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.     

Engineering a mimicry of bone marrow tissue ex vivo

Hematopoietic stem cells reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. The use of cytokines alone for the ex vivo expansion of stem cells in flat, two-dimensional culture flasks, dishes or bags is inadequate and, given the three-dimensionality of the in vivo bone marrow microenvironment, inappropriate. Three-dimensional culture conditions can therefore provide an ex vivo mimicry of bone marrow, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation. Choice of scaffold, manipulation and reproducibility of the scaffold properties and directed structuring of the niche, by choosing pore size and porosity may inform the resident stem cells of their fate in a directed fashion. The use of bioreactors for cultivation of hematopoietic cells will allow for culture control, optimization, standardization, scale-up, and a "hands-off" operation making the end-product dependable, predictable and free of contaminants, and therefore suitable for human use and therapeutic applications.     

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.     

Cell processing engineering for ex-vivo expansion of hematopoietic cells

The cell processing engineering for ex vivo expansion of hematopoietic cells is reviewed. All hematopoietic cells of different lineages and/or at various stages of differentiation are derived from the same precursor, pluripotent hematopoietic stem cells. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. Although stroma-mediated hematopoiesis has been studied in vitro using the Dexter culture system in tissue culture flasks, hematopoiesis in the Dexter culture system is almost limited to a granulocyte lineage and the system could not expand primitive cells. The addition of large amounts of cytokines to the culture of hematopoietic cells enabled their expansion, but is too expensive. Some clonal stromal cell lines have been established from the Dexter culture of murine bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. In order to solve the problem regarding the usage of exogeneic stromal cell lines, a novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was proposed. In order to mimic the contact between stromal and hematopoietic cells in vivo in the bone marrow, several types of three-dimensional (3-D) culture of hematopoietic cells were developed. The 3-D coculture of hematopoietic cells with spatial development of stromal cells in nonwoven fabrics enabled the expansion of progenitors without cytokine addition. Progenitors in cord blood mononucleated cells were also successfully expanded without the addition in the 3-D coculture with primary human bone marrow stromal cells in 3-D. Heparin addition to the 3-D coculture and coating the nonwoven fabrics with N-(O-beta-(6-O-sulfogalactopyranosyl)-6-oxyhexyl)-3,5-bis(dodecyloxy)-benzamide further increased the number of progenitors.     

When nano meets stem: the impact of nanotechnology in stem cell biology

Nanotechnology and biomedical treatments using stem cells are among the latest conduits of biotechnological research. Even more recently, scientists have begun finding ways to mate these two specialties of science. The advent of nanotechnology has paved the way for an explicit understanding of stem cell therapy in vivo and by recapitulation of such in vivo environments in the culture, this technology seems to accommodate a great potential in providing new vistas to stem cell research. Nanotechnology carries in its wake, the development of highly stable, efficient and specific gene delivery systems for both in vitro and in vivo genetic engineering of stem cells, use of nanoscale systems (such as microarrays) for investigation of gene expression in stem cells, creation of dynamic three-dimensional nano-environments for in vitro and in vivo maintenance and differentiation of stem cells and development of extremely sensitive in vivo detection systems to gain insights into the mechanisms of stem cell differentiation and apoptosis in different disease models. The present review presents an overview of the current applications and future prospects for the use of nanotechnology in stem cell biology.     

Recovering vision in corneal epithelial stem cell deficient eyes

A healthy corneal epithelium, which is essential for proper vision and protection from external pathogens, is continuously replenished throughout life by stem cells located at the limbus. In diseased or injured eyes, however, in which stem cells are deficient, severe ocular problems manifest themselves. These are notoriously difficult to manage and as a result the last 20 or so years has seen a number of therapeutic strategies emerge that aim to recover the ocular surface and restore vision in limbal stem cell deficient eyes. The dominant concept involves the generation of laboratory cultivated epithelial cell sheets expanded from small biopsies of the epithelial limbus (for patient or donors) or another non-corneal epithelial tissue such as the oral mucosa. Typically, cells are grown on sterilised human amniotic membrane as a substrate, which then forms part of the graft, or specially formulated plastic culture dishes from which cells sheets can be released by lowering the temperature, and thus the adherence of the plastic to the cells. Overall, clinical results are promising, as is discussed, with new cultivation methodologies and different cell lineages currently being investigated to augment the treatment options for visual disturbance caused by a corneal epithelial limbal stem cell deficiency.     

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.     

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.     

Porcine uterus contains a population of mesenchymal stem cells

The uterus has a remarkable ability of cycling remodeling throughout the reproductive life of the female. Recent findings in the human and mouse indicate that adult stem/progenitor cells may play a prominent role in the maintenance of uterine endometrial and myometrial homeostasis. We aimed to characterize the prospective stem/progenitor cells in the porcine uterus and establish a new model for uterine stem cell research. In this study, we demonstrated that cells isolated from porcine uterus have capacity for in vitro differentiation into adipogenic and osteogenic lineages and express the mesenchymal stem cell (MSC) markers CD29, CD44, CD144, CD105, and CD140b as revealed by RT-PCR. Moreover, we showed that some cells isolated from the porcine uterus when cultured at low density produce large clones with an efficiency of 0.035%. Simultaneously, they were negative for hematopoietic stem cell markers such as CD34 and CD45. Low expression of nestin, which is specific for neural stem cells and various progenitor cells, was also detected. We conclude that the porcine uterus contains a small population of undifferentiated cells with MSC-like properties similar to human and mouse uteri.     

Reprogramming somatic cells into stem cells

Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into multi/pluripotent cells. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer (SCNT), as they may further instruct ex ovo strategies for cellular manipulation.     

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.     

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.     

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.     

Derivation, growth and applications of human embryonic stem cells

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES 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).     

植物细胞培养技术新领域——植物干细胞培养

本文综述了干细胞培养这一植物细胞培养新领域的研究进展,并对干细胞培养体系的建立方法、生长特性以及应用前景进行了综述和探索性展望。     

Germ cells from mouse and human embryonic stem cells

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.