Sunny Sun Kin Chan

Acquisition of a Quantitative, Stoichiometrically Conserved Ratiometric Marker of Maturation Status in Stem Cell-Derived Cardiac Myocytes

Summary There is no consensus in the stem cell field as to what constitutes the mature cardiac myocyte. Thus, helping formalize a molecular signature for cardiac myocyte maturation would advance the field. In the mammalian heart, inactivation of the “fetal” TNNI gene, TNNI1 (ssTnI), together in temporal concert with its stoichiometric replacement by the adult TNNI gene product, TNNI3 (cTnI), represents a quantifiable ratiometric maturation signature. We examined the TNNI isoform transition in human induced pluripotent stem cell (iPSC) cardiac myocytes (hiPSC-CMs) and found the fetal TNNI signature, even during long-term culture. Rodent stem cell-derived and primary myocytes, however, transitioned to the adult TnI profile. Acute genetic engineering of hiPSC-CMs enabled a rapid conversion toward the mature TnI profile. While there is no single marker to denote the mature cardiac myocyte, we propose that tracking the cTnI:ssTnI protein isoform ratio provides a valuable maturation signature to quantify myocyte maturation status across laboratories.

Chemistry and biological activities of naturally occurring phthalides

Abstract Phthalides are widely present in plants, fungi and liverworts. Many phthalide-containing plants have been used worldwide as herbal remedies in traditional and folk medicines, dietary supplements and food flavorings. In this review, the chemical structures, classifications, and plant sources of about 137 natural phthalides of plant origins are described. The recent advances in the biological activities of these pthalides, such as actions on the central nervous system, anti-platelet aggregation and anti-thrombosis, cardiac function modulation and anti-angina, inhibition of smooth muscle cell proliferation, protection against cerebral ischemia, and smooth muscle relaxation, are also discussed.

Fibroblast Growth Factor-10 Promotes Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells

Background The fibroblast growth factor (FGF) family is essential to normal heart development. Yet, its contribution to cardiomyocyte differentiation from stem cells has not been systemically studied. In this study, we examined the mechanisms and characters of cardiomyocyte differentiation from FGF family protein treated embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Methodology/Principal Findings We used mouse ES cells stably transfected with a cardiac-specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescent protein (EGFP) and mouse iPS cells to investigate cardiomyocyte differentiation. During cardiomyocyte differentiation from mouse ES cells, FGF-3, -8, -10, -11, -13 and -15 showed an expression pattern similar to the mesodermal marker Brachyury and the cardiovascular progenitor marker Flk-1. Among them, FGF-10 induced cardiomyocyte differentiation in a time- and concentration-dependent manner. FGF-10 neutralizing antibody, small molecule FGF receptor antagonist PD173074 and FGF-10 and FGF receptor-2 short hairpin RNAs inhibited cardiomyocyte differentiation. FGF-10 also increased mouse iPS cell differentiation into cardiomyocyte lineage, and this effect was abolished by FGF-10 neutralizing antibody or PD173074. Following Gene Ontology analysis, microarray data indicated that genes involved in cardiac development were upregulated after FGF-10 treatment. In vivo, intramyocardial co-administration of FGF-10 and ES cells demonstrated that FGF-10 also promoted cardiomyocyte differentiation. Conclusion/Significance FGF-10 induced cardiomyocyte differentiation from ES cells and iPS cells, which may have potential for translation into clinical applications.

Synergistic interaction between the Ligusticum chuanxiong constituent butylidenephthalide and the nitric oxide donor sodium nitroprusside in relaxing rat isolated aorta

ETHNOPHARMACOLOGICAL RELEVANCE Ligusticum chuanxiong Hort. (Umbelliferae), a traditional Chinese medicinal herb, is often prescribed together with nitric oxide donors for treating coronary heart diseases such as angina in China; however, studies concerning their pharmacological interaction are scarce. AIM OF THE STUDY The objective of the present study was to examine the interaction between the Ligusticum chuanxiong major active constituent butylidenephthalide (BDPH) and the nitric oxide donor sodium nitroprusside (SNP) in vasorelaxation. MATERIALS AND METHODS Vasorelaxation was examined in rat isolated aorta using an organ bath system. RESULTS BDPH and SNP interacted synergistically under 9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin H2 (U-46619)-induced tone. This synergism became greater with increasing U-46619 concentrations where Ca2+ sensitization contributed more significantly, and less when U-46619 was replaced with phenylephrine where participation of Ca2+ sensitization was minimal. BDPH-SNP synergism remained intact in the absence of external Ca2+, indicating that regulation of Ca2+ influx was not a requirement for the manifestation of this interaction. CONCLUSIONS The present study demonstrated the synergistic relaxation between BDPH and SNP in rat isolated aorta. This interaction is related to an enhancement of the effectiveness of SNP in producing relaxation under tone induced mainly by Ca2+ sensitization.

Salvianolic acid B-vitamin C synergy in cardiac differentiation from embryonic stem cells

Inefficient cardiomyocyte differentiation limits the therapeutic use of embryonic stem (ES) cell-derived cardiomyocytes. While large collections of proprietary chemicals had been screened to improve ES cell differentiation into cardiomyocytes, the natural product library remained unexplored. Using a mouse ES cell line transfected with a cardiomyocyte-specific alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein (EGFP) reporter, we screened 24 natural products with known cardioprotective actions. Salvianolic acid B (saB), while produced minimal effect on its own, concentration-dependently synergized with vitamin C in inducing cardiomyocyte differentiation, as demonstrated by an increase in EGFP(+) cells, beating area in embryoid bodies, and expression of cardiomyocyte maturity markers. This synergy is specific to cardiomyocyte differentiation, and is involved with collagen synthesis. The present study demonstrates the saB-vitamin C synergy in inducing ES cell differentiation into matured and functional cardiomyocytes, and this may lead to a practicable cocktail approach to generate ES cell-derived cardiomyocytes for cardiac stem cell therapy.

Development of Bipotent Cardiac/Skeletal Myogenic Progenitors from MESP1+ Mesoderm

Summary The branchiomeric skeletal muscles co-evolved with new chambers of the heart to enable predatory feeding in chordates. These co-evolved tissues develop from a common population in anterior splanchnic mesoderm, referred to as cardiopharyngeal mesoderm (CPM). The regulation and development of CPM are poorly understood. We describe an embryonic stem cell-based system in which MESP1 drives a PDGFRA+ population with dual cardiac and skeletal muscle differentiation potential, and gene expression resembling CPM. Using this system, we investigate the regulation of these bipotent progenitors, and find that cardiac specification is governed by an antagonistic TGFβ-BMP axis, while skeletal muscle specification is enhanced by Rho kinase inhibition. We define transcriptional signatures of the first committed CPM-derived cardiac and skeletal myogenic progenitors, and discover surface markers to distinguish cardiac (PODXL+) from the skeletal muscle (CDH4+) CPM derivatives. These tools open an accessible window on this developmentally and evolutionarily important population.

Qianliguang (Senecio scandens) safety dilemma: Dose is the key?

Qianliguang ( SENECIO SCANDENS) is a common Chinese medicinal herb. Qianliguang-containing herbal proprietary products are registered as over-the-counter remedies in China and exported to Western countries. The safety of using Qianliguang and its products has raised general concerns because of a potential risk of the presence of hepatotoxic pyrrolizidine alkaloids (PAs). A systematic toxicological study is thus required to verify this public concern. In the present article, we report, for the first time, that S. SCANDENS contains nine hepatotoxic PAs with a content of 6.95-7.19 microg/g. At a dose equivalent to the daily intake recommended by the Pharmacopoeia of China, the total content of toxic PAs in Qianliguang was determined to be 3.48 microg/kg/day, which is far below the lowest dose to cause hepatotoxicity (15 microg/kg/day) suggested by the International Program on Chemical Safety. No significant hepatotoxic effects were observed in rats fed with the extract at this human-equivalent dose for 14 consecutive days. However, a single overdose of the herbal water extract (6 g/kg), which was about 8-fold higher than the recommended dose, produced typical PA-induced hepatotoxicity in rats. Therefore, appropriate dosage guidelines should be implemented for the herbal industry, for export/import retailers, and for herbal medicine practitioners to ensure the safe and beneficial use of these herbal medicines.

Muscle pathology from stochastic low level DUX4 expression in an FSHD mouse model

Facioscapulohumeral muscular dystrophy is a slowly progressive but devastating myopathy caused by loss of repression of the transcription factor DUX4; however, DUX4 expression is very low, and protein has not been detected directly in patient biopsies. Efforts to model DUX4 myopathy in mice have foundered either in being too severe, or in lacking muscle phenotypes. Here we show that the endogenous facioscapulohumeral muscular dystrophy-specific DUX4 polyadenylation signal is surprisingly inefficient, and use this finding to develop an facioscapulohumeral muscular dystrophy mouse model with muscle-specific doxycycline-regulated DUX4 expression. Very low expression levels, resulting in infrequent DUX4 + myonuclei, evoke a slow progressive degenerative myopathy. The degenerative process involves inflammation and a remarkable expansion in the fibroadipogenic progenitor compartment, leading to fibrosis. These animals also show high frequency hearing deficits and impaired skeletal muscle regeneration after injury. This mouse model will facilitate in vivo testing of therapeutics, and suggests the involvement of fibroadipogenic progenitors in facioscapulohumeral muscular dystrophy.Facioscapulohumeral muscular dystrophy is a severe myopathy that is caused by abnormal activation of DUX4, and for which a suitable mouse model does not exist. Here, the authors generate a novel mouse model with titratable expression of DUX4, and show that it recapitulates several features of the human pathology.

Expansion and Purification Are Critical for the Therapeutic Application of Pluripotent Stem Cell-Derived Myogenic Progenitors

Summary Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.

Heterogeneity of Mesp1+ mesoderm revealed by single-cell RNA-seq.

Mesp1 is a transcription factor that promotes differentiation of pluripotent cells into different mesoderm lineages including hematopoietic, cardiac and skeletal myogenic. This occurs via at least two transient cell populations: a common hematopoietic/cardiac progenitor population and a common cardiac/skeletal myogenic progenitor population. It is not established whether Mesp1-induced mesoderm cells are intrinsically heterogeneous, or are simply capable of multiple lineage decisions. In the current study, we applied single-cell RNA-seq to analyze Mesp1+ mesoderm. Initial whole transcriptome analysis showed a surprising homogeneity among Mesp1-induced mesoderm cells. However, this apparent global homogeneity masked an intrinsic heterogeneity revealed by interrogating a panel of early mesoderm patterning factors. This approach enabled discovery of subpopulations primed for hematopoietic or cardiac development. These studies demonstrate the heterogeneic nature of Mesp1+ mesoderm.