Alignment and elongation of human adipose-derived stem cells in response to direct-current electrical stimulation

Abstract

In vivo, direct current electric fields are present during embryonic development and wound healing. In vitro, direct current (DC) electric fields induce directional cell migration and elongation. For the first time, we demonstrate that cultured human adipose tissue-derived stem cells (hASCs) respond to the presence of direct-current electric fields. Cells were stimulated for 2-4 hours with DC electric fields of 6 V/cm that were similar to those encountered in vivo post-injury. Upon stimulation, hASCs were observed to elongate and align perpendicularly to the applied electric field, disassemble gap junctions, and upregulate the expression of genes for connexin-43, thrombomodulin, vascular endothelial growth factor, and fibroblast growth factor. In separate related studies, human epicardial fat-derived stem cells (heASCs) were also observed to align and elongate. It is interesting that the morphological and phenotypic characteristics of mesenchymal stem cells derived both from liposuction aspirates and from cardiac fat can be modulated by direct current electric fields. In further studies, we will quantify the effects of the electrical fields in the context of wound healing.

Fig. 1

Experimental Setup for applying direct-current electrical stimulation. (A) The well-defined chamber geometry permits application of a prescribed electrical field strength, and two salt bridges prevent electrolysis products from contaminating the chamber by providing a pathway for current from each media reservoir to an Ag-AgCl electrode (one anode, one cathode). (B) A view of an assembled chamber. Scale bar corresponds to 1 cm.

Fig. 2

Morphological changes associated with direct-current stimulation. (A) bright-field images taken at time 0, 2, and 4 hrs for hASCs either unstimulated (−ES) or exposed to an electric field of 6 V/cm (+ES) for 2 or 4 hrs. Scale bar corresponds to 200 um, and arrows correspond to direction of electric field. (B) Average cell length. (C) Orientation angle of cells with respect to the horizontal axis. * Significantly different mean value from all other groups; ж significantly different variance from all other groups; § significantly different variance from unstimulated controls (p < 0.01)

Fig. 3

Ultrastructural and phenotypic changes associated with direct-current stimulation of hASCs. Flourescent images taken at time 0 (A, C) and 4 h (B,D) of cells immunostained for actin with phalloidin (red) (A, B) and connexin-43 (green) (C, D) and DAPI (A-D), for cells either unstimulated (A, C) or stimulated with an electric field of 6 V/cm for 4 h (B, D). Scale bar corresponds to 100um, and arrows indicate the direction of the electric field. (E) Density of gap junctions (p<0.01) (F) Gene expression results for RNA levels as compared to housekeeper gene (Cyclo-B) (* p<0.05)

Fig. 4

Preliminary results with electrical stimulation and epicardial adipose-derived heASCs. Bright-field images (A,B), Flourescent images taken of cells immunostained for actin with phalloidin (red) (C, D), connexin-43 (green) (E, F) and DAPI (C-F), for cells either unstimulated (A, C, E) or stimulated with an electric field of 6 V/cm for 4 h (B, D, F). Scale bar corresponds to 100um, and arrows indicate the direction of the electric field.