Fine structure of neurally differentiated iPS cells generated from a multiple sclerosis (MS) patient: a case study.

We compared the characteristics of neural cells derived from induced pluripotent stem (iPS) cells from a patient with multiple sclerosis versus neurally differentiated control iPS cells of a healthy individual. The iPS cells were differentiated toward the oligodendrocyte lineage using a four-step protocol established for the differentiation of embryonic stem cells. The resulting cell population was immunostained on day 112 of differentiation for the presence of oligodendrocytes and analyzed by transmission electron microscopy (TEM). Both patient and control samples resembled a mixed population of neural cells rather than oligodendroglia of high purity, including neural stem cell-like cells and possibly oligodendrocytes demonstrable by TEM.

Fine structure of progenitor cells in early ectopic human embryos.

This study has documented the major types of lineage progenitor cells at the second level of cell differentiation after the establishment of the primary germ layers in ectopic human embryos in vivo. These correspond to stages 8 and 9 of embryogenesis (weeks 3-4) in the Carnegie collection. The aim of this study was to provide images of fine structure of tissue progenitor cells to compare them with current imaging of their equivalent stem cells identified using fluorescent stem cell markers. These include neural, mesenchymal, endodermal, ectodermal (epidermal) and haematopoietic progenitor cells, including those for amniotic, yolk sac and chorionic tissues that are used in current stem cell research. Neural induction by the notochord has been imaged. This study should give valuable clues to understand the pattern of cell differentiation of embryonic stem cells (ESC) in vitro, which are more or less mimicked in ESC colonies, embryoid bodies and neurospheres as documented in the literature. The fine structure of week-3 and week-4 human ectopic embryos is presented to demonstrate progenitor tissue cells that will eventually form the brain, spinal cord, skin, gut, heart, blood, muscle, bone and other tissues of the human body later on in development. These images should help stem cell researchers using fluorescent markers and other techniques to identify embryonic and adult stem cells in culture.

The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro.

The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique.

Structural changes of the zona pellucida during fertilization and embryo development.

The zona pellucida (ZP) is a unique extracellular coat surrounding the maturing oocyte, during ovulation, fertilization, and early embryo development. It is formed by three/four glycoproteins. Ultrastructural data obtained with transmission (TEM) and scanning electron microscopy (SEM) were compared with molecular data on the glycoproteins network from ovulation to blastocyst formation. Molecular models are quite different to the morphology obtained with TEM, which shows a microfibrillar architecture, or with SEM, which shows a spongy or smooth surface. The saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide technique allows to show the ZP real microfilamentous structure and the related functional changes. These results support an ultrastructural supramolecular model, more similar and comparable to molecular models related with the glycoprotein network. A detailed mapping of single mammalian ZP proteins and their relationship within the supramolecular architecture of the zona matrix would clearly supply insights into the molecular basis of sperm-egg recognition. Differences in ZP glycoproteins among mammals do not affect structural morphology; further studies are needed to clarify the relationships between ultrastructural and molecular organizations.

Outlook: Derivation of embryonic stem cells for therapy: new technologies.

Embryonic stem cells are currently derived from the inner cell mass of human blastocysts, generated from spare embryos donated for research. To overcome ethical concerns raised by destruction of the embryo, two groups of workers have attempted to derive these cells from isolated blastomeres of 8- to 10-cell stage embryos using the embryo biopsy method akin to that used in preimplantation diagnosis. This paper briefly discusses these two techniques in relation to the routine derivation of stem cells from blastocysts. Some embryological aspects of using the inner cell mass of blastocysts in preference to early embryonic cells are presented. The paper also considers some pitfalls in therapeutic cloning, especially in non-human primates, since legislation to allow this procedure for stem cell research is currently being passed in Australia.

Critical evaluation of human blastocysts for assisted reproduction techniques and embryonic stem cell biotechnology.

Critical examination of 30 blastocysts by transmission electron microscopy (TEM) reveals cellular features not usually evident, including abnormalities of cell structure and aberrations such as multinucleation, internal fragmentation, phagocytic or degenerating cells. Invariably, such blastocysts are inactive and delay or fail to expand and hatch in vitro. Hatching seems to be a major problem in ageing blastocysts due to inactivity of the surface epithelium of trophoblast cells that do not stretch and expand. These lack surface microvilli and contractile tonofilaments that anchor on to specialized cell junctions such as desmosomes. Trophoblast expansion and consequent thinning of the zona is a prerequisite to proper hatching aided by the hydrostatic pressure in the blastocoele and by specialized cells at hatching points. Proper assessment of the inner cell mass is required if a healthy population of cells is to be harvested for embryonic stem cell culture. An inactive blastocyst is obviously not good material and could have a defective inner cell mass (ICM). Normally approximately 3-5% of cells are mitotic in blastocysts and arrested cell division is also an indicator of inactivity. An attempt has been made to evaluate blastocyst internal structure for both assisted reproduction techniques and embryonic stem cell biotechnology.

Mechanics of human blastocyst hatching in vitro.

Critical examination of 30 blastocysts by transmission electron microscopy at various stages of blastulation and hatching, has revealed the presence of specialized, plump, trophoblastic cells at the points of hatching, which seem to aid in initial breaking of the zona pellucida (ZP) and then widen its opening to permit the progressive emergence of the embryo in amoeboid fashion, when it acquires a characteristic dumb-bell shape. These cells are named 'zona-breaker' cells and their characteristics are described. Normally, trophoblast cells in expanding blastocysts are flattened (squamous), forming a continuous robust epithelium with specialized cell junctions. Bundles of tonofilaments anchor onto desmosomes, forming a terminal web. Proper expansion of blastocysts by intake of fluid into the blastocoele causes an increase in internal hydrostatic pressure that stretches the trophoblast epithelium leading to an enlargement of its volume two- to three-fold, consequently thinning the zona prior to hatching. This is an important prerequisite to normal hatching. The blastocysts usually hatch out at the pole opposite the inner cell mass (ICM), though a few hatch out at the embryonal pole or elsewhere. In all cases zona-breakers seem to play a vital role in the hatching process.

The fine structure of human embryonic stem cells.

The fine structure of human embryonic stem (ES) cell colonies was analysed by transmission electron microscopy (TEM) after 35 passages of in-vitro culture. Most cells formed compact, saucer-shaped colonies with epithelioid cells on the periphery and polygonal cells within the colony. Three morphological types of cells were identified based on their fine structure: undifferentiated cells resembling inner cell mass (ICM) cells of blastocysts; protein-synthesizing cells at the onset of cellular differentiation; and compact masses of secretory cells resembling unicellular goblet cells of the intestine. The predominant cell type was the undifferentiated ES cells resembling ICM cells of blastocysts. These cells had large nuclei containing reticulated nucleoli, well-developed rough endoplasmic reticulum (RER), Golgi complexes, elongated tubular mitochondria, lysosomes and typical centrosomes with centrioles associated with microtubules and microfilaments, organizing the cytoskeleton. Some ES cells have very large nuclei and scanty cytoplasms with fewer organelles. The isolated or attached protein-synthesizing cells at the onset of differentiation had extensive RER and large Golgi complexes. The morphologically differentiated cells formed compact colonies and resembled goblet-like cells in microstructure. They had RER and large Golgi complexes associated with secretory vesicles. The epithelioid cells at the periphery were columnar and largely polarized by centrosomes associated with Golgi complexes. Epithelioid cells in all three categories had specialized cell junctions (desmosomes), anchored by tonofilaments, and surface blebs. Isolated cells were seen on the surface, towards the centre of the colony, and their free surfaces had microvilli and larger blebs. Approximately 3-5% of all cells were mitotic, with typical bipolar spindles organized by centrosomes, pivotally located at the poles, and appeared to resemble typical somatic cells.