Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies

Various types of embryoid body (EB) that were formed from mouse embryonic stem (ES) cells under various culture conditions were characterized in terms of gene expression pattern to estimate the differentiation status of the bodies. The gene expression of typical markers (i.e., GATA-4, GATA-6, transthyretin [TTR], alpha-fetoprotein [AFP], Nkx2.5, and alpha-myosin heavy chain [alpha-MHC]) was quantitatively analyzed in various types of EB, and the gene expression pattern of those marker genes was graphically shown for each EB. The gene expression pattern accurately represented the differentiation status of the EBs. The gene expression pattern indicated that the Nkx2.5 and alpha-MHC genes were highly expressed in the EBs formed from 1000 ES cells in a low-adherence 96-well plate. By transferring the EBs into an attachment culture, cardiomyocytes were more efficiently generated in the outgrowth of the EBs. When we increased the seeding cell number from 1000 to 4000 ES cells, the gene expression pattern changed, that is, the expression levels of the TTR and AFP genes increased, whereas those of the Nkx2.5 and alpha-MHC genes decreased, and the trend of differentiation changed from cardiomyogenesis to visceral yolk-sac-like structure formation.     

Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips

The proliferation and differentiation properties of embryoid bodies (EB) from mouse embryonic stem (ES) cells were compared under two microchip conditions: microwell chip and micropatterned chip. The microwell chip contained 270 microwells (diameter, 600 μm; depth, 600 μm) on a polymethylmethacrylate plate and was surface-modified with polyethylene glycol (PEG) to render it non-adhesive. The micropatterned chip contained 270 gelatin spots (diameter, 200 μm) as the cell adhesion area on a glass plate; the region lacking these spots was PEG-modified to render it non-adhesive. The ES cells spontaneously formed the EBs from cell aggregates in each microwell in the chip. In contrast, cells inoculated onto the patterned chip formed a monolayer on the gelatin spots and gradually proliferated to form EBs. The EBs in the patterned chip maintained the high cell growth rate and the expression of endoderm (TTR and AFP) and mesoderm (Nkx2.5, αMHC, Flk1, and PDGFRβ) markers was increased, and these cell properties were similar to the previous methods (hanging drop and round-bottomed 96-well plate cultures). In contrast, the proliferation of ES cells in the microwell chip was lower than in the patterned chip and previous methods, and the EB differentiation proceeded slowly and only formed a small amount of endoderm. These results indicate that the difference of EB generating process in the microchip cultures may affect to the proliferation and differentiation of ES cells, and the existence of microwell structure in the microchip downregulates the cell proliferation and the differentiated progress of ES cells.     

Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells

When cultured in suspension without antidifferentiation factors, embryonic stem (ES) cells spontaneously differentiate and form three-dimensional multicellular aggregates called embryoid bodies (EBs). EBs recapitulate many aspects of cell differentiation during early embryogenesis, and play an important role in the differentiation of ES cells into a variety of cell types in vitro. There are several methods for inducing the formation of EBs from ES cells. The three basic methods are liquid suspension culture in bacterial-grade dishes, culture in methylcellulose semisolid media, and culture in hanging drops. Recently, the methods using a round-bottomed 96-well plate and a conical tube are adopted for forming EBs from predetermined numbers of ES cells. For the production of large numbers of EBs, stirred-suspension culture using spinner flasks and bioreactors is performed. Each of these methods has its own peculiarity; thus, the features of formed EBs depending on the method used. Therefore, we should choose an appropriate method for EB formation according to the objective to be attained. In this review, we summarize the studies on in vitro differentiation of ES cells via EB formation and highlight the EB formation methods recently developed including the techniques, devices, and procedures involved.     

CoCl(2) inhibits neural differentiation of retinoic acid-treated embryoid bodies

The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.     

Differentiation of mouse embryonic stem cells into neurons using conditioned medium of dorsal root ganglia

Mouse embryonic stem (ES) cells, which are continuously growing cell lines, have a pluripotent ability to differentiate into various cell lineages in vitro including neurons. We investigated the effects of chick dorsal root ganglion (DRG) conditioned medium (CM) and nerve growth factor (NGF) on the directed differentiation of ES cells into neurons. Because DRGs from 8-day-old chick embryos are often used in bioassays of neurotrophic factors, DRGs may release soluble factors that can induce ES cell differentiation into neurons in a culture broth. When cultivated in a Dulbecco's modified Eagle's medium (DMEM)/F-12K medium containing DRG-CM or NGF, the ES cell colonies clearly showed neurite outgrowths. Of particular significance, the immunofluorescence analysis of ES cell colonies using an anti-betaIII-tubulin antibody indicated that the addition of DRG-CM effectively promoted the differentiation of ES cells into neurons. We confirmed the effect of DRG-CM addition on ES cell differentiation into neurons via neuronal stem cells by the immunofluorescence analysis of ES cell colonies. Thus, DRG-CM appeared to effectively promote ES cell differentiation into neurons.     

Immobilization of leukemia inhibitory factor (LIF) to culture murine embryonic stem cells

Murine embryonic stem (ES) cells were cultured on a material containing immobilized leukemia inhibitory factor (LIF). To immobilize LIF, we synthesized photoreactive gelatin mixed with LIF and cast the mixture on a polystyrene plate, which was then dried. LIF was immobilized by photoirradiation in the presence or absence of a photo mask. The plate was washed until LIF was no longer released. Murine ES cells were cultured on the immobilized LIF. Activation of STAT3 was maintained on the immobilized LIF for 6 d even after removing soluble LIF. Oct-3/4 was also expressed in the cells cultured on the immobilized LIF. As a result, the mouse ES cells were cultured without differentiating on the immobilized LIF for 6 d. It was possible to culture murine ES cells without adding soluble LIF at each medium change. We conclude that our material containing immobilized LIF might be useful in the culture of murine ES cells.     

Characterization of neurons differentiated from mouse embryonic stem cells using conditioned medium of dorsal root ganglia

Mouse embryonic stem (ES) cells have the pluripotent ability to differentiate in vitro into various cell lineages, including neurons. Adding chick dorsal root ganglion (DRG) conditioned medium (CM) to the culture medium promotes the differentiation of ES cells into neurons. We determined the types of neurons that differentiate from ES cells. The addition of DRG-CM caused nearly half of all ES cells on the periphery of the colony sphere to differentiate into neurons. Immunofluorescence analysis showed that the neurons that differentiated from ES cells were mainly motor, GABAergic, serotonergic, and cholinergic neurons. Of particular note, flow cytometry showed that approximately 50% of betaIII-tubulin-positive neurons were motor neurons. This indicates that DRG-CM induces ES cells to differentiate into motor neurons as target of DRG neurons (sensory neurons).     

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.     

Simultaneous induction of calcium transients in embryoid bodies using microfabricated electrode substrates

Precise control of differentiation processes of pluripotent stem cells is a key component for the further development of regenerative medicine. For this purpose, combining a cell-aggregate-size treatment for regulating intercellular signal transmissions and an electrical stimulation technique for inducing cellular responses is a promising approach. In the present study, we developed microfabricated electrode substrates that allow simultaneous stimulation of embryoid bodies (EBs) of P19 cells. Mouse embryonal carcinoma P19 cells can be induced to differentiate into three germ layers and serve as a promising stem cell model. Microcavity–array patterns were fabricated onto indium–tin–oxide (ITO) substrates using a standard photo-lithography technique, and uniform-sized EBs of P19 cells were inserted into each microcavity. Electrical stimulation was applied to the EBs through substrate electrodes and stimulus-induced intracellular calcium transients were monitored. We confirmed that the developed electrode device could simultaneously stimulate smaller (200 μm diameter) and larger (500 μm diameter) EBs inserted in the microcavities and induce specific spatio-temporal patterns of intracellular calcium transients in the EBs with fine reproducibility. We concluded that the developed microcavity array with embedded electrodes could simultaneously and effectively stimulate uniform-sized EBs inserted in it. Therefore, it is a promising experimental tool for precisely controlling cell differentiation processes.     

Enhancement of cell function through heterotypic cell-cell interactions using E-cadherin-expressing NIH3T3 cells

An epithelial cell adhesion molecule, E-cadherin was expressed in NIH3T3 fibroblasts, and cell-cell interactions between keratinocytes and fibroblasts, or between hepatocytes and fibroblasts were artificially engineered. When the E-cadherin-expressing NIH3T3 cells were co-cultured with rat hepatocytes, the cell-cell contacts were formed with high frequency, and enhanced albumin secretion was observed.     

Effect of ectopic expression of homeoprotein EGAM1C on the cell morphology, growth, and differentiation in a mouse embryonic stem cell line, MG1.19 cells

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, while Egam1c transfectants formed compacted colonies with increased E-CADHERIN expression. In Egam1c transfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genes Fgf5 (epiblast), T (mesoderm), Gata6 (primitive endoderm), and Cdx2 (trophectoderm) in -LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, including Tpbpa (spongiotrophoblast) and Plat (parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.     

Cell microarray for screening feeder cells for differentiation of embryonic stem cells

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.     

Accumulation of neurons differentiated from mouse embryonic stem cells in particular areas of culture plate surface

Nanoscale magnetic beads coated with nerve growth factor (NGF) allow us to accumulate neurons differentiated from mouse ES cells in a selected area of the culture plate surface using a magnet. Neurons with neurite outgrowths within a particular area expressed TrkA and incorporated beads in the soma.     

Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression

Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Since in vitro culture may influence Oct-4 expression, we investigated to what extent blastocysts cultured in vitro from the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We compared in vivo with in vitro derived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells' differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of the in vitro derived blastocysts, 17% gave rise to ES cells vs 38% of the in vivo blastocysts. Six-day old outgrowths from in vivo developed blastocysts expressed Oct-4 in 55% of the cases vs 31% of the in vitro derived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collected in vivo blastocysts compared to in vitro cultured blastocysts. In vitro cultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level than in vivo blastocysts.     

Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.     

Improvement of neuronal cell adhesiveness on parylene with oxygen plasma treatment

We improved adhesiveness of a neuron-like cell, PC12, on a Parylene-C surface by O(2) plasma treatment which changes the surface from hydrophobic to hydrophilic. Neural cell adhesiveness on the plasma-treated Parylene-C was more than twenty times better compared to non-treated Parylene-C and it was close to that on a conventional polystyrene tissue-culture dish.     

Neurite outgrowths of neurons using neurotrophin-coated nanoscale magnetic beads

Neurotrophin-coated nanoscale magnetic beads were used to regulate the differentiation and survival of neurons. The beads coated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) promoted neurite outgrowths of neurons in the same manner as soluble NGF or soluble BDNF, but beads coated with bovine serum albumin did not promote neurite outgrowths. When the volume of NGF-coated bead solution was increased, the number of neurons with neurite outgrowths increased. The addition of anti-NGF antibodies decreased the numbers of neurons with neurite outgrowths in proportion to the volume of anti-NGF antibodies added. NGF-coated beads appeared to bind to soma with neurite outgrowths as determined using fluorescence. In addition, hybrid beads coated with both NGF and BDNF promoted neurite outgrowths of PC12h cells, although the cells did not produce neurite outgrowths in response to BDNF. Neurons with neurite outgrowths could be concentrated within a particular area when NGF-coated beads were immobilized in a particular area of the culture plate surface using a magnet. The results demonstrate that neurotrophin-coated nanoscale magnetic beads allow us to cultivate neurons in a selected area of the culture plate surface by using a magnet. Thus, neurotrophin-coated nanoscale magnetic beads are applicable to micro-integrated systems and biosensors.