Function Of Time In Culture Research Articles

1alpha,25(OH)2D3 regulation of integrin expression is substrate dependent.

Osteoblasts are attachment-dependent cells that interact with their surface through integrin-mediated mechanisms. Their differentiation is regulated by 1,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D(3)] and is affected by substrate chemistry and microtopography, suggesting that 1alpha,25(OH)(2)D(3) may regulate integrin expression in a surface-specific manner. To test this hypothesis, osteoblast-like human MG63 cells were grown on tissue culture plastic and on grit-blasted and acid-etched titanium disks with a complex microtopography to induce osteoblast differentiation. Expression of alpha(2), alpha(5), alpha(v), beta(1), and beta(3) integrins were quantified by real-time polymerase chain reaction (PCR) as a function of time in culture and treatment with 1alpha,25(OH)(2)D(3). Results were correlated with expression of osteocalcin, a marker of a differentiated osteoblast. Osteocalcin mRNA increased with time and 1alpha,25(OH)(2)D(3) treatment and these changes were greater in cultures on the titanium disks. Integrin expression varied with time in culture and this was also surface dependent. At each time point, beta(1) and alpha(2) mRNAs were greater on titanium than on plastic, whereas alpha(5) expression was reduced and alpha(v),beta(3) expression was unaffected. 1alpha,25(OH)(2)D(3) increased beta(1) mRNA on both surfaces at all time points, but it increased alpha(2) expression only in 8-d cultures. 1alpha,25(OH)(2)D(3) caused reduced alpha(5) expression only in cultures grown on plastic for 8 d, and had no effect on either alpha(v) or beta(3) expression regardless of surface. These results show that integrin expression in human osteoblast-like cells is differentially modulated by 1alpha,25(OH)(2)D(3) in a time-dependent manner that is sensitive to the surface on which the cells are grown.

Effects of osteogenic protein-1 (OP-1, BMP-7) on gene expression in cultured medial collateral ligament cells.

Osteogenic protein-1 (OP-1, also called BMP-7), a member of the BMP family and the TGF-beta superfamily, induces formation of new bone and cartilage, but also regulates a wide array of processes. In the present study, the expression of several characteristic biochemical markers of ligaments, such as Six1, Scleraxis, aggrecan, and type I collagen in primary cultures of adult rat medial collateral ligament (MCL) cells was determined. The effects of OP-1 on cell proliferation and on gene expression were subsequently examined. OP-1 stimulated cell proliferation, alkaline phosphatase (AP) activity, and the steady-state mRNA levels of the transcription factor Runx2/Cbfa1 in a dose- and time-dependent manner. The mRNA levels of type I collagen only increased slightly, but the activity of the cloned collagen promoter increased by 2-fold in transiently transfected MCL cells. OP-1 also stimulated aggrecan mRNA expression. The mRNA levels of Six1 and Scleraxis were not detectably altered by OP-1. In control cultures, the steady-state mRNA levels of ActR-I, BMPR-IA, BMPR-IB, and BMPR-II increased as a function of time in culture. The mRNA levels of BMP-1 and -4 increased significantly after 12 days, but those of BMP-2 and -6 did not change. The GDF-1, -3, -5, -6, and -8 mRNA levels in the control cultures also increased as a function of time. OP-1 treatment stimulated mRNA expression of BMPR-IA and BMPR-II, but had little effect on ActR-I and BMPR-IB mRNA expression. OP-1 lowered the BMP-1, -2, and -6 mRNA levels without changing the BMP-4 mRNA level. OP-1 treatment also reduced the mRNA levels of GDFs detected. In summary, the present study demonstrated that OP-1 stimulated cell proliferation and mRNA expression of several biochemical markers in this ligament cell culture model and established the spatial and temporal appearance of several members of the TGF-beta superfamily.

Insulin impairs the maturation of chondrocytes in vitro.

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 microM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [ 3H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Biomechanical and optical characteristics of a corneal stromal equivalent.

Cell matrix interactions are important in understanding the healing characteristics of the cornea after refractive surgery or transplantation. The purpose of this study was to characterize in more detail the evolution of biomechanical and optical properties of a stromal equivalent (stromal fibroblasts cultured in a collagen matrix). Human corneal stromal fibroblasts were cultured in a collagen matrix. Compaction and modulus were determined for the stromal equivalent as a function of time in culture and matrix composition. The corneal stromal fibroblasts were stained for alpha-smooth muscle actin expression as an indicator of myofibroblast phenotype. The nominal modulus of the collagen matrix was 364 +/- 41 Pa initial and decreased initially with time in culture and then slowly increased to 177 +/- 75 Pa after 21 days. The addition of chondroitin sulfate decreased the contraction of the matrix and enhanced its transparency. Cell phenotype studies showed dynamic changes in the expression of alpha-smooth muscle actin with time in culture. These results indicate that the contractile behavior of corneal stromal cells can be influenced by both matrix composition and time in culture. Changes in contractile phenotype after completion of the contraction process also indicate that significant cellular changes persist beyond the initial matrix-remodeling phase.

Time course of actin cytoskeleton stiffness and matrix adhesion molecules in human bronchial epithelial cell cultures

Human bronchial epithelial (HBE) cells adhere to underlying extracellular matrix (ECM) via integrin-type transmembrane receptors. Integrins link the ECM to the cytoskeleton (CSK), establishing a mechanical continuum by which forces are transmitted between the outside and the inside of the cells. The present study investigates the time course of global and actin CSK stiffness of HBE cells (16HBE14o-) growing on various matrix substrates as a function of culture time until confluence, and the concomitant time course of F-actin and adhesion molecule distribution. Our results showed a progressive increase in actin CSK stiffness from cell seeding to confluence, related to acquisition of highly polymerized cortical and cytosolic F-actin organization and up-regulation of certain matrix ligands, such as β1-, α5-, and αv-integrin subunit expression. Moreover, compared to fibrillar type I collagen, reticular type IV collagen used as matrix substrate, appeared to amplify actin CSK stiffness of HBE confluent cells probably in relation to up-regulation of α3-integrin subunit. Taken together, these results support the concept of a close interaction among actin CSK stiffness, structural actin organization, specific integrin molecule involvement, cell spreading, and extracellular matrix.

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms.

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells.

The multipotential C3H10T1/2 mesenchymal cells undergo chondrogenic differentiation only when seeded as high-density micromass cultures, particularly upon treatment with bone morphogenetic protein-2 (BMP-2). The molecular mechanism(s) responsible for the cell density-dependent onset of cartilage-specific gene expression is presently unknown. Interestingly, a number of recent studies have indicated that activating protein-1 (AP-1), a well known downstream target of the mitogenic activated protein kinase (MAP kinase) signaling pathway, is a target of chondrogenic/osteogenic growth factors such as BMP-2, and plays a role in osteogenic gene regulation as well as in chondrogenic differentiation. The aim of this study is to examine the density-dependent alteration in the level and binding activity of AP-1 and its functional involvement in C3H10T1/2 mesenchymal chondrogenesis. To measure the activity of the AP-1 transcription factor, we generated a pool of stable C3H10T1/2 cell lines harboring a luciferase expression vector driven by a concatamer of an efficient AP-1 response element (AP1-10T1/2 cells). Luciferase activity of AP1-10T1/2 cultures was found to decrease sharply with increase in cell density, either as a function of culture time or initial cell seeding densities. In C3H10T1/2 micromass cultures undergoing chondrogenesis, AP-1 activity was further reduced and then maintained at a low, steady level for the entire 3-4 day culture period. AP-1 activity in micromass cultures was not significantly affected by BMP-2 treatment, but chondrogenesis was compromised upon competitive inhibition of AP-1 activity with a double-stranded AP-1 binding oligonucleotide. The level of AP-1 binding correlated with the activity of its response element but not with the levels of its leucine-zipper containing subunits, c-Jun and c-Fos. These findings suggest that a cell density-dependent, low but steady level of AP-1 binding and activity is required for promoting the chondrogenic potential of C3H10T1/2 cells.

The migration of cells from the ruptured human anterior cruciate ligament into collagen-glycosaminoglycan regeneration templates in vitro

Guided tissue regeneration of the ruptured anterior cruciate ligament (ACL) offers the potential benefits of retaining the complex footprints of the ACL and the proprioceptive nerve fibers of the tissue. For this approach to be successful, ACL cells must retain the ability to migrate into an adjacent regeneration template, or scaffold, after ligament rupture. Ruptured ACLs were obtained from the knees of four men, ages 25–35, at the time of ACL reconstruction. Explants of ACL tissue were taken from three locations along the longitudinal axis of the remnant: the rupture site, the middle of the remnant, and far from the rupture site. These three areas have been found to be distinct histologically, with the region far from the rupture site having a histologic appearance similar to the intact ligament. Explants from each area were cultured in conventional tissue culture dishes (2-D culture) and on porous collagen-glycosaminoglycan (CG) scaffolds. Two-dimensional outgrowth was measured 3 times a week, and the 3-D explant/scaffold constructs were examined at 1, 2, 3 and 4 weeks to assess outgrowth of cells into the scaffold. The cell number density and expression of α-smooth muscle actin (SMA) were determined at each time point. The decrease in the diameter of the scaffolds and non-seeded controls were determined as a function of time in culture. The outgrowth of cells onto the tissue culture dishes was observed to begin as early as 3 days and as late as 21 days, with outgrowth first detected at an average of 6.8±2.0 days after explantation. In general, there was a larger area of outgrowth at the 2-week time point from explants with higher cell number density and higher blood vessel density. The 2-week area of outgrowth also correlated with the percentage of SMA-positive cells in the explant. In the experimental constructs with CG scaffolds, fibroblasts were noted to migrate from the human ACL explants into the templates at the earliest time point recorded (1 week). The migration and proliferation of cells from the explants in the CG matrices resulted in an increase in the cell density in the scaffolds with time. There was a significant effect of the location from which the explant was taken on cell density in the scaffold, with a higher density of cells migrating from the explants from the rupture site of the ACL specimens. The percentage of cells staining positive for the SMA isoform varied from 0 to 50% of cells in the scaffold. Scaffolds co-cultured with explants showed a reduction in diameter that was significantly affected by time in culture and the location in the ACL from which the explant was taken. The percentage contraction attributed to the cells was 15% at 2 weeks, and increased to 27% for the injury-zone explant at 4 weeks. There was a significant correlation of the cell-mediated contraction of the matrices at 4 weeks with the cell density in the scaffolds, but not with the number of SMA-positive cells in the scaffolds. These data demonstrate that cells in the human ACL retain their ability to migrate into an adjacent CG scaffold in vitro, weeks after complete rupture. Moreover, the ACL-derived cells can express a contractile actin isoform and can contract a CG analog of extracellular matrix.

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures.

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

NGF-independent survival of postganglionic sympathetic neurons in neuronal-vascular smooth muscle cocultures.

The present study tests the hypothesis that vascular cells promote the survival of postganglionic sympathetic neurons in the absence of nerve growth factor (NGF). To test this hypothesis, neurons isolated from superior cervical ganglia of 2- to 4-day-old rat pups were grown in the absence of NGF and in the absence and presence of vascular smooth muscle cells (VSM). Neuronal survival was assessed as a function of time in culture. At all time points studied, VSM promoted the survival of the neurons. After 5 days in the absence of NGF, 7 +/- 2% of neurons survived in the absence and 28 +/- 7% survived in the presence of VSM. An endothelin receptor antagonist reduced neuronal survival in cocultures grown in the absence of NGF. These data indicate that VSM produce factors other than NGF that promote the survival of cultured postganglionic sympathetic neurons. The data also indicate that endothelin contributes to this effect and suggest that endothelin as well as other VSM-derived factors may play a role in the development of sympathetic innervation to the vasculature.

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells.

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.

Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)

The human placenta has a remarkable capacity to aromatize C19-steroids, produced by the fetal adrenals, to estrogens. This reaction is catalyzed by aromatase P450 (P450arom), encoded by the CYP19 gene. In placenta, CYP19 gene expression is restricted to the syncytiotrophoblast layer. Cytotrophoblasts isolated from human placenta, when placed in monolayer culture in 20% O2, spontaneously fuse to form syncytiotrophoblast. These morphological changes are associated with a marked induction of aromatase activity and CYP19 gene expression. When cytotrophoblasts are cultured in an atmosphere containing 2% O2, they manifest increased rates of DNA synthesis and fail to fuse and form syncytiotrophoblast. The objective of the present study was to utilize cytotrophoblasts isolated from midterm human placenta to analyze the effects of O2 on CYP19 gene expression and the molecular mechanisms that mediate these effects. We observed that when trophoblast cells were maintained in 2% O2, there was only a modest induction of CYP19 expression as a function of time in culture, and aromatase activity was barely detectable. However, when cytotrophoblasts that had been maintained in 2% O2 for 3 days were placed in a 20% O2 environment, there was a rapid onset of cell fusion and induction of P450arom mRNA and aromatase activity. In addition, mRNAs for the helix-loop-helix factors Mash-2 (mammalian achaete-scute homologous protein-2) and Id1 (inhibitor of differentiation 1) were readily detectable in freshly isolated cytotrophoblasts and were markedly decreased upon differentiation to syncytiotrophoblast in 20% O2. By contrast, when cytotrophoblasts were cultured in 2% O2, mRNA levels for Mash-2 and Id1 remained elevated. Interestingly, overexpression of Mash-2 in primary cultures of human trophoblast cells markedly inhibited cell fusion and the spontaneous induction of P450arom mRNA levels and caused a marked decrease in expression of co-transfected fusion gene constructs containing either 125, 201, 246, or 501 bp of DNA flanking the 5'-end of the placenta-specific exon (exon I.1) of the human CYP19 gene linked to the human GH (hGH) structural gene, as reporter. In studies using BeWo, a human choriocarcinoma cell line, overexpression of Mash-2 also inhibited expression of cotransfected CYP19I.1:hGH fusion gene constructs. The findings that Mash-2 had no effect on the expression of a CYP19I.1(-42):hGH fusion gene in primary cultures of human trophoblast and BeWo cells suggest that Mash-2 exerts its inhibitory effects directly or indirectly though CYP19I.1 5'-flanking sequences that lie between -42 and -125 bp. By contrast, neither Id1 nor Id2 had an effect on CYP19I. 1 promoter activity in the transfected BeWo cells. These findings suggest that Mash-2 may serve as a hypoxia-induced transcription factor that prevents differentiation to syncytiotrophoblast and aromatase induction in human trophoblast cultured under low O2 conditions.

Marrow stromal stem cells.

Multipotential marrow stromal stem cells were known as early as 1968 (1) through the work of Friedenstein and his coworkers, who established that cells that are adherent, clonogenic, nonphagocytic, and fibroblastic in habit (defined as colony-forming units–fibroblastic; CFU-Fs) can be isolated from the bone marrow stroma of postnatal organisms. CFU-Fs, as these investigators found, can give rise under appropriate experimental conditions to a broad spectrum of fully differentiated connective tissues, including cartilage, bone, adipose tissue, fibrous tissue, and myelosupportive stroma (2, 3). Evidence for the physiological relevance of the stromal system and stem cells rests primarily on the in vivo transplantation of marrow stromal cell strains obtained from marrow cell suspensions and expanded in culture. Transplantation of such cells in open systems, such as the space under the kidney capsule, results in the generation of a chimeric ossicle, that is, a structure replicating the histology and architecture of a miniature bone and comprising tissues of both donor and host origin. In these systems, bone trabeculae and cortices, myelosupportive stroma, and adipocytes are of donor origin, whereas the hematopoietic cells that colonize the ossicle and reach full maturity within it are of host origin (4). This outcome can be viewed as the mirror image of bone marrow transplantation, in which host stromal cells provide the structures within which donor cells undergo hematopoiesis. In addition to transplantation in open systems, stromal cell strains can also be transplanted in diffusion chambers that exclude the immigration of host hematopoietic cells into the forming stromal tissues. Under these conditions, an array of differentiated connective tissues — cartilage, bone, fibrous tissue, and adipocytes — develops, all of donor origin (3). In the nonvascularized diffusion chambers, cartilage is more frequently observed than in open transplants and is regularly distributed at sites of predicted low oxygen tension. This principle is reflected in current micromass culturing techniques for obtaining cartilage formation from stromal cells in vitro (5). Cell strains derived from the ex vivo expansion of a single clone (i.e., the progeny of a single CFU-F) are endowed with the same multipotentiality under the same or similar experimental conditions. Thus, a single CFU-F can give rise to ossicles identical to those generated by transplanted nonclonal stromal cell strains, which may include cells of multiple differentiated phenotypes (6). Based on such observations, Friedenstein, Owen, and others developed the concept that cartilage, fat, bone, and other connective tissues derive from a common ancestor, the stromal stem cell. Their studies also established that the stromal stem cell persists within the bone marrow of postnatal and even adult organisms. However, remarkable differences are observed between individual CFU-Fs. Cell morphology and rates of proliferation vary dramatically, as does the ability to form multilayer or nodular structures. Expression of various markers of the osteoblastic, chondrogenic, and adipogenic phenotypes is variable not only between different cell strains, but also within a cell strain, as a function of time in culture. Furthermore, upon transplantation, some CFU-Fs form bone and support hematopoiesis and adipogenesis, some only form bone, while others form only connective tissue (6). To date, no clear-cut phenotypic characteristics have been identified that allow CFU-F subsets to be isolated with predictably broad or restricted potential. Recent attempts, employing monumental numbers of putative markers to purify the true marrow stromal stem cell (inappropriately termed the “mesenchymal stem cell”) from a heterogeneous population of adherent stromal cells, have identified cells that are neither indefinitely self-renewing nor homogeneously multipotential (7). These mesenchymal stem cells, although supposedly purified, reproduce all of the known virtues and vices of the marrow CFU-F population as a whole, as known from Friedenstein’s studies and others’, except that these cells are obtained with considerably lower efficiency than with the earlier protocols. Ironically, the rediscovery of the widely known properties of marrow stromal cells in 1999 was celebrated in the scientific and lay press as the happy product of an extraordinary and successful hunt.

Influence of preculture on the prefreeze and postthaw characteristics of hepatocytes.

Recent studies performed in our laboratory have shown that a brief period of preculture prior to cryopreservation improves the postthaw viability of hepatocytes. The purpose of this investigation is to characterize specific metabolic and biochemical characteristics of the hepatocytes (both frozen and nonfrozen) to help elucidate the role of preculture on the postthaw viability. Fresh and thawed hepatocytes were cultured in a bioartificial liver (BAL) to determine albumin secretion as a function of time in culture. In addition, cell extracts were analyzed using nuclear magnetic resonance (NMR) spectroscopy to quantify changes in cell membrane composition and energetics as a function of time in culture prefreeze and postthaw. The results of these studies showed an increase in albumin concentration in the culture medium with time in culture for the period tested for both fresh and frozen and thawed hepatocytes. NMR spectroscopy of lipid extracts indicates that in vitro culture of hepatocytes results in an increase in cholesterol relative to membrane phospholipid. Moreover, the NMR results also indicate phospholipid interconversion, via specific lipases in cultured hepatocytes, and these changes are consistent with water permeability measurements performed previously. Significant changes in phosphoenergetics were also observed, with the net energy charge for the cells increasing significantly with time in culture. In addition, NMR spectra show increased levels of 6-phosphogluconate, another indicator of the cellular response to the stresses of isolation and ex vivo culture. These results suggest that energetic considerations may be a significant factor in the ability of hepatocytes to survive the stresses of freezing and thawing. Significant shifts in membrane phospholipids may also influence membrane permeability and postthaw survival.

Effect of Extracellular Matrix Composition on the Expression of Glutathione S-transferase Isoenzymes in Organotypical Hepatocyte Cultures

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

High density micromass cultures of embryonic limb bud mesenchymal cells: an in vitro model of endochondral skeletal development.

To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.

Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation.

Calpain is a ubiquitous neutral calcium-activated thiol protease that is implicated in various cellular functions including exocytosis, cell fusion, apoptosis and proliferation. The calpain system is composed of the enzymes mu-calpain and m-calpain and their endogenous inhibitor, calpastatin. We employed the spontaneously immortalized human HaCaT keratinocytes, which retain their ability to differentiate in vitro and in vivo, to study the modulation of the calpain system during keratinocyte differentiation. The cellular levels of keratinocyte differentiation markers and of the components of the calpain system were monitored by immunoblotting. Three established differentiation stimuli: increase in cell density as a function of time in culture, elevation of extracellular calcium concentration and exposure to 1,25-dihydroxyvitamin D3 enhanced the expression of the three keratinocyte differentiation markers keratin 10, involucrin and transglutaminase. The differentiation of HaCaT cells was accompanied by elevation of the components of the calpain system, although the pattern of increase varied according to the specific differentiation stimulus. A higher increase in calpains as compared with the increase in calpastatin suggests an increase in net calpain activity during differentiation. Such an increase may play a part in the differentiation process itself and/or in the regulation of key events in differentiating keratinocyte metabolism.

Characterization of the Regulatory Regions of the Human Aromatase (P450arom) Gene Involved in Placenta-Specific Expression

Aromatase P450 (P450arom), a product of the CYP19 gene, catalyzes the conversion of C19-steroids to estrogens. Human P450arom is expressed in placental syncytiotrophoblast, ovarian granulosa cells, and adipose stromal cells by use of tissue-specific promoters that are located 5′ of unique untranslated first exons. Mononuclear cytotrophoblasts isolated from midterm human placenta spontaneously fuse in culture to form multinucleated syncytiotrophoblast. These morphological changes are associated with a marked induction of P450arom gene expression. The majority of P450arom transcripts in placental syncytiotrophoblast contain sequences encoded by exon I.1, which lies more than 35 kb upstream of the translation initiation site in exon II. To functionally map genomic sequences required for placenta-specific P450arom expression, fusion genes containing various amounts of DNA flanking the 5′-end of placenta-specific exon I.1 linked to the human GH (hGH) gene, as reporter, were introduced into primary cultures of human trophoblast cells and other cell types. Since the trophoblast cells manifest high levels of aromatase P450 expression, we believe that this provides a physiologically relevant system for characterizing the regulatory regions of this gene. Expression of the fusion genes increased as a function of time in culture in concert with syncytiotrophoblast differentiation and induction of aromatase activity and of P450arom gene expression. P450arom-hGH fusion genes containing 923 and 501 bp of exon I.1 5′-flanking DNA were expressed at comparable levels; these levels were more than 3-fold greater than those of fusion genes containing 2400 bp of exon I.1 5′-flanking DNA, suggesting the presence of an upstream silencer element(s). Expression of these fusion genes was undetectable in cell lines that do not express aromatase or that express aromatase utilizing a nonplacental P450arom promoter. By contrast, P450arom I.1-hGH fusion genes containing 246, 201, or 125 bp of exon I.1 5′-flanking sequence were expressed both in trophoblast cells and in other cell lines. These findings demonstrate that 501 bp of exon I.1 5′-flanking DNA contain response elements required for trophoblast-specific expression of P450arom. These results also suggest the presence of regulatory elements between −501 bp and −246 bp of exon I.1 5′-flanking sequence that bind inhibitory transcription factors expressed in nontrophoblast cells. Deletion and site-directed mutagenesis experiments further suggest that cis-acting elements, including a GC box and two hexameric sequences present within 246 bp of sequence flanking the 5′-end of exon I.1, contribute to the high levels of P450arom promoter activity in primary cultures of placental cells. By competitive and supershift electrophoretic mobility shift assays, it was observed that the ubiquitously expressed transcription factor Sp1 comprises one of the proteins binding to the GC box in the 5′-flanking sequence of P450arom exon I.1.

Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroids and growth hormone in co-cultured adult male rat hepatocytes

To investigate the hormonal control of the expression of flavin-containing monooxygenase (FMO; EC 1.14.13.8) under defined in vitro conditions, adult male rat hepatocytes were isolated by collagenase perfusion and co-cultured with rat liver epithelial cells of primitive biliary origin. The direct effect of 17β-estradiol, testosterone, 5α-dihydrotestosterone (5α-DHT) and human growth hormone (hGH) on FMO activity was studied using this in vitro model. Optimal, non-cytotoxic hormonal concentrations were determined by measuring the lactate dehydrogenase (LDH) index. In addition, the microsomal protein content of the cultured hepatocytes was determined as a function of culture time. The female sex hormone 17β-estradiol caused a significant decrease in FMO as a function of culture time. After 14 days of exposure, FMO activity decreased by 56%. Neither of the male sex hormones or human growth hormone had an effect on FMO activity. These results in co-cultured male rat hepatocytes support in vivo observation that 17β-estradiol is a potent hormone involved in the negative regulation of the expression of FMO in male rat liver.

Modulation of cardiac Na+ current phenotype by beta1-subunit expression.

Na+ current (INa) is smaller, activates and inactivates more slowly, and displays less negative voltage dependence of inactivation in the neonatal rat than in the adult rat. We have observed very similar changes when INa is recorded as a function of time in culture in mouse atrial tumor (AT-1) cells. The differences between mature and immature INa are reminiscent of those observed when skeletal muscle Na+ channel alpha subunits are expressed alone (immature) or with the beta1 subunit (mature). In the present experiments, we tested the hypothesis that suppression of beta1-subunit expression by antisense oligonucleotides would prevent the development of a mature INa. The mouse beta1 subunit was cloned from an AT-1 cDNA library and found to be identical to that in the rat at 216/218 amino acids. AT-1 cells exposed to anti-beta1 antisense oligonucleotides displayed an immature INa at day 8 in culture, whereas untreated cells or cells exposed to sense oligonucleotides displayed a mature INa. This result was observed with 2 different oligonucleotides, and neither affected the rapidly activating component of the delayed rectifier K+ current, another current recorded in AT-1 cells. These findings indicate that in these cells, the gating of INa is modulated by beta1 expression and that alpha-beta1 coexpression is required for the development of a mature cardiac INa phenotype.