Marcela García

CIRCADIAN RHYTHM OF DNA SYNTHESIS AND MITOTIC ACTIVITY IN TONGUE KERATINOCYTES

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S‐phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M‐phase), by the colchicine‐arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5‐BrdU for S‐phase determination. Animals given both treatments were divided into six groups and killed at 4h intervals until 20:00h. Tongue samples were processed for histology and immuno‐histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S‐phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.

A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement

IntroductionMesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes.MethodsIn this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement.ResultsWe showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum.ConclusionsThis protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3β- and 17β-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour-bearing mice

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S‐phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S‐strain mice. After an appropriate period of synchronization, the C3H/S‐histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animals flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour‐bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour‐bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.

Circadian rhythm of VEGF expression in the liver of hepatectomized tumor bearing mice

In this study we analyzed VEGF-C expression in regenerating liver (after partial hepactectomy) of ES2 hepatocellular carcinoma bearing mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h throughout one complete circadian cycle from 26 to 50 h post-hepatectomy. Tumor samples were processed for immunohistochemistry. The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 cells (quantitative analysis). The results show that controls and tumor bearing mice have statistical differences at all analysed time points, but the maximun value of VEGF expression of treated animals is significantly higher (p < 0.01) than the control group. We can conclude that when a partial hepatectomy is made with the purpose of eradicating a hepatic tumor, the presence of possible metastasis could release factors related with cellular proliferation that could increase the possibilities of tumoral recidives.

Changes in DNA synthesis circadian rhythms in a hepatocellular carcinoma after hepatectomy

The effect of partial hepatectomy on the DNA synthesis rhythms of ES12a hepatocellular carcinoma grafted into C3H/S male mice was studied and compared with tumor growth in unoperated animal controls. The subtotal hepatectomy also changes the concentration of substances related to cellular proliferation to generate a compensatory hyperplasia in the liver. Thus, we analyzed the interaction between liver regeneration after hepatectomy and the DNA synthesis activity of the tumor. The animals were sacrificed every 4 h throughout one complete circadian cycle. All experimental groups of animals received an intraperitoneal dose of 50 mg of 5-bromodeoxiuridine per kg of body weight before being killed. Tumor samples were processed for histology and immunohistochemistry. DNA synthesis indices were expressed as labeled nuclei per 1000 nuclei. The results show that the ES12a tumor displays the DNA synthesis activity rhythm which, as in normal hepatocytes, passes through a peak during the nocturnal phase, and the surgery alters the temporal structure of the DNA synthesis indices. This fact should be considered in both neoplastic drug treatments and in growth related experiments.

VEGF EXPRESSION IN HEPATECTOMIZED TUMOR-BEARING MICE

The experiments were designed in order to study the VEGF expression in intact (group I), hepatectomized (group II), and hepatectomized-tumor bearing mice (group III) throughout one complete circadian time span. Adult male mice were used for the VEGF expression study. The statistical analysis was performed using analysis of variance (ANOVA). The results showed statistical differences in the VEGF expression between groups I and II, but the most significant differences were found between groups I and III. In conclusion, these expressions have a circadian rhythm in all groups; moreover, in group III, this expression was higher and appeared before than in the others.

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice

Tongue keratinocytes have high S‐phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S‐phase index in cell populations in tumor‐bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor‐free and tumor‐bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4‐h intervals until a circadian cycle was completed (from 26 until 50 h post‐surgery in the operated animals). Each animal was injected with 5‐bromodeoxyuridine (50 mg/kg) 1 h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean ± S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student—Newman—Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.

Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation

Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell–substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.Stem cells: Proteins in secreted vesicles offer potential therapyTiny protein-containing vesicles released by partially differentiated stem cells contain a suite of therapeutic proteins that make them a promising alternative to cell-based treatments. Carlos Luzzani from LIAN-CONICET in Buenos Aires, Argentina, and colleagues reprogrammed skin cells to an embryonic-like state, and then coaxed these induced pluripotent stem cells (iPSCs) to form mesenchymal stem/stromal cells (MSCs), a type of adult stem cell that can give rise to bone, muscle and other tissues. The researchers analyzed all the proteins produced by the iPSCs, MSCs and the sub-micron sized bubbles known as extracellular vesicles that each secretes. They found that vesicles from MSCs, but not iPSCs, included a small set of proteins involved in regeneration and immune modulation. These vesicles may provide the regenerative benefits of MSCs without the safety risks of a cell-based therapy.

Circadian rhythm of VEGF expression in the liver of hepatectomized-tumor-bearing adult and young mice

Abstract We analyzed VEGF expression in regenerating liver (after partial hepatectomy) of tumor-bearing adult and young mice, throughout one complete circadian cycle. The animals were sacrificed every 4 h, from 26 to 46 h post-hepatectomy. Liver samples were processed for immunohistochemistry. The results showed circadian variation of VEGF expression in hepatectomized mice (control group) and hepatectomized-tumor-bearing mice. The maximum value of VEGF expression was found at 16:00/30 h of day/hour post-hepatectomy (HD/HPH) in tumor-bearing young mice, and at 20:00/34 HD/HPH in the controls. In adult mice the maximum values of VEGF expression were at 16:00/30 HD/HPH in tumor-bearing mice, and at 08:00/46 HD/HPH in the controls. Young tumor-bearing mice showed significantly higher mean values than the controls. In conclusion, the presence of the tumor in the animals induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression.