Oxygen concentration regulates 5-azacytidine-induced myogenesis in C3H/10T1/2 cultures.

This study reports that changing the oxygen concentration within a physiologic range has a striking effect on myogenesis induced by the cytidine analog 5-azacytidine. Reducing oxygen from 20% to 2.5% increases 7-fold the number of myocytes that appear in cultures of C3H/10T1/2 mouse embryo cells 10 days after they receive a 24-h exposure to 5-azacytidine. Reducing oxygen does not alter the extent to which a 24-h exposure to 5-azacytidine inhibits cytosine methylation in newly synthesized DNA. Instead, the oxygen-sensitive step in myogenesis occurs after 5-azacytidine is removed from the culture medium. Reducing oxygen increases the rate of logarithmic growth in C3H/10T1/2 cultures after 5-azacytidine exposure, suggesting that survival and proliferation of myocyte stem cells (morphologically indistinguishable from uncommitted C3H/10T1/2 cells) may be the oxygen-sensitive steps in myogenesis.     

Effects of extracellular matrices derived from different cell sources on chondrocyte functions

Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM.     

DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax

HPLC analysis was used to examine the cytosine methylation of total DNA extracted from four early-flowering lines that were induced by treating germinating seeds of flax (Linum usitatissimum) with the DNA demethylating agent 5-azacytidine. In the normal lines that gave rise to the induced early-flowering lines, flowering usually begins approximately 50 days after sowing. The early-flowering lines flower 7–13 days earlier than normal. The normal level of cytosine methylation was approximately 14% of the cytosines and 2.7% of the nucleosides. In the early-flowering lines, these levels were 6.2% lower than normal in DNA from the terminal leaf clusters of 14-day-old seedlings and 9.7% lower than normal in DNA from the cotyledons and immature shoot buds of 4-day-old seedlings. This hypomethylation was seen in lines that were five to nine generations beyond the treatment generation. The level of hypomethylation was similar in three of the four early-flowering lines, but was not as low in the fourth line, which flowers early but not quite as early as the other three lines. Unexpectedly, the degree of hypomethylation seen in segregant lines, derived by selecting for the early-flowering phenotype in the F₂ and F₃ generations of out-crosses, was similar to that seen in the early-flowering lines. Analysis of the methylation levels in segregating generations of out-crosses between early-flowering and normal lines demonstrated a decrease in methylation level during the selection of early-flowering segregants. The results suggest an association between hypomethylation and the early-flowering phenotype, and that the hypomethylated regions may not be randomly distributed throughout the genome of the early-flowering lines.     

Inhibitory effect of caffeine on 5-azacytidine-induced digital malformations in the rat

The effect of caffeine on 5-azacytidine (5-AC)-induced digital malformations in rat fetuses was investigated. Caffeine suppressed all types of digital defects in the fore- and hindlimbs except for syndactyly induced by 1.0 mg/kg of 5-AC; it was still effective when administered 24 hours after 5-AC treatment. However, fetal mortality increased as the frequency of malformations decreased. While the malformation results support the view that caffeine inhibits the processes leading to malformation expression, the relation between its suppressive effect on malformations and its enhancing effect on fetal mortality is unclear.     

Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

The in vitro lifespan of MRC-5 cells is shortened by 5-azacytidine-induced demethylation

The minor base 5-methylcytosine (5mC) in DNA may be important for the regulation of gene expression. Random loss of 5mC may occur during pre-replicative DNA synthesis in mortal cell strains, and thus give rise to biochemical aberrations in aging cells. 5-Azacytidine (5azaC) was used to induce loss of 5mC in DNA of human diploid fibroblasts (MRC-5) in an attempt to accelerate in vitro senescence. The 5mC content of DNA was measured by incorporation of [3H]uridine into dividing cells, hydrolysis of DNA and separation of bases by HPLC. In untreated MRC-5 cells, 5mC was 3.6% of the total cytosine (C+5mC) at population doubling (PD) 20 (28% of lifespan) and fell to 1.6% at PD 67 (97% of lifespan). A single pulse treatment with 5azaC (1 microgram/ml) induced demethylation and shortened the lifespan by 10% (6.8 PDs loss). Pulse-treated cells showed temporary growth inhibition, though they subsequently regained normal growth rate and morphology. However, uniform treatment with 0.1 microgram/ml 5azaC between PD 20 and 23 produced no immediate growth inhibition, but a 22% loss of 5mC and 25% decrement in lifespan (16.6 PDs loss). The present results indicate that 5mC levels fall during normal aging of MRC-5 cells and accelerated 5mC loss shortens the in vitro lifespan of these cells. Hypomethylation may thus be responsible for some aspects of in vitro aging.     

Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres.

The effects of extracellular matrices and growth factors on the development of isolated blastomeres derived from intact 4-, 8-, and 16-cell porcine embryos (termed, respectively, 1/4, 1/8, and 1/16 blastomeres) were investigated in vitro and in vivo. Blastomeres were incubated in extracellular matrix components fibronectin (FIN) or swine skin gelatin (SSG)-precoated culture dishes containing either modified Krebs' Ringer Bicarbonate solution (mKRB) supplemented with 10% heat-inactivated lamb serum, or Hanks' solution supplemented with 10% heat-inactivated newborn calf serum (NBCS) or Waymouth medium supplemented with 10% NBCS or in noncoated dishes in mKRB supplemented with either insulin (10, 100, or 1,000 micrograms/ml), transferrin (10, 100, or 1,000 micrograms/ml), or cAMP (0.2 or 2.0 micrograms/ml). Cultures observed at 24-h intervals and morphological development was recorded. Blastomeres were classified into three categories according to their morphology: (1) regular blastocysts, (2) trophectodermal vesicles, or (3) no development. After 96 h, culture was determined; the overall diameter of the blastocysts was determined and the nuclei were counted. Blastomeres/blastocysts did not adhere to the bottom of the culture dishes coated with extracellular matrices. Blastocyst formation rate was highest when FIN/mKRB was used and reached 44.3%, 41.8%, and 36.5% for 1/4, 1/8, and 1/16 blastomeres, respectively. The respective blastocysts contained an average of 31.2 +/- 5.8, 58.2 +/- 8.4, and 18.5 +/- 3.5 nuclei and had an overall diameter of 250.0 +/- 10.1, 235.0 +/- 12.8, and 172.5 +/- 13.7 microns, 1/8 blastomeres displayed a better (p less than 0.05) growth rate than 1/4 and 1/16 blastomeres, and 1/8 blastomeres in FIN/mKRB grew better (p less than 0.01) when cultured in an open system than in a microdrop under oil (35.5% vs. 5.0% blastocysts). Neither cAMP nor transferrin had a significant stimulating effect on blastocyst development of 1/8 blastomeres when mKRB plus serum was used as the medium. Insulin supplementation (10 or 100 micrograms/ml) to mKRB plus lamb serum significantly (p less than 0.05) stimulated blastocyst formation rate compared with controls (58.6% and 38.9% vs. 17.7%, respectively; 32.6 +/- 2.5, 58.5 +/- 11.8, and 45.1 +/- 4.6 nuclei, respectively). Transfer of 457 blastocysts grown for 24 h in FIN/mKRB to 16 recipients gilts led to three pregnancies, and two litters were born from 1/8 blastomere-derived blastocysts following 116 days of gestation.4+ is     

Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro

To elucidate the effect of extracellular matrices (ECMs) and related and nonrelated-limbal feeder cells as substitutes for the in vivo niche on the phenotype and genotype of the limbal stem cell (SC) expansion in vitro, human limbal SCs were used. The limbus explants were expanded on human amniotic membrane (AM), commercial ECMs including matrigel (MAT), collagen (COL), and control (no ECM) in presence and absence of feeder cells including human limbal fibroblasts (LFs), a limbus-specific cell and mouse embryonic fibroblasts (MEFs). Proliferation, cell death, immunocytochemistry, expression of specific genes, ultrastructural characteristics, and size and granularity of expanded human limbal SCs in different groups were evaluated. The growth, cell proliferation, and survival of limbal SCs were enhanced by AM and MAT matrices. Ultrastructure and expression of stemness markers revealed that there was no significance difference between AM and MAT. However, flow cytometric analysis showed that the size and granularity of cultured cells increased in the presence of MAT and COL as well as in no ECM group. Moreover, co-culturing of limbal explants with LFs and MEFs on AM and MAT groups, enhanced the expansion and survival of cultured cells in comparison with others. In conclusion, the cultivation of human limbal explants on AM co-culturing with human LFs promises to be a good model for preparing undifferentiated epithelial sheets suitable for transplantation.     

Effects of ascorbate on myogenesis in micromass culture

Summary Micromass cultures from stage 23 and 24 chick wing mesenchyme were grown in serum-containing medium with or without additional ascorbic acid. It was found that ascorbic acid administered as a single pulse or present continuously throughout culture, in concentrations as low as 25 μg/ml, was sufficient to abolish 80% of myogenesis as assessed by immunolocalization using muscle-specific antibodies. This effect was not significantly altered when cultures were maintained in a serum-free medium that promotes myogenesis. In contrast to the above findings, spectrophotometric analysis of accumulated sulphated glycosaminoglycans, an indicator of chondrogenesis, was elevated by ascorbate treatment. Furthermore, a similar level of glycosaminoglycan stimulation was found in ascorbate treated stage 23 distal-tip limb cultures that were essentially free of myogenic cells. We conclude, therefore, that the presence of myoblasts in whole-limb cultures has no appreciable inhibitory effects on chondrogenesis. This work was supported by the Nuffield Foundation, England.     

5-azacytidine-induced tumorous transformation and DNA hypomethylation in Nicotiana tissue cultures

The phenomenon of habituation is considered in plant tissue cultures to be a real process of chemical tumorogenesis; the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca x N. langsdorffii nontumorous hybrid (NNT) during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.     

Cell transport in model extracellular matrices

The rapid transport of cells has been shown to occur by ordered countercurrent convection. This convection can be created by mixtures of macromolecules which make up the extracellular matrix and by the degradation and aggregation products of these macromolecules. The ordered countercurrent convection is manifested in the form of structured flows and arises in isothermal systems with small concentration gradients of solutes. The flows are gravity driven but may rapidly move at angles close to the horizontal axis if they are mechanically constrained to do so. These flows have been shown to rapidly transport cells at rates ranging from 1 to 100 mm h-1, depending on the conditions of the experiment. The transport of cells is nonspecific in that various cell types (chondrocytes, fibroblasts, endothelial cells, and red blood cells) as well as inert particles of similar size (latex beads 6-microns diam) are transported at similar rates. Latex bead transport by structured flow has also been demonstrated to occur in confined spaces in the form of Teflon tubing down to 200 microns in diameter and at angles in the range of 45-90 degrees to the horizontal axis. The flows may also occur over relatively long distances for a prolonged period of time. The conditions for flow formation are simple and widespread. It is suggested that it may contribute to the forces involved in the movement of cells in the extracellular matrix in vivo especially during remodeling and embryogenesis.     

Inhibitory effects of cobalt chloride and cinnamaldehyde on 5-azacytidine-induced digital malformations in rats.

The effect of two DNA repair inhibitors in bacteria, cobalt chloride and cinnamaldehyde, on 5-azacytidine (5-AC)-induced digital malformations was studied. Both agents inhibited the induced digital malformations. The effect of cobalt chloride was significant 3 hr before to 1 hr after the 5-AC treatment, and the effect of cinnamaldehyde was significant 3 hr before to 24 hr after the treatment. However, an increase in fetal mortality was observed with the latter agent. The mechanisms underlying the suppressive effects of both agents may be different, but their natures require elucidation.     

Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.     

Effects of triploidy on rainbow trout myogenesis in vitro

To examine the effects of triploidy on rainbow trout myogenesis in vitro , mononuclear cells were liberated enzymatically from the lateralis muscles of diploid and triploid trout. The muscle of diploids yielded 1 × 10⁶± 1 × 10⁵ (± s.e.m. ) mononuclear cells g⁻¹ muscle compared to 0.7 × 10⁶± 8 × 10⁴ cells g⁻¹ from triploids ( P <0.01). The plating efficiencies of diploid and triploid mononuclear cells on Matrigel™ following 18 h of culture in Leibovitz's L-15 + 10% foetal bovine serum were not significantly different, 35.0 ± 3.5% and 33.0 ± 2.9%, respectively. For most time points examined, the proportion of nuclei in multinucleated cells and the proportion of nuclei in myosin positive cells were not significantly different for diploid and triploid trout. Taken together, these data suggest that diploid and triploid myogenic cells will differentiate similarly when compared under identical, in vitro conditions.     

Spontaneous and 5-azacytidine-induced reexpression of ornithine carbamoyl transferase in hepatoma cells.

Rat hepatoma cells that do not synthesize the hepatic enzyme ornithine carbamoyl transferase spontaneously give rise to producing cells at a low frequency. Reexpression of this differentiation trait is strongly increased by 5-azacytidine treatment, suggesting that hypermethylation plays a critical role in the impaired expression of the ornithine carbamoyl transferase gene in hepatoma cells.     

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.