First Few Days Of Culture Research Articles

Chondrogenic Differentiation of Human Mesenchymal Stem Cells and Macrophage Polarization on 3D-Printed Poly(ε-caprolactone)/Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Blended Scaffolds with Different Secondary Porous Structures.

This study was aimed to evaluate the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and polarization of THP-1-derived macrophages cultured on poly(ε-caprolactone) (PC)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PH) blended scaffolds with dual primary (PP) and secondary (SP) pores, which were fabricated via a 3D printing technique, i.e., fused deposition modeling, followed by a salt-leaching process at 50 °C for varied times, i.e., 15, 30, and 60 min. Sodium chloride (SC), a porogen, was initially incorporated in the blend at varied weight percentages, i.e., 0, 25, and 50%, whereas 1 M NaOH solution and deionized water were used as salt-leaching agents. To elucidate the surface properties of the developed scaffolds, directly governed by the amount of the salt originally mixed and the salt-leaching efficiency, several characterization techniques, e.g., scanning electron microscopy, X-ray microcomputed tomography, mercury intrusion porosimetry, atomic force microscopy, and contact angle measurement, were used. Meanwhile, the salt-leaching efficiency was determined by means of weight loss measurement and thermogravimetric analysis. It was found that the alkaline solution could satisfactorily leach out the salt particles in 60 min with a mild etching of the polymer framework. The most immensely and homogeneously pitted filament surface was observed in the NaOH-treated scaffold initially integrated with 50% salt, i.e., 60B_PC/PH/50SC; the SP structure was mostly open and interconnected. The size of most of micropores was about 0.14 μm. With its suitable microsurface roughness and hydrophilicity, 60B_PC/PH/50SC could properly support the initial attachment and lamellipodia formation of hMSCs, which was favorable for chondrogenesis. Consequently, a significantly increased ratio of glycosaminoglycans/deoxyribonucleic acid and a superior expression of the COL2A1 gene were detected when cells were grown on this material. Although 60B_PC/PH/50SC induced the macrophages to secrete a slightly high level of IL-1β during the first few days of culture, the polarized M1 cells could return to a nearly normal stage at Day7, suggesting no unfavorable chronic inflammation caused by the material.

The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.

Spontaneous Formation of 3D Breast Cancer Tissues on Electrospun Chitosan/Poly(ethylene oxide) Nanofibrous Scaffolds.

Three-dimensional (3D) tissue culture has attracted a great deal of attention as a result of the need to replace the conventional two-dimensional cell cultures with more meaningful methods, especially for understanding the sophisticated nature of native tumor microenvironments. However, most techniques for 3D tissue culture are laborious, expensive, and limited to spheroid formation. In this study, a low-cost and highly effective nanofibrous scaffold is presented for spontaneous formation of reproducible 3D breast cancer microtissues. Experimentally, aligned and non-aligned chitosan/poly(ethylene oxide) nanofibrous scaffolds were prepared at one of two chitosan concentrations (2 and 4 wt %) and various electrospinning parameters. The resulting fabricated scaffolds (C2P1 and C4P1) were structurally and morphologically characterized, as well as analyzed in silico. The obtained data suggest that the fiber diameter, surface roughness, and scaffold wettability are tunable and can be influenced based on the chitosan concentration, electrospinning conditions, and alignment mode. To test the usefulness of the fabricated scaffolds for 3D cell culture, a breast cancer cell line (MCF-7) was cultured on their surfaces and evaluated morphologically and biochemically. The obtained data showed a higher proliferation rate for cells grown on scaffolds compared to cells grown on two-dimensional adherent plates (tissue culture plate). The MTT assay revealed that the rate of cell proliferation on nanofibrous scaffolds is statistically significantly higher compared to tissue culture plate (P ≤ 0.001) after 14 days of culture. The formation of spheroids within the first few days of culture shows that the scaffolds effectively support 3D tissue culture from the outset of the experiment. Furthermore, 3D breast cancer tissues were spontaneously formed within 10 days of culture on aligned and non-aligned nanofibrous scaffolds, which suggests that the scaffolds imitate the in vivo extracellular matrix in the tumor microenvironment. Detailed mechanisms for the spontaneous formation of the 3D microtissues have been proposed. Our results suggest that scaffold surface topography significantly influences tissue formation and behavior of the cells.

Transient Expression of GATA3 in Hematopoietic Stem Cells Facilitates Helper Innate Lymphoid Cell Differentiation.

Helper Innate lymphoid cells (ILCs) are tissue resident lymphocytes that play a critical role in a number of biological processes. Several transcription factors are required for the differentiation of hematopoietic stem cells (HSCs) into ILCs. Recent studies demonstrate GATA3 as a transcriptional regulator that plays an essential role in ILC development. We aimed to modulate the differentiation of human cord blood-derived CD34+ cells into ILCs by transient and ectopic expression of mRNA encoding transcription factors known to be important for ILC lineage differentiation, including GATA3, TOX, NFIL3, ID2, and RORγt. Using this experimental protocol, only GATA3 significantly modulated HSCs to differentiate into helper ILCs. Transient overexpression of GATA3 drove the emergence of CD34+α4β7+ early ILC progenitors during the first few days of culture. These ILC progenitors further acquired IL-7Rα and CD117 to give rise to immediate ILC precursors. In support of these findings, analysis of the genes induced by GATA3 in HSCs showed an upregulation of those associated with ILC development. Moreover, we show GATA3 also acts on more committed progenitors and significantly shifts the differentiation of progenitors away from the ILC1/NK lineage to the ILC2 and ILC3 lineage. In summary, transient overexpression of GATA3 mRNA in CD34+ HSCs enhances the differentiation of HSCs into the helper ILC lineages, at the expense of NK cell development.

Analysis of in vitro follicle development during the onset of premature ovarian insufficiency in a mouse model.

Premature ovarian insufficiency (POI) occurs in 1% of women under 40 years of age and is predominantly idiopathic. In a transgenic mouse model of follicular POI, the Double Mutant (DM), female mice are fertile at 6 weeks of age, become infertile by 9 weeks and exhibit POI by 3 months. DM female mice generate oocytes lacking mucin O-glycans and complex N-glycans due to deletion of core 1 synthase, glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1galt1) and mannoside acetylglucosaminyltransferase 1 (Mgat1) respectively (DM, C1galt1F/FMgat1F/F:ZP3Cre; Control, C1galt1F/FMgat1F/F). To determine whether DM follicle development could be improved in a controlled environment, follicles from DM and Control mice were cultured individually and follicle growth, morphology, survival and antrum formation were evaluated. DM ovaries were more rigid than Control ovaries at 3, 6 and 9 weeks, which was exacerbated with age, resulting in a failure to isolate follicles from 9 week-old DM females. DM follicles had decreased survival compared with Control follicles from females at 3 and 6 weeks of age. Furthermore, survival rate of DM follicles decreased with age between 3 and 6 weeks. DM follicles at both 3 and 6 weeks had accelerated follicle growth and altered antrum formation during the first few days of culture but, after 6 days, follicles were equivalent in size to the Controls. In conclusion, a population of DM follicles retain the potential to develop in vitro, and therefore follicle culture offers a reliable method to generate antral follicles from preantral follicles after the onset of POI in these female mice.

Mesenchymal Stem Cells and Endothelial Cells: A Common Ancestor?

Bone marrow mesenchymal stem cells (BM-MSCs) are commonly known as nonhematopoietic-nonendothelial cells based on in vitro expressed markers and properties. Despite the massive research on ex vivo expanded MSCs, their in vivo identity remains elusive. In this study, we report the existence of large multinuclear CD31 positive cells in the beginning of human BM-MSCs culture. Interestingly, the adjacent multinuclear cells occasionally formed tube-like structures. The large multinuclear cells then gave rise to mononuclear cells that fulfilled the criteria for BM-MSCs and were negative for CD31 but positive for other endothelial markers, CD54, CD106, and CD144. These observations, although primitive, imply that MSC ancestors may directly participate in the formation of new vessels. Further studies on BM-MSCs in the first few days of culture are definitely required to investigate the exact role of these cells in the vascular system.

Stiffness of the substrate influences the phenotype of embryonic chicken cardiac myocytes

We examined the effect of substrate stiffness on the beating rate, force of contraction, and cytoskeletal structure of embryonic chicken cardiac myocytes by culturing them on laminin-coated polyacrylamide (PA) substrates. Cells cultured on PA substrates with elasticity comparable to that of the native myocardium (18 kPa) exhibited the highest beating rate during the first few days of culture. The initial beating rate of individual cells on all the substrates varied significantly but began to converge within 5 days. We also examined the focal adhesions (FAs) and cytoskeletal structure on different substrates via confocal microscopy and found a higher percentage of FAs on tissue culture (TC) plastic dishes compared with the softer PA gels. Furthermore, highly aligned sarcomeric striations were clearly visible on 18 kPa, 50 kPa, and TC dish, whereas cells on 1 kPa only showed nonaligned diffused striations. The force of contraction on these substrates was measured using a micro-electromechanical system force sensor, which showed that the force of contraction for the cells on TC dishes (F = 71.30 ± 6.38 nN) was significantly larger than those cultured on the 18-kPa PA gel (F = 30.16 ± 3.83 nN). This is most likely due to the formation of higher percentage of FAs on the TC dishes compared with fewer FAs on the softer gels. Our cumulative findings can have a significant impact on the design of 3D cardiac tissue engineered scaffolds.

Up-regulation of uPARAP/Endo180 during culture activation of rat hepatic stellate cells and its presence in hepatic stellate cell lines from different species

BackgroundThe urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation.ResultThis study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently.ConclusionThese results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.

Amino acid‐mediated heterotypic interaction governs performance of a hepatic tissue model

Tissue-engineered models that mimic in vivo tissue organization offer the potential of capturing complex signaling pathways in vitro. In the liver, hepatocytes and endothelial cells are closely associated but separated by the extracellular matrix of the space of Disse. This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying the matrix with endothelial cells. We demonstrate that during the first few days of culture, the secretion of albumin and fibrinogen was 2-fold higher in cocultures compared to hepatocytes alone. Hepatocyte function in both cultures stabilized to a similar level during the second week, suggesting that endothelial cells can induce the early recovery of hepatocytes after isolation and seeding. Endothelial cell-conditioned medium reproduced the effect of coculture in a dose-dependent fashion, suggesting a role for endothelial cell-derived soluble factors. Endothelial cell-conditioned medium increased mRNA levels of various acute-phase proteins such as albumin, fibrinogen, transferrin, and alpha-macroglobulin in hepatocytes. Surprisingly, the effect of endothelial cell-conditioned medium was not mediated by growth factors or cytokines, or by secreted extracellular matrix, but by the release of the amino acid proline, which mediates endogenous collagen synthesis by hepatocytes. These findings suggest an important role for proline secretion by endothelial cells as a paracrine factor regulating hepatocyte function.

The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) entrains to non-photic maternal rhythms in the fetal and neonatal periods of rodents but this capacity disappears in later life. In order to understand the mechanism behind the non-photic entrainment in the early postnatal period, the phase response of the clock gene (Bmal1) expression rhythm to external stimuli was examined in cultured SCN harvested at postnatal day 6. The SCN was obtained from transgenic mice carrying a bioluminescence reporter for Bmal1 expression. Phase-dependent phase shifts of circadian rhythm were detected in the pup as well as in the adult for culture medium exchange but the amount of phase shift was significantly larger in the pup than in the adult SCN. Half of the pup SCNs did not show integrated circadian rhythmicities in the first few days in culture. In pups, the circadian period of Bmal1 expression rhythm was shorter and the amplitude of circadian rhythm was much lower than in adults. It is concluded that the responsiveness of cultured SCN to medium exchange is much larger in pups than in adult mice. Immaturity of the structural organization in the circadian system seems to underlie the high responsiveness of the pup SCN.

Non-adhesive organ culture of human biliary epithelium with stroma

Objective. Explanted tissue has been shown to keep adult human cells in organ culture with a preserved morphology for at least one month as spheres in a non-adhesive organ culture. In the present study, we explored whether also human biliary epithelium can be grown in this manner, because the result may be of interest in studies of hepato-biliary-pancreatic carciogenesis. Material and methods. Small tissue samples were obtained from the gallbladder wall of patients who had been operated upon with cholecystectomy. Fragments of about 300 µm in diameter from each patient were cultured and investigated with light microscopy at the time of explantation and after 5, 10, 20, 30 and 40 days of culture. Scanning and transmission electron microscopy were performed to demonstrate the ultrastructure. Incubation of cultured fragments with the vital dyes revealed a viable epithelium. Results. At the time of explantation, all the tissue fragments had a rough appearance with an uneven, torn periphery, while during the first few days of culture they became rounder with a smooth-looking surface covering the entire circumference. This spheroid morphology persisted for the remainder of the culture period. The core of the fragments harboured connective tissue with vascular elements, fibroblasts and leucocytes. Immunostaining for cytokeratin 7, 19 and 20 revealed a strong positive staining of the epithelium. Conclusions. These results show that biliary epithelium can be grown in vitro in a non-adhesive organ culture with their stroma.

4 GENE EXPRESSION IN CULTURES OF INNER CELL MASSES ISOLATED FROM IN VITRO-PRODUCED AND IN VIVO-DERIVED BOVINE BLASTOCYSTS

Genetic modification of embryonic stem (ES) cells derived from domestic species could be exploited to produce transgenic animals; however, fully validated ES have not been obtained in domestic species. Recent findings regarding key transcription factors and regulation of pluripotency and self-renewal in murine ES cells may provide keys to enable the derivation of ES in domestic species. The aim of this study was to identify and monitor the expression of candidate genes, which are known to be involved in the maintenance of self-renewal and pluripotency in mouse and human ES cells, during the critical first steps in establishment of primary cultures. Inner cell masses (ICMs) were isolated via manual dissection of 25 to 30 commercial in vitro-produced (IVP) blastocysts (Bomed, Inc., Madison, WI, USA) in each of three separate replicates and from 10 in vivo-derived Day 7-8 bovine blastocysts. On the day of ICM isolation (Day 0), 4-5 ICM clumps were collected for RT-PCR analysis. The remaining isolated ICMs were cultured (4-5 ICM clumps per well) on mitomycin C (Sigma-Aldrich, St. Louis, MO, USA)-inactivated mouse embryonic fibroblasts (STO, ATCC, Manassas, VA, USA). The ICM clumps were cultured in 12-well tissue culture dishes in ES medium consisting of Knockout DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% FCS (Hyclone, Logan, UT, USA), 2 mM l-glutamine (Invitrogen), 0.1 mM 2-mercaptoethanol (MP Biomedicals, Irvine, CA, USA), and non-essential amino acids (Sigma). Two to four cultured ICM clumps were collected for RT-PCR analysis on Days 1-4 from IVP embryos and on Days 2, 4, and 6 from in vivo-derived embryos. Total RNA was extracted from the collected samples using the Absolutely RNA Nanoprep Kit (Strategene, La Jolla, CA, USA). First-strand DNAs were synthesized using Superscript III (Invitrogen) and cDNAs were amplified with PfuUltra hotstart PCR mastermix (Stratagene). Primers were designed based on homology between human and mouse sequences and were validated using bovine tissues. In experiments spanning these critical first few days of culture, the pluripotency-related genes (Nanog, Oct-4, Sox-2) and components of the LIF (LIFR, Gp130), BMP (Bmpr1a, Id-1), and Wnt (Beta-catenin, Frizzled) pathways were expressed in the ICM cultures over the 4 days of IVP-ICM cultures and the 6 days of in vivo-derived ICM cultures. These results indicate that the markers of pluripotency and the components of signaling pathways implicated in the maintenance of murine embryonic stem cells are present in ICMs of Day 7-8 bovine blastocysts and continue to be expressed at least during the initial days of culture. Genes (NCAM, Lef1) associated with early differentiation, however, were also expressed. Whether their expression is an indicator of ICM differentiation or of residual contamination with trophectoderm remains to be determined. Further studies will determine whether stimulation of these pathways can facilitate efficient derivation and maintenance ruminant ES cells.

Development of Neuronal Sensitivity to Toxins in Cerebrospinal Fluid from HIV-Type 1-Infected Individuals

HIV infection of the immature nervous system generally results in a rapid progression of neurological disease that cannot easily be explained by the severity of encephalitis, viral burden, systemic immune deficiency, or developmental changes in utero. Rather than the viral infection dictating disease progression, we explored the possibility that immature neurons might be particularly sensitive to toxins secreted in response to HIV. Primary cultures of rat cortical neurons were exposed to toxic cerebrospinal fluid (CSF) from HIV-infected individuals (CSF(tox)) and evaluated for changes in intracellular calcium and cell death. CSF(tox) had no detectable effect on early neurite outgrowth, calcium regulation, or cell death during the first few days in culture. Starting at Day 4, delayed increases in intracellular calcium appeared in response to CSF(tox). The magnitude of the delayed calcium rise and cell death increased with the age of the culture and correlated with the appearance of synaptophysin immunoreactive varicosities. A similar gradual development of sensitivity was seen during exposure of feline neurons to toxins generated by choroid plexus macrophages after exposure to feline immunodeficiency virus. The possibility that toxin sensitivity is dependent on the presence of synaptic activity is consistent with the rapid pathogenesis in the CNS seen during the first postnatal year. Emerging synaptic activity coupled with other factors such as high metabolic demand in the young nervous system may combine to increase the likelihood of calcium overload and neuronal dysfunction in response to HIV-associated toxins.

Interaction between Metabotropic and NMDA Subtypes of Glutamate Receptors in Sprout Suppression at Young Synapses

Recently, NMDA receptors (NMDARs) have been implicated in a cell contact-dependent suppression of sprouting in cultured Xenopus tectal neurons during an early period when neither AMPA/kainate (KA) receptors nor action potentials play a prominent role in cell-cell communication. We asked how the NMDA receptors function in the absence of the depolarizing effect of AMPA/KA receptor activity. We show that type II metabotropic glutamate receptors (mGluRs) can operate synergistically with NMDA receptors in the absence of AMPA/KA receptor function to suppress an early neurite sprouting response of the tectal neurons. Calcium imaging with fluo-3 AM and morphological analyses after exposure to glutamate receptor antagonists show that a combination of AMPA/KA receptor and type II mGluR blockers mimics the decrease in intracellular free calcium in response to glutamate and the structural effects produced by NMDA receptor antagonists in these cultures. Patch-clamp analyses confirmed a decrease in NMDA receptor-mediated currents with type II mGluR blockade, and 8-bromo cAMP application produced a decrease in NMDA receptor-mediated calcium influx. These data suggest that type II mGluRs potentiate NMDA receptor function by decreasing cAMP levels in tectal neurons. We also show that NMDARs exhibit low magnesium sensitivity in tectal neurons during the first few days in culture. Thus both metabotropic and ionotropic glutamate receptors can play a role in the contact-mediated suppression of ongoing sprouting at early neuron-neuron contacts before action potential activity.

Response Differences between Human CD4+ and CD8+ T-Cells during CD28 Costimulation: Implications for Immune Cell-Based Therapies and Studies Related to the Expansion of Double-Positive T-Cells during Aging

Since CD28 costimulation is critical for T-cell activation, there is great interest in CD28 as a target for immuntherapeutic approaches. We show that stimulation of human CD4(+) and CD8(+) T-cells differs in their responsiveness to stimulation with anti-CD3/CD28-coated beads, as surrogate antigen-presenting cells. While the CD4(+) subset responded with sustained proliferation, CD8(+) T-cells grew for a limited period only and failed to produce IL-2 beyond the first few days in culture. This decrease is accompanied with an increased rate of apoptosis in CD8(+) T-cells despite Bcl-x(L) expression. The CD8(+) but not the CD4(+) subset developed a reversible double-positive phenotype during CD28 costimulation. This finding may have some bearing on the appearance of double-positive T-cells in human peripheral blood. This double-positive subset was shown to undergo a statistically significantly increase during aging in humans. Taken together, the above data have important implications for immunotherapy and immune senescence.

Dedifferentiation of intrinsic response properties of motoneurons in organotypic cultures of the spinal cord of the adult turtle.

Explant cultures from the spinal cord of adult turtles were established and used to study the sensitivity of the intrinsic response properties of motoneurons to the changes in connectivity and milieu imposed by isolation in culture. Transverse sections 700 microm thick were explanted on cover slips and maintained in roller-tube cultures in medium containing serum and the growth factors brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT3), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF). The gross morphology of acute sections was maintained after 4 weeks in culture. Cell bodies of motoneurons remained stainable in fixed cultures with an antibody against choline acetyltransferase (ChAT) throughout the culture period. During culture, motoneurons maintained stable resting membrane potentials and were contacted by functional synapses. The ability to generate action potentials was also preserved as was delayed inward rectification and generation of calcium spikes in the presence of tetra-ethyl ammonium (TEA). In response to depolarization, however, motoneurons presented strong outward rectification, and only 41% of the cells recorded from maintained the ability to fire repetitively. By the second week in culture, a fraction of motoneurons displayed fast and slow transient outward rectification and low-threshold calcium spikes, features not seen in turtle motoneurons in acute slices. On the other hand, properties mediated by L-type Ca2+ channels disappeared during the first few days in culture. Our observations show that the phenotypical intrinsic response properties of mature spinal motoneurons are modified in explant cultures. The properties acquired resemble the properties in juvenile motoneurons in several species of terrestrial vertebrates.

Developmental appearance of nuclear GM1 in neurons of the central and peripheral nervous systems

Previous studies demonstrated expression of GM1 ganglioside in the nuclear envelope of differentiating neuroblastoma cells and cultured cerebellar granule cells from neonatal rat brain. In the present study, relatively few of the latter cells were shown to possess a nucleus with appreciable GM1 during the first few days in culture, but increasing numbers of such cells possessed GM1-expressing nuclei as morphological differentiation progressed. This phenomenon reached a plateau by the 8th day in culture, approximately 90% of observed nuclei showing cytochemical evidence of GM1 at that time. Cerebral cortical neurons from embryonic rat brain in culture also gave clear evidence of GM1 in the nuclear membrane. Similar results were obtained with cultured neurons from the superior cervical ganglion from embryonic rats, demonstrating developmental appearance of GM1 in the nuclear envelope of PNS neurons. Cytochemical evidence for GM1 in purified nuclei from freshly isolated cortical neurons of neonatal rat brain indicated that expression of nuclear GM1 is not an artifact of cell culture. Study of NG108-15 neuroblastoma×glioma hybrid cells showed upregulation of nuclear GM1 to lag somewhat behind neurite outgrowth, suggesting nuclear GM1 to have a functional role subsequent to onset of morphological differentiation.

Exposure of cartilage to a fibronectin fragment amplifies catabolic processes while also enhancing anabolic processes to limit damage.

The addition of fibronectin fragments to cultured cartilage causes an initial suppression of proteoglycan synthesis, induction of matrix metalloproteinases, and resultant decrease in proteoglycan content by about 50% during the first few days in culture. Because the proteoglycan loss appears to be limited, we investigated whether the fibronectin fragments induce anabolic responses that might counter the damage. The effects of various lengths of exposure of cultured cartilage to the fibronectin fragment on proteoglycan content, proteoglycan synthesis rates, stromelysin-1 release, and tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-6 release were investigated. The results showed that about 7 days of exposure of cultured cartilage to the fibronectin fragment was required for maximal cytokine release, proteoglycan depletion, and stromelysin-1 release. However, nearly maximal suppression of proteoglycan synthesis occurred within 1 day of the addition of the fibronectin fragment and, after its removal, the rates increased to supernormal levels. Decreasing exposure to 3 days caused only a small decrease in cartilage proteoglycan content, although stromelysin-1 release still occurred. Decreasing exposure to 1 day caused an immediate increase in proteoglycan synthesis and an increase to supernormal proteoglycan contents. The effect of first treating cartilage with the fibronectin fragment for various periods and then allowing a recovery was to make the cartilage more resistant to secondary exposures. This study shows that cartilage damage can be caused by short exposures to the fibronectin fragment and that exposures either optimal or suboptimal for damage additionally amplify anabolic processes to make the cartilage resistant to further damage and, thus, condition it against pending amplification of damage.

Embryonic rat spinal cord neurons change expression of glycine receptor subtypes during developmentin vitro

The expression of functional glycine receptors (GlyRs) by embryonic rat spinal cord neurons during development in vitro was investigated using whole-cell patch-clamp recordings. Functional GlyRs were expressed by most neurons within 1 day in vitro, and by all neurons from 4 days onward. However, the extent to which responses to glycine were blocked by the antagonist strychnine differed significantly between the first few days and 8 days in culture. Responses to glycine by neurons during the first few days in culture exhibited significantly less blockade by strychnine than those in neurons after 1 week in culture. Responses to glycine at both ages reflected an increased conductance to chloride ions, ruling out involvement of N-methyl-D-aspartate type glutamate receptors, and were not due to cross activation of gamma-aminobutyric acid receptors. Monoclonal antibody 4a, which recognizes multiple subtypes of rat GlyR alpha subunits, labeled most neurons as early as 1 day in vitro, confirming that neurons express some form of GlyR alpha subunits by the first day in culture. These results show that rat spinal cord neurons express GlyRs early in their differentiation in vitro, and they suggest that individual neurons express as functional, cell-surface GlyRs a strychnine-insensitive isoform of the GlyR, possibly the previously described alpha 2* subunit. In addition, these results indicate that the expression of GlyR isoforms changes from predominantly a strychnine-insensitive isoform to other, strychnine-sensitive isoform(s) GlyR during development in vitro.

Living adult rat cardiomyocytes in culture: evidence for dissociation of costameric distribution of vinculin from costameric distributions of attachments.

Adult rat cardiomyocytes were placed in tissue culture to determine the relationships of their vinculin positive costameres, their attachments associated with the costameres, the fate of their myofibrils. The costameric structures were detected using interference contrast microscopy and the visualization of the fluorescently labeled vinculin and alpha-actinin molecules. The cardiomyocytes isolated from the heart retained their myofibrils upon attachment to the cell surfaces. One group of cells then rounded up, only to respread after 6 days in culture. These cells initially demonstrated costameric distributions of attachments and vinculin. These relationships were lost during the rounding-up process only to be regained as the cells respread. The second group of freshly isolated cardiomyocytes did not round up but began to spread on the substratum by sending out lamellipodia from their rectangularly shaped body. These newly cultured cardiomyocytes initially exhibited costameric distributions of close attachments detected by interference microscopy. Over the next 3 days although the cells remain attached to the substratum, the costameric attachments were gradually lost. Nevertheless, when similar cells were injected with fluorescently labeled vinculin, costameric distributions of vinculin could be detected in the absence of costameric attachments. Cardiomyocytes, injected with fluorescent alpha-actinin, revealed that during the first few days in culture the existing myofibrils disassembled from the edges of the cell towards the middle. The center group of myofibrils was retained as the cells began to spread. Our observations of living cells support the hypothesis that proteins in addition to vinculin are needed for cardiomyocytes to generate costameric attachments to the cell surfaces. We speculate that the ability of the vinculin-attached Z-lines of adult cardiomyocytes to dissociate from the extracellular matrix may aid in the remodeling of the adult heart in the repair process after myocardial infarction and also in stress induced hypertrophic growth.