Abstract
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human ...induced pluripotent stem cells (hiPSCs) carrying a TNNI1
EmGFP
and TNNI3
mCherry
double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To ...identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.
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•Human PSC hematopoietic commitment capacity correlates with IGF2 expression•IGF2 expression depends on signaling-dependent chromatin accessibility•Maturation capacity is associated with reprogramming-related DNA methylation•Epigenetic features can help identify human PSC lines with differential capacities
Nishizawa et al. integrate analysis of differentiation outcome and molecular characterization to identify features of human iPSCs associated with high and low capacities for hematopoietic specification and maturation. Prospective use of this type of information could help in choosing iPSC lines best suited to different applications.
Research on cardiomyopathy models using engineered heart tissue (EHT) created from disease‐specific induced pluripotent stem cells (iPSCs) is advancing rapidly. However, the study of restrictive ...cardiomyopathy (RCM), a rare and intractable cardiomyopathy, remains at the experimental stage because there is currently no established method to replicate the hallmark phenotype of RCM, particularly diastolic dysfunction, in vitro. In this study, we generated iPSCs from a patient with early childhood‐onset RCM harboring the TNNI3 R170W mutation (R170W‐iPSCs). The properties of R170W‐iPSC‐derived cardiomyocytes (CMs) and EHTs were evaluated and compared with an isogenic iPSC line in which the mutation was corrected. Our results indicated altered calcium kinetics in R170W‐iPSC‐CMs, including prolonged tau, and an increased ratio of relaxation force to contractile force in R170W‐EHTs. These properties were reversed in the isogenic line, suggesting that our model recapitulates impaired relaxation of RCM, i.e., diastolic dysfunction in clinical practice. Furthermore, overexpression of wild‐type TNNI3 in R170W‐iPSC‐CMs and ‐EHTs effectively rescued impaired relaxation. These results highlight the potential efficacy of EHT, a modality that can accurately recapitulate diastolic dysfunction in vitro, to elucidate the pathophysiology of RCM, as well as the possible benefits of gene therapies for patients with RCM.
In this study, we demonstrated that R170W induced pluripotent stem cell‐derived cardiomyocytes (R170W‐iPSC‐CMs) and engineered heart tissue (EHTs) exhibit prolonged relaxation time and relaxation impairment and established an in vitro evaluation model. Furthermore, gene correction and overexpression of TNNI3 improved relaxation impairment in iPSC‐CMs and ‐EHTs. The EHT modality can precisely recapitulate the impaired relaxation of restrictive cardiomyopathy (RCM) in vitro and therefore may serve as a gateway to insight into RCM pathogenesis and the development of novel therapeutic strategies.
Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported ...in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day 4 mesodermal cells, and day 8, day 20, and day 30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day 20 CMs was significantly higher compared to other cells. Transplantation of day 20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day 20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.
Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, ...specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.
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•Synthetic miRNA switches can purify target cell populations based on miRNA activity•miR-1-, -208a-, and -499a-5p-switches highly purify hPSC-derived cardiomyocytes•miR-Bim switches enrich for cardiomyocytes without the need for cell sorting•miRNA switches can isolate desired cell types without significant side effects
Miki et al. develop synthetic miRNA switches for isolating cell populations that are otherwise difficult to purify. Several miRNA-responsive switches precisely and efficiently isolate human pluripotent stem cell (hPSC)-derived cardiomyocytes, and miRNA switches can be programmed for purification of various cell types including hPSC-derived endothelial cells, hepatocytes, and insulin-producing cells.
The incomplete differentiation of human induced pluripotent stem cells (iPSCs) poses a serious safety risk owing to their potential tumorigenicity, hindering their clinical application. Here, we ...explored the potential of phospho-D-peptides as novel iPSC-eliminating agents. Alkaline phosphatases overexpressed on iPSCs dephosphorylate phospho-D-peptides into hydrophobic peptides that aggregate and induce cell death. We isolated a peptide candidate, D-3, that selectively and rapidly induced toxicity in iPSCs within 1 hr but had little influence on various non-iPSCs, including primary hepatocytes and iPSC-derived cardiomyocytes. Two hours of D-3 treatment efficiently eliminated iPSCs from both single cultures and co-cultures spiked with increasing ratios of iPSCs. In addition, D-3 prevented residual iPSC-induced teratoma formation in a mouse tumorigenicity assay. These results suggest the enormous potential of D-3 as a low-cost and effective anti-iPSC agent for both laboratory use and for the safe clinical application of iPSC-derived cells in regenerative medicine.
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•D-3 induces toxicity in iPSCs and ESCs within 1 hr of incubation•D-3 has little influence on various non-iPSCs, including hepatocytes and neurons•D-3 prevents residual iPSC-induced teratoma formation in an animal model•Alkaline phosphatase activity on the cell surface is required for D-3's toxicity
Yi Kuang et al. discovered a phosphor-peptide D-3 that responds to the activity of alkaline phosphatase on the cell surface to selectively and effectively remove iPSCs and ESCs within 1–2 hr. D-3 is a promising low-cost iPSC-eliminating agent and is simple to use.
Hematopoietic differentiation from human induced pluripotent stem (iPS)/embryonic stem (ES) cell attracts much attention due to its huge potential for regenerative medicine. As indicated by some ...earlier papers, there is large variation in differentiation potential among pluripotent stem cell (PSC) lines, and this is one of major concerns in clinical application of PSCs. If it becomes possible to predict which PSC line has high differentiation potential without real differentiation experiment, it would greatly contribute to clinical application of PSCs. Although some papers reported about presence of epigenetic memories of parental somatic cells in iPS cells, the amount of the influence on differentiation potential remains to be known. Furthermore, especially in studies using human PSCs, genetic difference among individual donors of iPS/ES cells seems to be large, thus the study using many PSC lines from many donors is warranted. To address these issues, we planned to collect data of many iPS/ES cell lines on genome-wide gene expression and genomic DNA methylation, and differentiation potentials of individual lines, and identify the factors which affected difference in differentiation potential among PSC lines. The final goal of this study is to create data base about gene expression and DNA methylation profile and differentiation potentials of many PSC lines. We believe that this dataset will allow us to predict differentiation potentials of individual PSC lines, and accelerate clinical application of PSC lines in hematology field.
We utilized 39 iPS/ES lines (iPS 35 lines, ES 4 lines) in this study. The iPS cell lines were derived from dermal fibroblast (n = 16), cord blood (n = 3), peripheral blood (n = 10), keratinocyte (n = 3), and dental pulp cell (n = 3), and were generated by retrovirus vector (n = 9), episomal vector (n = 25), and sendai virus vector (n = 1). The iPS cells were derived from 15 donors, and the ES cells were derived from 4 donors. We assessed hematopoietic differentiation potential by investigating hematopoietic differentiation efficiency for the first 15 days from start of differentiation, and colony forming potential of hematopoietic precursor cells (CD34+CD38-CD43+lineage marker- population) generated from PSC lines using semi-solid methylcellulose based-media. In addition, we collected genome-wide mRNA expression and DNA methylation profile of PSC lines, parental lines of iPS cells, hematopoietic precursor cells generated from PSCs by using mRNA microarray, genomic methylation beads array, and next generation sequencers, and analyzed correlation of these data with differentiation potentials of individual PSC lines.
We have found that there is large variation in hematopoietic differentiation efficiency and colony forming ability as reported previously. Genome-wide investigation of gene expression and genomic DNA methylation revealed that expression of some genes or some factors were significantly correlated with hematopoietic differentiation efficiency or colony forming ability of hematopoietic precursor cells. Importantly, the factors affecting differentiation efficiency for first 15 days and those affecting colony-forming ability were absolutely different. More importantly, by combining several factors discovered in this analysis, we can predict hematopoietic differentiation potential of individual iPS/ES cell lines regardless of what parental cell lines iPS cells are derived or whether it is an iPS cell or ES cell.
From genome-wide analysis of gene expression and genomic DNA methylation, and hematopoietic differentiation experiments, we discovered the factors that were associated with difference in differentiation potential among PSC lines. Now, we are focusing on investigating molecular mechanisms by which the discovered factors are responsible for the difference in hematopoietic differentiation potentials among PSC lines. We believe that our findings will contribute not only to clinical application of hematopoietic cells generated from human PSCs, but also to further understanding of human developmental hematopoiesis.
No relevant conflicts of interest to declare.
Sugar content is one of the most important quality traits of tomato. Cell wall invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source ...leaves and fruits, while invertase inhibitor (INVINH) regulates this process. In this study, knock-out of cell wall INVINH in tomato (SlINVINH1) was performed by genome editing using, CRISPR/Cas9 and Target-AID technologies. Most of the genome-edited lines set higher soluble solid content (SSC) fruit than the original cultivar 'Suzukoma', while fruit weight was different among the genome-edited lines. From these genome-edited lines, three lines (193-3, 199-2, and 247-2), whose SSC was significantly higher than 'Suzukoma' and fruit weight were almost the same as the original cultivar, were selected. The fruit weight and overall plant growth of the two lines were comparable to those of the original cultivar. In contrast, the fructose and glucose contents in the mature fruits of the two lines were significantly higher than those of the original cultivar. The mature fruits of genome edited line 193-3 showed the highest sugar content, and the fructose and glucose contents were 29% and 36% higher than that of the original cultivar, respectively. Whole genome sequence data showed no off-target mutations in the genome-edited lines. Non-target metabolome analysis of mature fruits revealed that fructose was the highest loading factor in principal component analysis (PCA) between the genome-edited line and the original cultivar, and no unexpected metabolites appeared in the genome-edited line. In this study, we succeeded in producing tomato lines with high sugar content without a decrease in fruit weight and deterioration of plant growth by knock-out of SlINVINH1 using genome editing technology. This study showed that functional disruption of SlINVINH1 is an effective approach to produce tomato cultivars with high sugar content.
Introduction
Assessing dysarthria features in patients with neurodegenerative diseases helps diagnose underlying pathologies. Although deep neural network (DNN) techniques have been widely adopted in ...various audio processing tasks, few studies have tested whether DNNs can help differentiate neurodegenerative diseases using patients’ speech data. This study evaluated whether a DNN model using a transformer architecture could differentiate patients with Parkinson’s disease (PD) from patients with spinocerebellar degeneration (SCD) using speech data.
Methods
Speech data were obtained from 251 and 101 patients with PD and SCD, respectively, while they read a passage. We fine-tuned a pre-trained DNN model using log-mel spectrograms generated from speech data. The DNN model was trained to predict whether the input spectrogram was generated from patients with PD or SCD. We used fivefold cross-validation to evaluate the predictive performance using the area under the receiver operating characteristic curve (AUC) and accuracy, sensitivity, and specificity.
Results
Average ± standard deviation of the AUC, accuracy, sensitivity, and specificity of the trained model for the fivefold cross-validation were 0.93 ± 0.04, 0.87 ± 0.03, 0.83 ± 0.05, and 0.89 ± 0.05, respectively.
Conclusion
The DNN model can differentiate speech data of patients with PD from that of patients with SCD with relatively high accuracy and AUC. The proposed method can be used as a non-invasive, easy-to-perform screening method to differentiate PD from SCD using patient speech and is expected to be applied to telemedicine.
Deficiency of citrin due to mutations of the SLC25A13 gene causes adult-onset type II citrullinemia (CTLN2) and one type of neonatal intrahepatic cholestasis (NICCD). About half of the NICCD patients ...are detected based on high galactose, phenylalanine, and/or methionine concentrations on newborn mass screening (NMS). To clarify the perinatal and neonatal effects and the inconsistent results on NMS, we examined aminograms, the levels of bile acids and galactose in dried blood spots for NMS from 20 patients with NICCD. Birth weight was low for gestational age (-1.4 +/- 0.7 SD). Affected fetuses may have suffered intrauterine citrin deficiency. The first abnormality detected after birth was citrullinemia, and 19 of 20 patients had citrulline levels higher than +2 SD of controls. Tyrosine, phenylalanine, methionine, galactose, and bile acids were less affected than citrulline on d 5 after birth. Galactose and bile acids levels were increased at 1 mo in comparison with d 5 after birth due to impairment of the cytosolic NADH reducing-equivalent supply into mitochondria of hepatocytes. Patients with negative findings on NMS had low levels of total 20 amino acids. Citrulline/serine, citrulline /leucine plus isoleucine, and citrulline/total amino acids ratios, controlled for the confounding effect of low amount of total amino acids, were higher in all patients than +2 SD, +2 SD, and +3 SD of controls, respectively. NMS for citrin deficiency (frequency of homozygote with SLC25A13 mutation: 1/10,000-1/38,000 in East Asia) will be useful for clarification of the clinical course, treatment, and prevention of this disease.
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