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PubMed2006

In vivo MR imaging of magnetically labeled human embryonic stem cells.

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.

Animals↗

PubMed2005

Genomic alterations in cultured human embryonic stem cells.

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Cell Culture Techniques↗

PubMed2005

The cryopreservation of human embryonic stem cells.

hES (human embryonic stem) cells hold tremendous potential in the newly emerging field of regenerative medicine, in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening. However, an essential prerequisite for the future widespread application of hES cells are the development of efficient cryopreservation protocols to facilitate their storage and transportation. This review summarizes the current state of progress in the field of hES cell cryopreservation, by critically examining and comparing the various cryopreservation protocols that have been developed. These can be classified into two categories: (1) conventional slow-cooling protocols and (2) vitrification protocols. Previously, the application of slow-cooling cryopreservation protocols to freely-suspended hES cell clumps yielded extremely dismal results. However, a recent study demonstrated that post-thaw survivability was markedly improved when slow-cooling protocols were applied instead to adherent hES colonies. Vitrification protocols have been shown to be much better than the standard slow-cooling protocol for the cryopreservation of freely suspended hES cell clumps. However, no study has yet attempted to apply vitrification protocols to adherent hES colonies. It must be noted that vitrification protocols are extremely labour-intensive and tedious to perform manually. Additionally, the use of cryostraws in vitrification protocols is unsuited for handling bulk quantities of hES cells, in addition to posing serious technical difficulties in developing machine automation for cryopreservation. These are some of the major challenges that have to be overcome if further progress is to be made in this field.

Animals↗

PubMedDate not supplied

Four moral questions for human embryonic stem cell research.

Human embryonic stem cell research offers great promise for the treatment of many serious disease conditions in a variety of medical areas, including wound healing. Before this promise can be realized, society and individual researchers, clinicians, and patients will have to answer four ethical questions: 1) Can we ever intentionally destroy a human embryo? 2) Can we benefit from others' destruction of embryos? 3) Can we create an embryo to destroy it? 4) Can we clone human embryos? After outlining the issues raised by each question, the author concludes by indicating his own affirmative answer to each of these questions.

Bioethics↗

PubMed2006

The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells.

As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).

Benzimidazoles↗

PubMed2005

X-inactivation status varies in human embryonic stem cell lines.

Human embryonic stem cells (hESCs) derived from human blastocysts have an apparently unlimited proliferative capacity and can differentiate into ectoderm, mesoderm, and endoderm. As such, hESC lines have enormous potential for use in cell replacement therapies. It must first be demonstrated, however, that hESCs maintain a stable karyotype and phenotype and that gene expression is appropriately regulated. To date, different hESC lines exhibit similar patterns of expression of markers associated with pluripotent cells. However, the evaluation of epigenetic status of hESC lines has only recently been initiated. One example of epigenetic gene regulation is dosage compensation of the X chromosome in mammalian females. This is achieved through an epigenetic event referred to as X-chromosome inactivation (XCI), an event initiated upon cellular differentiation. We provide the first evidence that undifferentiated hESC lines exhibit different patterns of XCI.

Aneuploidy↗

PubMed2006

Diverse epigenetic profile of novel human embryonic stem cell lines.

Human embryonic stem cells (hESCs) are a promising model for studying mechanisms of regulation of early development and differentiation. OCT4, NANOG, OCT4-related genes and some others were recently described to be important in pluripotency maintenance. Lesser is known about molecular mechanisms involved in their regulation. Apart from genetic regulation of gene expression epigenetic events, particularly methylation, play an important role in early development. Using RT-PCR we studied the expression of pluripotency-related genes OCT4, NANOG, DPPA3 and DPPA5 during hESCs differentiation to embryoid bodies. Analysis of methylation profiles of promoter or putative regulatory regions of the indicated genes demonstrated that expression of the pluripotency-maintaining genes correlated with their methylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. We propose that DNA methylation underlies the developmental stage-specific mechanisms of pluripotency-related genes expression and reactivation and may have an impact on differentiation potential of hESC lines.

5' Flanking Region↗

PubMed2006

Propagation of human embryonic stem cells on human feeder cells.

Human embryonic stem (hES) cell lines are usually derived and propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and culture ingredients of animal origin for hES cell support increases the risk of cross-contamination of the hES cells with infectious animal agents from the MEFs and animal-based culture medium. This thus makes such hES cells lines undesirable for clinical application. This chapter describes several protocols used in the propagation of hES cells on human fibroblast feeder cells. Two culture methods, the bulk enzymatic culture protocol and the microdissection "cut and paste" protocol are described. Only certain human fetal and adult fibroblast feeders support hES cell growth. Methods for the characterization of pluripotent undifferentiated hES cells grown on human feeders including cell surface marker staining and real-time polymerase chain reaction are also described.

Alkaline Phosphatase↗

PubMed2006

[The effects of embryonic bodies on differentiation of mouse embryonic stem cells to neuronal cells in vitro].

This study was directed at assessing the effects of embryonic bodies on differentiation of the embryonic stem cells. The embryonic bodies of different days gained by means of hanging, suspending and hung-suspended were processed by all-trans retinotic acids for 4 days. Then they were detected by immunocytochemistry methods and were measured to detect the fluorescence intensity changes of calcium after Glu stimulation. During this period, we observed the differentiation process and compared the differentiation ratio of embryonic stem cells processed by means of hanging, suspending for several days. It was found that the differentiation ratio of EBs obtained by suspending for 3 days, or by hanging for 3 days and suspending for 1 day, is relatively higher, which can help us find out the intrinsic differentiation mechanism of embryonic stem cells.

Animals↗

PubMed2005

Embryonic stem cells: a perspective.

Embryonic stem (ES) cells are proliferating tissue-culture cells which are embryonic in origin and retain the full lineage and differentiative capacity of the early mammalian embryo. Only mouse ES cells have been fully characterized but similar cultures have been isolated from numbers of other mammalian embryos including, notably, human. ES cells can differentiate either in vivo in the context of an embryo or equivalently in vitro. Their ability to undergo genetic alteration and selection in vitro followed by embryonic development in vivo has led to their extensive use as a vector for mammalian genetic engineering. This is not an application for human ES cells but their ability to differentiate in vitro into a plethora of specific tissue precursors has led to scenarios for their use in cellular and tissue engineering therapies. Ethical and practical problems for the provision of ad hominem human ES cells remain. Knowledge from the mouse system may aid our understanding of the relationship between ES cells and embryos.

Animals↗

PubMed2006

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.

Animals↗

PubMed2005

Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis.

We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.

Alternative Splicing↗

PubMed2006

Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events.

BACKGROUND: An effective embryonic-maternal interaction is crucial for successful human pregnancy. Failure of this process is a major cause of infertility and can lead to placental dysfunction resulting in recurrent miscarriage, fetal retardation and pre-eclampsia. Research is severely constrained by ethical and practical considerations; therefore, we aimed to generate cytotrophoblast stem (CTBS) cell lines from human embryonic stem cells (HESCs). METHOD: Beta-HCG was used as a marker of viable trophoblast cells. In defined culture, embryoid bodies were generated from HESCs and selected for trophoblast enrichment by rounds of cellular aggregation and disaggregation. Distinct CTBS cell lines were isolated and characterized. Spheroid cytotrophoblast bodies were generated and their interaction with luteal-phase endometrial stroma was analysed by real-time image analysis. RESULTS: Three CTBS cell lines were derived, which were maintained in the absence of residual HESCs, fibroblast feeder cells or extracellular matrix. CTBS cells displayed typical cytotrophoblast and syncytiotrophoblast characteristics and exhibited further differentiation to invasive endovascular cell phenotype. One cell line was generated with constitutive expression of enhanced green fluorescent protein (eGFP). Spheroid trophoblast bodies mimicked closely the early invasive stages of implantation when incubated with human endometrial stromal preparations in vitro. CONCLUSION: These human CTBS cell lines are a significant new model for investigating human placentation and may have considerable potential in cell therapy applications.

Cell Differentiation↗

PubMed2007

Expansion of mouse embryonic stem cells on microcarriers.

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large-scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling-up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage-dependent cells, a proper support is necessary. Cytodex-3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5-3.9 x 10(6) cells/mL. Improved expansion was achieved using an inoculum of 5 x 10(4) cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real-time PCR and immunostaining analysis.

Animals↗

PubMed2006

Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients.

Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis.

Animals↗

PubMed2000

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.

Animals↗

PubMed2005

Comparative characteristics of three human embryonic stem cell lines.

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.

Animals↗

PubMed2002

Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

BACKGROUND AND AIMS: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ-stained cellular clusters originating from ES cells. METHODS: Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to form outgrowths in the culture. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/ml ) for 15 minutes before being examined microscopically. The gene expression of endocrine pancreatic markers was also analyzed by reverse transcriptase-polymerase chain reaction. In addition, insulin production was examined immunohistochemically, and its secretion was examined using enzyme-linked immunosorbent assay. RESULTS: DTZ-stained cellular clusters appeared after approximately 16 days in the EB culture and became more apparent by day 23. They were found to be immunoreactive to insulin and expressed pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1, proinsulin 2, glucagon, pancreatic polypeptide, glucose transporter-2 (GLUT2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) mRNA. They were also able to secrete detectable amounts of insulin. CONCLUSIONS: ES cell-derived DTZ-positive cellular clusters possess characteristics of the endocrine pancreas, including insulin secretion. Further, DTZ staining is a useful method for the identification of differentiated pancreatic islets developed from EBs in vitro.

Animals↗