Presence Of Differentiation Factors Research Articles

Murine osteoclastogenesis suppression using conditioned media produced by melanoma or activated and non-activated Jurkat-E6 cells.

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non-activated Jukart-E6 cells, as well as from one murine (B16-F10) and one human melanoma cell line (SK-MEL-28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony-stimulating factor, prostaglandin E2, receptor activator of nuclear factor-κB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor-α and interferon γ (IFN-γ) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat-E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat-E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat-E6 cells shows higher secretion of IFN-γ, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16-F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.

Differentiation of adult human mesenchymal stem cells into dopaminergic neurons.

The striatal dopamine (DA) deficiency is known as the main cause of the clinical picture of Parkinson’s disease (PD). The disease is a progressive degeneration of dopaminergic neurons in the striatum. The treatment of PD is based on compensation for the brain’s supply of DA lost by drug therapy, deep brain stimulation, surgery, gene and cell therapies. Clinical studies have focused on the utility of stem cell-based therapies in PD. Embryonic and mesenchymal stem cells (MSCs) are widely used. Recently, human adipose derived stem cells (hADSCs) have been considered as a suitable source of tissue for this purpose. In this project, hADSCs differentiated into dopaminergic neurons and the specificity of the cell preparations was examined. Human adipose tissues were collected from healthy volunteers undergoing liposuction and hADSCs were isolated by collagenase-based enzymatic method. Flow cytometry was performed using the surface cluster of differentiation (CD) markers to confirm the cell typical properties. Then hADSCs were differentiated to dopaminergic neurons in neurobasal medium in the presence of differentiation factors and confirmed by immunocytochemistry via neuronal and dopaminergic markers. The isolated hADSCs were cultured and identified by the expression of MSCs surface markers including CD90, and CD44. These cells did not express hematopoietic surface markers such as CD45 and CD14. Differentiated cells express neuronal marker NeuN and dopaminergic marker tyrosine hydroxylase (TH). It is concluded that hADSCs can be easily taken from the patient’s own body and differentiated into dopaminergic cells having a lower risk of transplant rejection.

SU-8-Derived Carbon Nanopillars Enhance Stem Cell Differentiation into Dopaminergic Neurons

Parkinson’s disease is a serious problem, especially as the life span increases in the general population, mostly because there is no cure and the current treatment options are far from being optimal. The disease is characterized by insufficient dopamine in the brain, a neurotransmitter involved in the motor function. Recent approaches recognize the need for personalized treatment, and one option is cell replacement therapy. This requires obtaining a large population of mature, dopaminergic neurons. Our group has previously shown that SU-8-derived carbon surfaces (either flat carbon surfaces or carbon micropillars) induce spontaneous differentiation of human neural stem cells (hNSCs) into dopaminergic neurons1. Here we report 3D carbon nanopillars, which show a high increase in the currents measured from dopamine exocytosis from hNSc-derived dopaminergic neurons. The carbon surfaces mentioned above can be employed as substrates for cell growth, but also as electrodes for monitoring dopamine release. Fabrication of the nanopillars is done by colloidal lithography and pyrolysis. A silicon wafer with a 200 µm SiO2layer on top is first spin-coated with 5 µm of SU-8 2005. Then, a monolayer of polystyrene beads is deposited on the SU-8 film and serves as etching mask during the plasma treatment. The diameter of the nanopillars can be tuned using beads of different sizes and the height can be controlled by adjusting the etching time. After etching, we use ultrasonication in isopropanol as lift-off technique to remove the beads. Finally, the samples are pyrolysed (1h at 900°C, with 2°C/min for both heating and cooling) to obtain the carbon nanopillar electrode chips. A schematic representation of the fabrication process is shown in figure 1A. The nanopillars employed for the measurements described in this abstract were obtained by using 1µm beads and an etching time of 20 min, which leads to structures with a height of 1.2 µm and a diameter of 450 nm (before pyrolysis, figure 1B) and a height of 600 nm and a width of 200 nm after pyrolysis (figure 1C). The carbon structures were employed as substrate for cell growth and differentiation (figure 1D shows a high-magnification SEM image of a cell growing on the nanopillars) after plasma treatment (to improve surface wettability) and poly-L-lysine modification (to improve cell adhesion). Even without adding differentiation factors to the system, differentiation into dopaminergic neurons was observed, with a yield of about 80% dopaminergic neurons. SEM imaging, immunocytochemistry and amperometry were employed as techniques to confirm this. The immunostaining was done for tyrosine hydroxylase (TH) and nuclei and shows that most cells are TH-positive and thus potentially dopaminergic. The dopaminergic phenotype was confirmed by amperometric monitoring of the dopamine released (exocytosis) from cells induced by KCl stimulation. Amperometric measurements were conducted on electrodes with nanopillars i) 48 h after cell seeding, in the absence of differentiation factors and ii) 10 days after cell seeding, in the presence of differentiation factors. For comparison, flat carbon surfaces and carbon micropillars were fabricated and tested in parallel. Compared to flat carbon surfaces, both the micropillars and the nanopillars show increased currents when measuring released dopamine using amperometry. We calculated the charges for exocytosis on micropillars as being 4 times higher than in the case of flat carbon, although the surface area increase is of just 1.9 times. On nanopillars, the measured charges are even higher, 12 times larger than for the flat carbon and 3 times larger than for micropillars, although in this case, the surface area increase is of only 1.4 times compared to the flat carbon. This leads to an improved detection and quantification of released dopamine when using nanopillar electrodes. The electrode chips used in all 3 cases have the same basic footprint size and were seeded with the same number of cells, in the same conditions. This work shows that carbon surfaces with nanopillars induce spontaneous differentiation of human neural stem cells into dopaminergic neurons and can be employed as electrodes for monitoring dopamine release from mature dopaminergic cells, with a much better electrochemical response compared to flat carbon surfaces or micropillar arrays. L. Amato et. al., Pyrolysed 3D-Carbon Scaffolds Induce Spontaneous Differentiation of Human Neural Stem Cells and Facilitate Real Time Dopamine Detection, Advanced Functional Materials, 2014, Vol. 24, Issue 44, 7042-7052. Figure 1

Histogenesis of odontogenic tumors

A theoretical presentation concerning the histogenesis of mixed odontogenic tumors in relation to tooth germ embryogenesis is discussed. It is proposed that the individual tumors that make up this group are solely and totally dependent upon the presence of differentiation factors which are or are not elaborated by a particular tumor. The probability of sequential differentiating events resulting in the progression of an immature entity (ameloblastic fibroma) to a highly differentiated entity (odontoma) has been discounted, with reservations. Rather, it is postulated that individual mixed odontogenic tumors arise at comparable stages observed during normal odontogenesis and, as such, are incapable of further differentiation and thus proliferate within the histologic confines of their innate differentiated capacity.