Easy and robust electrotransfection protocol for efficient ectopic gene expression and genome editing in human B cells.

B-cell lines and primary PBMCs are notoriously hard to transfect, thus making genome editing, ectopic gene expression, or gene silencing experiments particularly tedious. Here we propose a novel efficient and reproducible protocol for electrotransfection of lymphoblastoid, B-cell lymphoma, leukemia cell lines, and B cells from PBMCs. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. Transfection and viability rates of about 79% and 58%, respectively, have been routinely achieved by optimizing the salt concentration in the electrotransfection medium and the amount of plasmid used. A validation of the protocol was obtained via the generation of a TP53-/- RPMI8866 lymphoblastoid cell line which should prove useful in future hematological and blood cancer studies.

In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

Experimental and Theoretical Brownian Dynamics Analysis of Ion Transport During Cellular Electroporation of E. coli Bacteria.

Escherichia coli bacterium is a rod-shaped organism composed of a complex double membrane structure. Knowledge of electric field driven ion transport through both membranes and the evolution of their induced permeabilization has important applications in biomedical engineering, delivery of genes and antibacterial agents. However, few studies have been conducted on Gram-negative bacteria in this regard considering the contribution of all ion types. To address this gap in knowledge, we have developed a deterministic and stochastic Brownian dynamics model to simulate in 3D space the motion of ions through pores formed in the plasma membranes of E. coli cells during electroporation. The diffusion coefficient, mobility, and translation time of Ca2+, Mg2+, Na+, K+, and Cl- ions within the pore region are estimated from the numerical model. Calculations of pore's conductance have been validated with experiments conducted at Gustave Roussy. From the simulations, it was found that the main driving force of ionic uptake during the pulse is the one due to the externally applied electric field. The results from this work provide a better understanding of ion transport during electroporation, aiding in the design of electrical pulses for maximizing ion throughput, primarily for application in cancer treatment.

Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior.

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered.

Specificity of cancer-related chromosomal translocations is linked to proximity after the DNA double-strand break and subsequent selection.

Most cancer-related chromosomal translocations appear to be cell type specific. It is currently unknown why different chromosomal translocations occur in different cells. This can be due to either the occurrence of particular translocations in specific cell types or adaptive survival advantage conferred by translocations only in specific cells. We experimentally addressed this question by double-strand break (DSB) induction at MYC, IGH, AML and ETO loci in the same cell to generate chromosomal translocations in different cell lineages. Our results show that any translocation can potentially arise in any cell type. We have analyzed different factors that could affect the frequency of the translocations, and only the spatial proximity between gene loci after the DSB induction correlated with the resulting translocation frequency, supporting the 'breakage-first' model. Furthermore, upon long-term culture of cells with the generated chromosomal translocations, only oncogenic MYC-IGH and AML-ETO translocations persisted over a 60-day period. Overall, the results suggest that chromosomal translocation can be generated after DSB induction in any type of cell, but whether the cell with the translocation would persist in a cell population depends on the cell type-specific selective survival advantage that the chromosomal translocation confers to the cell.

Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role.

BACKGROUND: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. METHODS: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. RESULTS: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. CONCLUSIONS: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.

Aluminum particles generated during millisecond electric pulse application enhance adenovirus-mediated gene transfer in L929 cells.

Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.

A Setup for Microscopic Studies of Ultrasounds Effects on Microliters Scale Samples: Analytical, Numerical and Experimental Characterization.

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells' classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

Electrophoresis-assisted accumulation of conductive nanoparticles for the enhancement of cell electropermeabilization.

The use of conductive nanoparticles (NPs) was previously proposed as a way to locally amplify the electric field (EF) intensity at the cell membrane to enhance cell electroporation. To achieve this, a close distance between the NPs and the cell membrane is mandatory. Here, a new method to improve the contact between NPs and cell surface using the effects of electric pulses (electrophoretic forces) is explored. The effects of two types of electric pulses are analyzed alone or combined in a two-pulse-train protocol on Chinese hamster DC-3F cells. Particularly we used 100 µs duration pulses, low intensity-millisecond pulses and combinations of both. Finally, we studied the use of surface coated NPs (PEGylated) for this application. Our results demonstrate that the delivery of an electric field prior to the electroporation pulses increases the accumulation of NPs around the cell membrane suggesting that NPs are pushed towards the cell surface through electrophoretic forces. This allowed reducing the need for long incubations between cells and NPs to observe an enhancement of electroporation mediated by conductive NPs. Thus low intensity-millisecond pulses can be used to increase the accumulation of either aggregated or individual (i.e. PEGylated) NPs supporting the electrophoretic nature of the observed effects.

BDNF-Gene Transfected Schwann Cell-Assisted Axonal Extension and Sprouting on New PLA-PPy Microfiber Substrates.

The work here reported analyzes the effect of increased efficiency of brain-derived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA-PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth.

Cell Electropermeabilisation Enhancement by Non-Thermal-Plasma-Treated PBS.

The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects.

Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane.

Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is approximately 1nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

In vitro osteoblastic differentiation of mesenchymal stem cells generates cell layers with distinct properties.

BACKGROUND: Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties. METHODS: Through the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers. RESULTS: The cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization. CONCLUSIONS: MSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Real time electroporation control for accurate and safe in vivo non-viral gene therapy.

In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested in luciferase gene electrotransfer to mice muscles. Controlled pulses resulted in protection of the tissue and high levels of luciferase in gene transfer experiments where uncorrected excessive applied voltages lead to intense muscle damage and consecutive loss of luciferase gene expression.

Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields.

BACKGROUND: Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions. METHODS: One or several electric pulses of 100 µs were used to induce Ca2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked. RESULTS: According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability. CONCLUSIONS: An easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.

Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions.

Microsecond pulsed electric fields (µsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, µsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.

Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro.

Gene electrotransfer is a safe and efficient nonviral technique for the transfer of nucleic acids of all sizes. Using a small reporter plasmid (3.5 kbp), electrotransfer of more than 90% of the cells, with ~70% viability, can be routinely achieved even in primary cells like mesenchymal stem cells. However, under the same experimental conditions, electrotransfer of larger plasmids (from 6 to 16 kbp) results in very low viability and transfection efficacy. Here, we show that these strong decreases are directly linked to the physical size of the plasmid molecule. Moreover, large plasmids are toxic only when the cells are exposed to electrotransfer pulses. This specific toxicity of large plasmids during electrotransfer is not due to transgene expression and occurs within less than 45 minutes. Indeed, postpulses recovery times of up to 45 minutes are able to entirely abolish the specific toxicity of large plasmid electrotransfer, resulting in a survival and transfection efficacy identical to that of small plasmids. Finally, electrotransfer of small and large plasmids can reach 90-99% of transfection with 60-90% survival considering the findings here reported.

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells.

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 µs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate.

Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer.

Efficient DNA electrotransfer can be achieved with combinations of short high-voltage (HV) and long low voltage (LV) pulses that cover two effects of the pulses, namely, target cell electropermeabilization and DNA electrophoresis within the tissue. Because HV and LV can be delivered with a lag up to 3000 sec between them, we considered that it was possible to analyze separately the respective importance of the two types of effects of the electric fields on DNA electrotransfer efficiency. The tibialis cranialis muscles of C57BL/6 mice were injected with plasmid DNA encoding luciferase or green fluorescent protein and then exposed to various combinations of HV and LV pulses. DNA electrotransfer efficacy was determined by measuring luciferase activity in the treated muscles. We found that for effective DNA electrotransfer into skeletal muscles the HV pulse is prerequisite; however, its number and duration do not significantly affect electrotransfer efficacy. DNA electrotransfer efficacy is dependent mainly on the parameters of the LV pulse(s). We report that different LV number, LV individual duration, and LV strength can be used, provided the total duration and field strength result in convenient electrophoretic transport of DNA toward and/or across a permeabilized membrane.

The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment.

Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC.