Permissive ureter specification by TBX18-mediated repression of metanephric gene expression.

The murine kidney and ureter develop in a regionalized fashion from the ureteric bud and its surrounding mesenchyme. Whereas the factors that establish the metanephric cell lineages have been well characterized, much less is known about the molecular cues that specify the ureter. Here, we have identified a crucial patterning function in this process for Tbx18, a T-box transcription factor gene specifically expressed in the mesenchymal primordium of the ureter. Using misexpression and loss-of-function mice combined with molecular profiling approaches, we show that Tbx18 is required and sufficient to repress metanephric mesenchymal gene programs. We identify Wt1 as a functional target of TBX18. Our work suggests that TBX18 acts as a permissive factor in ureter specification by generating a mesenchymal domain around the distal ureteric bud where SHH and BMP4 signaling can occur.

Single-cell RNA sequencing reveals the transcriptional heterogeneity of Tbx18-positive cardiac cells during heart development.

The T-box family transcription factor 18 (Tbx18) has been found to play a critical role in regulating the development of the mammalian heart during the primary stages of embryonic development while the cellular heterogeneity and landscape of Tbx18-positive (Tbx18+) cardiac cells remain incompletely characterized. Here, we analyzed prior published single-cell RNA sequencing (scRNA-seq) mouse heart data to explore the heterogeneity of Tbx18+ cardiac cell subpopulations and provide a comprehensive transcriptional landscape of Tbx18+ cardiac cells during their development. Bioinformatic analysis methods were utilized to identify the heterogeneity between cell groups. Based on the gene expression characteristics, Tbx18+ cardiac cells can be classified into a minimum of two distinct cell populations, namely fibroblast-like cells and cardiomyocytes. In terms of temporal heterogeneity, these cells exhibit three developmental stages, namely the MEM stage, ML_P0 stage, and P stage Tbx18+ cardiac cells. Furthermore, Tbx18+ cardiac cells encompass several cell types, including cardiac progenitor-like cells, cardiomyocytes, and epicardial/stromal cells, as determined by specific transcriptional regulatory networks. The scRNA-seq results revealed the involvement of extracellular matrix (ECM) signals and epicardial epithelial-to-mesenchymal transition (EMT) in the development of Tbx18+ cardiac cells. The utilization of a lineage-tracing model served to validate the crucial function of Tbx18 in the differentiation of cardiac cells. Consequently, these findings offer a comprehensive depiction of the cellular heterogeneity within Tbx18+ cardiac cells.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Tbx18 promoted the conversion of human-induced pluripotent stem cell-derived cardiomyocytes into sinoatrial node-like pacemaker cells.

Sinoatrial node (SAN) pacemaker cells originate from T-box transcription factor 18 (Tbx18)-expressing progenitor cells. The present study aimed to investigate whether overexpression of human transcription factor Tbx18 could reprogram human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into SAN-like pacemaker cells (SANLPCs) in vitro. In the study, hiPSCs were first differentiated into hiPSC-CMs through regulating the Wnt/ß-catenin pathway, then purified hiPSC-CMs were transfected by Tbx18 adenovirus (Tbx18-CMs group) or green fluorescent protein (GFP) adenovirus (GFP-CMs group). The beating frequency of the Tbx18-CMs group was significantly higher than that of the hiPSC-CMs group and GFP-CMs group. Compared with the other two groups, the expression levels of hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4, connexin-45 in the Tbx18-CMs group were markedly upregulated, while the expressions of transcription factor NKX2.5, CX43 were significantly downregulated. Whole-cell patch-clamp results illustrated that action potential and "funny" current (If ) similar to SAN pacemaker cells could be recorded in the Tbx18-CMs group. In conclusion, this present study demonstrated that overexpression of Tbx18 promoted the conversion of hiPSC-CMs into SANLPCs.

TBX18 transcription factor overexpression in human-induced pluripotent stem cells increases their differentiation into pacemaker-like cells.

BACKGROUND: The discovery of gene- and cell-based strategies has opened a new area to investigate novel approaches for the treatment of many conditions caused by cardiac cell failure. The TBX18 (T-box 18) transcription factor is considered as a prominent factor in the sinoatrial node (SAN) formation during the embryonic development. In this in vitro study, the effect of TBX18 gene expression on human-induced pluripotent-stem-cell-derived cardiomyocytes (hiPS-CMs) to induce pacemaker-like cells was examined. METHODS: The human-dermal-fibroblast-derived iPSCs were transfected using chemical, physical, and Lentiviral methods of TBX18 gene delivery during differentiation into cardiomyocytes (CMs). After the differentiation process through small-molecule-based temporal modulation of the Wnt signaling pathway, the hiPSC-CMs were analyzed using the real-time polymerase chain reaction, immunocytochemistry, immunofluorescence, whole-cell patch-clamp recording, and western blotting to investigate the accuracy of differentiation and identify the effect exerted by TBX18. RESULTS: The hiPS-CMs showed spontaneous beating and expressed specific markers of cardiac cells. The lentiviral-mediated TBX18 delivery was the most efficient method for transfection. The results showed the increment in Connexin 43 expression among untransfected hiPS-CMs, whereas this protein was significantly downregulated followed by TBX18 overexpression. TBX18-hiPSCMs were detected with pacemaker cell features. CONCLUSIONS: It was demonstrated that the TBX18 gene is able to conduct hiPSCs to differentiate into pacemaker-like cells. The TBX18 gene delivery seems to have the potential for the development of biological pacemakers; however, more investigations are still needed to assess its usefulness to fix arrhythmic conditions with SAN failure basis.

Functional biological pacemaker generation by T-Box18 protein expression via stem cell and viral delivery approaches in a murine model of complete heart block.

Despite recent advances in the treatment of cardiac arrhythmia, the available options are still limited and associated with some complications. Induction of biological pacemakers via Tbx18 gene insertion in the heart tissue has been suggested as a promising therapeutic strategy for cardiac arrhythmia. Following a previous in vitro study reporting the production of Tbx18-expressing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), we aimed to investigate the efficacy of these engineered cells to generate pacemaker rhythms in a murine model of complete heart block. We also attempted to generate a functional pacemaker by Tbx18 overexpression in native cardiac cells of rat heart. The hiPSC-derived pacemaker cells were produced by lentiviral delivery of Tbx18 gene to stem cells during a small molecule-based differentiation process. In the present study, 16 male albino Wistar rats were randomly assigned to Tbx18-lentivirus (n = 4) and Tbx18-pacemaker cells (n = 4) administered via injection into the left ventricular anterolateral wall. The control rats received GFP-lentiviruses (n = 4) and GFP-pacemaker cells (n = 4). Fourteen days after the injection, the rats were sacrificed and analyzed by electrocardiography (ECG) recording using a Langendorff-perfused heart model following complete heart block induced by hypokalemia and crashing. Immunofluorescence staining was used to investigate the expression of Tbx18, HCN4 and connexin 43 (Cx43) proteins in Tbx18-delivered cells of heart tissues. The heart rate was significantly reduced after complete heart block in all of the experimental rats (P < 0.05). Heart beating in the Tbx18-transduced hearts was slower compared with rats receiving Tbx18-pacemaker cells (P = 0.04). The duration of ventricular fibrillation (VF) was higher in the lentiviral Tbx18 group compared with the GFP-injected controls (P = 0.02) and the Tbx18-pacemaker cell group (P = 0.02). The ECG recording data showed spontaneous pacemaker rhythms in both intervention groups with signal propagation in Tbx18-transduced ventricles. Immunostaining results confirmed the overexpression of HCN4 and downregulation of Cx43 as a result of the expression of the Tbx18 gene and spontaneously contracting myocyte formation. We confirmed the formation of a functional pacemaker after introduction of Tbx18 via cell and gene therapy strategies. Although the pacemaker activity was better in gene-received hearts since there were longer VF duration and signal propagation from the injection site, more data should be gathered from the long-term activity of such pacemakers in different hosts.

Pericytes in the Retina.

Pericytes (PCs) are specialized cells located abluminal of endothelial cells (ECs) on capillaries, embedded within the same basement membrane. They are essential regulators of vascular development, remodeling, and blood-retina-barrier (BRB) tightness and are therefore important components to maintain tissue homeostasis. The perivascular localization and expression of contractile proteins suggest that PCs participate in capillary blood flow regulation and neurovascular coupling. Due to their ability to differentiate into various cell types in vitro, they are regarded as potential cells for tissue repair and therapeutic approaches in regenerative medicine. Altered function or loss of PCs is associated with a multitude of CNS diseases, including diabetic retinopathy (DR). In this chapter, we will provide a short overview of retinal vascular development, the origin of PCs, and focus on PCs in retinopathy of prematurity (ROP) and in the diabetic retina. Further, animal models to study the fate of PCs and the potential role of (retinal) PCs in regeneration and wound healing will be discussed.

TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.

KEY POINTS: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. ABSTRACT: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10-5  ± 2.2 × 10-6 to 2.8 × 10-5  ± 4.3 × 10-6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.

Pericytes and cardiac stem cells: Common features and peculiarities.

Clinical data and basic research indicate that the structural and functional alterations that characterize the evolution of cardiac disease towards heart failure may be, at least in part, reversed. This paradigm shift is due to the accumulation of evidence indicating that, in peculiar settings, cardiomyocytes may be replenished. Moving from the consideration that cardiomyocytes are rapidly withdrawn from the cell cycle after birth, independent laboratories have tested the hypothesis that cardiac resident stem/progenitor cells resided in mammalian hearts and were important for myocardial repair. After almost two decades of intensive investigation, several (but partially overlapping) cardiac resident stem/progenitor cell populations have been identified. These primitive cells are characterized by mesenchymal features, unique properties that distinguish them from mesodermal progenitors residing in other tissues, and heterogeneous embryological origins (that include the neural crest and the epicardium). A further layer of complexity is related to the nature, in vivo localization and properties of mesodermal progenitors residing in adult tissues. Intriguingly, these latter, whose possible perivascular pericyte/mural cell origin has been shown, have been identified in human hearts too. However, their exact anatomical localization, pathophysiological role, and their relationship with cardiac stem/progenitor cells are emerging only recently. Therefore, aim of this review is to discuss the different origin, the distinct nature, and the complementary effect of cardiac stem cells and pericytes supporting regenerative strategies based on the combined use of both myogenic and angiogenic factors.

Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells.

A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco's modified Eagle's medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

Misexpression of Tbx18 in cardiac chambers of fetal mice interferes with chamber-specific developmental programs but does not induce a pacemaker-like gene signature.

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.

Ureter growth and differentiation.

The mammalian ureter is a slender tube that connects the renal pelvis with the bladder. It allows the unidirectional movement of urine by means of a peristaltically active smooth muscle layer that together with fibroelastic material ensheathes a water-impermeable multilayered urothelium. The ureteric urothelium as well as the outer mesenchymal coat arise from undifferentiated precursor tissues, the distal ureteric bud and its surrounding mesenchyme, respectively. Specification, growth and differentiation of these ureteric precursor tissues are tightly linked to each other, and are highly integrated with those of the adjacent rudiments of kidney and bladder. Here, we review the current knowledge on the cellular mechanisms as well as the molecular players that guide development of the tissue architecture of the ureter and its peristalsis.

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney.

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18(-/-) kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRß+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18(-/-) kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.

Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells.

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF-AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury.

The Notch-PDGFRß axis suppresses brown adipocyte progenitor differentiation in early post-natal mice.

De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively explored. Here, through in vivo lineage tracing and mouse modeling, we observed that platelet-derived growth factor receptor beta (PDGFRß)+ pericytes give rise to developmental brown adipocytes but not to those in adult homeostasis. By contrast, T-box 18 (TBX18)+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFRß+ pericytes promotes brown adipogenesis by downregulating PDGFRß. Furthermore, inhibition of Notch signaling in PDGFRß+ pericytes mitigates high-fat, high-sucrose (HFHS)-induced glucose and metabolic impairment in mice during their development and juvenile phases. Collectively, these findings show that the Notch/PDGFRß axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health.

Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease.

During embryonic heart development, the transcription factors Tcf21, Wt1, and Tbx18 regulate activation and differentiation of epicardium-derived cells, including fibroblast lineages. Expression of these epicardial progenitor factors and localization of cardiac fibrosis were examined in mouse models of cardiovascular disease and in human diseased hearts. Following ischemic injury in mice, epicardial fibrosis is apparent in the thickened layer of subepicardial cells that express Wt1, Tbx18, and Tcf21. Perivascular fibrosis with predominant expression of Tcf21, but not Wt1 or Tbx18, occurs in mouse models of pressure overload or hypertensive heart disease, but not following ischemic injury. Areas of interstitial fibrosis in ischemic and hypertensive hearts actively express Tcf21, Wt1, and Tbx18. In all areas of fibrosis, cells that express epicardial progenitor factors are distinct from CD45-positive immune cells. In human diseased hearts, differential expression of Tcf21, Wt1, and Tbx18 also is detected with epicardial, perivascular, and interstitial fibrosis, indicating conservation of reactivated developmental mechanisms in cardiac fibrosis in mice and humans. Together, these data provide evidence for distinct fibrogenic mechanisms that include Tcf21, separate from Wt1 and Tbx18, in different fibroblast populations in response to specific types of cardiac injury.

TBX18 knockdown sensitizes esophageal squamous cell carcinoma to radiotherapy by blocking the CHN1/RhoA axis.

OBJECTIVE: Radioresistance is a challenge in the effective treatment of esophageal squamous cell carcinoma (ESCC). Herein, this research ascertained whether TBX18 reduced the radiosensitivity of ESCC. METHODS: Bioinformatics analysis was utilized to retrieve differentially expressed genes. Then, the expression of corresponding candidate genes was tested using qRT-PCR in ESCC clinical specimens, and TBX18 was selected for subsequent experiments. The binding between TBX18 and CHN1 was evaluated by dual-luciferase reporter and ChIP assays, and the relationship between CHN1 and RhoA was identified by GST pull-down. Ectopic expression or knockdown experiments and radiation treatment were performed in cells and the nude mouse xenograft model to clarify the impacts of TBX18, CHN1, and RhoA on radiosensitivity in ESCC. RESULTS: Bioinformatics analysis and qRT-PCR retrieved upregulated TBX18 in ESCC for the follow-up study. Additionally, TBX18 was positively correlated with CHN1 in ESCC clinical specimens. Mechanistically, TBX18 bound to the CHN1 promoter region to transcriptionally activate CHN1, thus elevating RhoA activity. Moreover, TBX18 knockdown reduced ESCC cell proliferation and migration while augmenting their apoptosis after radiation, which was negated by further overexpressing CHN1 or RhoA. CHN1 or RhoA knockdown diminished ESCC cell proliferation and migration, as well as enhanced cell apoptosis, subsequent to radiation. Likewise, TBX18 overexpression increased ESCC cell autophagy after radiation, which was partially reversed by knockdown of RhoA. The results of in vivo xenograft experiments in nude mice were concurrent with the in vitro results. CONCLUSION: TBX18 knockdown lowered CHN1 transcription and thus reduced RhoA activity, which sensitized ESCC cells to radiotherapy.

MicroRNA-dependent suppression of biological pacemaker activity induced by TBX18.

Chemically modified mRNA (CMmRNA) with selectively altered nucleotides are used to deliver transgenes, but translation efficiency is variable. We have transfected CMmRNA encoding human T-box transcription factor 18 (CMmTBX18) into heart cells or the left ventricle of rats with atrioventricular block. TBX18 protein expression from CMmTBX18 is weak and transient, but Acriflavine, an Argonaute 2 inhibitor, boosts TBX18 levels. Small RNA sequencing identified two upregulated microRNAs (miRs) in CMmTBX18-transfected cells. Co-administration of miR-1-3p and miR-1b antagomiRs with CMmTBX18 prolongs TBX18 expression in vitro and in vivo and is sufficient to generate electrical stimuli capable of pacing the heart. Different suppressive miRs likewise limit the expression of VEGF-A CMmRNA. Cells therefore resist translation of CMmRNA therapeutic transgenes by upregulating suppressive miRs. Blockade of suppressive miRs enhances CMmRNA expression of genes driving biological pacing or angiogenesis. Such counterstrategies constitute an approach to boost the efficacy and efficiency of CMmRNA therapies.

Epicardium-Derived Tbx18+ CDCs Transplantation Improve Heart Function in Infarcted Mice.

Cardiosphere-derived cells (CDCs) constitute a cardiac stem cell pool, a promising therapeutics in treating myocardial infarction (MI). However, the cell source of CDCs remains unclear. In this study, we isolated CDCs directly from adult mouse heart epicardium named primary epicardium-derived CDCs (pECDCs), which showed a different expression profile compared with primary epicardial cells (pEpiCs). Interestingly, pECDCs highly expressed T-box transcription factor 18 (Tbx18) and showed multipotent differentiation ability in vitro. Human telomerase reverse transcriptase (hTERT) transduction could inhibit aging-induced pECDCs apoptosis and differentiation, thus keeping a better proliferation capacity. Furthermore, immortalized epicardium CDCs (iECDCs) transplantation extensively promote cardiogenesis in the infracted mouse heart. This study demonstrated epicardium-derived CDCs that may derive from Tbx18+ EpiCs, which possess the therapeutic potential to be applied to cardiac repair and regeneration and suggest a new kind of CDCs with identified origination that may be followed in the developing and injured heart.