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

Abstract

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.

Keywords: Adipogenic; Cell separation; Dielectrophoresis; Differentiation; Mesenchymal stem cells; Osteogenic.

Figure 1

Microscopic evidence of MSCs transformation towards osteogenic (first column) and adipogenic cells (second column). For the osteogenic transformation cells were stained with Alizarin red and observed under phase contrast microscopy, where calcium deposits can be seen as black spots. For the adipogenic transformation, staining was done with Bodipy, and the formation of lipid droplets was observed under fluorescence microscopy as bright spots.

Figure 2

DEP spectra of MSCs (red trace), fully differentiated adipogenic (yellow trace) or osteogenic (blue trace) cells obtained from two donors (A and B). Standard deviations are represented as low opacity areas.

Figure 3

Computed CO frequencies (A and B), and electric parameters (C, D, E) of MSCs (black), fully differentiated adipogenic (grey), and fully differentiated osteogenic (white) cells from the same donor (*, **, *** for p < 0.05, 0.01, 0.001, respectively).

Figure 4

DEP spectra of osteogenic (A) and adipogenic (B) cells from the same donor as a function of the number of weeks in the differentiation process: MSCs (red), week 2 (yellow) and week 4 (blue). Standard deviations are represented as low opacity areas.

Figure 5

First (A) and second (B) CO frequencies of osteogenic (continuous trace) and adipogenic (dashed trace) cells from the same donor as a function of the week of differentiation (*, **, *** for p < 0.05, 0.01, 0.001, respectively; #, $ refers to osteogenic and adipogenic cells, respectively).

Figure 6

Membrane permittivity (A), membrane conductivity (B), and cytoplasm conductivity (C) of osteogenic (continuous trace) and adipogenic (dashed trace) cells from the same donor as a function of the week of differentiation (*, **, *** for p < 0.05, 0.01, 0.001, respectively; #, $ refers to osteogenic and adipogenic cells, respectively).

Figure 7

Simulations of the DEP based microfluidic separation device. (A) Velocity of the cell suspension and DEP buffer, (B) electric field intensity distribution along the central channel. In (C–F) black comets represent undifferentiated MSCs, and grey comets represent differentiated MSCs. (C) MSCs mixed with osteogenic cells at the first week of differentiation, (E) MSCs mixed with fully differentiated osteogenic cells, (D) MSCs mixed with adipogenic cells at the first week of differentiation, (F) MSCs mixed with fully differentiated adipogenic cells. Size distribution and electrical parameters of cells used in the simulations were within measured ranges (Supplementary information Table 2).