Spontaneously Formed Spheroids from Mouse Compact Bone-Derived Cells Retain Highly Potent Stem Cells with Enhanced Differentiation Capability

The results from our recent study showed the presence of two distinct spheroid-forming mechanisms, i.e., spontaneous and mechanical. In this study, we focused on the spontaneously formed spheroids, and the character of spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) was explored. Cells from (C57BL/6J) mouse leg bones were isolated, and compact bone-derived cells were cultured after enzymatic digestion. Spontaneous spheroid formation was achieved on a culture plate with specific water contact angle as reported. The expression levels of embryonic stem cell markers were analyzed using immunofluorescence and quantitative reverse transcription polymerase chain reaction. Then, the cells from spheroids were induced into osteogenic and neurogenic lineages. The spontaneously formed spheroids from CBDCs were positive for ES cell markers such as SSEA1, Sox2, Oct4, and Nanog. Additionally, the expressions of fucosyltransferase 4/FUT4 (SSEA1), Sox2, and Nanog were significantly higher than those in monolayer cultured cells. The gene expression of mesenchymal stem cell markers was almost identical in both spheroids and monolayer-cultured cells, but the expression of Sca-1 was higher in spheroids. Spheroid-derived cells showed significantly higher osteogenic and neurogenic marker expression than monolayer-cultured cells after induction. Spontaneously formed spheroids expressed stem cell markers and showed enhanced osteogenic and neurogenic differentiation capabilities than cells from the conventional monolayer culture, which supports the superior stemness.

DO MRPS18-2 AND RB PROTEINS COOPERATE TO CONTROL CELL STEMNESS AND DIFFERENTIATION, PREVENTING CANCER DEVELOPMENT?

In childhood tumors, including retinoblastoma, osteosarcoma, and neuroblastoma, the RB-E2F1 pathway is inactivated, as a rule. These tumors arise from precursor cells that fail to undergo the terminal differentiation. Noteworthy, the RB1-encoded protein (RB) does not control the cell cycle in embryonic stem cells. It has not been yet well understood how RB controls cell stemness and differentiation. The question arises why “inactive” RB is required for the survival and stemness of cells? Recently, we have found that overexpression of the RB-binding protein MRPS18-2 (S18-2) in primary fibroblasts leads to their immortalization, which is accompanied by the induction of embryonic stem cell markers and, eventually, malignant transformation. We suggest that cell stemness may be associated with high expression levels of both proteins, RB and S18-2. There must be a strict regulation of the expression levels of S18-2 and RB during embryogenesis. Disturbances in the expression of these proteins would lead to the abnormalities in development. We think that the S18-2 protein, together with the RB, plays a crucial role in the control on cell stemness and differentiation. We hope to uncover the new mechanisms of the cell fate determination. The S18-2 may serve as a new target for anticancer medicines, which will help to improve human health.

Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

Increased Stem Cell Marker Expressions during the Peri-Implantation Period in the Rat Endometrium: Constructive Role of Exogenous Zinc and/or Progesterone

Background. The aim of this study is to determine the effects of zinc and/or progesterone via the expression ofαvβ5 integrins and Vitronectins and embryonic stem cell markers during the peri-implantation period.Methods. Four experimental groups were organized. All subjects were mated with males of the same strain to induce pregnancy; after 5 days, zinc and/or progesterone were administered. Blood levels of zinc and progesterone were determined on the sixth day and endometrial tissues were obtained in order to evaluate the immunohistochemical expression of integrins and embryonic stem cell markers.Results. Theαvβ5 integrin and vitronectin expression increased in the zinc group compared with the control group and no difference in the progesterone group and zinc + progesterone group. Expression of Klf-4, Sox-2, and c-Myc was found to be increased in the zinc group compared to controls, while no difference was determined between the progesterone, zinc + progesterone, and control groups. Distinctively, expression of the embryonic stem cell marker Oct-4 was increased in all of the experimental groups.Conclusions. Expression ofαvβ5 integrin, vitronectin, and embryonic stem cell markers might be increased by the administration of zinc. Our results suggest that zinc could be useful in the induction of implantation rates.

184 ISOLATION AND CHARACTERIZATION OF BOVINE ENDOMETRIAL STEM CELLS

Adult mesenchymal stem cells had been isolated from various tissues of different species, including endometrial tissue of humans, mice, and pigs, but not from cattle. The aim of our work was to identify such cells in the bovine endometrium and to establish a model system in which to test inducers of differentiation and recruiters of stem cell niches, for potential therapeutic use in this and other species, such as horses. We searched for endometrial stem cells in healthy cycling cows and in cattle with clinical (C) or subclinical (SC) endometritis. For this, the uterine tracts of slaughtered cows were collected at early (Days 2 to 5; ELF) and late luteal phases (Days 11 to 15; LLF) of the oestrus cycle of healthy cows. For endometritis-diseased cattle, uterine biopsies were taken in live animals. In all cases, markers of stemness, inflammation, uterine function, and housekeeping were studied both at mRNA and protein level, by RT-qPCR and Western blot/immunohistochemistry respectively. In addition, cell primary cultures were established in vitro from all the animals (n = 4 for ELF, n = 4 for LLF; n = 4 for C and n = 4 for SC). We found that the endometrium of the majority of studied animals expressed embryonic stem cell markers, OCT4 and SOX2, but not or little NANOG, as well as CD44, c-Kit, and STAT3, all markers of mesenchymal stem cells. The expression profile of these markers was not related to the stage of the oestrus cycle; however there was a statistically significant reduction in the expression of embryonic stem cell markers in ill animals, being the lowest in clinically ill and intermediate in subclinical endometritis, (P < 0.05 and Pearson correlation coefficient 0.92). For markers of multipotency (mesenchymal), the expression was lower in clinical endometritis (P < 0.05). In conclusion, the expression profile of stem cell markers is indicative of the presence of stem cells in the bovine endometrium. At the protein level, we verified our findings for OCT4, SOX2, and CD44 using Western blot and immunohistochemistry. In general, there was a concordance between mRNA and protein profiles. Inflammatory markers showed a pattern characteristic for each of the studied stages. In order to have an ultimate criterion of the presence of stem cells, we tested the differentiation potential of the isolated cell lines, upon induction towards chondrogenic, osteogenic, and adipogenic lineages. We found that all the cell lines tested (n = 8) displayed mesenchymal differentiation potential as demonstrated by specific staining and gene expression markers. At present, work is in progress to isolate pure stem cell populations from these primary cultures to further characterise these cells. Conclusion: we showed for the first time the presence and differentiation potential of endometrial stem cells in cattle. This can have an effect on the development of new therapeutic approaches to combat uterine diseases, such as endometritis or endometriosis (in horses). This work was supported by grant FONDECYT REGULAR 1110642, from the Government of Chile.