Elimination of [PSI+] Prion and Influence on Yeast Cell Growth

Background Prions are proteinaceous infectious particles that act as pathogens and cause the development of lethal neurodegenerative diseases in humans and other animals. Yeast Saccharomyces cerevisiae is a widespread model system in which mechanisms of prion induction and elimination have been identified. New and safe substances and methods are being sought to cure cells of prion proteins. It is particularly important that by treating cells from prions and restoring them from the [PSI+] to the [psi−] form, the primary growth of the cells is restored. One of the main objectives of this study was to determine the growth dynamics of S. cerevisiae cells with different [PSI+] prion variants, cells that have lost [PSI+] prion variants, and cells that never had [PSI+] prion variants. Results In this research, we applied GuHCl and combined GuHCl and PEF treatment against [PSI+] prion. We evaluated cells culture growth dynamics – optical density and doubling time and determined that method of [PSI+] prion elimination does not affect cell doubling time. Also, we found that both elimination methods affect the optical density reached by [psi−] cells. However, the cells in which the [PSI+] prion has been eliminated by GuHCl alone are able to reach the same optical density as unaffected [psi−] cells and higher optical density than the affected [psi−] cells by GuHCl alone. Conclusions These findings indicate the potential long-term positive effect of [PSI+] prion on cell growth, which persists after [PSI+] removal.

Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem cells

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17β signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte–macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.

Postnatal Changes of Neural Stem Cells in the Mammalian Auditory Cortex

Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.

Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation

Bone tissue engineering-based therapy for bone lesions requires the expansion of seeding cells, such as autologous mesenchymal stem cells (MSCs). A major obstacle to this process is the loss of the phenotype and differentiation capacity of MSCs subjected to passage. Recent studies have suggested that primary cilia, primordial organelles that transduce multiple signals, particularly hedgehog signals, play a role in senescence. Therefore, we explored the relationships among senescence, primary cilia, and hedgehog signaling in MSCs. Ageing of MSCs by expansion in vitro was accompanied by increased cell doubling time. The osteogenic capacity of aged MSCs at passage 4 was compromised compared to that of primary cells. P4 MSCs exhibited reductions in the frequency and length of primary cilia associated with decreased intensity of Arl13b staining on cilia. Senescence also resulted in downregulation of the expression of hedgehog components and CDKN2A. Suppression of ciliogenesis reduced the gene expression of both Gli1, a key molecule in the hedgehog signaling pathway and ALP, a marker of osteoblastic differentiation. This study demonstrated that the senescence of MSCs induced the loss of osteoblastic differentiation potency and inactivated hedgehog signaling associated with attenuated ciliogenesis, indicating that primary cilia play a mediating role in and are biomarkers of MSC senescence; thus, future antisenescence strategies involving manipulation of primary cilia could be developed.

Hyperglycemia and hyperinsulinemia stimulate cervical cancer cell proliferation in vitro and in vivo study

Background: Diabetes mellitus and malignant tumor are the second and third causes of women death in Mexico. Hyperglycemia, insulin and insulin-like growth factor 1 are the main risk factors involved in cancer development in patient with diabetes. The aim of this study was to evaluate the effect of hyperglycemia and hyperinsulinemia over cell proliferation and tumor growth in cervical cancer. Methods: Cell proliferation, apoptosis and cell cycle of cervical cancer cell lines (HeLa, SiHa and CaSki) in presence of hyperglycemia and/or insulin were evaluated. Xenograft model for cervical cancer was done in diabetic female nu/nu mice; biochemical parameters, body weight, tumoral volume and cell doubling time were evaluated. Results: Hyperglycemia and hyperinsulinemia significantly increase cell proliferation and decreases apoptosis with no change in cell cycle. Insulin treatment increase tumor volume and diminish cell doubling time, this group also developed hyperinsulinemia and in Langerhans pancreatic islet hypertrophy; whereas, hyperglycemic groups show the same effects but in lesser degree than the insulin treated group. Conclusion: Glucose and insulin stimulates both, proliferation and tumoral growth of cervical cancer, so this should be a possible explanation for the low survival of diabetic patients with cervical cancer in compare to non-diabetic patients with cervical cancer.

Cytotoxicity, Antiproliferative and Apoptotic Effect of n-Hexane Fraction of Lime Parasite (Dendrophtoe pentandra)

Breast cancer is one of the biggest causes of death in women in the world. Lime parasite (Dendrophtoe pentandra (L.) Miq.), a folk remedy used by Indonesian people, is believed to be efficacious as anticancer drug. This research aims to know the activity of n-hexane fractions of lime parasite in inhibiting the proliferation and apoptosis of T47D cells in vitro. Cytotoxic test with MTT method assay from n-hexane fractions used a multilevel concentration. Antiproliferative test was carried out by the method of MTT assay and cell doubling time was calculated at the time of duplication. Apoptotic test was done with concentration of 1 IC50and ½ IC50which was analyzed by flow cytometry. The results reveals that fractions of lime parasite have cytotoxic activity with concentration of IC50is included in moderatecytotoxic level. The result of the doubling time of the optimum fraction of n-hexane is in 31 hours with the concentration of ¼IC50. Results for the flow cytometry shows the fraction of n-hexane does not induce apoptosis in cells of T47D. Those results show that the active fraction of lime parasite has cytotoxic activity which is able to inhibit proliferation, but does not induce apoptosis of T47D cell.