Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells

ABSTRACT

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3β with IC₅₀ values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Fivefold increase in derivation rates of mouse embryonic stem cells after supplementation of the media with multiple factors

The objective of this study was to determine the ability of multiple-factor supplementation to augment derivation of mouse embryonic stem (mES) cells. Three factors, leukemia inhibitory factor (LIF), Parke-Davis 98059 (PD98059), and 6-bromoindirubin-3′-oxime (BIO), were added as supplements (individually or in a combination of all three) at two consecutive stages of culture; that is, from the start of blastocyst culture to the outgrowth stage, and from putting disaggregated outgrowth into culture medium to generation of primary mES colonies, respectively. The main outcome measure was the percentage of derivable mES cell lines, based on the number of blastocysts initially cultured. Three experiments demonstrated the following: (1) For the addition of individual single factor, only LIF yielded mES cell lines (6.2%), whereas a combination of all three factors resulted in the greatest number of mES cell lines (31.3%). (2) The advantages of a combination of multiple factors (LIF + PD98059 + BIO) were manifested only when they were used during the first stage of the culture and not during the second stage (31.6% vs. 6.2%, respectively). (3) The quality of the inner cell mass (ICM) outgrowth obtained from first-stage culture was studied. After alkaline phosphatase and Oct-4 staining, which documented pluripotency of the embryonic stem cells, outgrowths cultured in multiple factors (LIF + PD98059 + BIO) stained much stronger and in higher proportions than did those obtained after supplementation only with LIF (80% vs. 30%, respectively).     

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.     

Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.     

Expansion of mouse embryonic stem cells on microcarriers

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large-scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling-up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage-dependent cells, a proper support is necessary. Cytodex-3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5-3.9 x 10(6) cells/mL. Improved expansion was achieved using an inoculum of 5 x 10(4) cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real-time PCR and immunostaining analysis.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.     

Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis

Whereas most mammalian cells require extracellular signals to suppress apoptosis, preimplantation embryos can survive and develop to the blastocyst stage in defined medium without added serum or growth factors. Since cells of these embryos are capable of undergoing apoptosis, it has been suggested that their lack of dependence upon exogenous growth factors results from the production of endogenous growth factors that suppress apoptosis by an autocrine signaling mechanism. In the present study, we have examined the growth factor requirements and intracellular signaling pathways that suppress apoptosis in both mouse preimplantation embryos and embryonic stem (ES) cells, which are derived from the blastocyst inner cell mass. Cultured ES cells, in contrast to intact embryos, required serum growth factors to prevent apoptosis. Suppression of ES cell apoptosis by serum growth factors required the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, since apoptosis was rapidly induced by inhibition of PI 3-kinase with LY294002. In contrast, inhibition of MEK/ERK signaling with U0126 or of mTOR with rapamycin had no detectable effect on ES cell survival. Thus, like most mammalian cells, the survival of ES cells is mediated by growth factor stimulation of PI 3-kinase signaling. Treatment with LY294002 (but not with U0126 or rapamycin) similarly induced apoptosis of mouse blastocysts in serum-free medium, indicating that intact preimplantation embryos are also dependent upon PI 3-kinase signaling for survival. These results demonstrate that PI 3-kinase signaling is required to suppress apoptosis of both ES cells and intact preimplantation embryos, consistent with the hypothesis that survival of preimplantation embryos is maintained by endogenous growth factors that stimulate the PI 3-kinase pathway.     

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.     

The effect of extracellular matrix on the differentiation of mouse embryonic stem cells

Embryonic stem cells (ESCs) are promising research materials to investigate cell fate determination since they have the capability to differentiate. Stem cell differentiation has been extensively studied with various microenvironment mimicking structures to modify cellular dynamics associated with the cell-extracellular matrix (ECM) interactions and cell-cell communications. In the current study, our aim was to determine the effect of microenvironmental proteins with different concentrations on the capacity and differentiation capability of mouse ESCs (mESCs), combining the biochemical assays, imaging techniques, Fourier transform infrared (FTIR) spectroscopy, and unsupervised multivariate analysis. Based on our data, coating the surface of mESCs with Matrigel, used as an acellular matrix substrate, resulted in morphological and biochemical changes. mESCs exhibited alterations in their phenotype after growing on the Matrigel-coated surfaces, including their differentiation capacity, cell cycle phase pattern, membrane fluidity, and metabolic activities. In conclusion, mESCs can be stimulated physiologically, chemically, or mechanically to convert them a new phenotype. Thus, identification of ESCs’ behavior in the acellular microenvironment could be vital to elucidate the mechanism of diseases. It might also be promising to control the cell fate in the field of tissue engineering.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.