We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

The high rate of mortality and frequent incidence of recurrence associated with hepatocellular carcinoma (HCC) reveal the need for new therapeutic approaches. In this report, we evaluated the efficacy of a novel chemo-immunotherapeutic strategy to control HCC and investigated the underlying mechanism that increased the antitumor immune response. We developed a novel orthotopic mouse model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice. These MTD2-derived hepatocytes form Tag expressing HCC tumors specifically within the liver. This approach provides a platform to test therapeutic strategies and antigen specific immune-directed therapy in an immunocompetent murine model. Using this model, we tested the efficacy of a combination of oral sunitinib, a small molecule multi-targeted receptor tyrosine kinase (RTK) inhibitor, and adoptive transfer of tumor antigen-specific CD8+ T cells to eliminate HCC. Sunitinib treatment alone promoted a transient reduction in tumor size. Sunitinib treatment combined with adoptive transfer of tumor antigen-specific CD8+ T cells led to elimination of established tumors without recurrence. In vitro studies revealed that HCC growth was inhibited through suppression of STAT3 signaling. In addition, sunitinib treatment of tumor-bearing mice was associated with suppression of STAT3 and a block in T cell tolerance. Conclusion These findings indicate that sunitinib inhibits HCC tumor growth directly through the STAT3 pathway and prevents tumor antigen-specific CD8+ T cell tolerance, thus defining a synergistic chemo-immunotherapeutic approach for HCC.

Background-We have shown that genetic overexpression of cell cycle proteins can increase the proliferation of transplanted cardiomyocytes derived from human induced-pluripotent stem cells (hiPSC-CMs) in animal models of myocardial infarction (MI). Here, we introduce a new, nongenetic approach to promote hiPSC-CM cell cycle activity and proliferation in transplanted human cardiomyocyte patches (hCMPs).Methods-Mice were randomly distributed into 5 experimental groups (n = 10 per group). One group underwent Sham surgery, and the other 4 groups underwent MI induction surgery followed by treatment with hCMPs composed of hiPSC-CMs and nanoparticles that contained CHIR99021 and FGF1 (the NP CF -hCMP group), with hCMPs composed of hiPSC-CMs and empty nanoparticles (the NP E -hCMP group); with patches containing the CHIR99021/FGF-loaded nanoparticles but lacking hiPSC-CMs (the NP CF -Patch group), or patches lacking both the nanoparticles and cells (the E-Patch group). Cell cycle activity was evaluated via Ki67 and Aurora B expression, bromodeoxyuridine incorporation, and phosphorylated histone 3 levels (immunofluorescence); engraftment via human cardiac troponin T or human nuclear antigen expression (immunofluorescence) and bioluminescence imaging; cardiac function via echocardiography; infarct size and wall thickness via histology; angiogenesis via isolectin B4 expression (immunofluorescence); and apoptosis via TUNEL and caspace 3 expression (immunofluorescence).

Stat3 alters the expression of its downstream genes and is associated with tumor invasion and metastasis in several human cancers. Its role in esophageal squamous cell carcinoma (ESCC) has not been well characterized. We examined the tumor sections of 100 cases of ESCC by immunohistochemistry and observed significant overexpression of Stat3 in the cytoplasm of 89% of ESCC cells and of phosphorylated Stat3 (p-Stat3) in the nuclei of 71% of ESCC when compare with normal esophageal mucosa (72%, p = 0.02; and 31%, p = 0.001). Overexpression of Stat3 and p-Stat3 positively correlated with that of matrix metalloproteinase-2 (MMP2), a known regulator for cell migration, in 65% of ESCC while only 26% shown in benign esophageal mucosa. To further investigate the association of Stat3 with tumor metastasis in vitro, invasion of EC-1 cells (a human ESCC cell line) were investigated with Boyden chambers. The results showed that transfection of Stat3 not only promoted invasion of EC-1 cells but also significantly induced MMP2 expression in a dose-dependent manner. In contrast, suppressing expression of endogenous Stat3 mRNA and protein by Stat3 siRNA significantly reduced EC-1 cell invasion and MMP2 expression. A high-affinity Stat3-binding element was localized to the positions of 648-641 bp (TTCTCGAA) in the MMP2 promoter with electrophoretic mobility shift assay. Our results suggest that Stat3, p-Stat3, and MMP2 were overexpressed in ESCC and associated with invasion of ESCC; and Stat3 up-regulated expression of MMP2 in ESCC through directly binding to the MMP2 promoter.

Aims In regenerative medicine, cellular cardiomyoplasty is one of the promising options for treating myocardial infarction (MI); however, the efficacy of such treatment has shown to be limited due to poor survival and/or functional integration of implanted cells. Within the heart, the adhesion between cardiac myocytes (CMs) is mediated by N-cadherin (CDH2) and is critical for the heart to function as an electromechanical syncytium. In this study, we have investigated whether the reparative potency of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) can be enhanced through CDH2 overexpression. Methods and results CDH2-hiPSC-CMs and control wild-type (WT)-hiPSC-CMs were cultured in myogenic differentiation medium for 28 days. Using a mouse MI model, the cell survival/engraftment rate, infarct size, and cardiac functions were evaluated post-MI, at Day 7 or Day 28. In vitro, conduction velocities were significantly greater in CDH2-hiPSC-CMs than in WT-hiPSC-CMs. While, in vivo, measurements of cardiac functions: left ventricular (LV) ejection fraction, reduction in infarct size, and the cell engraftment rate were significantly higher in CDH2-hiPSC-CMs treated MI group than in WT-hiPSC-CMs treated MI group. Mechanistically, paracrine activation of ERK signal transduction pathway by CDH2-hiPSC-CMs, significantly induced neo-vasculogenesis, resulting in a higher survival of implanted cells. Conclusion Collectively, these data suggest that CDH2 overexpression enhances not only the survival/engraftment of cultured CDH2-hiPSC-CMs, but also the functional integration of these cells, consequently, the augmentation of the reparative properties of implanted CDH2-hiPSC-CMs in the failing hearts.

NPs encapsulated with CHIR + FGF1 exerted substantial myocardial protective effects and represents a potentially novel strategy for improving postischemic myocardial protection. Results Identification of chemicals that promote cell cycle activity of human induced pluripotent stem cell-derived cardiomyocytes. Using the BrdU incorporation assay, we screened several chemicals for their capacity to enhance the cell cycle activity of cultured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We picked hiPSC-CMs because they are easy to handle and represent a practical system for drug testing and screening (5). These chemicals included Ly294002 (PI3K inhibitor) (6), FGF1 (6, 7), SB203580 and VX702 (p38 MAPK inhibitors) (6), KN93 (Ca 2+ /calmodulin-dependent protein kinase II inhibitor) (8), Su1498 (Flk-1 inhibitor) (8), and CHIR99021 (Wnt activator and GSK3α and 3β inhibitor) (8, 9). We found a combination of 5 μM CHIR99021 and 100 ng/mL FGF1 was the most potent treatment to induce cell cycle in hiPSC-derived cardiomyocytes (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.132796DS1). Characterization of CHIR-FGF1-NPs. We have previously shown that PLGA NPs can be used as a platform for slow release (up to 4 weeks) of chemicals to injured animal hearts and that they provide cardioprotection (2). To characterize the long-term cardioprotective function of the FGF/CHIR combination in vivo, we formulated the PLGA NPs with these 2 factors. The size of PLGA NPs was measured using scanning electron microscopy, for both CHIR99021-and FGF1-loaded NPs (Supplemental Figure 2, A and B). Quantification of particle diameter for CHIR-NPs (Supplemental Figure 2C) and FGF1-NPs (Supplemental Figure 2D) yielded values of 123.63 ± 44.48 nm and 129.57 ± 45.94 nm, respectively. The size and shape of CHIR-NPs and FGF1-NPs were uniform. The encapsulation efficiency of CHIR-NPs and FGF1-NPs, i.e., (the amount encapsulated/total amount available) × 100%, was 50.41% and 62.8 ± 1.6%, respectively. The concentration of encapsulated CHIR and FGF1 was 8.07 μg/mg and 1.26 ± 0.03 μg/mg, respectively. Determination of release kinetics of CHIR-and FGF1-loaded NPs as a function of time, using either NanoDrop via UV-Vis spectrophotometer (for CHIR) or ELISA (for FGF1), and the cumulative percentage of CHIR and FGF1 released from NPs, are shown in Supplemental Figure 2, E and F. When 1000 μg of CHIR-and FGF1-loaded NPs were incubated in 1000 μL of Dulbecco's phosphate-buffered saline (DPBS), pH 7.4 at 37°C, 55% of the encapsulated CHIR was released during the first day and 85% by day 15 (Supplemental Figure 2E). In contrast, 55% of the encapsulated FGF1 was released during the initial 3 days, and 63% was released by day 10. Notably, between day 1 and day 30, the release kinetics strictly followed the Korsmeyer-Peppas model for FGF1-NPs (Supplemental Figure 2F). Fitting this model, C t /C 0 = kt n , where C t = concentration at time t; C 0 = equilibrium concentration; ...

No abstract

Expression of PRL-1 protein was examined with immunohistochemistry in 60 cases of esophageal squamous cell carcinoma (ESCC) with matched lymph node metastasis (n=40) and 6 cases of esophageal adenocarcinoma. Its association with ESCC metastasis was also explored. The results showed that PRL-1 protein was more frequently expressed in ESCC with metastasis (31/40, 77.5%) than in ESCC without metastasis (10/20, 50%, P=.031) and more in metastatic ESCC (39/40, 97.5%) than primary ESCC (68.3%, P=.003). PRL-1 protein expression significantly correlated with the stage of ESCC, 79.4% (27/34) in stage III ESCC and 33.3% (1/3) in stage 1 ESCC (P=.003). PRL-1 protein was characteristically expressed in the nuclei, in addition to the cytoplasm, of 3 out of 4 (66.7%) poorly differentiated esophageal adenocarcinoma. These findings suggest that PRL-1 may serve as the potential biomarker to predict the metastasis of ESCC and a better biomarker than PRL-3 in the differential diagnosis of poorly differentiated ESCC.