Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.     

Human ES and iPS cells as cell sources for the treatment of Parkinson's disease: Current state and problems

Cell therapy using human embryonic stem cells (hESCs) is a promising therapeutic option for Parkinson's disease (PD), an incurable neurodegenerative disease. A prerequisite for clinical application of hESCs for PD is an efficient and strict differentiation of hESCs into midbrain dopamine (mDA) neuron‐like cells, which would be directly translated into high effectiveness of the therapy with minimum risk of undesirable side effects. Due to fruitful efforts from many laboratories, a variety of strategies for improving efficiency of dopaminergic differentiation from hESCs have been developed, mostly by optimizing culture conditions, genetic modification, and modulating intracellular signaling pathways. The rapid advances in the fields of dopaminergic differentiation of hESCs, prevention of tumor formation, and establishment of safe human induced pluripotent stem cells (hiPSCs) would open the door to highly effective, tumor‐free, and immune rejection‐free cell therapy for PD in the near future. J. Cell. Biochem. 109: 292–301, 2010. © 2009 Wiley‐Liss, Inc.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.     

Phenotype dictates the growth response of vascular smooth muscle cells to pulse pressure in vitro.

The objective of this study was to determine the effect of phenotype on pulse pressure-induced signaling and growth of vascular smooth muscle cells in vitro. Using a perfused transcapillary culture system, cells were exposed to increases in pulsatile flow and hence pulse pressure and maintained for 72 h before cells were harvested. Cell proliferation was determined by cell number, DNA synthesis, and proliferating cell nuclear antigen expression. Mitogen-activated protein kinase (MAPK) levels were determined by immunoblot and kinase activity by phosphorylation of myelin basic protein. Cell phenotype was determined by immunoblot and immunocytofluorescence using antisera specific for the differentiation markers alpha-actin, myosin, calponin, osteopontin, and phospholamban. In cells that highly expressed these differentiation markers, there was a significant increase in cell growth in response to chronic increases in pulse pressure without a significant change in MAPK activity in these cells. In contrast, in cells that weakly expressed SMC differentiation markers, there was a significant decrease in cell growth concomitant with a significant decrease in MAPK signaling in these cells. We conclude that SMC phenotype dictates the growth response of SMC to mechanical force in vitro.     

Lentiviral-RNA-interference system mediating homogenous and monitored level of gene silencing in human embryonic stem cells

Genetic modifications of human embryonic stem cells (hESCs) that will efficiently promote stable homogenous gene silencing, and will also allow monitoring of the silencing level, may be invaluable for the study of function of genes in early human embryogenesis, differentiation, and maintenance of pluripotency of hESCs. RNA-mediated interference (RNAi) emerges as a highly efficient tool for specific knockdown of gene expression. Lentiviruses are efficient vectors for the delivery and stable expression of transgenes in hESCs. We sought to develop a lentiviral-RNAi-based system that will efficiently induce homogenous gene silencing and will allow the monitoring of its relative level in hESCs. Dual-promoter lentiviral vectors coexpressing an RNAi cassette and a reporter gene were initially used for efficient and stable induction of heterogeneous levels of gene silencing in polyclonal hESCs. This step was further combined with the isolation of transduced clones with different homogenous levels of gene silencing. The level of silencing in each of the clones correlated and could be monitored by the level of expression of the vector's reporter transgene. Thus, our system allows easy identification of clones with relatively different homogenous levels of gene silencing. Our approach would be valuable for the study of function of genes, in particular those whose role in hESCs biology depends on their level of expression.     

Transplantation of human embryonic stem cell‐derived endothelial cells for vascular diseases

Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self‐renewal and pluripotency, hESCs and their derived endothelial cells (hESC‐ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However, hESC‐ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases, as well as the monitoring of transplanted cells' fate via molecular imaging. Finally, cell enhancement strategies to improve the engraftment efficiency of hESC‐ECs will be discussed. J. Cell. Biochem. 106: 194–199, 2009. © 2008 Wiley‐Liss, Inc.     

Trolox‐induced cardiac differentiation is mediated by the inhibition of Wnt/β‐catenin signaling in human embryonic stem cells

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/β‐catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6‐Hydroxy‐2,5,7,8‐tetramethylchromane‐2‐carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time‐ and dose‐dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/β‐catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/β‐catenin regulator.     

Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells

Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs.     

Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.     

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment

Background  

We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.     

Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing the VEGF(165) gene

Endothelial progenitors derived from human embryonic stem cells (hESCs) hold much promise in clinical therapy. Conventionally, lineage-specific differentiation of hESCs is achieved through supplementation of various cytokines and chemical factors within the culture milieu. Nevertheless, this is a highly inefficient approach that is often limited by poor replicability. An alternative is through genetic modulation with recombinant DNA. Hence, this study investigated whether transduction of hESCs with an adenoviral vector expressing the human VEGF(165) gene (Ad-hVEGF(165)) can enhance endothelial-lineage differentiation. The hESCs were induced to form embryoid bodies (EBs) by culturing them within low-attachment plates for 7 days, and were subsequently trypsinized into single cells, prior to transduction with Ad-hVEGF(165). Optimal transduction efficiency with high cell viability was achieved by 4-h exposure of the differentiating hESCs to viral particles at a ratio of 1 : 500 for three consecutive days. ELISA results showed that Ad-hVEGF(165)-transduced cells secreted human vascular endothelial growth factor (hVEGF) for more than 30 days post-transduction, peaking on day 8, and the conditioned medium from the transduced cells stimulated extensive proliferation of HUVEC. Real-time PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165)-transduced cells. Additionally, flow cytometric analysis of CD133 cell surface marker revealed an approximately 5-fold increase in CD133 marker expression in Ad-hVEGF(165)-transduced cells compared to the non-transduced control. Hence, this study demonstrated that transduction of differentiating hESCs with Ad-hVEGF(165) facilitated expression of the VEGF transgene, which in turn significantly enhanced endothelial differentiation of hESCs.     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

脐带血来源干细胞神经分化的研究进展

中枢神经系统损伤后的自身修复能力有限,因而研究者致力于寻找一种合适的细胞进行移植以代替受损的神经细胞修复神经损伤。近年来的研究表明,脐带血干细胞能够在体外诱导条件下向神经样细胞分化,并在动物体内实验中促进神经损伤的恢复,有可能作为一种有效的细胞资源,应用于人类中枢神经系统疾病的细胞替代治疗以及神经保护与支持。     

人胚胎干细胞向内皮祖细胞诱导分化及其应用研究进展

近年来,内皮细胞的应用价值不断提高,应用领域不断拓宽,但其来源有限,成为研究应用的主要障碍.胚胎干细胞在体外可分化为多种组织细胞系,有可能成为获取内皮细胞的另一来源.就人胚胎干细胞向内皮祖细胞分化、分离方法、相关分子机制及内皮祖细胞应用价值等进行阐述,以期能够引起更多的关注,推动其研究的进展.     

Uterine smooth muscle cells in primary culture

Summary Smooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45–55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.     

Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.