Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.     

Evidence of specific characteristics and osteogenic potentiality in bone cells from tibia

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes.     

Bone-like nodules formed in vitro by rat periodontal ligament cells

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in α-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 μg/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-β-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.     

Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway

Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through activation of Caspase-3 and induced autophagy through inhibition of phospho-mammalian target of rapamycin (p-mTOR). Both 3-methyladenine (3MA) and U0126 led to increase of apoptosis in osteoblasts with serum deprivation. Estradiol failed to over-ride the inhibitory effect of 3MA on phosphorylation of AKT but directly led to dephosphorylation of mTOR and upregulation of LC3 protein expression. However, the estradiol-enhanced LC3 protein expression was significantly suppressed by U0126 through inhibition of phosphorylation of extracellular signal-regulated kinase (ERK). Estradiol rescued osteoblast apoptosis via promotion of autophagy through the ER–ERK–mTOR pathway.     

Effect of phenol red and steroid hormones on cystic fibrosis transmembrane conductance regulator in mouse endometrial epithelial cells

Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions. The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique. When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05). The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed. Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro. Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

Effect of culture media on expansion properties of human umbilical cord matrix-derived mesenchymal cells

Background aims. Mesenchymal stromal cells (MSC) have been isolated from a number of different tissues, including umbilical cord. Because of the lack of a uniform approach to human umbilical cord matrix-derived mesenchymal (hUCM) cell expansion, we attempted to identify the optimum conditions for the production of a high quantity of hUCM cells by comparing two media. Methods. We compared the ability of Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and Alpha Minimum Essential Medium (α-MEM) with Glutamax (GL) (α-MEM/GL) to expand hUCM cells. For this purpose, hUCM cells were cultured in plates containing different culture media supplemented with 10% fetal bovine serum (FBS). Culture dishes were left undisturbed for 10-14 days to allow propagation of the newly formed hUCM cells. The expansion properties, CD marker expression, differentiation potential, population doubling time (PDT) and cell activity were compared between the two groups. Results. The hUCM cells harvested from each group were positive for MSC markers, including CD44, CD90 and CD105, while they were negative for the hematopoietic cell surface marker CD34. Differentiation into adipogenic and osteogenic lineages was confirmed for both treatments. Cell activity was higher in the α-MEM/GL group than the DMEM/F12 group. PDT was calculated to be 60 h for the DMEM/F12 group, while for the α-MEM/GL group it was 47 h. Conclusions. Our data reveal that α-MEM/GL with 10% FBS supports hUCM cell growth more strongly than DMEM/F12 with 10% FBS.     

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.     

Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.     

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0.5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.     

Effect of a continuously applied compressive pressure on mouse osteoblast-like cells (MC3T3-E1) in vitro

Bone metabolism is often affected by a variety of mechanical forces, but the cytological basis of their action is not known. In this study, we examined the effect of a continuously applied compressive pressure (CCP) on the growth and differentiation of clonal mouse osteoblast-like cells (MC3T3-E1) cultured in a specifically devised culture chamber. The gas phase of the chamber was maintained at a pressure of 2 atmospheres (atm) above ambient (3 atm total, 3.1 kg/cm2; 3.0 x 10(5) Pa) by continuously infusing a compressed mixed gas (O2: N2:CO2 = 7.0%:91.3%:1.7%). The pO2, pCO2, and pH in the culture medium at 37 degrees C under 3 atm were maintained at the same levels as those under 1 atm. MC3T3-E1 cells were cultured in alpha-minimal essential medium containing 10% fetal bovine serum under either 3 atm in the CCP culture chamber or 1 atm in an ordinary CO2 incubator. Alkaline phosphatase activity, a marker of osteoblasts, was greatly suppressed by the CCP treatment. The inhibition of alkaline phosphatase activity was rapidly restored when the cells were transferred to an ordinary CO2 incubator under 1 atm, indicating that the inhibition of alkaline phosphatase activity by CCP is reversible. Cell growth was not altered under CCP. The CCP treatment greatly increased the production and secretion of prostaglandin E2 (PGE2). Adding either conditioned medium from the CCP culture or exogenous PGE2 to the control culture under 1 atm suppressed alkaline phosphatase activity dose-dependently. The CCP treatment also suppressed collagen synthesis and calcification. These results suggest that CCP causes the cells to produce and secrete PGE2, which, in turn, inhibits differentiation of osteoblasts and the concomitant calcification.     

Interferon stimulated gene 15 (ISG15): Molecular characterization and expression profile in endometrium of buffalo (Bubalus bubalis)

A sequential medium was evaluated on the survival, activation and growth rates of caprine preantral follicles submitted to a long-term culture period, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with hormones (GH and/or FSH) added sequentially on different days of culture. Ovarian fragments were cultured in the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+), FSH/FSH, FSH/GH, FSH/FSH+GH, GH/GH, GH/FSH, GH/FSH+GH, FSH+GH/FSH+GH, FSH+GH/FSH and FSH+GH/GH. Follicle morphology, viability and ultrastructure were analyzed. After day 1 of culture, FSH treatments maintained the percentage of normal follicles similar to the fresh control. At day 16 of culture, the treatment FSH/GH showed the highest (P<0.05) percentage of normal follicles. The ultrastructure of follicles was preserved in the fresh control and FSH/GH treatment. Follicles cultured with FSH/GH had a higher (P<0.05) viability than α-MEM(+); however the viability was lower (P<0.05) when compared to the fresh control. The FSH/GH treatment showed the highest (P<0.05) percentage of follicular activation and secondary follicle formation and produced the largest (P<0.05) mean follicular diameter after 16 days of culture. In conclusion, a sequential medium supplemented with FSH followed by GH during a long-term culture maintains the survival, viability and ultrastructure of goat preantral follicles, and promotes activation and secondary follicles.     

Comparative Effect of Human Platelet Derivatives on Proliferation and Osteogenic Differentiation of Menstrual Blood-Derived Stem Cells

Menstrual blood has been recognized as an easily accessible and inexpensive source of stem cells, in recent years. To establish a safe and efficient protocol for development of menstrual blood-derived stem cells (MenSCs) into osteoblasts, the effect of substitution of fetal bovine serum (FBS) with human platelet derivatives (HPDs) was evaluated during proliferation and osteogenic differentiation of MenSCs. To this aim, parallel experiments were carried out on cultured MenSCs in the presence of platelet-rich plasma, platelet-poor plasma, platelet gel supernatant, or human platelet releasate (HPR), and compared with cells cultured in conventional growth medium containing FBS. There was no significant difference between growth curves of cultured MenSCs in presence of different fortified media. However, the MenSCs demonstrated variant differentiation patterns in response to FBS replacement with HPDs. Mineralization, as judged by Alizarin red staining, was significantly higher in cells differentiated in the presence of HPR compared to cells that were fortified with other medium supplements. A greater osteocalcin production level, alkaline phosphatase activity, and mRNA expression of osteogenic-specific genes in differentiated MenSCs under HPR condition further confirmed our previous findings. Based on our data, FBS substitution by HPDs not only allows for successful MenSCs proliferation, but also promotes MenSCs development into osteoblasts. The effectiveness of HPR on osteogenic differentiation of MenSCs represents an important novel step toward safe and applied stem cell therapy of bone diseases.     

Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture

This study examined the effects of 17-beta-estradiol (E₂) on chondrocyte differentiation in vitro. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used to determine whether the effects of E₂ were dependent on the stage of chondrocyte maturation and whether they were sex-specific. [³H]-incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators of differentiation. Alakaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. Specificity of the E₂ effects was assessed using 17-alpha-estradiol. The role of fetal bovine serum and phenol red in the culture medium was also addressed. The results demonstrated that E₂ decreases cell number and [³H]-incorporation in female chondrocytes, indicating that it promotes differentiation of these cells. Alkaline phosphatase specific activity is stimulated in both growth zone and resting zone cells, but the effect is greater in the less mature resting zone chondrocytes. The increase in enzyme activity is targeted to the matrix vesicles in both cell types, but the fold increase is greater in the growth zone cells. In male chondrocytes, there was a decrease in [³H]-incorporation at high E₂ concentrations in resting zone cells at the earliest time point examined (12 hours) and a slight stimulation in alkaline phosphatase activity in growth zone cells at 24 hours. Cells cultured in serum-free medium exhibited a dose-dependent inhibition in alkaline phosphatase activity when cultured with E₂, even in the presence of phenol red. E₂-stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. E₂ increased percent collagen production in female cells only; the magnitude of the effect was greatest in the resting zone chondrocyte cultures. The results of this study indicate that the effects of E₂ are dependent on time of exposure, presence of serum, and the sex and state of maturation of the chondrocytes. E₂-stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles. © 1993 Wiley-Liss, Inc.     

Isolation and expansion of equine umbilical cord-derived matrix cells (EUCMCs)

Stem cells from extra-embryonic sources can be obtained by non-invasive procedures. We have standardized a method for the expansion of equine umbilical cord-derived matrix cells (EUCMCs) for potential therapy.EUCMCs were isolated from the umbilical cord of five mares immediately after delivery. For expansion, cells were grown in α-MEM and MSCBM. Moreover, to measure the effect of growth factor supplementation, epidermal growth factor (EGF) was added to α-MEM.α-MEM and MSCBM media performed similarly in terms of population doubling and CFU number value. EGF supplementation of α-MEM determined a significant increase of the population doubling value. EGF supplementation did not affect the adipogenic and chondrogenic differentiation while bone nodule sizes an increased with the osteogenic protocol.Both α-MEM and MSCBM can be used to cultivate EUCMCs. α-MEM supplemented with EGF might represent an advantage for EUCMCs expansion. The results could be useful in choosing the culture medium since α-MEM is more cost-effective than MSCBM.     

Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton’s jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4–6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro.     

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.     

Kinetics of cellular proliferation and hormonal receptors in EVSA-T breast cancer cell line.

The hormone-independent human breast cancer cell line EVSA-T, originally described as negative estrogen and progesterone receptors is shown to become positive hormone receptors when the cellular proliferation rate is slowed down. The experimental procedure included the following steps: 1) EVSA-T cells were seeded in minimum essential medium supplemented with 10% fetal bovine serum and kept undisturbed for 2 days; 2) culture medium was replaced with Dulbecco's solution and Ham's F-12 and cells were incubated in serum-free media for another 24 h; 3) then, cells were "rescued" with 10% FBS supplemented medium and estrogen (ER) and progesterone receptors (PgR) were measured immediately, time 0, and 6, 12, 18, 24 and 30 h after the media were changed. Cell yield was quantified at the same times. Experimental data indicate that changing the proliferation kinetics makes it possible to detect estradiol and progesterone receptors on EVSA-T cells. Estrogen receptor appeared at 18 h after rescue, 6 hours before progesterone receptor could be detected. Immunohistochemical analysis of ER content confirmed this observation, showing maximal positive stain at 18 h. Furthermore, ER disappeared when cells recovered their normal proliferation rate.