Generation of Functional Human 3D Cortico-Motor Assembloids.

Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functional circuits that could be used to understand development and disease.

Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.

Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)-positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.

[Morphohistochemical and electron microscopic characteristics of malignant tumors of the upper respiratory tract after combined local hyperthermia and radiotherapy]. / Morfogistokhimicheskaia i élektronno-mikroskopicheskaia kharakteristika zlokachestvennykh opukholei verkhnikh dykhatel'nykh putei pri sochetanii lokal'noi gipertermii i luchevoi terapii.

The paper discusses morphohistochemical and electron microscopic characteristics of 15 primary tumors of the upper respiratory tract removed following preoperative irradiation in combination with local hyperthermia as a radio-modifying factor. Eighteen tumors removed after irradiation alone served as controls. Application of local microwave hyperthermia resulted in a higher degree of irreversible tumor cell dystrophia and damage. Hyperthermia--induced damage included disorders in ultrastructure of intracellular membranes and cell organelles mainly due to derangement of membrane--binding proteins, protein--lipid and nucleotide complexes. Thermoradiotherapy stimulated protective reaction of surrounding tissue which took the form of increase in fraction of leukocytes and macrophages with high acid phosphatase level in areas of tumor cell damage.

Ultrastructural and immunohistochemical characterization of submandibular duct cells in culture and modification of outgrowth differentiation by manipulation of calcium ion concentration.

The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.

Organelle dynamics in lobster axons: anterograde and retrograde particulate organelles.

Particulate organelles in isolated axons from the walking legs of the lobster were detected with differential interference contrast optics and video microscopic techniques. The motion of the organelles was studied in normal axons, in axons whose surface membrane was rendered permeable with saponin, and in axoplasm extruded from the axons. In normal axons at 20-22 degrees C, organelles moved more rapidly in the anterograde direction than in the retrograde direction (respective mean velocities 1.73 micron/s and 0.63 micron/s). The instantaneous velocities of both sets of organelles were variable: those of the anterograde organelles varied less than those of retrograde organelles. The variation in instantaneous velocity was patterned; all organelles studied had velocities that fluctuated slowly with a major frequency at about 0.1 Hz. Some organelles oscillated about a fixed position at a similar major frequency. In axons with a permeabilized surface membrane there was no organelle motion unless adenosine 5'-triphosphate (ATP) was present in the bathing medium. Organelle motion reactivated with ATP was patterned in a way similar to that in normal intact axons. In extruded axoplasm in the presence of ATP, organelles moved along transport filaments that were assumed to be microtubules. Movement of organelles from one transport filament to another was not accompanied by changes in motion that could explain the normal fluctuation in velocity. The evidence indicates that the variable, or oscillatory, character of organelle motion in lobster axons is caused by an active component of the mechanisms of axonal transport.

Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.

To study the roles played by cardiac valvular endothelium in normal and pathologic conditions, we have established and characterized a system of bovine valvular endothelial cells (VEC) in culture. Viable VEC from calf atrioventricular valves were obtained by a non-enzymatic procedure using 3 mM ethylenediamine-tetraacetic acid (EDTA) as dissociating agent. The cells grown in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, vitamins and 20% fetal calf serum, developed as monolayers of closely apposed polygonal cells which were subcultured for up to seven passages. VEC maintained in culture the general ultrastructure displayed in vivo, expressed von Willebrand factor, presented angiotensin converting enzyme activity and synthesized a rich extracellular matrix. VEC preserved the cell surface anionic sites (detected with cationized ferritin, pI 8.4) and cationic sites (visualized with haemeundecapeptide pI 4.85), and took up, especially by adsorptive endocytosis, albumin-gold conjugate. The cells were coupled by functional communicating (gap) junctions, as demonstrated by microinjection of 6-carboxyfluorescein. VEC in culture produced fibronectin, prostacyclin, hyaluronic acid and heparin-like glycosaminoglycans (identified by electrophoresis, enzyme digestion, and deaminative cleavage of molecules). These properties render cultured VEC a suitable model for investigating their functions and involvement in normal and pathologic heart valves.

An electron microscopic and spectroscopic study of murine epiphyseal cartilage: analysis of fine structure and matrix vesicles preserved by slam freezing and freeze substitution.

Newborn mice epiphyseal growth plates were preserved by slam freezing/freeze substitution and examined by conventional electron microscopy, stereopsis, high voltage electron microscopy, and electron spectroscopic imaging (ESI). To illustrate the improved ultrastructure of this cryogenic procedure, conventional, aqueously fixed growth plates were included showing collapsed hypertrophic chondrocytes surrounded by a depleted and condensed extracellular matrix. In contrast, the cryogenically prepared epiphyses contain chondrocytes and extracellular matrix vesicles both in direct contact with proteoglycan filaments retained in an expanded state. ESI is an electron microscopic technique which enables the direct localization of atomic elements superimposed over fine structural details. This technique was used to examine the colocalization of calcium and phosphorus within matrix vesicles and within their associated extracellular environments. Matrix vesicles appeared in three distinct diameter ranges. The integrity of the matrix vesicles was examined at various stages of mineralization and also within the mineralized zone of provisional calcification.

Three-dimensional electron microscopy of the internal nucleolus-associated chromatin and of the nucleolar vacuoles during early germination of Sinapis alba.

Spatial relationships between the internal nucleolus-associated chromatin (NAC) and the numerous nucleolar vacuoles that appear during early germination have been studied in nucleoli of quiescent (non-germinated) and early germinating embryos of Sinapis using serial sections. In quiescent non-vacuolated nucleoli, the transcriptionally inactive internal NAC is a short strand about 900 nm thick that in cross-section appears as heterogeneous fibrillar centres (FCs). At 4 and 6 h after germination one or several large networks of interconnected nucleolar vacuoles develop around the dispersing internal NAC. Clumps of dense chromatin are still present within the nucleolar vacuoles and are probably unfolding into deoxyribonucleoprotein (DNP) fibres (about 110 nm thick), which rapidly intrude within the nucleolar body and form thin chromatin threads. At 24 h after germination the internal NAC is more dispersed and forms, for its greatest part, a long thread (about 240 nm in diameter) wrapped up with a few dense fibrillar component, the whole forming the first outline of a nucleolonema. In cross-section most of the internal NAC appears as homogeneous FCs but short portions remain more condensed and appear as heterogeneous FCs always associated with a nucleolar vacuole. From 48 h the internal NAC is a longer thinner strand (about 160 nm in diameter), probably continuous and surrounded entirely by a homogeneous muff of dense fibrillar component, the whole forming a typical nucleolonema (about 950 nm thick) meandering throughout the nucleolus. Small amounts of the internal NAC still remain undispersed in the form of heterogeneous FCs associated with a nucleolar vacuole. The repeated association of nucleolar vacuoles and dispersing internal NAC suggests that they could play a role in chromatin dispersion and, or, activation by creating a favourable microenvironment.

Ultrastructure of aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall).

Six types of setae and one type of cuticular depression were examined on the lateral antennule of the spiny lobster Panulirus interruptus using scanning electron microscopy. The organization and ultrastructure of the innervation of the most numerous setal type, the aesthetasc, were investigated using light- and transmission electron microscopy. Each aesthetasc is innervated by approximately 300 bipolar neurons whose sensory dendrites penetrate the hair and extend toward the tip, and whose axons project towards the central nervous system. The neuronal somata and two types of glia form a cluster within the antennular lumen. The inner sheath-cell somata encircle the dendritic tract distal to the sensory somata. These cells appear to extend distal processes which wrap the dendritic tract to the base of the aesthetasc. Elongate outer sheath cells are interposed between the glia-wrapped dendritic tract and the hypodermis which underlies the antennule cuticle. A continuous investment of neural lamella separates the hypodermis, the entire cluster of somata, and sensillar nerve from the antennule lumen. The organization of the neuronal somata and their association with outer and inner sheath cells in this marine species appear similar to those of crustaceans from freshwater and terrestrial habitats.

Structural changes induced in capillary endothelial cells by growth factors: altered distribution of cytoskeletal elements and organelles in cells spreading in the presence of tumor conditioned medium and hypothalamus derived growth factor.

Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.

A quantitative ultrastructural evaluation of the cell organelle specificity of the uranaffin reaction in normal human platelets.

The purpose of this study was to determine the normal variation in the number of uranaffin-positive organelles in normal human platelets and to study the effect of pH and fixation on the uranaffin reaction. The normal variation in uranaffin organelles was determined by studying platelets from nine normal subjects. At pH 3.9, the mean number of reactive sites/platelet profile was 0.43 +/- 0.10 when platelets were fixed with lower glutaraldehyde concentrations and 0.56 +/- 0.24 with higher glutaraldehyde concentrations. Fixed platelets were reacted at pH 2.8, 3.9, 5.0, and 7.0 with four different uranaffin procedures that varied in the extent of fixation and rinse steps (isotonic saline vs. cacodylate buffer). The number of uranaffin-positive sites in 200 platelet profiles was scored under the electron microscope. There was a progressive increase in the number of reactive sites/platelet profile as the pH increased from 2.8 to 7.0. In general, higher pH favored granule matrix and core staining, whereas low pH favored both the staining of granule membranes and their contents. The uranaffin reaction showed organelle specificity when run under certain experimental conditions. At low pH and using isotonic saline in the rinse steps, only the dense bodies and ribosomes stained. The biochemical content of the dense body responsible for uranaffin reactivity is discussed.

The effects of heat on Sporothrix schenckii in vitro and in vivo.

In order to clarify the mechanism of action of topical thermotherapy on sporotrichosis, the effects of heat on Sporothrix schenckii in vitro and in vivo were investigated by observing the percentage germination and the ultrastructure. When the spores were heated to 42 degrees C, it took 10 hr with the conidia, 2 hr with the yeast-like cells and 1 hr with the spores in vivo to reduce the germination rates to 10%. The percentage germination curves were reduced slowly at first but later exponentially. Changes in the ultrastructure became evident in 2 hr with the yeast-like cells and in 8 hr with the conidia. The ribosome count declined and amorphous dense materials appeared in the cytoplasm and mitochondria. In vivo, the outstanding feature of the heated spores was the diversity of internal ultrastructural changes encountered and morphological changes. These were observed at 1 hr post treatment.

Electron microscopical studies on the nerve endings of the outer hair cells in acoustically exposed rabbits.

The afferent and efferent nerve endings of the outer hair cells of the rabbit after acoustic exposure were investigated under the electron microscope. After acoustic stimulation, the afferent and efferent nerve endings of normal range were respectively 57 per cent and 64 per cent. This result suggests that half of the afferent and efferent nerve endings maintain their function under the conditions of this experiment. An abnormal inclusion body forming an oblong dense body and cistern in addition to the postsynaptic cisterna was observed. The active zone and the vesicular gap-structure of the synapse are discussed.

An ultrastructural evaluation of the cell organelle specificity of the uranaffin reaction in two human endocrine neoplasms.

The organelle specificity of the uranaffin reaction was determined by subjecting two human neoplasms (a pheochromocytoma and islet cell carcinoma) to four different experimental conditions. In Uranaffin Procedure (UP) I, fixed tissue was immersed in 0.9% sodium chloride (NaCl) before reacting with 4% uranyl acetate (pH 3.9) for 24 hours. In UP II, the tissue was prepared as in UP I with the exception that the tissue was immersed in uranyl acetate for 48 hours. In UP III, fixed tissue was prepared as in UP I with the exception that tissue was immersed in 0.1M cacodylate buffer (pH 7.2) instead of 0.9% NaCl. In UP IV, fixed tissue was prepared as in UP III with the exception that the tissue was immersed in uranyl acetate for 48 hours instead of 24 hours. When UPs I and II were utilized, only three cell organelles showed electron-dense reactivity: the nucleus, ribosomes, and cytoplasmic neurosecretory-like granules. In the nucleus, the nuclear chromatin, nucleolus, interchromatinic granules and perichromatinic granules were intensely stained. The reaction product in all of the uranaffin-positive organelles had a finely granular appearance. When fixed tissue was immersed in cacodylate buffer instead of isotonic saline, a non-specific reactivity was observed. The reaction product in some areas had a distinct crystalline appearance and filled some areas of the cytosol, the cisternae of the endoplasmic reticulum, the Golgi apparatus, the perinuclear cisternae, the nucleus, nucleolus, mitochondria, neurosecretory-like granules and larger lysosome-like bodies. There was a statistically significant (p less than 0.05) increase in the number of uranaffin-positive granules/mu2 when both endocrine neoplasms were reacted with uranyl acetate for 48 hours instead of 24 hours. The increase in uranaffin-positive granules using UP II did not result in an increase in non-specificity of the staining reaction.

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.

Events in the cytoplasm during male meiosis in Lilium.

An electron microscopic investigation of the events associated with meiosis in Lilium has revealed a number of changes in both the organellar population and the other cytoplasmic components. Ribosome numbers decrease significantly in early prophase and are later replenished in the tetrads, a process most likely involving the newly arising cytoplasmic nucleoloids. The organelles show a cycle of de- and redifferentiation and later in meiosis unusual internal structures can be seen before these organelles enter a division phase resulting in increased numbers. The localization of acid phosphatase during these changes has also been studied using electron microscopic cytochemical methods. In early prophase, considerable amounts of acid phosphatase are found in vesicles scattered through the cytoplasm; activity is also found in association with most membranous surfaces and often markedly associated with condensing mitochondria. Later in prophase the enzyme activity decreases to normal levels. Electron microscopic autoradiography revealed that DNA is synthesized in both plastids and mitochondria during meiotic prophase with activity reaching a peak during zygotene and ceasing by diakinesis and tetrad formation. These changes point to a certain independence of organelles from nuclear control during meiosis. The events are also evaluated in relation to a cytoplasmic clearing mechanism, which may occur in preparation for the changeover from sporophytic to gametophytic control and the development of gametes.

The effect of cycloheximide on dictyosome activity in Tradescantia pollen tubes determined using cytochalasin D.

Dictyosome activity in Tradescantia pollen tubes has been determined using a recently developed method based on the assumption that the rate of vesicle accumulation around the dictyosomes, after treatment with cytochalasin D, is equivalent to the actual rate of vesicle production. In tubes germinated in the presence of 1.0 micrograms/ml cycloheximide, reduced dictyosome activity could be detected as early as 10 min after sowing, although tube extension was not halted until later. After 30 min vesicle production had completely ceased. These observations are discussed in relation to previous reports on the effect of cycloheximide on pollen tube growth, and in relation to the synthesis and transfer of membrane proteins to secretory vesicles and the plasma membrane. It is concluded that the ability of pollen to germinate and produce short tubes in the presence of cycloheximide, does not necessarily indicate that protein synthesis is not a requirement for early pollen tube growth, as protein shortages would not be expected to become apparent over time periods less than the dictyosome turnover time and the secretory vesicle residence time.

Uraniosomes produced in the synovial membrane by uranyl acetate.

Uranyl acetate was injected into the rabbit knee joint. This produced single-membrane-bound presumably lysosmal bodies (called 'uraniosomes') containing electron-dense crystals in Type A and Type B synovial intimal cells, subsynovial macrophages and lipocytes. Uranium deposits were also seen in the extracellular matrix. All uraniosomes and extracellular deposits analysed by electron-probe X-ray analysis were found to contain uranium, potassium and phosphorus. Traces of calcium and sulphur were also found in some of the uraniosomes and extracellular uranium deposits.

Phenoxy acid herbicides cause peroxisome proliferation in Chinese hamsters.

An increase in either the size or amount of peroxisomes was obtained in the liver cells of Chinese hamsters after the animals were exposed to the phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chloro-2-methylphenoxyacetic acid (MCPA). At the dose level studied, 2,4-D was found to be more potent than MCPA in increasing the number of peroxisomes. A phenoxy acid derivative, clofibrate, one of the peroxisome proliferators known to possess carcinogenic properties in rodents, appeared to be still more potent in inducing peroxisome proliferation than either of the herbicides studied. Further investigations are warranted to clarify the significance of peroxisome proliferation to the toxicity of phenoxy herbicides.