Electrically Guiding Migration of Human Induced Pluripotent Stem Cells
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.
KeywordsHuman induced pluripotent stem (hiPS) cellElectric field (EF)Cell migrationWound healingGalvanotaxis
Human induced pluripotent stem (hiPS) cells show great potential for stem cell based therapies, as they are very similar to human embryonic stem (hES) cells in their ability to self-renew and to differentiate into many types of cell within the body, and are free from the ethical issues to immunological barriers associated with the use of hES cells . While there has been a great deal of interest in the pluripotency and differentiation of hiPS cells, little is known about their migration and guidance cues.
Amongst the various challenging hurdles to successful stem cell therapy, is the poor homing to and integration of transplanted stem cells with target tissue. Poor healing responses and lack of regeneration may be partly due to the fact that very few therapeutic stem cells actually migrate to the damaged/diseased tissue to mount a reparative response [2, 3]. One example of such poor homing responses comes from a model of renal ischemia/reperfusion injury, in which intravenously injected mesenchymal stem cells (MSCs) sequestered mainly in the lung and liver, with less than 1% of injected MSCs reaching the injured kidney . Also, homing of MSCs to infracted areas in heart has been shown to be very poor, and this contributes to poor survival rate . Effective techniques to stimulate and guide migration of stem cells may therefore offer new strategies to improve the efficiency of stem cell therapies.
Signals that guide and regulate the migration of stem cells include growth factors (GFs), chemokines, molecules of the extracellular matrix (ECM), and their respective receptors. The importance of stromal-derived factor-1 (SDF-1), a well studied chemokine expressed by a variety of cell types including stromal cells and stem cells, and its unique receptor CXCR4 in migration and homing of stem cells has been demonstrated. This SDF-1/CXCR4 signaling plays a critical role in the homing of hematopoietic stem cells , the directional migration of neural stem cells to sites of central nervous system (CNS) injury , and the directional migration of endothelial progenitor cells . In addition to SDF-1/CXCR4, the flucosylated CD44 has been shown to promote efficient adhesive interactions of MSCs with marrow vasculature and subsequent homing to endosteal surfaces . Also, it has recently been suggested that insulin-like growth factor 2 (IGF-2)/IGF-2 receptor signaling is essential for homing of endothelial progenitor cells and vasculogenesis at ischemic or tumour sites . In contrast, CD26 has been shown to be a negative regulator of hematopoietic stem cell homing .
Applied electric fields (EFs) may be another effective means to guide directional migration of hiPS cells. Damaged tissue and wounds produce weak, direct current (DC) EFs, which direct migration of many cell types. This phenomenon is known as galvanotaxis or electrotaxis . With the aim of developing novel techniques to guide the migration of stem cells, we tested whether EFs direct the migration of hiPS cells. We found that: 1) applied EFs guide migration of isolated or clustered hiPS cells toward the anode, with a threshold voltage of <30 mV/mm; 2) EFs significantly increase migration speed of hiPS cells by 20% at 50 mV/mm, and 40% at 75 mV/mm; 3) EFs directed migration of hiPS cells in three-dimensional (3D) culture; 4) exposure to an EF did not alter expression of the stem cell markers SSEA-4 and Oct-4 in hiPS cells; 5) galvanotaxis of hiPS cells differed from that of hES cells in migratory speed, time-course of migration, to direction of migration; 6) Rho kinase (ROCK) inhibition significantly inhibited the directedness of migration, but increased the speed of migration in hiPS cells.
Materials and Methods
Human iPS cells (foreskin-derived iPS cell line, clone #3, a kind gift from Dr. James Thomson), were maintained on mouse embryonic fibroblasts (MEFs) (Globalstem Rockville, MD) in 6-well tissue culture plates (Thermo Fisher Scientific, Waltham, MA) with hESC medium: DMEM/F12 supplemented with glutamine, 20% knockout serum replacement, non-essential amino acids, 0.1 mM β-mercaptoethanol (all from Invitrogen, Inc., Carlsbad, CA), to fibroblast growth factor 2 (FGF2) (20 ng/ml, Chemicon). For experiments using feeder-free conditions, human iPS cells were transitioned from fibroblast feeders to hESC-qualified matrigel-coated galvanotaxis chamber with small colony clusters. Cells were allowed to grow in mTeSR™ maintenance medium (Stem Cell Technologies, Vancouver, BC, Canada) for approximately 12 h at 37°C with 5% CO2 prior to experiments. For ROCK inhibition experiments, Y-27632 (Tocris Bioscience, Ellisville, MO) was added to the cells 30 min before exposure to an EF with the final concentration of 2 μM. The foreskin-derived fibroblast (CRL-2522™, ATCC, Manassas, VA) was used as the control for hiPS cells.
Human embryonic stem cell line H7 (WiCell Research Institute, Madison, WI) was cultured in mTeSR™ maintenance medium at 37°C with 5% CO2 with daily medium change. One day prior to the EF experiments, hESCs were treated with 1 mg/ml dispase (Invitrogen, Carlsbad, CA) and manually detached with a cell scraper. Small colony clusters were then plated in the hESC-qualified matrigel-coated electrotactic chamber in mTeSR™ culture medium, at a variety of cell densities up to 3~5 × 103 cells/cm2. Cells were allowed to attach and grow for approximately 12 h at 37°C with 5% CO2 prior to experiments.
3D Matrigel Culture
For 3D culture of human iPS cells, a stepped dilution using a combination of hiPS cell-qualified matrigel and mTeSR™ medium was tested to optimize the culture condition. One day prior to the EFs experiments, hiPS cells were treated with 1 mg/ml dispase (Invitrogen, Carlsbad, CA) for 8 min and manually detached with a cell scraper. Cells at density of 0.5~1.0 × 105/ml were transferred to a 0.5-ml tube and mixed well with matrigel at a volume ratio of 1:1 by gently pipetting up and down to distribute the colonies. 20 μl of matrigel and cells mixtures were then transfer to a custom designed 3D electrotactic chamber in 60-mm dishes, and placed in an incubator for 20 min to allow matrigel to polymerize. Once gel was formed, 4.5 to 5.0 ml mTeSR™ medium was added into the dishes. Cells were allowed to grow overnight at 37°C with 5% CO2.
EFs Stimulation and Time Lapse Image Recording
Prior to EF exposure, mTeSR™ containing 25 mM HEPES buffer was added to one well of the chamber and the culture medium was observed to fill the opposite well assuring that the chamber was not clogged. EFs were applied to cells through two silver electrodes immersed in Steinberg’s solution-filled reservoirs that were connected to wells at each end of the galvanotaxis chamber by two agar bridges (2% agar in Steinberg’s solution). hiPS or hES cells were exposed to an EF with strength ranging from 30 to 200 mV/mm for 3~8 h at 37°C. Field strengths were measured directly at the beginning and the end of the experiment. Phase contrast (for 2D) and DIC (for 3D) time lapse imaging was performed using Nikon Eclipse imaging system equipped with a CO2 and temperature control chamber. 3D scanning was performed at various Z focal planes across the whole thickness of matrigel containing hES or hiPS cells colonies. Data analysis was performed using the NIH Image J software.
Quantitative Analysis of Cell Behavior
Cell migration was analyzed as previously reported [36, 37]. We used the NIH Image J software to quantify migration directedness and trajectory speed by tracing the position of cell nuclei at frame intervals of 10 min. The directedness of migration was assessed as cosine θ, where θ is the angle between the EFs vector and a straight line connecting the start and end position of a cell. A cell moving directly along the field lines toward the anode would have a directedness of −1; a cell moving directly toward the cathode would have a directedness of 1. A value close to 0 represents random migration. The cosine θ will range from −1 to +1, and an average of all the individual cells yields the directedness value for a population of cells, giving an objective quantification of the direction of cell migration. The trajectory speed (Tt/T) is the total migration distance of cells per minute; Dx/T is an x-axis displacement speed, which represents the migration along the EF vector.
hiPS cells were seeded onto chambered glass-bottom dishes (Thermo Fisher Scientific) that were pre-coated with matrigel. Before or after EF stimulation, cells were washed with phosphate buffered saline (PBS), fixed with 2% paraformaldehyde for 15 min at room temperature. After 3 washes with PBS, cells were blocked with 3% bovine serum albumin (BSA) (Sigma-Aldrich). Cells were then incubated with Alexa Fluor 568 phalloidin (Invitrogen, USA) for 20 min at room temperature or primary antibodies against monoclonal mouse anti-SSEA-4 (Developmental Studies Hybridoma Bank, Iowa) at 1:50 dilution and rabbit anti-Oct-4 (Chemicon) at 1:500 dilution overnight at 4°C. After washes with PBS, cells were incubated with Alexa Fluor conjugate-secondary antibodies (5 μg/ml; Invitrogen) for 30 min at 4°C, stained with Hoechst 33342 (2 μg/ml; Invitrogen) for 5 min, mounted using Fluoro-Gel with Tris Buffer (Electron Microscopy Sciences, Hatfield, PA), and imaged using an Olympus BX51 fluorescence microscope (Olympus, Center Valley, PA).
Data are expressed as mean ± SEM. Statistical differences between groups were assessed with unpaired, two-tailed Student’s t-test, with statistical significance set at p < 0.05. Statistical analysis was performed using the software of SPSS 10.0.
EF-Directed Migration of hiPs Cells
To determine whether the density of hiPS cells affects EF-directed migration, we analyzed migration of hiPS cells in dissociated culture and culture of large colonies (>300 cells). Both the dissociated cells and the large colonies migrated toward the anode in an EF (Fig. S2; supplemental movie 4, 5). Galvanotaxis of hiPS cells therefore remained very similar between dissociated cells and tightly connected cells in colonies, although their responses varied in terms of speed (dissociated cells: 0.50 ± 0.02 μm/min vs. large colonies: 0.04 ± 0.01 μm/min, p < 0.05) and directedness (dissociated cells: −0.61 ± 0.06 vs. large colonies: −0.96 ± 0.01, p < 0.01). Cells in colonies migrated more directionally, and the larger the colony, the more uniformly they migrated toward the anode (Fig. 1f, S2 D, S2 H).
Voltage-Dependent Galvanotaxis of hiPs Cells
In order to determine the threshold voltage required to induce galvanotaxis, we analyzed the voltage-dependent galvanotaxis of hiPS cells. Significant directional migration was observed at a field strength of 30 mV/mm with directedness of −0.37 ± 0.08 (Fig. 1g) (p < 0.01, when compared with the no EF control). The threshold voltage required to induce galvanotaxis of hiPS cells was therefore <30 mV/mm. EFs of higher voltage significantly increased directedness, which doubled in an EF of 75 mV/mm. Applied EFs also significantly increased trajectory speed. The increase in speed was dependent on field strength, with an EF of 75 mV/mm increasing the speed by 40% and this leveled out at higher voltages (Fig. 1h). Consistent with the directional migration, displacement along the χ axis (the field line toward the anode) in hiPS cells significantly increased following EF stimulation (Fig. S3). These results indicate that a robust galvanotaxis of hiPS cells occurs in vitro in an EF of 75 mV/mm, under our experimental conditions, with a threshold of <30 mV/mm.
Galvanotaxis of hiPs Cells in 3D Matrigel
EF Exposure Did Not Alter Expression of Stem Cell Markers in hiPs Cells
In order to investigate whether EF exposure had any effect on the pluripotency of hiPS cells, we stained two stem cell markers, SSEA-4 and Oct-4, in hiPS cells following 5 h of EF exposure (100 mV/mm). Both SSEA-4 and Oct-4 were highly expressed in hiPS cells exposed to an EF (Fig. S4). The expression levels were similar to untreated control cells, indicating that EFs have no significant effect on the pluripotency of hiPS cells.
Galvanotaxis of hiPs Cells was Different from That of hES Cells
Rho/ROCK Signaling was Required for Galvanotaxis of hiPs Cells
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 ; 2) increasing the migratory potential of stem cells by cytokine pretreatment or its receptor overexpression [16, 17]; 3) removing the extracellular matrix (ECM) barrier by matrix metalloprotease stimulation to accelerate the migration of stem cells . 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 . 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 [20–22]. 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 .
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, 24]. 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. 1a, 2a and e). 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. 2a and e). 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 [25–27]. 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 . Surprisingly, hES (H7) cells migrated in a direction opposite to that of hiPS cells in an EF; hES cells migrated toward the cathode (Fig. 3). 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 and 7; supplemental movies 16, 17, 18 and 19). 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. 3b). 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 . 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 [29–31]. 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, 32]. The ROCK inhibitor therefore is considered as a promising molecule to aid in the expansion of pluripotent stem cells for regenerative medicine . We and others have previously found an important role for ROCK in galvanotaxis [15, 34]. 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. 4e and f). Reduced cell-cell contact and increased filopodium formation in hiPS cells also occurred following ROCK inhibition in hiPS cells (Fig. 4a and b, Fig. S5). Our results are consistent with a previous report suggesting ROCK inhibition promoted cell motility in mouse ES cells . 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.
This work is supported by a grant from the California Institute of Regenerative Medicine RB1-01417 (to MZ). MZ is also supported by NIH 1R01EY019101, NSF MCB-0951199, and the UC Davis Dermatology Developmental Fund. WD is in part supported by grants from the National Institutes of Health (RO1 NS059043 and RO1 ES015988), National Multiple Sclerosis Society, Feldstein Medical Foundation, and Shriners Hospitals for Children. We thank James Thomson for providing iPS cells and Jan Nolta for her advice and support. We also thank Bing Song for sharing technical expertise, and other members from the Zhao, Deng to Li labs for their help.
MZ and YH designed the research; JZ, MC, QZ and XZ performed the research; WD and RL contributed reagents and cell lines; JZ and MZ analyzed the data; JZ and MZ wrote the paper with help from WD to XZ.
Conflicts of interest
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