Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.     

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。     

Expression of phase I and phase II genes in mouse embryonic stem cells cultured in the presence of 2,3,7,8-tetrachlorodibenzo-para-dioxin

Embryonic stem (ES) cells have features that resemble the pluripotent cells of peri-implantation embryos and have been used as an in vitro model to assess the effects of test substances on these stages of development. Here, for the first time, we report on the effects of the xenobiotic 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on mouse ES cells cultured with TCDD at concentrations ranging from 0.0001 to 100 nM for 15 min to 48 h. TCDD effects were determined by analysing the induction of Cyp1A1, Cyp1A2, Cyp1B1 (phase I) and Nqo1, Gsta1, Ugt1a6 (phase II) genes. Cyp1A1 was the phase I gene most rapidly induced (4 h at 1 nM); Cyp1B1 was induced at 48 h (1 nM), whereas Cyp1A2 expression was not affected. TCDD did not alter phase II gene expression, which remained at basal levels throughout the 48 h of culture. We studied more accurately the expression of Cyp1A1, the earliest gene to respond to the presence of TCDD. We found that: 1) Cyp1A1 gene induction is dependent on the duration of exposure (precisely it is first induced after 3 h of culture at 1 nM, the minimum effective-dose); 2) Cyp1A1 induction requires the continuous presence of TCDD, being interrupted 4 h after removal of the xenobiotic; and 3) induced expression of CYP1A1 protein is dependent on TCDD concentration, the higher the concentration the earlier the production of the enzyme. Furthermore, after 48 h of treatment, TCDD did not promote either apoptosis or changes to the differentiation status of the ES cells. These results are the first important step to investigate the effects of dioxin on the very early stages of mammalian development.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Selection of RNA aptamers against mouse embryonic stem cells

Embryonic stem cells (ESCs) are capable of unlimited self-renewal and differentiation into multiple cell types. Recent large-scale analyses have identified various cell surface molecules on ESCs. Some of them are considered to be beneficial markers for characterization of cellular phenotypes and/or play an essential role for regulating the differentiation state. Thus, it is desired to efficiently produce affinity reagents specific to these molecules. In this study, to develop such reagents for mouse ESCs (mESCs), we selected RNA aptamers against intact, live mESCs using several selection strategies. The initial selection provided us with several anti-mESC aptamers of distinct sequences, which unexpectedly react with the same molecule on mESCs. Then, to isolate aptamers against different surface markers on mESCs, one of the selected aptamers was used as a competitor in the subsequent selections. In addition, one of the selections further employed negative selection against differentiated mouse cells. Consequently, we successfully isolated three classes of anti-mESC aptamers that do not compete with one another. The isolated aptamers were shown to distinguish mESCs from differentiated mouse cell lines and trace the differentiation process of mESCs. These aptamers could prove useful for developing molecular probes and manipulation tools for mESCs.     

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.     

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

Engineering tissue from human embryonic stem cells

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.     

Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells

Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.     

Betacellulin and nicotinamide sustain PDX1 expression and induce pancreatic beta-cell differentiation in human embryonic stem cells

The major obstacle in cell therapy of diabetes mellitus is the limited source of insulin-producing β cells. Very recently, it was shown that a five-stage protocol recapitulating in vivo pancreatic organogenesis induced pancreatic β cells in vitro; however, this protocol is specific to certain cell lines and shows much line-to-line variation in differentiation efficacy. Here, we modified the five-stage protocol for the human embryonic stem cell line SNUhES3 by the addition of betacellulin and nicotinamide. We reproduced in vivo pancreatic islet differentiation by directing the cells through stages that resembled in vivo pancreatic organogenesis. The addition of betacellulin and nicotinamide sustained PDX1 expression and induced β-cell differentiation. C-peptide—a genuine marker of de novo insulin production—was identified in the differentiated cells, although the insulin mRNA content was very low. Further studies are necessary to develop more efficient and universal protocols for β-cell differentiation.     

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.     

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.