Federico Ferro

Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics

Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum.

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.

Adipose tissue-derived stem cell in vitro differentiation in a three-dimensional dental bud structure.

Tooth morphogenesis requires sequential and reciprocal interactions between the cranial neural crest-derived mesenchymal cells and the stomadial epithelium, which regulate tooth morphogenesis and differentiation. We show how mesenchyme-derived single stem cell populations can be induced to transdifferentiate in vitro in a structure similar to a dental bud. The presence of stem cells in the adipose tissue has been previously reported. We incubated primary cultures of human adipose tissue-derived stem cells in a dental-inducing medium and cultured the aggregates in three-dimensional conditions. Four weeks later, cells formed a three-dimensional organized structure similar to a dental bud. Expression of dental tissue-related markers was tested assaying lineage-specific mRNA and proteins by RT-PCR, immunoblot, IHC, and physical-chemical analysis. In the induction medium, cells were positive for ameloblastic and odontoblastic markers as both mRNAs and proteins. Also, cells expressed epithelial, mesenchymal, and basement membrane markers with a positional relationship similar to the physiologic dental morphogenesis. Physical-chemical analysis revealed 200-nm and 50-nm oriented hydroxyapatite crystals as displayed in vivo by enamel and dentin, respectively. In conclusion, we show that adipose tissue-derived stem cells in vitro can transdifferentiate to produce a specific three-dimensional organization and phenotype resembling a dental bud even in the absence of structural matrix or scaffold to guide the developmental process.

Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation

Background: Self-renewal properties are attributed to critical amounts of the OCT4A transcription factor, and little is known about its post-translational regulation. Results: OCT4A interacts with ERK1/2 and is phosphorylated at Ser-111, increasing its ubiquitination and degradation. Discussion: These results suggest an increase in OCT4A degradation downstream of MEK1 activation and FGF2 treatment. Significance: Controlling the mechanism by which cells balance self-renewal would advance our knowledge of stem cells. Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111. To investigate the possibility that ERK1/2 activation can enhance OCT4A degradation, we analyzed endogenous ubiquitination in cells transfected with FLAG-OCT4A alone or with constitutively active MEK1 (MEK1CA), and we observed that the extent of OCT4 ubiquitination was clearly increased when MEK1CA was coexpressed and that this increase was more evident after MG132 treatment. These results suggest an increase in OCT4A ubiquitination downstream of MEK1 activation, and this could account for the protein loss observed after FGF2 treatment and MEK1CA transfection. Understanding and controlling the mechanism by which stem cells balance self-renewal would substantially advance our knowledge of stem cells.

FRTL-5 experiment during ENEIDE mission

The FRTL-5 experiment was performed during the 10 day Italian Soyuz Mission “ENEIDE” (from April 15 to April 25, 2005) on the International Space Station. The main objectives were: 1) the validation of the FRTL5 cells as a biological system to evaluate space environment effects; 2) the investigation of the space environment-related pathophysiological mechanisms of cellular damage and/or behaviour; 3) to verify if fastgrowing cells could be differently sensitive to space environment-related effects as compared to cells in physiological standby. Because of the limited available space in the dedicated facilities and the restrictive requirements imposed by ESA, RSA and NASA, and because no pre-qualified equipment existed, all of the equipment and the procedures have been subjected to structural failure test and to severe qualification tests. Results were: 1) all the qualification procedures and tests were successful 2) Overall cell number is lower in the cultures exposed to space environment as compared to the controls reproducing the temperature conditions during the ENEIDE mission; 3) This phenomenon is most likely related to a slower growth rate in proliferative state; 4) This slow growth rate is: a) reversible, as demonstrated by the results of the growth curves, the plating and cloning efficiencies measured on the samples once they have been returned to our laboratory in Udine; b) mostly related to space effects as indicated by additional control in a clinostat. More experiments of this kind are needed to verify and validate these data and to investigate the molecular mechanisms underling the phenomenon.