With the aim of developing novel techniques to guide human stem cells, to facilitate stem cell therapy, we investigated the application of EFs to guide hiPS cell migration in both 2D and 3D cultures. We reported that: 1) a DC EF guided migration of hiPS cells in 2D culture; colonies showed significantly greater directionality than dissociated cells; 2) hiPS cells remained immotile in a 3D environment in the absence of an EF, while a small EF induced significant directional migration; 3) electrically guided migration of hiPS cells showed voltage dependence with a threshold voltage of <30 mV/mm; 4) EF exposure did not affect the expression of stem cell markers; 5) hiPS cells migrated toward the anode with greater sensitivity and directedness compared to hES cells which migrated toward the cathode; 6) ROCK inhibition by Y-27632 significantly reduced the directedness of galvanotaxis but increased motility.
Several potential methods to direct migration of stem cells have been explored, in order to overcome the barrier of poor homing and integration of stem cells with the targeted tissues. These methods include: 1) enhancing the chemotaxis of stem cells by genetic manipulation of chemokines and their receptors such as SDF-1/CXCR-4 [8
]; 2) increasing the migratory potential of stem cells by cytokine pretreatment or its receptor overexpression [16
]; 3) removing the extracellular matrix (ECM) barrier by matrix metalloprotease stimulation to accelerate the migration of stem cells [18
]. In addition to those biochemical methods, a biophysical approach using a magnetic field has been shown to offer a guidance cue for the migration of neural stem cells [19
]. However, effective and practical techniques to guide stem cell migration have not yet been established.
We have shown here that small applied EFs can be an effective signal to stimulate and direct migration of hiPS cells. Endogenous EFs occur in the body at sites of wounds and damaged tissue [20
]. Applied EFs guide migration of many types of cells. We have recently provided evidence that EFs could be a predominant guidance cue for corneal epithelial wound healing, over-riding contact inhibition release, free edge, injury stimulation and other well-accepted guidance cues [12
There are several important points to consider when developing methods of electrical stimulation and guidance of hiPS cells for possible practical use. First is the threshold of applied EFs. 3D tissues are volume conductors in which EFs dissipate exponentially. To maintain a constant high-voltage gradient in electrically conductive tissue inevitably involves issues such as electrode products, heat generation, effects on other types of cells, and permeability of blood vessels. Low voltage offers many advantages. In our experiments, an EF of 30 mV/mm induced significant directional migration. This is a voltage below the thresholds for galvanotaxis of fibroblasts and endothelial cells [23
]. Application of an EF of this strength may therefore selectively guide migration of hiPS cells without significant effects on fibroblasts and endothelial cells. The second point is the migration of clusters of cells. Large numbers of hiPS cells will need to be used for transplantation. Clusters of cells are normally immotile, although some cells may move within the clusters (Figs. , ). This poses a problem for the transplanted cells to integrate with the host tissue. Very encouragingly, we have shown that an applied EF was able to stimulate and induce directional migration of clusters of hiPS cells. Thirdly, transplanted cells have to migrate in a 3D environment. We have shown that hiPS cells did not migrate in 3D in matrigel in the absence of an EF (Fig. ). Excitingly, applied EFs stimulated and guided migration of hiPS cells. Exogenously applied EFs have been safely used in the treatment of chronic wounds, neurorehabilitation, and spinal cord injury [25
]. Our results thus provide a basis for developing stimulation techniques for guiding migration of transplanted hiPS cells in vivo
where they need to migrate in 3D.
There are a number of important differences in galvanotaxis between hiPS cells and hES cells, although the biological potency and epigenetic state of iPS cells are indistinguishable from those of ES cells [28
]. Surprisingly, hES (H7) cells migrated in a direction opposite to that of hiPS cells in an EF; hES cells migrated toward the cathode (Fig. ). Since cathodal migration is the most popular migratory response in many different types of cells, we employed the foreskin dermal fibroblasts, from which the hiPS cells were generated in this study, as the cell control, and found that the non-programmed fibroblast also migrate toward the anode in EFs, same to that observed in hiPS cells (supplemental figures 6
; supplemental movies 16
). In addition, hES cells were less sensitive to an EF. The same voltage (100 mV/mm) took 3–4 times longer time to induce a directional response in hES cells and the response was much weaker (Fig. ). Thus, these results revealed new differences in cellular behavior between iPS and ES cells. Whether these differences are due to the different gene expression signatures will need to be studied [13
]. Nevertheless, electrical stimulation might be particularly suitable in therapies using iPS cells compared to ES cells.
ROCK is known to be important to maintain iPS and ES cell cultures and we found that it critically influenced galvanotaxis of hiPS cells. ROCK is the downstream effector kinase of Rho A, a family member of the Rho GTPases. Rho A/ROCK signaling has been implicated in cytoskeleton remodeling, cell adhesion, membrane protrusion, and cell migration [29
]. Recent studies demonstrated that inhibition of ROCK with Y-27632 was an effective means of increasing the survival rate of stem cells including hES and hiPS cells [14
]. The ROCK inhibitor therefore is considered as a promising molecule to aid in the expansion of pluripotent stem cells for regenerative medicine [33
]. We and others have previously found an important role for ROCK in galvanotaxis [15
]. Y-27632 at 2 μM, a concentration much lower than that normally used for enhancing the survival rate of stem cells, dramatically decreased the directedness of hiPS cells in an EF by 75%. ROCK activation thus is involved in galvanotaxis of hiPS cells. A significant decline in directional migration following ROCK inhibition was associated with an increase in the migratory speed of hiPS cells (Fig. ). Reduced cell-cell contact and increased filopodium formation in hiPS cells also occurred following ROCK inhibition in hiPS cells (Fig. , Fig. S5
). Our results are consistent with a previous report suggesting ROCK inhibition promoted cell motility in mouse ES cells [35
]. When considering guiding stem cell migration electrically, the effect of ROCK inhibition will be an important factor.
In summary, a very small EF (30 mV/mm) is an effective cue to guide and stimulate migration of hiPS cells. Most importantly, applied EFs can direct migration of large clusters of hiPS cells which are normally stationary in a 3D environment. This response depends on Rho/ROCK signaling. The size, duration and direction of applied EFs can be relatively easily controlled. Electrical stimulation may offer a practical approach to facilitate stem cell therapy, where guided cell migration and integration with host tissue are needed. These results may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells.