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.     

Culture systems for pluripotent stem cells

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application.     

Available human feeder cells for the maintenance of human embryonic stem cells

Mouse embryonic fibroblasts (MEFs) have been previously used as feeder cells to support the growth of human embryonic stem cells (hESCs). In this study, human adult uterine endometrial cells (hUECs), human adult breast parenchymal cells (hBPCs) and embryonic fibroblasts (hEFs) were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells. Cultured hUECs, hBPCs and hEFs were mitotically inactivated and then plated. hESCs (Miz-hES1, NIH registered) initially established on mouse feeder layers were transferred onto each human feeder layer and split every 5 days. The morphology, expression of specific markers and differentiation capacity of hESCs adapted on each human feeder layer were examined. On hUEC, hBPC and hEF feeder layers, hESCs proliferated for more than 90, 50 and 80 passages respectively. Human feeder-based hESCs were positive for stage-specific embryonic antigen (SSEA)-3 and -4, and Apase; they also showed similar differentiation capacity to MEF-based hESCs, as assessed by the formation of teratomas and expression of tissue-specific markers. However, hESCs cultured on hUEC and hEF feeders were slightly thinner and flatter than MEF- or hBPC-based hESCs. Our results suggest that, like MEF feeder layers, human feeder layers can support the proliferation of hESCs without differentiation. Human feeder cells have the advantage of supporting more passages than when MEFs are used as feeder cells, because hESCs can be uniformly maintained in the undifferentiated stage until they pass through senescence. hESCs established and/or maintained under stable xeno-free culture conditions will be helpful to cell-based therapy.     

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.     

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.     

Possibility of insulin-producing cells derived from mouse embryonic stem cells for diabetes treatment

Insulin injection therapy is the principal current treatment of type 1 diabetes. Patients, however, suffer from various complications generated by insufficient control of blood glucose levels over a long period. Therefore, a method which can infuse insulin in response to changes of blood glucose levels is eagerly desired. Transplantation of insulin releasing cells derived from embryonic stem (ES) cells has been expected to be one of promising approaches to realize this requirement. In this study, ES cell progeny which were derived in culture media with/without fetal calf serum contained two distinct kinds of cells immunostained by anti-insulin and anti-C-peptide antibodies. The cytoplasm and nuclei of one type of cell were immunoreactive against antibodies for insulin, while the other kind of cell only had the cytoplasm stained by the anti-insulin antibody. The first cell type was the major population of insulin-positive cells in serum-free medium, while the latter kind of cells was the major population in medium containing serum. Interestingly, the latter insulin-positive cells could be also immunostained by anti-C-peptide antibodies, and was observed even after nine subcultures in medium containing serum. Although there still remain many issues to be addressed in order to definitely demonstrate that insulin-positive cells derived from ES cells to be truly beta cells in the islets, these properties of the obtained cells are believed to promising cells for treatment of type 1 diabetes.     

金枪鱼鱼骨活性钙对鼠胚胎成骨细胞前体细胞(MC3T3-E1)活力和凋亡作用

目的:以金枪鱼骨为原料,制备柠檬酸-苹果酸钙剂(CMC),研究其对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的细胞活力、凋亡的影响,并探讨其作用机制。方法:在小鼠胚胎成骨细胞前体细胞(MC3T3-E1)培养液中,分别加入高浓度(1 μg/mL)CMC和低浓度(0.1 μg/mL)CMC,利用MTT法检测对MC3T3-E1细胞活力的影响,流式细胞仪检测血清饥饿诱导的细胞凋亡。结果:高浓度和低浓度的钙对MC3T3-E1细胞活力均有显著的促进作用,24 h的高钙组的细胞增长率(9.67%)优于低钙组(8.95%);36 h和48 h后低钙组的增长率分别为14.96%和20.33%,明显优于36 h和48 h作用后的高钙组(6.23%和1.73%),两组细胞凋亡率与空白组比较差异较小,且均显著低于对照组(p<0.01)。结论:CMC高钙组和CMC低钙组在一定浓度范围内,能促进MC3T3-E1细胞活力显著增强,并且改善血清饥饿诱导的成骨前体细胞凋亡。     

Electrospun zein/PVA fibrous mats as three-dimensional surface for embryonic stem cell culture

This paper reports a new method of producing electrospun zein/polyvinyl alcohol (PVA) mats as three-dimensional (3D) cell culture surface material. The electrospun structure has many advantages and protein-based biomaterials possess unique properties preferred for cell biocompatibility. However, electrospun fiber matrices developed from proteins have poor mechanical properties and morphological stability in the aqueous environments required for cell culture. Efforts have been made to improve the properties of electrospun protein scaffolds, including dry mechanical for handling and surface hydrophilicity, but the current methods have major limitations, such as cytotoxicity and low efficiency. In this research, experimental data showed that the adding different proportion of PVA influenced the properties of the zein mats. Considering the properties of the mats and mouse embryonic stem cells growth behavior on the various electrospun mats, the EM3 (zein/PVA with mass ratios of 3:1) showed better growth status than any other mats. EM3 affixed to cover-glass could be autoclaved for 30?min at 120°C. In addition, embryonic stem cells cultured on chosen electrospun zein/PVA mats in vitro proved that culture products were easily attached onto mat and mat could be used for induction of stem cell differentiation and therefore promising mats for 3D cell culture surface applications.     

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.