Light activated cell migration in synthetic extracellular matrices
Introduction
Cell migration is fundamental to many biological processes including tissue development, regeneration, and repair [1], [2]. It is a highly integrated multi-step process involving cell polarization, protrusion, adhesion, translocation, and rear retraction [3]. A full understanding of the local driving forces and regulating factors of cell migration remains a challenge today, particularly in three dimensional (3D) environments. The primary, non-contact techniques available for single cell manipulation are field gradient traps including optical tweezers [4], magnetic tweezers [5], and dielectrophoretic traps [6]. Unfortunately, these methods for cell manipulation are limited to a liquid medium in a two dimensional (2D) environment which bears little resemblance to the native cell surrounding of the extracellular matrix. In the case of engineered 3D environments, chemical and mechanical gradients can stimulate migration of cell populations but do not allow manipulation of single cells [7], [8], [9]. Less is known about the effects of cell adhesion, matrix stiffness, or geometry on cell motility in 3D [10]. Here, we present a strategy for manipulating individual cells in a synthetic extracellular matrix through selective optical activation of signaling inside the cell to investigate stem cell motility with single cell resolution.
Biomaterial scaffolds are efficient tools for culturing cells in a 3D environment that mimics, to varying degrees, the native in vivo environment [11]. The physical structure and chemical functionality of a synthetic biomaterial scaffold can be manipulated to create highly defined microenvironments in which to evaluate cell response. While cell migration is observed in and around many biomaterials in vivo, similar in vitro cell migration in these 3D environments has not been achieved. Hence, the use of optical tools to precisely control the migration of single cells [12], [13] holds great promise to probe materials response.
In this work, a visible laser was applied to guide directional migration of mammalian stem cells in 3D hydrogels. Mesenchymal stem cells (MSCs) were transfected with a genetically encoded photoactivatable Rac1 (PA-Rac) [14]. Rac is a subfamily of Rho GTPases that plays a pivotal role in cell migration by stimulating actin polymerization [15]. On exposure to visible light locally in defined regions of the cell, subcellular activation of the PA-Rac occurs. The locally augmented Rac activation then stimulates migration of the cell towards the light. Repeated application of the light promotes continuous cell migration. The unique ability to direct the motility of mammalian stem cells in defined, synthetic environments allows investigation into how the local adhesive context, stiffness, and geometry in a material influences migration with single cell resolution to elucidate both fundamental biological principles and materials design parameters.
Section snippets
Cell culture
Human mesenchymal stem cells (hMSCs) were derived from human embryonic stem cells (hESCs) (Hues9) as reported previously [16]. The hMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone), 2 mm l-glutamine (GIBCO), 100 units/ml of penicillin, and 100 μg/ml of streptomycin (GIBCO). Cells were incubated at 37 °C and 5% CO2 and passaged every 4–5 days using 0.025% trypsin-n-EDTA (Clonetics Biowhittaker) when the cells
Photoactivated cell migration by periodic subcellular activation
Photoactivated cell migration was accomplished by periodic subcellular activation of PA-Rac within stem cells encapsulated in hydrogels. Cells transfected with PA-Rac responded to light of 458 nm rapidly and reversibly (Fig. 1A). Precise spatiotemporal control of the Rac activity was obtained by modulating the Rac1-LOV interactions using focal illumination (Fig. 1B) [14]. In this way, the overall Rac activity in the cellular region exposed to light was increased due to the local activation of
Conclusion
We have used a combination of new techniques to manipulate single stem cells in synthetic 3D matrices using a photoactivatable Rac. Photoactivated cell migration in biomaterials allows for detailed characterization of fundamental cell responses to local environmental physical and biological cues critical in processes such as development, regeneration, and cancer. These studies demonstrate that cell motility can be tuned and guided by adjusting both internal signaling events and external
Acknowledgments
The authors gratefully acknowledge the generous support from the Arthritis Foundation Postdoctoral Fellowship (QG), the Jules Stein Professorship (JE), and NIH GM46425 (DM).
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These authors contributed equally to this work.