Hiraku Doi

The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells

Developing effective drug therapies for arrhythmic diseases is hampered by the fact that the same drug can work well in some individuals but not in others. Human induced pluripotent stem (iPS) cells have been vetted as useful tools for drug screening. However, cardioactive drugs have not been shown to have the same effects on iPS cell-derived human cardiomyocytes as on embryonic stem (ES) cell-derived cardiomyocytes or human cardiomyocytes in a clinical setting. Here we show that current cardioactive drugs affect the beating frequency and contractility of iPS cell-derived cardiomyocytes in much the same way as they do ES cell-derived cardiomyocytes, and the results were compatible with empirical results in the clinic. Thus, human iPS cells could become an attractive tool to investigate the effects of cardioactive drugs at the individual level and to screen for individually tailored drugs against cardiac arrhythmic diseases.

Identification of cardiac stem cells with FLK1, CD31, and VE-cadherin expression during embryonic stem cell differentiation

We evaluated the expression of the FLK1, one of the lateral mesoderm early markers where cardiogenesis occurs, to characterize and isolate cardiac stem/progenitor cells from ES cells. Dissociated cells from embryoid bodies (EBs) on day 3, 4, or 5 were collected into two subpopulations with or without FLK1 expression and coculture on OP9 stromal cells was continued to examine whether contracting colonies came out or not. FLK1+ cells from EBs at days 3 and 4 formed spontaneous contracting colonies more efficiently than FLK1− cells on the same days, but not at day 5. Most contracting cardiac colonies derived from FLK1 cells mainly on day 4 were detected on endothelial cells along with hematopoietic cells. Further characterization of cells with these capabilities into three lineages revealed the FLK1+ CD31−VE‐cadherin‐ phenotype. Our findings indicate that FLK1+ cells, especially FLK1+ CD31−VE‐cadherin− cells, could act as cardiohemangioblasts to form cardiac cells as well as endothelial cells and hematopoietic cells.—Iida, M., Heike, T., Yoshimoto, M., Baba, S., Doi, H., Nakahat, T. Identification of cardiac stem cells with FLK1, CD31, and VE‐cadherin expression during embryonic stem cell differentiation. FASEB J. 19, 371–378 (2005)

Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice

AIMS Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors. METHODS AND RESULTS During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17. CONCLUSIONS Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.

Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%–40% of each cell type differentiated into Flk1+ cells. The sorted Flk1+ cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1+ mGS cells generated more contractile colonies than did the Flk1+ ES cells, whereas the Flk1+ EG cells generated equivalent numbers as the Flk1+ mGS cells. Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis of differentiation markers such as Rex1, FGF‐5, GATA‐4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0–4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT‐PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1+ mGS cells express mature cardiac cell‐specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells.

Molecular and clinical analysis of Japanese patients with persistent congenital hyperinsulinism: predominance of paternally inherited monoallelic mutations in the KATP channel genes.

BACKGROUND Preoperative identification of the focal form of congenital hyperinsulinism is important for avoiding unnecessary subtotal pancreatectomy. However, neither the incidence nor the histological spectrum of the disease is known for Japanese patients. AIMS The aim of the study was to elucidate the molecular and histological spectrum of congenital hyperinsulinism in Japan. SUBJECTS Thirty-six Japanese infants with persistent congenital hyperinsulinism were included in the study. METHODS All exons of the ATP-sensitive potassium channel (K(ATP) channel) genes (KCNJ11 and ABCC8), the GCK gene, and exons 6 and 7 and 10-12 of the GLUD1 gene were amplified from genomic DNA and directly sequenced. In patients with K(ATP) channel mutations, the parental origin of each mutation was determined, and the results were compared with the histological findings of surgically treated patients. In one of the patients with scattered lesions, islets were sampled by laser capture microdissection for mutational analysis. RESULTS Mutations were identified in 24 patients (66.7%): five in GLUD1 and 19 in the K(ATP) channel genes. Sixteen had a paternally derived, monoallelic K(ATP) channel mutation predictive of the focal form. In 10 patients who underwent pancreatectomy, the molecular diagnosis correctly predicted the histology, more accurately than [18F]-3,4-dihydroxyphenylalanine positron emission tomography scans. Three patients showed focal lesions that occupied larger areas of the pancreas. Preferential loss of the maternal allele was observed in these islets. CONCLUSION The majority of the Japanese patients with K(ATP) channel hyperinsulinism (84.2%) demonstrated paternally inherited monoallelic mutations that accurately predicted the presence of the focal form.

Successful treatment of pulmonary hypertension secondary to congenital extrahepatic portocaval shunts (Abernethy type 2) by living donor liver transplantation after surgical shunt ligation

In this report, we describe a living donor liver transplantation (LDLT) in a patient (7‐year‐old boy) with Abernethy type 2 congenital extrahepatic portocaval shunts (CEPS). This patient underwent a surgical shunt ligation as the first treatment for pulmonary hypertension; pulmonary hypertension was improved and controlled successfully 4 years after the first operation. However, pulmonary hypertension recurred gradually because of multiple intrahepatic portosystemic shunts; therefore, LDLT was performed as a radical treatment of intrahepatic portosystemic shunts. His pulmonary arterial pressure was also controlled 22 months after LDLT, the postoperative continuous intravenous prostaglandin I2 (PGI2) treatment could be withdrawn successfully. We suggest that clinicians carefully follow up the recurrent portosystemic shunt and cardiopulmonary disorders secondary to Abernethy type 2 CEPS.

Development of pulmonary hypertension in 5 patients after pediatric living‐donor liver transplantation: De novo or secondary?

The development of portopulmonary hypertension (PH) in a patient with end‐stage liver disease is related to high cardiac output and hyperdynamic circulation. However, PH following liver transplantation is not fully understood. Of 617 pediatric patients receiving transplants between June 1990 and March 2004, 5 (median age 12 yr, median weight 24.5 kg) were revealed to have portopulmonary hypertension (PH) after living‐donor liver transplantation (LDLT), as confirmed by echocardiography and/or right heart catheterization. All children underwent LDLT for post‐Kasai biliary atresia. In 2 patients with refractory biliary complications, PH developed following portal thrombosis; 2 with stable graft function, who had had intrapulmonary shunting (IPS) before LDLT, were found to have PH in spite of overcoming liver dysfunction due to hepatitis. PH developed shortly after distal splenorenal shunting in 1 patient, who suffered liver cirrhosis due to an intractable outflow blockage. The onset of PH ranged from 2.8 to 11 yr after LDLT, and mean pulmonary artery pressure (mPAP) estimated by echocardiography at the time of presentation ranged from 43 to 120 mmHg. Three of the 5 patients are alive under prostaglandin I2 (PGI2) treatment. Of these, 1 is prepared for retransplantation for an intractable complications of liver allograft, while the other 2 with satisfactory grafts are being considered for lung transplantation. Even after LDLT, PH can develop with portal hypertension. Periodic echocardiography is essential for early detection and treatment of PH especially in the recipients with portal hypertension not only preoperatively but also postoperatively. Liver Transpl 12:870–875, 2006.

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function

Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), cardiovascular complications, thrombocytopenia and Ehlers–Danlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-function mutation in FLNA. They presented with multiple complications, including valvulopathy, intestinal malrotation and chronic intestinal pseudo-obstruction (CIPO). However, these siblings had atypical clinical courses, such as a lack of PVNH and a spontaneous improvement of CIPO. Trio-based whole-exome sequencing revealed a 4-bp deletion in exon 40 that was predicted to cause a lethal premature protein truncation. However, molecular investigations revealed that the mutation induced in-frame skipping of the mutated exon, which led to the translation of a mutant FLNA missing an internal region of 41 amino acids. Functional analyses of the mutant protein suggested that its binding affinity to integrin, as well as its capacity to induce focal adhesions, were comparable to those of the wild-type protein. These results suggested that exon skipping of FLNA partially restored its protein function, which could contribute to amelioration of the siblings’ clinical courses. This study expands the diversity of the phenotypes associated with loss-of-function mutations in FLNA.

Lasting 18F-DOPA PET Uptake after Clinical Remission of the Focal Form of Congenital Hyperinsulinism

Background: Positron emission tomography (PET) using 18F-DOPA is a useful tool for detecting the focal forms of congenital hyperinsulinism. 18F-DOPA is taken up by aromatic L-amino acid decarboxylase in pancreatic β-cells. However, the role of this enzyme in insulin secretion is unknown. Subjects and Methods: A Japanese boy who presented with symptomatic hyperinsulinemic hypoglycemia at the age of 2 days and spontaneous resolution at 1 year and 10 months was subjected to mutational analysis and repeated 18F-DOPA PET scans. Results: Mutational analysis revealed a paternally inherited monoallelic mutation, c.4186G>T (p.D1396Y), in the ABCC8 gene, and an 18F-DOPA PET scan revealed focal uptake in the body of the pancreas. The patient was successfully treated with frequent feeding. A follow-up PET scan revealed virtually identical uptake to that observed previously. However, in the arterial stimulation-venous sampling procedure, no significant insulin release was observed. He was placed on a normal diet, and no hypoglycemia recurrence was observed. Conclusion: This case demonstrates two important findings. Firstly, the uptake of 18F-DOPA does not correlate with the insulin-secreting capacity of the lesion. Secondly, clinical remission could be a functional process not necessarily accompanied by the apoptotic death of abnormal β-cells.

Results of living donor liver transplantation in five children with congenital cardiac malformations requiring cardiac surgery

Abstract:  In the pediatric population, the concomitant presentation of end‐stage liver disease and congenital cardiac malformation occurs rarely. Determining the surgical priority in these cases is a challenge due to the presence of hemodynamic alterations that increase surgical risks. We examined five cases that received living‐donor liver transplantation. In four patients that had congenital heart disease with a left to right shunt, two had cardiac surgery first, one had both heart and liver surgery simultaneously, and one underwent liver transplantation first. Both of the patients that received heart surgery before liver transplantation needed emergency liver transplantation because of post‐operative liver failure. All five patients had a good outcome. Meticulous surgery, close monitoring, and adequate volume management, in addition to tailoring management decisions to the patients specific condition, make it possible to correct both the liver and the heart abnormalities with satisfactory results.