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 α-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.     

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 μm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.     

Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival

Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2–20 μM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the anti-apoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Neuronal differentiation of NTE-deficient embryonic stem cells

Organophosphates induce neurological disorders. One of the enzymes inhibited by these compounds is neuropathy target esterase (NTE). In vitro, inhibition of NTE activity by organophosphates is correlated with inhibition of neurite initiation and reduction of neurite length, supporting the hypothesis that organophosphate-induced neurological disorders are caused by inhibition of NTE activity. However, there is no direct evidence for the involvement of NTE in organophosphate-induced impairment of neurites in vitro. To examine the role of NTE, we have generated NTE-deficient mouse embryonic stem cells. These cells can differentiate into neuron-like cells. Although NTE-deficient cells exhibited a delay in neurite initiation in vitro, both the proportion of neuron-like cells which initiated neurites and the elongation of these neurites occurred at the normal rate. These results demonstrate that NTE activity is not required for neurite initiation or elongation per se, but is essential for the optimal rate of neurite initiation.     

Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells

For myocardial regeneration therapy, the low differentiation capability of functional cardiomyocytes sufficient to replace the damaged myocardial tissue is one of the major difficulties. Using Nkx2.5-GFP knock-in ES cells, we show a new efficient method to obtain cardiomyocytes from embryonic stem (ES) cells. The proportion of GFP-positive cells was significantly increased when ES cells were cultured with a conditioned medium from aortic endothelial cells (ECs), accompanied by upregulation of cardiac-specific genes as well as other mesodermal genes. The promotion was more prominent when EC-conditioned medium was added at an early stage of ES cell differentiation culture (Day 0-3). Inhibitors of bone morphogenic protein (BMP), cyclooxygenase (COX), and nitric oxide synthetase (NO) prevented the promotion of cardiomyogenesis by EC-conditioned medium. These results suggest that supplementation of EC-conditioned medium enables cardiomyocytes to be obtained efficiently through promotion of mesoderm induction, which is regulated by BMP, COX, and NOS.     

Telomerase inhibition promotes an initial step of cell differentiation of primate embryonic stem cell

Embryonic stem (ES) cell is well known as a totipotent cell, which is derived from a blastcyst and has potential to differentiate into every kind of somatic cell. ES cell bears self-renewal characteristic as well as differentiation potential. ES cell bears telomerase activity to avoid telomere shortening, which is a characteristic of differentiated somatic cells. As the differentiation of ES cells proceeds, their telomerase activity is losing. However, it has not been convinced whether suppression of the telomerase activity promotes progression of ES cell differentiation. The effect of telomerase inhibitor on the differentiation potential of marmoset ES cell was assessed, counting cells expressing embryonic markers (alkaline phosphatase and TPA-1-60) under existence of a telomerase inhibitor. Telomerase inhibitor showed a promotional effect for the marmoset ES cell differentiation. This result suggests that exogenous inhibition of telomerase activity leads to induction of an early differentiation of primate ES cell.     

胚胎干细胞分化过程中的表观遗传调控

作为一类既有自我更新能力,并具有多向分化潜能的细胞,胚胎干细胞具有非常重要的理论研究意义和临床应用前景。近期以胚胎干细胞为模型,研究有关干细胞分化的表观遗传调控已成为新的研究热点。本文就胚胎干细胞分化过程中DNA甲基化、组蛋白修饰、非编码RNA调控以及与胚胎干细胞分化密切相关的表观遗传学动态变化做一概述,对表观遗传学改变与胚胎干细胞分化关系的基础研究进行探讨。     

胚胎干细胞诱导分化为雄性生殖细胞的研究进展

胚胎干细胞（embryonic stem cells,ES细胞）具有自我更新及无限分化潜能,理论上可以分化为生殖细胞。目前,在人及鼠中已有体外诱导ES细胞分化为成熟精子的报道。系统阐述影响ES细胞分化为雄性生殖细胞的内源性及外源性因素,并结合国内外最新研究进展总结其诱导分化方法,展望应用前景,期望为从事相关研究的学者提供参考。     

Neural differentiation of human embryonic stem cells

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes.     

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment

Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.     

从胚胎干细胞到视网膜

视网膜退行性病变影响着全世界数百万人。然而,视网膜是人体再生能力很差的一类组织,成年机体无法自我更新那些病变中丢失的视网膜细胞,导致视网膜退行性病变的不可逆性。因此,恢复患者视觉将依赖于引入外源细胞替代丢失的视网膜神经元。胚胎干细胞（ES细胞）具有无限的自我更新能力和形成机体所有类型细胞的巨大分化潜力。这两个特性使得ES细胞成为细胞替代疗法的理想供体细胞。近年来,人们在探索将ES和诱导多能干细胞（iPS细胞）体外定向诱导分化为视网膜神经元,甚至整个视网膜方面已取得多项进展,并且体外形成的视网膜细胞可以与宿主视网膜整合。在此篇综述中,首先简要概括哺乳动物视网膜的组织结构、发育过程和调控机制,然后,重点阐述近年来科研工作者探索ES/iPS细胞体外诱导分化为视网膜细胞和组织的研究进展。     

The cardiac differentiation of bone marrow stem cells might be not important for heart repair

Lots of evidence showed that bone marrow stem cells can differentiate into cardiac myocytes so as to treat damaged hearts. However, the following studies revealed that bone marrow stem cells also produced protective effects on hearts by releasing some beneficial cytokines and suppressing inflammatory effects and so on. Therefore, we speculated that the cardiac differentiation of bone marrow stem cells did not play an important role in cardiac repair.     

人胚胎干细胞的体外定向分化

人胚胎干细胞（human embryonic stem cells,hESCs）由囊胚期胚胎内细胞团分离培养获得,具有保持未分化状态的无限增殖能力。hESCs具有多向分化潜能,在体内和体外均可分化形成所有三个胚层（外胚层、中胚层、内胚层）的衍生物。hESCs一般在鼠胚胎成纤维细胞（mouse embryonic fibroblast,MEF）饲养层上培养和扩增。为了优化培养条件,目前人们已发展了多种人类细胞饲养层和无饲养层、非条件培养基体系。hESCs可以在体外定向诱导分化为多种细胞类型,为揭示人胚早期发育机制和发展多种疾病的细胞移植治疗奠定了基础。hESCs可以在体外进行遗传修饰,将有助于揭示特定基因在发育过程中的调控和功能。对hESCs的深入研究将极大地推动医学和生命科学的进展,并将最终应用于临床,造福人类。