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.
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.
Human pluripotent stem cells (PSCs) express human endogenous retrovirus type-H (HERV-H), which exists as more than a thousand copies on the human genome and frequently produces chimeric transcripts ...as long-non-coding RNAs (lncRNAs) fused with downstream neighbor genes. Previous studies showed that HERV-H expression is required for the maintenance of PSC identity, and aberrant HERV-H expression attenuates neural differentiation potentials, however, little is known about the actual of function of HERV-H. In this study, we focused on ESRG, which is known as a PSC-related HERV-H-driven lncRNA. The global transcriptome data of various tissues and cell lines and quantitative expression analysis of PSCs showed that ESRG expression is much higher than other HERV-Hs and tightly silenced after differentiation. However, the loss of function by the complete excision of the entire ESRG gene body using a CRISPR/Cas9 platform revealed that ESRG is dispensable for the maintenance of the primed and naïve pluripotent states. The loss of ESRG hardly affected the global gene expression of PSCs or the differentiation potential toward trilineage. Differentiated cells derived from ESRG-deficient PSCs retained the potential to be reprogrammed into induced PSCs (iPSCs) by the forced expression of OCT3/4, SOX2, and KLF4. In conclusion, ESRG is dispensable for the maintenance and recapturing of human pluripotency.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis ...are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple-reporter human induced pluripotent stem cell lines that express HAND1mCherry, HAND2EGFP and either MYH6-driven iRFP670 or tagBFP constitutively and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry− CMs and identified a gene network of LEF1, HAND1, and HAND2 to regulate proliferation in CMs. Finally, we identified CD105 as a surface marker of highly proliferative CMs.
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•Expression of HAND1 marks cardiovascular progenitor cells•LEF1 is a key regulator of proliferating cardiomyocytes•CD105 expression marks highly proliferative cardiomyocytes
HAND1 and HAND2 are important cardiac transcriptional factors, but their functions in human cardiogenesis are difficult to analyze. Okubo et al. established HAND1mCherry and HAND2EGFP reporter induced pluripotent stem cells and showed that HAND1 marks cardiovascular progenitors and regulates the gene network of cardiomyocyte proliferation. They further showed that CD105 can be used as a surface marker for proliferative cardiomyocytes.
The effects of transcription factors on the maintenance and differentiation of human-induced or embryonic pluripotent stem cells (iPSCs/ESCs) have been well studied. However, the importance of ...posttranscriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 posttranscriptionally regulated genes with strict upregulation of the protein level in iPSCs/ESCs. Among them, we found 84 genes were vital for the survival of iPSCs and HDFs, including 20 genes that were specifically necessary for iPSC survival. These 20 proteins were upregulated only in iPSCs/ESCs and not in differentiated cells derived from the three germ layers. Although there are still unknown mechanisms that downregulate protein levels in HDFs, these results reveal that posttranscriptionally regulated genes have a crucial role in iPSC survival.
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•The posttranscriptionally regulated 20 genes are necessary for iPSC survival•The proteins of HSPA8, EIF3D, and NCBP2 are quickly degraded in HDFs•mRNA localization affects the protein amounts in most of the 20 genes•Translation is repressed in HDFs despite mRNA binding to ribosomes
Biological sciences; Stem cells research; Omics
Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens ...enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151
population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151
population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151
population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.
X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who ...are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.
•A patient-derived iPSC model recapitulates defective erythroid maturation in female XLSA.•Azacitidine reactivates the silent wild-type ALAS2 allele and ameliorates inefficient erythropoiesis in iPSC-derived HPCs from female XLSA.
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Early embryonic rodent ventricular cells exhibit spontaneous action potential (AP), which disappears in later developmental stages. Here, we used 3 mathematical models-the Kyoto, Ten ...Tusscher-Panfilov, and Luo-Rudy models-to present an overview of the functional landscape of developmental changes in embryonic ventricular cells. We switched the relative current densities of 9 ionic components in the Kyoto model, and 160 of 512 representative combinations were predicted to result in regular spontaneous APs, in which the quantitative changes in Na(+) current (I Na) and funny current (I f) made large contributions to a wide range of basic cycle lengths. In all three models, the increase in inward rectifier current (I K1) before the disappearance of I f was predicted to result in abnormally high intracellular Ca(2+) concentrations. Thus, we demonstrated that the developmental changes in APs were well represented, as I Na increased before the disappearance of I f, followed by a 10-fold increase in I K1.
Abstract only
Introduction:
In conventional cardiac differentiation methods, a heterogeneous cardiac population is generated from human induced pluripotent stem cells (hiPSCs). Purifying ventricular- ...or atrial cardiomyocytes (VCMs or ACMs, respectably) is valuable for drug discovery for region-specific heart diseases. Previously, we have identified a novel marker, CD151, which can separate advanced specified subtypes; CD151
high
VCMs in ventricular inducing condition (VIC) and CD151
low
ACMs in atrial inducing condition (AIC). Although CD151 is known as a cell adhesion molecule, the function in cardiac subtype specification is unclear. In this study, we addressed the mechanisms of how CD151 distinguishes cardiomyocyte subtypes in cardiac differentiation from hiPSCs.
Methods:
We performed RNA sequencing in VIC- and AIC- CD151
high/low
CMs. Pathway analysis using differential expression genes between CD151
high/low
CMs was conducted to identify prominent pathways in subtype specification.
Results:
In VIC, mitosis-related genes were enriched in CD151
high
VCMs. To clarify whether those genes affect cell proliferation or multi-nucleation, we performed the flow cytometric analysis of CD151
high/low
VCMs with KI-67 proliferation marker and Hoechst staining. Although the percentage of KI67-positive cells was almost the same, binucleated cells were significantly more in CD151
high
VCMs than in CD151
low
VCMs. In AIC, transcriptome analysis suggested that Notch signaling was activated in CD151
high
ACMs. To reveal that Notch signaling inhibits atrial specification, we analyzed ACMs treated with LY411575, a Notch signaling inhibitor. LY411575 induced atrial marker gene expression and increased the number of cells showing action potentials of ACMs. CD151
low
ACMs after LY411575 treatment contained ACMs with the highest efficiency.
Conclusion:
Our results demonstrate that the CD151 expression indicates mitotic activity in VCMs. CD151
high
VCMs are more advanced specified VCMs with binucleation without cell proliferation. On the other hand, in ACMs, CD151 works as an indicator of Notch signaling, which suppresses atrial specification. Our study provides a novel approach to generating and purifying hiPSCs-ACMs by Notch inhibition and CD151 separation.
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