Kazuhisa Chonabayashi

MicroRNA-33 Deficiency Reduces the Progression of Atherosclerotic Plaque in ApoE−/− Mice

Background Cholesterol 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 Results We 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. Conclusions These 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.

Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity

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.

Kpm/Lats2 is linked to chemosensitivity of leukemic cells through the stabilization of p73

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 damage-inducing agents. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in Kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73 was clearly observed in control cells but hardly detected in Kpm/Lats2-knockdown cells. Chromatin immunoprecipitation (ChIP) assay showed that p73 was recruited to the PUMA gene promoter in control cells but not in Kpm/Lats2-knockdown cells after DNA damage. The analyses with transient coexpression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. Our results suggest that Kpm/Lats2 is involved in the fate of p73 through the phosphorylation of YAP2 by Kpm/Lats2 and the induction of p73 target genes that underlie chemosensitivity of leukemic cells.

Presenilin 1 is involved in maturation and trafficking of N-cadherin to the plasma membrane

One pathological characteristic of Alzheimers disease (AD) is extensive synapse loss. Presenilin 1 (PS1) is linked to the pathogenesis of early onset familial Alzheimers disease (FAD) and is localized at the synapse, where it binds N‐cadherin and modulates its adhesive activity. To elucidate the role of the PS1/N‐cadherin interaction in synaptic contact, we established SH‐SY5Y cells stably expressing wild‐type (wt) PS1 and dominant‐negative (D385A) PS1. We show that the formation of cadherin‐based cell–cell contact among SH‐SY5Y cells stably expressing D385A PS1 was suppressed. Conversely, wt PS1 cells exhibited enhanced cell–cell contact and colony formation. Suppression of cell–cell contact in D385A cells was accompanied by an alteration in N‐cadherin subcellular localization; N‐cadherin was retained mainly in the endoplasmic reticulum (ER) and cell surface expression was reduced. We conclude that PS1 is essential for efficient trafficking of N‐cadherin from the ER to the plasma membrane. PS1‐mediated delivery of N‐cadherin to the plasma membrane is important for N‐cadherin to exert its physiological function, and it may control the state of cell–cell contact.

Enhanced engraftment, proliferation, and therapeutic potential in heart using optimized human iPSC-derived cardiomyocytes

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.

Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation

&NA; 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.

Primary large B-cell lymphoma of the bone marrow

A diagnosis of primary bone marrow lymphoma, classified as diffuse large B-cell lymphoma, was made in a 31-year-old man. He presented with persistent fever and pancytopenia and reported pain in the pelvis and femora. Computed tomography imaging showed hepatosplenomegaly but no lymphadenopathy. On magnetic resonance imaging (MRI), the right ilium and both femora demonstrated abnormal signals with low intensity on T1-weighted images and high intensity on T2weighted images (top left). A bone marrow biopsy from the right iliac crest showed diffuse infiltration by large CD20positive cells. Cytogenetic analysis of bone marrow cells was normal. A staging F-fluorodeoxyglucose positron emission tomography (PET) scan revealed striking disseminated bone marrow uptake without evidence of hepatic, splenic or lymph node involvement (top right). He underwent chemotherapy with six courses of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone), followed by high-dose chemotherapy combined with autologous peripheral blood stem cell transplantation. At the end of the treatment, the PET and MRI scans were repeated; while persistent bone marrow disease was suggested by MRI (bottom left), the PET scan was normal (bottom right). The patient remains in complete remission at 12 months. Although diffuse large B-cell lymphoma frequently involves the bone marrow, isolated bone marrow involvement is rare. PET scanning can be useful in diagnosis.

Successful allogeneic stem cell transplantation with long-term remission of ETV6/FLT3 -positive myeloid/lymphoid neoplasm with eosinophilia

Dear Editor, FLT3 is one of the most frequently mutated genes in hematological malignancies [1]. The most common FLT3 mutations are internal tandem duplications, which occur in 20– 30 % of patients with AML [2]. FLT3 fuses to ETV6 (TEL) and contributes to oncogenesis in a small number of patients with myeloid/lymphoid neoplasms with eosinophilia (MLNeo), carrying the translocation t(12;13)(p13;q12) [3, 4]. We previously identified a novel ETV6/FLT3 variant fusion transcript in a MLN-eo patient [5]. Here, we provide a clinical report of the aforementioned patient, who achieved long-term remission after allogeneic stem cell transplantation (allo-SCT). A 33-year-old man presented with systemic lymphadenopathy and elevated WBC counts of 17×10/L (4 % myelocytes, 6 %metamyelocytes, 49 % neutrophils, 18 % eosinophils, 13 % monocytes, and 10 % lymphocytes). Biopsy specimens from lymph nodes showed diffuse infiltration of lymphoblasts (cytoplasmic CD3, CD4, and CD8). A bone marrow (BM) exam revealed myeloid hyperplasia with eosinophilia (Fig. 1). Karyotypes of BM cells and lymph node cells mostly consisted of 46,XY,t(12;13)(p13;q12). Neutrophil-fluorescent in situ hybridization (FISH) analysis using the ETV6 break-apart probe showed a split signal. RT-PCR analysis revealed the expression of ETV6/FLT3 fusion transcript in both BM and lymph node cells. The patient was diagnosed with T-lymphoblastic lymphoma and myeloproliferative neoplasm with eosinophilia. Complete disappearance of the enlarged lymph nodes was observed after initiation of hyper-CVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone). However, karyotype analysis of BM cells after three cycles of chemotherapy showed continued presence of t(12;13)(p13;q12). Thus, matched unrelated allo-SCT was performed. The conditioning regimen included cyclophosphamide at 120 mg/kg and 1,200 cGy total body irradiation. GVHD prophylaxis was performed with tacrolimus and short-term methotrexate. Engraftment was achieved on day+24 without significant regimen-related toxicity. On day+28, engraftment of donor cells was confirmed by XY-FISH and translocation-positive BM cells were below the detection limit of ETV6-FISH. FISH analysis on day+60 indicated the reappearance of a split signal for ETV6 in 4 % of BM cells. At the same time, chromosomal analysis showed a complex karyotype, including t(12;13)(p13;q12) in 2 of the 20 metaphase BM cells. Tacrolimus administration was gradually tapered thereafter. The patient achieved normalization of the karyotype of BM cells on day+151 with no signs of GVHD and remained in complete molecular remission having a good performance status 6 years after transplantation. ETV6/FLT3-related neoplasm is considered a very rare subtype of MLN-eo (Table 1). A recent report described the therapeutic efficacy of FLT3 inhibitors in two patients with ETV6/FLT3-positive MLN-eo. However, relapse and resistance occurred in both patients [4]. To date, there have been no reports of long-term survival of patients with ETV6/FLT3-related neoplasm. Although the role of alloSCT for ETV6/FLT3-related neoplasm is uncertain, there is K. Chonabayashi :M. Hishizawa (*) :M. Matsui : T. Kondo : T. Ishikawa :A. Takaori-Kondo Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan e-mail: hishiza@kuhp.kyoto-u.ac.jp

Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of SCN5A-D1275N-Related Cardiac Sodium Channelopathy

BACKGROUND TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5. CONCLUSIONS This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.

Successful use of second cord blood transplantation to achieve long-term remission in cord blood donor cell-derived AML harboring a FLT3-ITD and an NPM1 mutation.

Successful use of second cord blood transplantation to achieve long-term remission in cord blood donor cell-derived AML harboring a FLT3-ITD and an NPM1 mutation