Nanotoxicology and influence of nanoparticles on human Mesenchymal Stem Cells
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Background and Aims:
Nanotechnology is expected to be promising in many fields including medical applications. While a large number of very attractive exploitations open their way to the clinics, the control agencies seem to be very careful in admitting newer nanomaterials to the use in humans, because of their potential toxicity. The very active research of new nanomaterials potentially useful in medicine has not been counterbalanced by an adequate knowledge of their pharmacokinetics and toxicity. Only recently, a systematic classification of nanomaterials has been proposed, posing the basis for dedicated modeling at nanoscale level. However, the use of in silico methods such as nano-QSAR and PSAR, are not alternative, but introductive to the mandatory experimental work.
Stem cells (SC), present in adult tissues, play an important role in the regeneration of damaged tissues and in the homeostasis of healthy ones; to interfere with their behavior could be dangerous. The nanoparticles (NPs) introduced into the body accidentally or deliberately, for diagnostic and therapeutic purposes, may encounter SC and modify their vitality, proliferation and differentiation.
One of the aim of the research conducted in our Lab is the evaluation of the effect of nanoparticles on viability, proliferation and differentiation of human cells with particular attention toward human mesenchymal stem cells (hMSCs) derived from adult adipose tissue.
NP characterization by TEM analysis and evaluation of dissolution.
Cytotoxicity: viability experiments are conducted using SKOV3, HCT116 e U87 cells lines and MSCs (primary culture). Cell will be seeded into 96-well assay plates and cultivated for 24 h at 37°C in 5% CO2 to equilibrate and become attached prior to the treatment. The cells will then be exposed, for different time at increasing concentrations NPs. Cell viability will be determined evaluating the ATP cell content by CellTiter Glo ®, or by MultiTox-Fluor Multiplex Cytotoxicity Assay ®, Promega, Milano, Italy).
NP uptake and intracellular localization: cells will be exposed to a sub lethal amount of NPs for and analyzed by electron microscopy (TEM and SEM). For TEM studies, cells will be harvested, fixed in 1% Karnowsky solution in 0.1M sodium cacodylate buffer (pH 7.4) for 6 hours at 4°C and postfixed with 1% osmium tetroxide in 0.1M Na-cacodylate buffer (pH 7.2), dehydrated and embedded in Epon-Araldite 812 resin.
Gene expression by Real Time PCR: we consider genes involved in the differentiation of MSCs (AdipoQ, FABP4, ACSS2), genes whose expression has been reported to be modified by stress condions(MT, HSPs), and housekeeping genes. For each of these genes, we design specific primers for real-time PCR analysis using the SybrGreen method and the CFX96 thermocycler (BioRad, Segrate, MI, Italy). The choice of housekeeping genes should be conducted with attention. The perfect control gene for real-time RT-PCR probably does not exist but the concurrent use of several housekeeping genes is a prerequisite for accurate and reliable gene profiling (Gornati et al., 2007). RT-PCR is also use to evaluate the expression of three specific markers positive for MSCs (CD44, CD90, CD105) and a negative marker (CD45) to confirm that the hMSCs are stem cells.
Telomerase activity: telomerase is an enzyme that has been attracting much attention in recent years because it is central in the processes of malignant transformation. The activity is measured in cellular extract examining, by quantitative PCR, the enzyme ability to synthesize telomere repeats on an oligo-nucleotidic substrate.
Morphology: hMSCs tare cultivated in specific media able to induce differentiation into osteogenic, adipogenic and chondrogenic lineages. The cell cultures are observed by phase-contrast microscope (Zeiss Axiovert 200, Arese, Milano, Italy) equipped with a data acquisition system (AxioCam The Cm1camera, Zeiss, Arese, Milan Italy) and the software (AxioVision Rel 4.8 Zeiss, Arese MI Italy). The same samples, stained with Von Kossa, Oil Red O and Alcian blue, are watch by bright field microscope equipped with a data acquisition system.