E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells

Conventionally, embryonic stem (ES) cells are cultured on a cell layer of mouse embryonic fibroblasts (MEFs) as feeder cells to support undifferentiated growth of ES cells. In this study, cell–cell interactions between mouse ES and feeder cells were artificially engineered via an epithelial cell adhesion molecule, E-cadherin, whose expression is considerable in ES cells. Mouse mesenchymal STO and NIH3T3 cells that were genetically engineered to express E-cadherin were used in ES cell cultures as feeder cells. ES cells cultured on the E-cadherin-expressing feeder cells maintained the expression of stem cell markers, alkaline phosphatase (AP), Oct3/4, Nanog and Sox2, and the efficiency of AP-positive colony formation was comparable to MEFs, and much better than parental STO and NIH3T3 cells. Furthermore, ES cells maintained on the E-cadherin-expressing feeder cells possessed the ability to differentiate into the three germ layers both in vitro and in vivo. The results indicated that E-cadherin expression in feeder cells could improve the performance of feeder cells, which may be further applicable to create new artificial feeder cell lines.     

Effects of Wnt-10b on proliferation and differentiation of adult murine skin-derived CD34 and CD49f double-positive cells

Although mouse Wnt-10b has been shown to play various roles in a wide range of biological actions, the effects on epithelial stem/progenitor cells in the skin have not been reported. In the present study, we investigated the effects of Wnt-10b on proliferation and differentiation of murine skin-derived CD34 and CD49f double-positive (CD34⁺CD49f⁺) cells, a supposed fraction as enriched epithelial stem/progenitor cells. The cells were prepared from dorsal skin samples obtained from young adult mice as α6 integrin (CD49f) and CD34 double-positive cells by fluorescent activated cell sorting (FACS), and they were cultured with or without Wnt-10b to investigate its effects on proliferation and differentiation. Involvement of canonical Wnt signaling pathway was confirmed by TOPFLASH assay, and differentiation of the CD34⁺CD49f⁺ cells was assessed by RT-PCR analysis and immunocytochemical examinations. The skin-derived CD34⁺CD49f⁺ cells were immunopositive for Lhx2 and expressed mRNA of classical markers for bulge stem cells, including Lhx2, keratin15, Sox9, S100a6, and NFATc1. Their proliferation was suppressed by Wnt-10b, and the markers for differentiated epithelial cells became to be expressed in the culture with Wnt-10b. These results suggest that Wnt-10b promotes differentiation of epithelial stem/progenitor cells in the skin.     

Chemically fixed nurse cells for culturing murine or primate embryonic stem cells

In current and past practice, murine or primate embryonic stem (ES) cells are usually cultured on live nurse cells for growth that keeps the cells in an undifferentiated state. It is troublesome, however, to prepare nurse cells for each cell culture and it is difficult to completely remove the nurse cells when they are transferred. In this study, mouse and monkey ES cells were therefore grown on chemically fixed mouse embryonic fibroblast (MEF) or human amniotic epithelial (HAE) cells. MEF cells were fixed by incubation in a glutaraldehyde or formaldehyde solution. HAE cells were immortalized by transfection of hTERT and chemically fixed with the same reagents. When mouse ES cells were cultured on these chemically fixed cells, the mouse ES cells grew well and expressed alkaline phosphatase, SSEA-1, and Oct-3/4 as their markers, indicating their undifferentiated state. The monkey ES cells also grew well and expressed alkaline phosphatase, SSEA-4, and Oct-4 as their markers, indicating their undifferentiated state. Freeze-drying HAE or MEF cells did not change their ability to support the undifferentiated growth of ES cells. Additionally, the chemically fixed cells could be utilized repeatedly in the culture of ES cells. These results demonstrate that chemically fixed nurse cells are useful for the maintenance of ES cells in an undifferentiated state in culture.     

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.     

Generation of human induced pluripotent stem cells from oral mucosa

Induced pluripotent stem (iPS) cells are one of the most promising sources for cell therapy in regenerative medicine. Using a patient's own genetically identical and histocompatible cells is the ideal way to practice personalized regenerative medicine. For personalized iPS cell therapy, the prerequisites for cell source preparation are a simple and safe procedure, no aesthetic or functional damage, and quick wound healing. Oral mucosa fibroblasts (OFs) may have high potential to fulfill these requirements. In this study, biopsy was performed in a dental chair; no significant incisional damage was recognized and rapid wound healing (within a week) was observed. We generated human iPS cells from the isolated OFs via the retroviral gene transfer of OCT4, SOX2, c-MYC, and KLF4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers such as OCT4, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Subsequent in vitro and in vivo analyses confirmed the pluripotency of resultant iPS cells, which matched the criteria for iPS cells. In addition, we found that the endogenous expression levels of c-MYC and KLF4 in OFs were similar to those in dermal fibroblasts. Taken together, we propose that OFs could be a practical source for preparing iPS cells to achieve personalized regenerative medicine in the near future.     

Development of single mouse blastomeres into blastocysts, outgrowths and the establishment of embryonic stem cells

The recently developed technique of establishing embryonic stem (ES) cell lines from single blastomeres (BTMs) of early mouse and human embryos has created significant interest in this source of ES cells. However, sister BTMs of an early embryo might not have equal competence for the development of different lineages or the derivation of ES cells. Therefore, single BTMs from two- and four-cell embryos of outbred mice were individually placed in sequential cultures to enhance the formation of the inner cell mass (ICM) and the establishment of embryonic outgrowth. The outgrowths were then used for the derivation of ES cell lines. Based on the expression of ICM (Sox2) and trophectoderm (Cdx2) markers, it was determined that ICM marker was lacking in blastocysts derived from 12% of BTMs from two-cell stage and 20% from four-cell stage. Four ES cell lines (5.6%; 4/72) were established ater culture of single BTMs from two-cell embryos, and their pluripotency was demonstrated by their differentiation into neuronal cell types. Our results demonstrate that sister BTMs of an early embryo are not equally competent for ICM marker expression. However, we demonstrated the feasibility of establishing ES cells from a single BTM of outbred mice.     

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).     

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.     

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.     

Developmental ability of cloned embryos from neural stem cells

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.     

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.     

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.     

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.     

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.     

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.