mRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy.

Synergistically improving T-cell responsiveness is promising for favorable therapeutic outcomes in immunologically cold tumors, yet current treatments often fail to induce a cascade of cancer-immunity cycle for effective antitumor immunity. Gasdermin-mediated pyroptosis is a newly discovered mechanism in cancer immunotherapy; however, cleavage in the N terminus is required to activate pyroptosis. Here, we report a single-agent mRNA nanomedicine-based strategy that utilizes mRNA lipid nanoparticles (LNPs) encoding only the N-terminus of gasdermin to trigger pyroptosis, eliciting robust antitumor immunity. In multiple female mouse models, we show that pyroptosis-triggering mRNA/LNPs turn cold tumors into hot ones and create a positive feedback loop to promote antitumor immunity. Additionally, mRNA/LNP-induced pyroptosis sensitizes tumors to anti-PD-1 immunotherapy, facilitating tumor growth inhibition. Antitumor activity extends beyond the treated lesions and suppresses the growth of distant tumors. We implement a strategy for inducing potent antitumor immunity, enhancing immunotherapy responses in immunologically cold tumors.

Immunopharmacological Properties of VitD3: 1, 25VitD3 Modulates Regulatory T Cells and Th17 Cells and the Cytokine Balance in PBMCs from Women with Unexplained Recurrent Spontaneous Abortion (URSA).

BACKGROUND: VitD3 may contribute to a successful pregnancy through modulation of immune responses. Therefore, VitD3 deficiency may have a role in the immunopathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the mechanisms of immunomodulatory actions of VitD3 in decreasing the risk of recurrent spontaneous abortion have not been understood well. OBJECTIVE: The purpose of this research was to investigate the influence of 1,25VitD3 on regulatory T cells /Th17 axis, the gene expressions and concentrations of related cytokines including, TGF-ß, IL-10, IL-6, IL-23, and IL-17A in peripheral blood mononuclear cells (PBMCs) of healthy women as a control group and women with URSA. METHODS: Isolation of PBMCs was performed from peripheral blood of the subjects of the studied groups (20 women with URSA as a case group, and 20 control women). The effects of 1,25VitD3 (50 nM, for 24 hours) on the studied parameters were evaluated and were compared to the positive and negative controls in vitro. Flow cytometry analysis was used to determine the percentages of regulatory T cells and Th17 cells. For gene expression measurement and cytokines assay, Realtime PCR and ELISA were carried out. RESULTS: The proportion of regulatory T cells was markedly lower, while the proportion of Th17 cells in women with URSA was considerably higher than in the control group (P=0.01, P=0.01). The ratio of the frequency of Tregs to the baseline (1,25VitD3/Untreated) increased, while the ratio of the frequency of Th17 cells to the baseline decreased in women with URSA relative to the controls (P= 0.01, P=0.04). 1,25VitD3 increased IL-10 expressions at both the protein and mRNA levels in PBMCs in women with URSA relative to the control group (P=0.0001, P=0.04). TGF-ß levels in the cultured supernatants decreased significantly in the case group in the presence of 1,25Vit- D3 relative to the controls (P=0.03). 1,25VitD3 treatment also significantly decreased gene expressions of IL-6, IL-17A, and IL-23 in PBMCs of women with URSA (P=0.01, P=0.001, P=0.0005), as well as the levels of those cytokines in cell culture supernatants (P=0.03, P=0.02, P=0.01, respectively) in women with URSA relative to the controls. CONCLUSION: According to the findings of this research, modulation of immune responses by 1,25VitD3 is accomplished by strengthening Tregs function and inhibiting inflammatory responses of Th17 cells, which may have a positive impact on pregnancy outcome. Thus, as an immunomodulating agent, VitD3 may be effective in reducing the risk of URSA.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Cardiac Sca-1+ Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

BACKGROUND: For more than a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. METHODS: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. RESULTS: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. CONCLUSIONS: Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed.

One-Step Biallelic and Scarless Correction of a ß-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection.

Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, ß-thalassemia (ß-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin ß (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including ß-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the ß-41/42 (TCTT) deletion mutation in ß thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal ß-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the ß-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of ß-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.

Multilevel analyses of SCN5A mutations in arrhythmogenic right ventricular dysplasia/cardiomyopathy suggest non-canonical mechanisms for disease pathogenesis.

AIMS: Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often associated with desmosomal mutations. Recent studies suggest an interaction between the desmosome and sodium channel protein Nav1.5. We aimed to determine the prevalence and biophysical properties of mutations in SCN5A (the gene encoding Nav1.5) in ARVD/C. METHODS AND RESULTS: We performed whole-exome sequencing in six ARVD/C patients (33% male, 38.2 ± 12.1 years) without a desmosomal mutation. We found a rare missense variant (p.Arg1898His; R1898H) in SCN5A in one patient. We generated induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) from the patient's peripheral blood mononuclear cells. The variant was then corrected (R1898R) using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology, allowing us to study the impact of the R1898H substitution in the same cellular background. Whole-cell patch clamping revealed a 36% reduction in peak sodium current (P = 0.002); super-resolution fluorescence microscopy showed reduced abundance of NaV1.5 (P = 0.005) and N-Cadherin (P = 0.026) clusters at the intercalated disc. Subsequently, we sequenced SCN5A in an additional 281 ARVD/C patients (60% male, 34.8 ± 13.7 years, 52% desmosomal mutation-carriers). Five (1.8%) subjects harboured a putatively pathogenic SCN5A variant (p.Tyr416Cys, p.Leu729del, p.Arg1623Ter, p.Ser1787Asn, and p.Val2016Met). SCN5A variants were associated with prolonged QRS duration (119 ± 15 vs. 94 ± 14 ms, P < 0.01) and all SCN5A variant carriers had major structural abnormalities on cardiac imaging. CONCLUSIONS: Almost 2% of ARVD/C patients harbour rare SCN5A variants. For one of these variants, we demonstrated reduced sodium current, Nav1.5 and N-Cadherin clusters at junctional sites. This suggests that Nav1.5 is in a functional complex with adhesion molecules, and reveals potential non-canonical mechanisms by which Nav1.5 dysfunction causes cardiomyopathy.

Transcriptional regulation of E-cadherin by small activating RNA: A new double-stranded RNA.

Recent studies have reported that chemically synthesized small activating RNA (saRNA) targeting the promoter regions of a gene can activate its expression in different cell lines. This technique can be a powerful therapeutic method for diseases caused by complete inactivation or reduced expression of specific genes. E-cadherin is a typical tumor suppressor gene. Loss of E-cadherin mediates the transition from benign lesions to invasive, metastatic cancer. In this study, several 21-nt small double-stranded RNAs (dsRNAs) targeting the promoter regions of human E-cadherin were designed and synthesized and the features of their function were investigated to study the regulatory role of dsRNA on E-cadherin expression. A new saRNA (dsEcad­661) that can enhance E-cadherin expression by targeting non-coding regulatory regions in gene promoters was identified. Using dsRNA with modified base quantity and cholesterol-conjugated dsRNA, we found the antisense strand may be the guide strand of saRNA in the upregulation of E-cadherin. These findings provide several important pieces of evidence that may improve understanding of the function of saRNA and may promote its development for clinical application.

Resident c-kit(+) cells in the heart are not cardiac stem cells.

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit(+) cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit(+) cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit(+) cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit(+) cells in the murine heart are endothelial cells and not CSCs.

Generation of a tamoxifen inducible Tnnt2MerCreMer knock-in mouse model for cardiac studies.

Tnnt2, encoding thin-filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2(MerCreMer/+)) knock-in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock-in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2(MerCreMer/+) mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre-LoxP technology. The Tnnt2(MerCreMer/+) mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury.

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Pluripotent stem cell-based heart regeneration: from the developmental and immunological perspectives.

Heart diseases such as myocardial infarction cause massive loss of cardiomyocytes, but the human heart lacks the innate ability to regenerate. In the adult mammalian heart, a resident progenitor cell population, termed epicardial progenitors, has been identified and reported to stay quiescent under uninjured conditions; however, myocardial infarction induces their proliferation and de novo differentiation into cardiac cells. It is conceivable to develop novel therapeutic approaches for myocardial repair by targeting such expandable sources of cardiac progenitors, thereby giving rise to new muscle and vasculatures. Human pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can self-renew and differentiate into the three major cell types of the heart, namely cardiomyocytes, smooth muscle, and endothelial cells. In this review, we describe our current knowledge of the therapeutic potential and challenges associated with the use of pluripotent stem cell and progenitor biology in cell therapy. An emphasis is placed on the contribution of paracrine factors in the growth of myocardium and neovascularization as well as the role of immunogenicity in cell survival and engraftment.

A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells.

Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine ESCs. We termed these hLR5 iPSCs because they require the expression of five ectopic reprogramming factors, Oct4, Sox2, Klf4, cMyc, and Nanog, to maintain this more naive state. The cells are "metastable" and upon ectopic factor withdrawal they revert to standard human iPSCs. Finally, we demonstrate that the hLR5 state facilitates gene targeting, and as such provides a powerful tool for the generation of recombinant human pluripotent stem cell lines.

[High-resolution analysis of chromosome 5 and identification of candidate genes in gastric cancer].

OBJECTIVE: To explore the relationship between loss of heterozygosity (LOH) of 5p with the histological phenotype in gastric cancer. METHODS: Eighty pairs of tumor and adjacent normal mucosa samples were collected and genomic DNA was extracted. Total of 17 polymorphic microsatellite markers for 5p were used for LOH analysis. A part of samples were fixed in 10% buffered formalin and stained with H&E. Histological type of gastric cancer was defined according to Lauren's classification. RESULTS: The average informative rate of all seventeen markers was 60.0%. The LOH at least in one locus was detected in 28 of the 80 (35.0%) cases. The highest LOH frequency occurring at D5S2849 (7.77 cM), with LOH frequency of 35.2% (19/54). The minimal LOH region was spanned from 6.67 to 9.41 cM (1.18 Mb, covering 2.7 cM), including D5S417, D5S2849, D5S1492 and D5S2088. In 28 with LOH, 24 (85.7%) cases were of intestinal type, and only 4 cases (14.3%) were of diffuse type. There is significant difference between LOH frequency in intestinal-type and diffuse-type gastric cancers (P < 0.01). Searching the NCBI database disclosed that this minimal deletion region at 5p15.33 covered 3 candidate genes, IRX1, IRX2, and CEI. CONCLUSION: The molecular events in 5p 15.33 may be related with the morphological differentiation and development of gastric cancer. Gastric cancer with LOH of 5p15.33 locus tends to develop in to intestinal type. The cluster of candidate genes in 5p15.33 may be closely implicated in carcinogenesis of intestinal type gastric carcinoma.

Identification of a new target region by loss of heterozygosity at 5p15.33 in sporadic gastric carcinomas: genotype and phenotype related.

Chromosome 5p, especially 5p15, involves in several cancers. To investigate its role in gastric cancer, we analyzed 46 intestinal-type and 34 diffuse-type gastric cancers by Loss of heterozygosity (LOH). We found a high frequent LOH at 5p15.33, and identified a minimal 2.7 cM candidate region of tumor suppressor gene, encompassing four loci (D5S417, D5S2849, D5S1492 and D5S2088). In total 80 cases, the highest LOH occurs at D5S2849 (35.19%). In intestinal-type cases, the highest LOH frequency is 50%, whereas in diffuse-type cases, the highest is only 16.67%. By statistical analysis we also observed an obvious genotype-phenotype correlation on 5p15.3 (P<0.01).

T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis.

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.