Rationale: Human pluripotent stem cell (hPSC)–derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. Objective: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. Methods and Results: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca 2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. Conclusions: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

SummaryHere, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs). Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue.

Rationale While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. Objective To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Methods and Results Using an antibiotic-selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1 and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPC) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. Conclusions This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium.

Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC‐CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic‐polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC‐CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC‐CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor‐stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC‐CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910–923

e16234 Background: Pancreatic ductal adenocarcinoma (PDAC) will be the second leading cause of cancer death by 2030 through early onset dedifferentiation and metastasis that results in limited treatments. FDA-approved targeted therapies–including proteasome, mitogen-activated protein kinase (MEK), histone deacetylase (HDAC), and mammalian target of rapamycin (mTOR) inhibitors–have shown preclinical benefit but failed in clinic. However, combination targeted therapy has been largely unexplored in clinical trials. We hypothesized based on prior drug screening efforts in the lab on aggressive subtypes of pancreatic cancer that combinations of these targeted therapies could be synergistically active in preclinical PDAC models. Methods: We optimized combinations of proteasome, MEK, mTOR, and HDAC inhibitors by performing cell viability experiments in human PDAC lines in 2D and 3D cell culture conditions in vitro. Optimized drug combinations from these experiments were then trialed in two different in vivo animal models–human FG xenografts in immunocompromised mice (NSG) and spontaneous primary PDAC tumors from transgenic pancreatic-specific Kras-overexpressing, P53-deficient mice (KPC). RNA-sequencing and Seahorse metabolic assays were utilized to analyze molecular mechanisms of drug synergy. Dedifferentiation was assessed by histology of KPC tumors. Results: The HDAC inhibitor Panobinostat and mTOR inhibitor Everolimus synergistically killed human pancreatic cancer cell lines grown in 2D and 3D cell culture in vitro. Synergy was highest at low concentrations of Panobinostat. In vivo, low-dose Panobinostat and Everolimus synergistically blocked growth of human FG xenografts in NSG mice. In vivo synergy of low-dose Panobinostat and Everolimus was highest in the KPC mouse model, where only combination therapy could significantly reduce tumor growth, stall dedifferentiation, and improve survival. Mechanistically, RNA-sequencing of Panobinostat/Everolimus-treated FG cells revealed that Panobinostat increased tumor suppressor genes opposing dedifferentiation such as p21, EGR1, and CCN2. Everolimus complemented Panobinostat by decreasing expression of enzymes metabolizing acetyl groups such as fatty acid synthase, stearoyl-CoA desaturase, and sterol response element binding factors. Functionally, Everolimus’ activity to reduce oxidative consumption increased histone acetylation specifically in the presence of Panobinostat. Conclusions: A combination of FDA-approved, targeted oral therapies (low-dose Panobinostat/Everolimus) is synergistically active in preclinical models of pancreatic cancer in vitro and in vivo. mTOR inhibitors create drug synergy by specifically increasing histone acetylation in HDAC inhibitor-treated cells by reversing HDAC inhibitor de-repression of acetyl/oxidative metabolism, illustrating a novel connection between cancer epigenetics and metabolism.