Stem cell membrane engineering for cell rolling using peptide conjugation and tuning of cell-selectin interaction kinetics

Abstract

Dynamic cell-microenvironment interactions regulate many biological events and play a critical role in tissue regeneration. Cell homing to targeted tissues requires well balanced interactions between cells and adhesion molecules on blood vessel walls. However, many stem cells lack affinity with adhesion molecules. It is challenging and clinically important to engineer these stem cells to modulate their dynamic interactions with blood vessels. In this study, a new chemical strategy was developed to engineer cell-microenvironment interactions. This method allowed the conjugation of peptides onto stem cell membranes without affecting cell viability, proliferation or multipotency. Mesenchymal stem cells (MSCs) engineered in this manner showed controlled firm adhesion and rolling on E-selectin under physiological shear stresses. For the first time, these biomechanical responses were achieved by tuning the binding kinetics of the peptide-selectin interaction. Rolling of engineered MSCs on E-selectin is mediated by a Ca(2+) independent interaction, a mechanism that differs from the Ca(2+) dependent physiological process. This further illustrates the ability of this approach to manipulate cell-microenvironment interactions, in particular for the application of delivering cells to targeted tissues. It also provides a new platform to engineer cells with multiple functionalities.

Fig. 1

Schematic illustration of conjugating peptides to the surfaces of stem cells to assist stem cell rolling under flow. E-selectin targeting peptides are conjugated on cell membranes by using NHS-PEG₂-maleimide as a linker molecule and the thiol-maleimide reaction for subsequent peptide conjugation, creating engineered stem cells. a) Unmodified stem cells do not bind to selectins and are quickly flushed away under flow. b) Stem cells modified with peptides that strongly bind to selectins remain bound under physiological flow and do not roll. c) Stem cells modified with peptides that bind to selectins with fast dissociation rate constant (k_off) roll in the direction of flow.

Fig. 2

Stem cells modified with peptides on their membranes retain their multipotency. a) Representative confocal images of 5-FAM-peptide K-modified hMSCs. The cells were detached and resuspended in medium after modification. Left: the confocal image was obtained by focusing in the middle of the cells. Middle and right: three-dimensional reconstructed confocal microscopy images of one engineered hMSC. The middle image is the top view, and the right image is the bottom view. b) Staining of differentiated adipocytes (red) and osteogenic cells (purple blue) from unmodified hMSCs and membrane-engineered hMSCs. c) RT-PCR analysis of RNA markers of adipogenesis and osteogenesis. Values are mean ± s.d. C: unmodified hMSCs in growth medium for three weeks; U: unmodified hMSCs in differentiation medium; E: engineered hMSCs in differentiation medium.

Fig. 3

hMSCs modified with peptide K strongly bound to E-selectin coated microfluidic channels under flow. a) Representative images of membrane-engineered hMSCs (GFP, green) and unmodified hMSCs (mCherry, red) in microfluidic channels before applying flow and 2 min after flow. Each column of images corresponds to a different shear stress that the cells were exposed to (left to right: 1.25 dyn/cm², 5 dyn/cm² and 10 dyn/cm²). b) Fraction of bound cells under different shear stresses. Values indicate mean ± s.e.m. (n=3 for each group). Blue-dashed box indicates the physiological range of shear stresses in human postcapillary venules.

Fig. 4

SPR sensorgrams of peptide binding on immobilized E-selectins. The red-colored lines are the adjusted data curves, and the black lines are the fitting curves using 1:1 binding model. The peptide concentrations of individual curves were from 10 μM to 0.013 μM as marked by the black arrow. a) Sensorgrams of peptide K binding on E-selectin. b) Sensorgrams of peptide M binding on E-selectin.

Fig. 5

MSCs modified with peptide M roll on E-selectin under physiologically relevant shear stresses. a) Time-lapse images of engineered hMSCs rolling on a 5 μg/mL E-selectin-coated surface at shear stress 10 dyn/cm². White arrows mark the flow direction. Green and red arrows mark two rolling cells respectively. Quantification of rolling velocity of engineered cells on surfaces coated with different concentrations of E-selectin, b) engineered hMSCs; c) engineered mMSCs and HL-60s. Values indicate mean ± s.d.(n=15 for each group).

Fig. 6

Tethering of membrane engineered stem cells and HL-60s on E-selectin coated substrate at different time points. hMSCs and mMSCs were modified at 100 μM NHS-PEG₂-maleimide and 6 μM peptide K. After trypsinization and resuspension in medium, cells at 500,000 cells/mL were flushed into microfluidic devices that were coated with 5 μg/mL E-selectin before use. Images were taken at shear stress 0.2 dyn/cm², 0.5 dyn/cm² and 0.25 dyn/cm² for hMSCs, HL-60s and mMSCs respectively. Black arrows mark some of the tethered cells.