Electrically guiding migration of human induced pluripotent stem cells

Abstract

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small, physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode, with a stimulation threshold of <30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary, whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF, while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition, a method to aid expansion and survival of stem cells, significantly increased the motility, but reduced directionality of iPS cells in an EF by 70-80%. Thus, our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.

Fig. 1

Electric fields guided migration of hiPS cells in a voltage dependent manner. a and b In the absence of an EF, a hiPS cell colony showed no migration over a period of 3 h. c Tracking of individual cells in the colony showed very little migration and any migration was in random directions. d and e An EF instigated and directed migration of the two colonies of hiPS cells toward the anode (left). f Tracking of individual cells showed the EF directed anodal migration, with increased motility. EF = 100 mV/mm; Scale bar, 50 μm. See Supplementary movies 1 and 2. g Migratory directedness of hiPS cells increased in a voltage dependent manner, with a threshold of 30 mV/mm or below. h EFs significantly increased the trajectory speed. #, P < 0.05; *, P < 0.01 compared to the no EF control

Fig. 2

EFs stimulated and guided migration of hiPS cells in 3D matrigel. a and e Small and large colonies of hiPS cells remained immotile in control culture without an EF over 5 and 8 h, respectively. b A small colony extended multiple protrusions to the anode and migrated in the same direction. f A large colony enlarged a leading process toward the anode and the whole colony migrated to the anode (left). Scale bar, 20 μm in A and B; 25 μm in E and F. See Supplementary movies 6, 7, 8 and 9. c and d EFs significantly increased trajectory speed (C) and migratory directedness (D) of hiPS cells in 3D matrigel in a voltage-dependent manner, with a threshold of <50 mV/mm. *, P < 0.01 compared to the no EF control

Fig. 3

Galvanotaxis of hiPS cells was different from that of hES cells. a No migration (left panel) of a colony of hES (H7) cells occurred in the absence of an EF. Right panel showed directional migration toward the cathode (right) of a hES cell colony in the presence of an EF. Scale bar, 20 μm. b hiPS cells and hES cells migrated in opposite directions in an EF. hiPS cells migrated toward the anode, whereas hES cells migrated toward the cathode. hiPS cells manifested robust galvanotaxis with directedness of −0.70 ± 0.08 within 1 h in an EF, whereas in the same field, hES cells presented a directeness value of only 0.14 ± 0.12 after 3 h. c EFs significantly increased the trajectory speed in both hiPS and hES cells. *, P < 0.01 when compared to the no EF control; #, P < 0.01 compared to hiPS cells. EF = 100 mV/mm. See Supplementary movies 10 and 11

Fig. 4

Rho A/ROCK signalling required for galvanotaxis of hiPS cells. a and b Y-27632, a ROCK inhibitor, induced a tendency for dissociation of cells from the colony to inhibited galvanotaxis. c and d Inhibition of Rock by Y-27632 increased the motility of hiPS cells and inhibited the directional migration. e and f Quantitative analysis showed a sharp decline in migratory directedness and a significant increase in the trajectory speed in Y-27632-treated hiPS cells. Scale bar, 25 μm. EF = 100 mV/mm. See Supplementary movies 12, 13, 14 and 15