Embryonic stem cells have huge potential in the field of tissue engineering and regenerative medicine as they hold the capacity to produce every type of cell and tissue in the body. In theory, the treatment of human disease could be revolutionized by the ability to generate any cell, tissue, or even organ, 'on demand' in the laboratory. This work reviews the history of murine and human ES cell lines, including practical and ethical aspects of ES cell isolation from pre-implantation embryos, maintenance of undifferentiated ES cell lines in the cell culture environment, and differentiation of ES cells in vitro and in vivo into mature somatic cell types. Finally, we discuss advances towards the clinical application of ES cell technology, and some of the obstacles which must be overcome before large scale clinical trials can be considered.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2184.2004.00298.x

Embryonic stem cells.

Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

https://www.pnas.org/content/pnas/101/34/12543.full.pdf

Derivation of midbrain dopamine neurons from human embryonic stem cells

What makes a stem cell is still poorly understood. Recent studies have uncovered that chromatin might hold some of the keys to how embryonic stem cells maintain their pluripotency, their ability to self-renew and induce lineage specification. Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.

Chromatin in pluripotent embryonic stem cells and differentiation

Global Transcription in Pluripotent Embryonic Stem Cells

This Review describes the diverse roles for histone-modifying and chromatin-remodelling enzymes in mammalian differentiation. These enzymes are involved in both maintaining pluripotency and specifying cell lineage commitment. Recent progress includes their functional characterization in mouse modelsin vivoand a new appreciation of their multifaceted molecular functions. Cellular differentiation is, by definition, epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodelling during differentiation, which results in a progressive transition from a fairly open chromatin configuration to a more compact state. Genetic studies in mouse models show major roles for a variety of histone modifiers and chromatin remodellers in key developmental transitions, such as the segregation of embryonic and extra-embryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation and the differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes that are driven by developmental transcription factors, there is emerging evidence that chromatin regulators have multifaceted roles in cell fate decisions.

/pdf/chromatin-modifiers-and-remodellers-regulators-of-cellular-1566pn9q2g.pdf

Chromatin modifiers and remodellers: regulators of cellular differentiation

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.

Background Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. Methods Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. Results Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. Conclusions This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.

/pdf/establishment-of-human-embryonic-stem-cell-lines-from-frozen-5calwvovmb.pdf

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

Developmental capacity of human multipronuclear (PN) zygotes cryopreserved using an ultra-rapid freezing method and electron microscope (EM) grids was studied. Multipronuclear zygotes obtained from a human IVF programme were used as an alternative to normal 2PN zygotes; they were divided into 3PN or >or =4PN zygotes and their in-vitro development and cryo-injury were compared according to PN number. EFS30, which consisted of 30% ethylene glycol, 18% Ficoll, 0. 5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline was used as the freezing solution. After ultra-rapid freezing and thawing 85.5% of multipronuclear zygotes survived. A comparison of cleavage rates between the control and cryopreserved groups showed no significant difference (3PN; 81.3 and 85.4% and > or =4PN; 90.0 and 95.7% respectively). Comparing the in-vitro development after thawing up to blastocyst formation on day 5 after IVF, the outcome of the frozen 3PN group (22.0%) was not different from that of control 3PN group (38.5%), while the outcome of the frozen > or =4PN group (4.5%) was significantly lower than that of control > or =4PN group (44.4%) (P < 0.05).

Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids

This study demonstrates that higher survival of vitrified-thawed bovine blastocysts can be obtained using electron microscope (EM) grids as embryo containers at freezing, rather than plastic straws. In-vitro produced day 7 bovine blastocysts after in-vitro fertilization (IVF) were vitrified on grids or in straws with EFS40 freezing solution and their survival after thawing was compared. Embryo survival was assessed as re-expanded and hatched rates at 24 and 48 h after thawing respectively. When the effects of exposure to vitrification solution and chilling injury from the freezing procedure were examined, embryo survival in the exposure group (24 h: 100, 48 h: 73.3%) was not different compared with that in the control group (100, 84.4%). After vitrification, the hatched rate of the EM grid group 48 h after thawing (67.8%) was significantly higher than that of the straw group (53.3%) (P < 0.05). Fast developing embryos (expanded blastocyst and early hatching blastocyst stage) showed better resistance to freezing than delayed ones (early blastocyst stage), irrespective of embryo containers (early: 24 h, 57.1 and 48 h, 24.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) (P < 0.001). When using expanded and early hatching blastocysts, embryo survival rates in the vitrification-EM grid group (67.8, 95.0% respectively) were significantly higher than that of the vitrification-straw group (53.0, 65.0%) at 48 h.

Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids

Ultrastructural and immunohistochemical studies of 4 groups of cells—(human embryonic stem cells (hES), embryoid bodies (EB), and spontaneously and retinoic acid (RA)-induced differentiating cells)—were carried out to investigate their detailed phenotype. Immunohistochemically, the EB cells showed strong immunoreactivity for CD34, CD117, and nestin. Differentiating cells expressed pancytokertin, vimentin, CD31, CD56, GFAP, nestin, and NeuN as well as CD34, and c-Kit. However, synaptophysin and neurofilaments were not present in these same differentiating cells. Transmission electron microscopy showed that hES and EB cells were very similar to germ cells or cells of the inner cell mass. Spontaneously and RA-induced differentiating cells exhibited epithelial, mesenchymal, endodermal, and neuronal phenotypes. The perikarya of the neuronal cells had rich RERs (Nissl substance) and long cytoplasmic processes filled with numerous neural tubules. However, neither synaptic junctions nor synaptic vesicles were dev...

https://www.tandfonline.com/doi/pdf/10.1080/01913120490515595

Se-Pill Park

Papers

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids

Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids

Ultrastructure of human embryonic stem cells and spontaneous and retinoic acid-induced differentiating cells.