRDE interactions did not repress miRDE expression. miRNA binding site mutations retargeted miRDE to other miR-17-92 cluster miRNAs, which are otherwise resistant to miRDE-mediated decay. miRDE function was required to accelerate virus production in the context of lytic HCMV infection. These results indicate a role for viral noncoding RNA in regulating cellular miRNAs during HCMV pathogenesis and suggest that noncoding RNAs may play a role in mature miRNA turnover.
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•A HCMV clinical strain selectively degrades miRNAs within the host miR-17-92 cluster•A HCMV intergenic noncoding RNA in UL144-145 region, miRDE, mediates host miRNA turnover•miRDE directs miRNA turnover via sequence-specific noncanonical miRNA-mRNA interactions•miRDE is specific to virus clinical strain, and its action accelerates HCMV lytic growth
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer ...(SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a ...15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b' sequences of the Toledo genome, targeted the 3'-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and ...end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.
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•Human iPSC-CMs recapitulate clinical features of iron overload cardiomyopathy•Iron overload impairs calcium kinetics leading to iPSC-CM contractile dysfunction•Ebselen, a selective DMT1 inhibitor, can prevent iron overload phenotypes
Rhee et al. model iron overload cardiomyopathy in a dish using human iPSC-CMs. They find significantly altered calcium kinetics mediating contractile dysfunction and arrhythmia. Ebselen, a selective DMT1 inhibitor and antioxidant, effectively prevents the observed iron overload phenotypes and therefore may provide a promising therapeutic solution for iron overload cardiomyopathy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Demineralized dentin matrix (DDM) is commonly used as a bone-graft substitute. This study measured and compared human hepatitis B viruses (HBV) DNA in fresh dentin to that of dentin processed into ...DDM extracted during dental treatment from HBV-infected patients. The hypothesis was that the processing procedure for DDM would inactivate or eliminate HBV in the dentin matrix obtained from infected patients.
Dentin from eighteen HBV-infected patients was collected and each dentin specimen was divided into two fragments. One fragment was used before processing as fresh dentin (control group) and the other was processed into DDM (experimental group). DNA was extracted and purified from each fresh and processed dentin specimen and the HBV DNA copy number quantitated by real time polymerase chain reaction. The HBV DNA copy number in the fresh dentin specimens were compared relative to serologic test results. The second parameter was to evaluate the effectiveness of the processing procedure (defatting, demineralization, freeze-drying, and sterilization) to inactivate or eliminate HBV by comparing the DNA copy number in the processed DDM with that in the matched fresh dentin specimens. All results were analyzed using Mann-Whitney U test to compare numerical measurements between groups and differences were considered statistically significant at P-values less than 0.05.
The presence of HBV DNA was detected in 55.56% (10/18) of the fresh dentin specimens. For the ten HBV DNA-positive fresh dentin specimens, HBV DNA was detected in two (20%) of the matched processed dentin specimens. The copy number of HBV DNA in the two positive processed dentin specimens was 1.79 and 4.03, which were statistically lower than that of the fresh dentin specimens (P = 0.0167).
The results from this study suggested that fresh dentin may be a carrier of HBV and that the procedure used to generate DDM extensively reduced the levels of HBV DNA. Further studies are needed to evaluate the infectivity of HBV in processed dentin.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
We examined whether it is possible to directly detect citrullinated antigens in the serum of rheumatoid arthritis (RA) patients using a monoclonal antibody (mAb) designed to be specific for ...citrullinated peptides. In order to confirm the potential of the mAb as a direct arthritis-inducing substance through experimental model of RA, a monoclonal antibody (mAb) 12G1 was generated using by immunization of mice with a challenging cyclic citrullinated peptide. Immunohistochemical analysis of RA-affected synovial tissue showed that our mAb 12G1 could indeed detect citrullinated proteins in target tissues. Subsequently, serum levels of citrullinated type II collagen and filaggrin were measured in healthy volunteers, patients with RA, ankylosing spondylitis (AS), and systemic lupus erythematosus (SLE) using a 12G1-based sandwich ELISA. This showed that citrullinated filaggrin showed 78.9% sensitivity and 85.9% specificity for RA diagnosis with a cutoff optical density (OD) value of 1.013, comparable with the results from a second-generation anti-citrullinated protein antibody (ACPA) test. Circulating citrullinated collagen and filaggrin were detected even in sera of RA patients who were negative for both rheumatoid factor (RF) and ACPA. ELISA results also showed that RF and ACPA titers showed significantly positive correlation with both citrullinated collagen and filaggrin OD values in sera of RA patients. 12G1 challenging aggravated the severity of murine arthritis. In summary, mAb 12G1 can directly detect citrullinated proteins in RA target tissue and in sera of RA patients and 12G1 showed direct arthritogenic potential
. This, 12G1 might be useful for diagnosis of RA including seronegative RA and may help to elucidate the pathophysiological role of citrullination in RA.
Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for differentiation into diverse tissues. We report a straightforward and highly efficient method for the generation of ...iPSCs from PBMCs. By plating the cells serially to a newly coated plate by centrifugation, this protocol provides multiple healthy iPSC colonies even from a small number of PBMCs. The generated iPSCs expressed pluripotent markers and differentiated into all three germ layer lineages. The protocol can also be used with umbilical cord blood mononuclear cells (CBMCs). In this study, we present a simple and efficient protocol that improved the yield of iPSCs from floating cells such as PBMCs and CBMCs by serial plating and centrifugation.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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