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Embryoid Bodies (embryoid + body)
Terms modified by Embryoid Bodies Selected AbstractsEpigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2006Noelia Andollo Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1-rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation-dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1-rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real-time reverse transcription,polymerase chain reaction analysis showed a higher expression of the U2af1-rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase-I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1-rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA-induced process of differentiation of ES cells. [source] Comparative evaluation of human embryonic stem cell lines derived from zygotes with normal and abnormal pronucleiDEVELOPMENTAL DYNAMICS, Issue 2 2010Qing Huan Abstract Human embryonic stem (hES) cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. To address this question, we generated nine hES cell lines from zygotes containing normal (2PN) and abnormal (0PN, 1PN, 3PN) pronuclei. A side-by-side comparison showed that all cell lines exhibited distinct identity and karyotypical stability. They expressed similar "stemness" markers and alkaline phosphatase activity and differentiated into three embryonic germ lineages in embryoid bodies and teratomas. Under neural differentiation-promoting conditions, they were directed into neural progenitors and neurons. However, a variation in cell cycle and the relative abundance of gene expression of undifferentiated and differentiated markers were observed. These variations were also seen among individually derived normal hES cell lines. Thus, normal hES cell lines can be developed from fertilized zygotes with abnormal pronuclei usually excluded from clinical use. Developmental Dynamics 239:425,438, 2010. © 2009 Wiley-Liss, Inc. [source] Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cellsDEVELOPMENTAL DYNAMICS, Issue 2 2005Sharon Gerecht-Nir Abstract The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large-scale gene screening. Two main clusters were identified,one of down-regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma-derived growth factor-1), LIN28, CD24, TERF1 (telomeric repeat binding factor-1), LEFTB (left,right determination, factor B), and a second of up-regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up-regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGF,3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGF,R2, and TGF,R3, other markers such as CD34, VCAM1, PECAM1, VE-CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development. Developmental Dynamics 232:487,497, 2005. © 2004 Wiley-Liss, Inc. [source] Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2007Malgorzata E. Rula Abstract The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm. genesis 45: 327,338, 2007. Published 2007 Wiley-Liss, Inc. [source] The Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide Exerts Anti-Apoptotic and Differentiating Effects during Neurogenesis: Focus on Cerebellar Granule Neurones and Embryonic Stem CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2007A. Falluel-Morel Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti-apoptotic and pro-differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor. [source] Stromal cell-secreted factors promote the survival of embryonic stem cell-derived early neural stem/progenitor cells via the activation of MAPK and PI3K-Akt pathwaysJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2010Seiji Ishii Abstract Neural stem/progenitor cells (NS/PCs) have been studied extensively with the hope of using them clinically to repair the damaged central nervous system. However, little is known about the signals that regulate the proliferation, survival, and differentiation of NS/PCs in early development. To clarify the underlying mechanisms, we took advantage of an in vitro ES cell differentiation system from which we can obtain neurospheres containing NS/PCs with characteristics of the early caudal neural tube, by treating embryoid bodies (EBs) with a low concentration of retinoic acid (RA). We found that conditioned medium from the PA6 stromal cell line (PA6CM) increased the efficiency of neurosphere formation by suppressing apoptosis and promoting the survival of the NS/PCs. PA6CM also induced the phosphorylation of Erk1/2 and Akt1 in cells derived from the EBs. Furthermore, inhibitors of the MAPK and PI3K-Akt signaling pathways, U0126 and LY294002, attenuated the effects of PA6CM, significantly increasing the number of apoptotic cells and decreasing the number of viable cells among the ES cell-derived NS/PCs. Thus, PA6CM appears to contain soluble factors that promote the survival of ES cell-derived early NS/PCs through the activation of the MAPK and PI3K-Akt pathways. © 2009 Wiley-Liss, Inc. [source] Effects of Ethanol on Mouse Embryonic Stem CellsALCOHOLISM, Issue 12 2009Alla Arzumanyan Background:, Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo. Methods:, To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1,6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin,dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining. Results:, Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation. Conclusion:, Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis. [source] Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia AJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2008S. KASUDA Summary.,Background:,Hemophilia A is an X-chromosome-linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives:,We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. Methods and results:,We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP),brachyury (Bry) and c-kit revealed that GFP,Bry+/c-kit+ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP,Bry+/c-kit+ cells were cultured under the liver cell-promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion:,Our findings suggest the potential for future development of an effective ES cell-based approach to treating hemophilia A. [source] Stable transgene expression in human embryonic stem cells after simple chemical transfectionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2009Jun Liu In this study we used plasmid-based vectors to investigate the transcriptional activities of three commonly used promoters in transient and stable transfection of MEL-1, a human embryonic stem (ES) cell line, using ExGen500, Fugene HD, and Lipofectamine. We demonstrated that cytomegalovirus (CMV), phosphoglycerate kinase (PGK) and human elongation factor-1, (EF1,) promoters all resulted in robust activity of a reporter gene in MEL-1 ES cell transient transfections regardless of the transfection reagent. Stable transfection outcomes varied, depending on the promoter and the transfection reagent used in the study. The phenomenon of transgene silencing was observed, most notably with the CMV vector, with which no positive stably transfected clones were obtained. Of the methods used in the study, Fugene HD resulted in the highest stable transfection rate, estimated by antibiotic selection, with plasmids containing genes under the control of the EF1, or PGK promoters. Stably transfected cells maintained typical hES cell morphology, with immunostaining exhibiting expression of the hES cell markers: Oct4, SSEA4, Tra-1-60, and Tra-1-81. Further, embryoid bodies formed by suspension culture retained reporter gene expression. Following injection into immunodeficient mice, the transfected cell lines showed robust formation of teratomas with cell types representative of the three germ layers. Mol. Reprod. Dev. 76: 580,586, 2009. © 2008 Wiley-Liss, Inc. [source] Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryoMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2007Hathaitip Sritanaudomchai Abstract Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, ,- skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and ,- fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, ,-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes. Mol. Reprod. Dev. 74: 1295,1302, 2007. © 2007 Wiley-Liss, Inc. [source] Isolation and culture of embryonic stem cells from porcine blastocystsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Ming Li Abstract This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7,9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent. Mol. Reprod. Dev. 65: 429,434, 2003. © 2003 Wiley-Liss, Inc. [source] Hyperpolarization-activated cyclic nucleotide-modulated ,HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Yang Qu The hyperpolarization-activated cation current (If), and the hyperpolarization-activated cyclic nucleotide-modulated ,HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 ,m) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation. [source] Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Sylvia Niebruegge Abstract The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called "embryoid bodies" (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493,507. © 2008 Wiley Periodicals, Inc. [source] Novel microfluidic platform for culturing neurons: Culturing and biochemical analysis of neuronal componentsBIOTECHNOLOGY JOURNAL, Issue 11 2009Jeong Won Park Abstract Neurons, one of the most polarized types of cells, are typically composed of cell bodies (soma), dendrites, and axons. Many events such as electric signal transmission, axonal transport, and local protein synthesis occur in the axon, so that a method for isolating axons from somata and dendrites is required for systematically investigating these axonal events. Based on a previously developed neuron culture method for isolating and directing the growth of central nervous system axons without introducing neutrophins, we report three modified microfluidic platforms: (1) for performing biochemical analysis of the pure axonal fraction, (2) for culturing tissue explants, and (3) a design that allows high content assay on same group of cells. The key feature of these newly developed platforms is that the devices incorporate a number of microgrooves for isolating axons from the cell body. They utilize an open cellculture area, unlike the enclosed channels of the previous design. This design has extended the axonal channel so that a sufficient amount of pure axonal fraction can be obtained to perform biochemical analysis. The design also addresses the drawback of the previous neuron culture device, which was not adaptable for culturing thick neuronal tissues such as brain explants, neurospheres, and embryoid bodies, which are essential model tissues in neuroscience research. The design has an open cellculture area in the center and four enclosed channels around open area, and is suitable for multiple drug screening assays. [source] Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier culturesBIOTECHNOLOGY PROGRESS, Issue 2 2010Paulo A. N. Marinho Abstract The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Bioassembly of three-dimensional embryonic stem cell-scaffold complexes using compressed gasesBIOTECHNOLOGY PROGRESS, Issue 2 2009Yubing Xie Abstract Tissues are composed of multiple cell types in a well-organized three-dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue-engineered constructs should have well-defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two-dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well-defined microstructure were constructed into a multilayer cell-scaffold complex using low pressure carbon dioxide (CO2) and nitrogen (N2). The mouse ES cells in the assembled constructs were viable, retained the ES cell-specific gene expression of Oct-4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO2 was replaced with N2. The compressed gas-assisted bioassembly of stem cell-polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] New blood for hemoglobin in the lens: roles in stem cell differentiation and fibre cell organelle loss?ACTA OPHTHALMOLOGICA, Issue 2008MA WRIDE Purpose Evidence is emerging for haemoglobin (Hb) expression outside the vascular system. We previously demonstrated Hb expression in the mouse lens during post-natal development and cataract progression. Here, we extended this work by carrying out a comprehensive spatio-temporal analysis of Hb subunit expression during mouse lens development and maturation. Methods We used RT-PCR, Western blotting and immunofluorescence to analyze Hb expression in mouse eyes (E16.5 to 9 wks). We also used a sensitive heme assay to test for the presence of heme in the lens by colourimetric assay and histological staining of paraffin-embedded sections. Results Hb subunits were expressed in lens epithelial cells and cortical lens fibre cells. However, the heme assay revealed negligible levels of this prosthetic group in the lens. Hb immunofluorescence was also observed in other regions of the developing eye including the cornea, the retinal ganglion cell layer and the retinal pigment epithelium. Finally, we also observed Hb expression in early embryos by microarray and during differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) in vitro. Conclusion These results suggest a paradigm shift: Hb subunits are expressed in the eye during development and in the adult and, therefore, may have novel roles in ocular development, physiology and pathophysiology. The absence of heme from the lens indicates that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of Hb in lens epithelial cells and cortical lens fibre cells may indicate an involvement for Hb subunits in lens epithelial cell differentiation into lens fibre cells and/or lens fibre cell organelle loss. [source] Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process controlBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Magnus Schroeder Abstract It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5,×,106 cells/mL after 9 days of differentiation. Approximately 30%,40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (,MHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28,×,109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc. [source] | ||||||||||||||||||||||