BackgroundCholesterol efflux from cells to apolipoprotein A-I (apoA-I) acceptors via the ATP-binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. MicroRNA (miR)-33 is known to target ABCA1 and ABCG1 in vivo.Methods and ResultsWe assessed the impact of the genetic loss of miR-33 in a mouse model of atherosclerosis. MiR-33 and apoE double-knockout mice (miR-33−/−Apoe−/−) showed an increase in circulating HDL-C levels with enhanced cholesterol efflux capacity compared with miR-33+/+Apoe−/− mice. Peritoneal macrophages from miR-33−/−Apoe−/− mice showed enhanced cholesterol efflux to apoA-I and HDL-C compared with miR-33+/+Apoe−/− macrophages. Consistent with these results, miR-33−/−Apoe−/− mice showed reductions in plaque size and lipid content. To elucidate the roles of miR-33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR-33−/−Apoe−/− bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR-33+/+Apoe−/− bone marrow, without an elevation of HDL-C. Some of the validated targets of miR-33 such as RIP140 (NRIP1) and CROT were upregulated in miR-33−/−Apoe−/− mice compared with miR-33+/+Apoe−/− mice, whereas CPT1a and AMPKα were not.ConclusionsThese data demonstrate that miR-33 deficiency serves to raise HDL-C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR-33-deficient mice, and detailed experiments are required to establish miR-33 targeting therapy in humans.

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, day4 mesodermal cells, and day8, day20, and day30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day20 CMs was significantly higher compared to other cells. Transplantation of day20 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 day20 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.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

Down-regulation of the Kpm/Lats2 tumor suppressor is observed in various malignancies and associated with poor prognosis in acute lymphoblastic leukemia. We documented that Kpm/Lats2 was markedly decreased in several leukemias that were highly resistant to conventional chemotherapy. Silencing of Kpm/Lats2 expression in leukemic cells did not change the rate of cell growth but rendered the cells more resistant to DNA damageinducing agents. Expression of p21 and IntroductionThe Warts (Wts) tumor suppressor gene (also termed Lats after large tumor suppressor) was first identified by mitotic recombination of somatic cells and screening for homozygous mutants with overproliferation phenotype in Drosophila melanogaster. 1,2 This discovery initiated a series of genetic studies in Drosophila that led to the delineation of a new signaling network named the Hippo pathway, which is now known to regulate cell growth, cell survival, and organ size in developing animals. [3][4][5] This pathway consists of a kinase cascade in its core where Hippo (Hpo) phosphorylates and activates Wts/Lats, 6 which then in turn phosphorylates and inactivates Yorkie (Yki), a transcription coactivator. Inactivation of Yki results in control of cell survival and cell growth through downregulation of Drosophila inhibitor of apoptosis 1 (Diap1) and Cyclin E. 7 Salvador (Sav), 8,9 a scaffold protein for Hpo, and Mats (mob as tumor suppressor), 10-12 a partner and potentiater of Wts, are also essential components of this pathway. Furthermore, recent evidence has placed Expanded (EX), Merlin (Mer), 13 both 4.1 family proteins, and Fat (FT), 14-17 the atypical cadherin, in the upstream of the Hippo pathway although their connection to the kinase cascade is largely based on genetic epitasis. The Hippo pathway is believed to be conserved throughout species because some of the mammalian homologues have been shown to compensate the corresponding defects in the Drosophila Hippo pathway. At present, however, only a small part of the mammalian Hippo pathway has been experimentally substantiated.Kpm (alternatively named Lats2) is one of the 2 human homologues of Drosophila Wts. 18,19 In parallel to Drosophila Wts, we and others have shown the critical involvement of Kpm/Lats2 in regulation of cell growth and survival. Kpm/Lats2 overexpression results in the cell cycle arrest in G2/M phase via inhibition of Cdc2-Cyclin B kinase activity leading eventually to apoptosis, 20 inhibition of G1/S transition via down-regulation of Cyclin E/Cdk2 kinase activity, 21 or apoptosis via down-regulation of Bcl-2 and Bcl-xL. 22 Kpm/Lats2 binds to Mdm2 and inhibits its E3 ubiquitin ligase activity, resulting in the stabilization of p53 and leading to the p53-dependent G1/S arrest in nocodazole-treated cells. 23 Moreover, Kpm/Lats2 is a target gene of p53 both in mammalian as well as in Drosophila cells, 24,25 suggesting that Kpm/Lats2 may be a positive-feedback-loop regulator of p53. Kpm/Lats2 knockout mice are embryonically lethal and fibroblasts isolated from these mice app...