Itsunari Minami

A Small Molecule that Promotes Cardiac Differentiation of Human Pluripotent Stem Cells under Defined, Cytokine- and Xeno-free Conditions

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, are potentially useful in regenerative therapies for heart disease. For medical applications, clinical-grade cardiac cells must be produced from hPSCs in a defined, cost-effective manner. Cell-based screening led to the discovery of KY02111, a small molecule that promotes differentiation of hPSCs to cardiomyocytes. Although the direct target of KY02111 remains unknown, results of the present study suggest that KY02111 promotes differentiation by inhibiting WNT signaling in hPSCs but in a manner that is distinct from that of previously studied WNT inhibitors. Combined use of KY02111 and WNT signaling modulators produced robust cardiac differentiation of hPSCs in a xeno-free, defined medium, devoid of serum and any kind of recombinant cytokines and hormones, such as BMP4, Activin A, or insulin. The methodology has potential as a means for the practical production of human cardiomyocytes for regeneration therapies.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Progressive maturation in contracting cardiomyocytes derived from human embryonic stem cells: Qualitative effects on electrophysiological responses to drugs

The field of drug testing currently needs a new integrated assay system, as accurate as systems using native tissues, that will allow us to predict arrhythmia risks of candidate drugs and the relationship between genetic mutations and acquired electrophysiological phenotypes. This could be accomplished by combining the microelectrode array (MEA) system with cardiomyocytes (CMs) derived from human embryonic stem cells (hESC) and induced pluripotential stem cells. CMs have been successfully induced from both types, but their maturation process is not systematically controlled; this results in loss of beating potency and insufficient ion channel function. We generated a transgenic hESC line that facilitates maintenance of hESC-CM clusters every 2 weeks by expressing GFP driven by a cardiac-specific alphaMHC promoter, thereby producing a compact pacemaker lineage within a ventricular population over a year. Further analyses, including quantitative RT-PCR, patch-clamp, and MEA-mediated QT tests, demonstrated that replating culturing continuously enhanced gene expression, ionic current amplitudes, and resistance to K(+) channel blockades in hESC-CMs. Moreover, temporal three-dimensional (3D) culturing accelerated maturation by restoring the global gene repressive status established in the adhesive status. Replating/3D culturing thus produces hESC-CMs that act as functional syncytia suitable for use in regenerative medicine and accurate drug tests.

Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved.

Resetting of peripheral circadian clock by prostaglandin E2

In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is thought to drive peripheral oscillators by controlling neuronal and humoral signals that can entrain the peripheral clocks. Here, we show that prostaglandin E2 (PGE2), a proinflammatory compound known to have diverse biological effects, is able to act as an in vivo clock‐resetting agent. We find that in cultured NIH3T3 fibroblasts, PGE2 is able to induce transient expression of Period 1 messenger RNA and the following circadian oscillation of clock gene expression. Furthermore, we demonstrate that intraperitoneal administration of PGE2 results in the phase shift of circadian gene expression in mouse peripheral tissues in a time‐dependent manner. This phase shift is also induced by the EP1/EP3 agonist sulprostone but not by the EP2 agonist butaprost. The PGE2‐induced phase shift is inhibited by the EP1 antagonist SC‐51322. These results suggest that PGE2 acts as an in vivo clock‐resetting factor by means of the EP1 subtype of PGE receptors.

A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

Summary Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

Long‐term potentiation of mGluR1 activity by depolarization‐induced Homer1a in mouse cerebellar Purkinje neurons

Metabotropic glutamate receptor 1 (mGluR1) plays a crucial role in synaptic plasticity and motor learning in the cerebellum. We have studied activity‐dependent changes in mGluR1 function in mouse cultured Purkinje neurons. Depolarizing stimulation potentiated Ca2+ and current responses to an mGluR1 agonist for several hours in the cultured Purkinje neurons. It also blocked internalization of mGluR1 and increased the number of mGluR1s on the cell membrane. We found that depolarization simultaneously increased transcription of Homer1a in Purkinje neurons. Homer1a inhibited internalization and increased cell‐surface expression of mGluR1 when coexpressed in human embryonic kidney (HEK)‐293 cells. Depolarization‐induced Homer1a expression in Purkinje neurons was blocked by a mitogen‐activated protein kinase (MAPK) inhibitor. Changes in internalization and mGluR1‐mediated Ca2+ response were also blocked by inhibition of MAPK activity, suggesting that localization and activity of mGluR1 were regulated in the same signalling pathway as Homer1a expression. It is thus suggested that depolarization of the Purkinje neuron leads to the increment in mGluR1 responsiveness through MAPK activity and induction of Homer1a expression, which increases active mGluR1 on the cell surface by blocking internalization of mGluR1.

A Chemical Probe that Labels Human Pluripotent Stem Cells

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

Differences in relationships among sleep apnoea, glucose level, sleep duration and sleepiness between persons with and without type 2 diabetes

Obstructive sleep apnoea is common in patients with diabetes. Recently, it was reported that short sleep duration and sleepiness had deleterious effects on glucose metabolism. Thereafter, several reports showed relationships between glucose metabolism and obstructive sleep apnoea, sleep duration or sleepiness. But the interrelationships among those factors based on recent epidemiological data have not been examined. We analysed data on 275 male employees (age, 44 ± 8 years; body mass index, 23.9 ± 3.1 kg m−2) who underwent a cross‐sectional health examination in Japan. We measured fasting plasma glucose, sleep duration using a sleep diary and an actigraph for 7 days, and respiratory disturbance index with a type 3 portable monitor for two nights. Fifty‐four subjects (19.6%) had impaired glucose metabolism, with 21 having diabetes. Of those 21 (body mass index, 25.9 ± 3.8 kg m−2), 17 (81.0%) had obstructive sleep apnoea (respiratory disturbance index ≥ 5). Regarding the severity of obstructive sleep apnoea, 10, four and three had mild, moderate and severe obstructive sleep apnoea, respectively. The prevalence of obstructive sleep apnoea was greater in those with than without diabetes (P = 0.037). Multiple regression analyses showed that the respiratory disturbance index independently related to fasting plasma glucose only in the diabetic subjects. In patients with diabetes, after adjustment for age, waist circumference, etc. sleep fragmentation had a greater correlation with fasting plasma glucose than sleep duration, but without significance (P = 0.10). Because the prevalence of obstructive sleep apnoea is extremely high in patients with diabetes, sufficient sleep duration with treatment for obstructive sleep apnoea, which ameliorates sleep fragmentation, might improve fasting plasma glucose.

Association Between Sleep Apnea, Sleep Duration, and Serum Lipid Profile in an Urban, Male, Working Population in Japan

BACKGROUND Dyslipidemia is often comorbid with obstructive sleep apnea (OSA), but few population-based studies have investigated their relationship. Short sleep duration is associated with hypertension and diabetes; however, its association with dyslipidemia is not well known. We investigated relationships among OSA, sleep duration, and the lipid profile in a community-based study. METHODS We measured the respiratory disturbance index (RDI) and sleep duration by a type 3 portable device and actigraph in 275 men in a Japanese company. Fasting blood parameters were obtained from periodic inspection data. RESULTS According to Japanese criteria, 143 subjects had dyslipidemia. Percent sleep time of oxygen saturation as measured by pulse oximetry (SpO2) < 90% and prevalence of severe OSA were greater and sleep duration and mean SpO2 during sleep were lower in subjects with dyslipidemia than in those without. Univariate analysis showed that the RDI was positively correlated with serum triglyceride (TG) levels (ρ = 0.20, P < .01), and sleep duration was negatively correlated with serum total cholesterol (TC) levels (γ = -0.13, P = .03) and serum low-density lipoprotein cholesterol levels (γ = -0.12, P = .04). Stepwise multiple regression analysis revealed that TG was correlated with RDI (β = 0.14, P = .02), BMI (β = 0.20, P < .01), and alcohol intake (β = 0.20, P < .01), and that TC was correlated with sleep duration (β = -0.13, P = .03), age (β = 0.15, P = .02), and waist/hip ratio (β = 0.15, P = .02). CONCLUSIONS Short sleep duration was associated with TC levels and RDI was positively associated with TG levels among working-aged men in an urban Japanese company. Correcting the status of OSA and/or short sleep duration might improve the lipid profile and cardiovascular consequences.