Hair follicle regeneration in vitro

Hair follicles (HF) are skin appendages that develop as a result of the epithelial-mesenchymal interactions. The HF mesenchymal component—dermal papilla (DP)—has an ability to induce HF growth after transplantation in afollicular epidermis; this ability is retained through several passages in culture. In the present study, early regeneration events of a human HF are designed in vitro, using DP cells and skin kerati-nocytes. An artificial hair germ model is developed to study the primary steps of the stem cell self-organiza-tion during HF regeneration.     

MORPHOLOGY AND ULTRASTRUCTURE OF EMBRYOGENIC CELL SUSPENSION CULTURES OF PANICUM MAXIMUM (GUINEA GRASS) AND PENNISETUM PURPUREUM (NAPIER GRASS)

Morphological and ultrastructural changes during the growth of embryogenic cell suspension cultures of Panicum maximum and Pennisetum purpureum have been studied. The suspensions consist almost entirely of cell aggregates of 50–75 embryogenic cells. The cell aggregates vary in size from 90–400 μm in P. maximum and from 70–340 μm in P. purpureum. Following the period of exponential growth starch grains gradually disappear and vacuolation increases. Ten to 16 days after subculture, P. maximum cells enlarge and separate from each other, and organized embryo-like structures appear. Ultrastructural studies show that the cell aggregates are made up of discrete, individual groups of 2–6 cells each. Each cell group appears to arise from a single cell and breaks away from the ‘mother group’ as cell divisions continue. The embryogenic cells are small (20 μm), isodiametric with many starch grains and contain a large nucleus with a prominent nucleolus. Extensive profiles of endoplasmic reticulum, many small peripheral vacuoles, and several amyloplasts are present. Plasmodesmatal connections exist only between cells within a cell group but not between cells of different cell groups in the large cell aggregates.     

Pros and cons of fish skin cells in culture: long-term full skin and short-term scale cell culture from rainbow trout, Oncorhynchus mykiss

Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines.     

The use of electric fields in tissue engineering: A review

The use of electric fields for measuring cell and tissue properties has a long history. However, the exploration of the use of electric fields in tissue engineering is only very recent. A review is given of the various methods by which electric fields may be used in tissue engineering, concentrating on the assembly of artificial tissues from its component cells using electrokinetics. A comparison is made of electrokinetic techniques with other physical cell manipulation techniques which can be used in the construction of artificial tissues.Key words: tissue engineering, electric field, microenvironment, electrokinetics, dielectrophoresis, polarity     

Selective bioparticle retention and characterization in a chip‐integrated confocal ultrasonic cavity

We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the chamber center, where the cells are trapped. We investigate the resonant modes in the expansion chamber and its connecting inlet channel by theoretical modeling and experimental verification during no‐flow conditions. Furthermore, by triple‐frequency ultrasonic actuation during continuous microfluidic sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass—injection—aggregation and retention—positioning. Finally, we demonstrate transillumination microscopy imaging of ultrasonically trapped COS‐7 cell aggregates. Biotechnol. Bioeng. 2009;103: 323–328. © 2009 Wiley Periodicals, Inc.     

Depletion of Wip1 phosphatase sensitizes murine skin cells to UV-B irradiation

UV irradiation is a major natural and artificial stress factor that may cause severe skin injury. UV irradiation induces DNA damage, which, eventually, may lead to cell death, senescence or oncogenic mutations and tumor growth. Wip1 is a phosphatase involved in the regulation of DNA damage response and oncogenic stress. Here, we studied response to UV-B irradiation in wild-type and Wip1-depleted murine cells of epidermal and mesenchymal lineages. We found that both cell types, skin keratinocytes and fibroblasts, responded to UV-B in a similar manner with increased cytotoxicity in Wip1–/–cells. The number of nuclear foci of histone γH2A-X, a DNA damage marker and aWip1 target protein, was higher in Wip1–/–cells before and after UV-B. We observed a twofold increase in cell number with active caspase-3 in Wip1-deficient keratinocytes. Thus, Wip1 deficiency sensitizes cells to UV-B irradiation by promoting cell death, possibly by caspase-3 dependent apoptosis.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Multiple three‐dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves

Single and multiple three‐dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega‐Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half‐wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross‐fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F‐actin at sites of cell–cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The cluster [Re<Subscript>6</Subscript>Se<Subscript>8</Subscript>I<Subscript>6</Subscript>]<Superscript>3−</Superscript> penetrates biological membranes: drug-like properties for CNS tumor treatment and diagnosis

Tumorigenic cell lines are more susceptible to [Re₆Se₈I₆]^3? cluster-induced death than normal cells, becoming a novel candidate for cancer treatment. Still, the feasibility of using this type of molecules in human patients remains unclear and further pharmacokinetics analysis is needed. Using coupled plasma optical emission spectroscopy, we determined the Re-cluster tissue content in injected mice, as a biodistribution measurement. Our results show that the Re-cluster successfully reaches different tissues, accumulating mainly in heart and liver. In order to dissect the mechanism underlying cluster biodistribution, we used three different experimental approaches. First, we evaluate the degree of lipophilicity by determining the octanol/water partition coefficient. The cluster mostly remained in the octanol fraction, with a coefficient of 1.86?±?0.02, which indicates it could potentially cross cell membranes. Then, we measured the biological membrane penetration through a parallel artificial membrane permeability assays (PAMPA) assay. The Re-cluster crosses the artificial membrane, with a coefficient of 122 nm/s that is considered highly permeable. To evaluate a potential application of the Re-cluster in central nervous system (CNS) tumors, we analyzed the cluster’s brain penetration by exposing cultured blood–brain-barrier (BBB) cells to increasing concentrations of the cluster. The Re-cluster effectively penetrates the BBB, reaching nearly 30% of the brain side after 24 h. Thus, our results indicate that the Re-cluster penetrates biological membranes reaching different target organs—most probably due to its lipophilic properties—becoming a promising anti-cancer drug with high potential for CNS cancer’s diagnosis and treatment.     

Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells.

Pluripotent embryonal carcinoma cells can be triggered to differentiate in vitro by allowing them to form multicellular aggregates. Nullipotent embryonal carcinoma cells form aggregates, but further development is blocked. Pluripotent and nullipotent embryonal carcinoma cell lines were co-cultured to form mixed aggregates in order to determine whether a developmental signal produced by the pluripotent cell could induce the nullipotent cells to differentiate. Unlike pure pluripotent cell aggregates, aggregates from cultures initiated with a 1:1 mixture of pluripotent (PSA-1) and nullipotent (F9) cells formed endoderm but failed to differentiate further. The nullipotent cells did not produce a detectable soluble inhibitor of differentiation. A hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent cell line was used so that the fate of both nullipotent and pluripotent cells could be followed in autoradiographs of histological sections of aggregates labeled with ³H-hypoxanthine. Seven day old aggregates of pure pluripotent cell cultures contained endoderm, ectoderm and embryonal carcinoma cells. On the other hand, in 7 day old mixed cell aggregates, almost all the pluripotent cells became endoderm located on the outer surface of the aggregate. The nullipotent cells in the mixed aggregates assumed an internal position and remained embryonal carcinoma cells. Following the efficiency of plating of pluripotential cells in pure and mixed aggregates as a function of time showed that viable pluripotent embryonal carcinoma cells were lost at a 10 fold greater rate in mixed cell aggregates than in pure pluripotent cell aggregates. We conclude that nullipotent embryonal carcinoma cells in mixed aggregates with pluripotent cells exert a limitation on the ability of these pluripotent cells to differentiate.     

Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell

A uniform electric field of 10 V/cm applied across the surface of embryonic toad Xenopus muscle cells results in the asymmetric accumulation of concanavalin A (Con A) receptors toward one side of the cells within 10 min, as visualized by postfield fluorescent Con A labeling. This field produces an extracellular voltage difference of 20 mV across these 20-microns wide cells. The effect is reversible in two respects: (a) Additional exposure of the cell to the same field of opposite polarity for 10 min completely reverses the asymmetric accumulation to the other side of the cell. (b) Relaxation occurs after the removal of the field and results in complete recovery of the uniform distribution in 30 min. Both the accumulation and the recovery movements are independent of cell metabolism, and appear to be electrophoretic and diffusional in nature. The threshold field required to induce a detectable accumulation by the present method is between 1.0 and 1.5 V/cm (corresponding to a voltage difference of 2-3 mV across a 20-microns wide cell). The electrophoretic mobility of the most mobile population of nonliganded Con A receptors is estimated to be about 2 x 10(-3) microns/s per V/cm, while their diffusion coefficient is in the range of 4-7 x 10(-10) cm2/s. Extensive accumulation of the Con A receptors by an electric field results in the formation of immobile aggregates. The Con A receptors appear to consist of a heterogeneous population of membrane components different in their charge properties, mobility, and capability in forming aggregates.     

Electronic distribution coding in cellular differentiation

The present theories of cell differentiation explain how different types of cells can be obtained, starting from a single cell or cell type, through the action of gene repressors. They do not, however, provide a model for the primary step, i.e. the control of the repressors themselves by the environmental factors that initiate differentiation. In this paper, it is proposed that changes in the distributions of charge and electric polarization in large molecules, induced by alteration of natural electric fields, are responsible for the primary step in cell differentiation. A discussion of important experiments concerned with the effects of artificial and natural electric fields is given in support of this hypothesis. As an illustration, a mechanism for the initiation of floral differentiation in higher plants by light stimulated electric fields is given in some detail from this point of view, and the problem of embryonic development is also discussed.     

Fibroblast aggregation by suspension with conjugates of poly(ethylene glycol) and RGD

Cell aggregates may be useful components of artificial organs and mammalian cell bioreactors, but many cells do not naturally aggregate. In a previous report,(4) we described a method for promoting neural cell aggregation by addition of water-soluble conjugates of cell adhesion peptides, containing the three amino acid sequence Arg-Gly-Asp (RGD), and poly(ethylene glycol) (PEG). Here, we examined the mechanism of conjugate-induced aggregation using fibroblasts and a variety PEG-peptide conjugates. Aggregation was monitored during rotation culture of fibroblasts in the presence of unconjugated GRGDY and PEG; monofunctional (PEG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctional conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY). GRGDY-PEG-GRGDY conjugates induced rapid and pronounced fibroblast aggregation that was dose-dependent; at the highest concentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were produced more quickly ( approximately 1 h) and were significantly larger at 24 h (mean radius approximately 66 mum) than at slightly lower concentrations (1.7 and 3.3 mg/mL). Aggregation with GRGDY-PEG-GRGDY was completely inhibited by dissolved GRGDY (1.7 mg/mL). Neither unmodified GRGDY, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to significant aggregation. The extent of aggregation depended on PEG molecular weight: conjugates with 3400 M(w) PEG produced aggregates with significantly larger mean radius than conjugates with 20,000 M(w) PEG. When 1N-8A fibroblasts, genetically engineered to produce recombinant nerve growth factor (NGF), were aggregated with GRGDY-PEG-GRGDY, aggregated cells produced more NGF per cell than nonaggregated cells. Aggregation of cells may lead to improved cell function, such as the increase in NGF production observed here, which could be useful in large-scale cell culture and construction of artificial organs or tissue transplants for tissue engineering. (c) 1996 John Wiley & Sons, Inc.     

Cytology of beet yellows virus infection inTetragonia

Summary This is the third paper of the series dealing with beet yellows virus infection ofTetragonia expansa Murr. It concerns the different kinds of aggregates of virus and the state of the virus particles in the different cells. In vascular parenchyma cells, the aggregates of virus are variable but are consistently intermingled with host cell components. In the sieve elements, the virus may fill the cell lumen solidly either without obvious order or in stacks of layers each as wide as the particle is long. The virus particles appear to be commonly disorganizing in parenchyma cells with degenerating protoplasts and in sieve elements solidly packed with virus. The factors possibly determining the conformation of viruses in plant cells and the terminological problems regarding designations of aggregates of virus particles and other products appearing in infected cells are discussed.This work was supported in part by National Science Foundation grant GB-5506.     

Cytosolic aggregates in presence of non‐translocated proteins perturb endoplasmic reticulum structure and dynamics

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non‐translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non‐translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three‐way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins—reticulons, and altered distribution of those associated with the three‐way ER junctions—Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non‐translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven‐pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non‐translocated precursor proteins—their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.     

Plasticity of epidermal adult stem cells derived from adult goat ear skin

Here we report the isolation and characterization of pluripotent stem cells from adult goat skin. We found that these primary cells have the properties of embryonic stem cells (ESC), including the expression of appropriate immunological markers and the capability of forming embryoid bodies. The subcultured cells also show the characteristics of stem cells, such as the expression of CK19, beta(1-)integrin, P63, and formation of holo-clones in culture. Therefore, we termed these cells epidermal adult stem cells (EpiASC), although their origin was not identified. We have shown that clones of individual EpiASC proliferate and differentiate in culture to produce neurons, cardiomyocytes, osteoblasts, and occytes. Further, we cultivated EpiASC on bioengineered dermis and denuded human amniotic membrane (HAM), to reconstruct artificial skin and corneal epithelium. We successfully transplanted those artificial tissues in goats with acute full-thickness skin defect (AFTSD) and limbal stem cell deficiency (LSCD), respectively. Our results showed that indeed EpiASC reconstructed the skin (hair was observed in restored areas), and repaired the damaged cornea of goats with total LSCD. These data confirm that EpiASC can differentiate into different functional cell types in vivo or in vitro. Due to their high degree of inherent plasticity, and to their easy accessibility for collection from the skin, EpiASC are excellent candidate sources for diverse cell therapies.     

The effect of temperature on cellular reactions of Crassostrea virginica to the injection of avian erythrocytes

Intracardial injection of chicken red blood cells (RBC) into the oyster, Crassostrea virginica, at 15–19°C elicited ingestion, digestion, and removal of the cells by the leukocytes. A rapid response of fibroblastlike cells and characteristic cell aggregates were also detected as early as 1 hr post-injection. While the former was engaged in an unsuccessful attempt to encapsulate red blood cell laden leukocytes intravascularly, the function of the latter cell type was undetermined. The process of disposing the red blood cells in the oysters under running seawater conditions was not noticeably different from that observed in the oysters under standing water conditions.In oysters previously sensitized with red blood cells, response to a challenge injection was similar to that observed following the first injection, and there was no indication that the activities of the leukocytes were enhanced. However, it was demonstrated that oyster leukocytes remained functionally viable for at least 22 days. There is evidence indicating that the number of the characteristic cell aggregates increases considerably after a secondary injection of red blood cells, although the specificity of this response is yet to be determined.At 6°C, although phagocytosis of the blood cells occurred quite extensively, leukocytes exhibited considerably low digestive and migratory activity which resulted in far less effective disposal of the blood cells. Preliminary observations also revealed possible adhesion of oyster leukocytes to the wall of blood vessels following the inoculation at this temperature.     

Novel method for measuring active tension generation by C2C12 myotube using UV‐crosslinked collagen film

We have developed a novel method for measuring active tension generated by cultured myotubes using UV‐crosslinked collagen film. Skeletal myoblasts cell line C2C12 or human primary skeletal myoblasts were seeded onto a thin (35 µm) collagen film strip, on which they proliferated and upon induction of differentiation they formed multinucleated myotubes. The collagen film–myotube complex contracted upon electric pulse stimulation which could be observed by light microscope. When collagen film–myotube complex were attached to force transducer, active tension generation was observed upon electric pulse stimulation. Measurement of active tension was possible for multiple times for more than 1 month with the same batch of collagen film–myotube complex. Active tension generation capability of C2C12 myotubes increased with progression of differentiation, reaching maximal value 6 days after induction of differentiation. Using this method, we measured the effect of artificial exercise induced by electric pulse on active tension generation capability of C2C12 myotubes. When the electric pulses of 1 Hz were continuously applied to induce artificial exercise, the active tension augmentation was observed. After 1 week of artificial exercise, the active tension reached ～10× of that before the exercise. The increased active tension is attributable to the formation of the sarcomere structure within the myotubes and an increased amount of myotubes on the collagen film. The increased amount of myotubes is possibly due to the suppressed atrophy of myotubes by enhanced expression of Bcl‐2. Biotechnol. Bioeng. 2010; 106: 482–489. © 2010 Wiley Periodicals, Inc.