Surface‐Anchored Nanogel Coating Endows Stem Cells with Stress Resistance and Reparative Potency via Turning Down the Cytokine‐Receptor Binding Pathways

Abstract Stem cell‐based therapy has great potential in regenerative medicine. However, the survival and engraftment rates of transplanted stem cells in disease regions are poor and limit the effectiveness of cell therapy due to the fragility of stem cells. Here, an approach involving a single‐cell coating of surface‐anchored nanogel to regulate stem cell fate with anti‐apoptosis capacity in the hypoxic and ischemic environment of infarcted hearts is developed for the first time. A polysialic acid‐based system is used to anchor microbial transglutaminase to the external surface of the cell membrane, where it catalyzes the crosslinking of gelatin. The single‐cell coating with surface‐anchored nanogel endows mesenchymal stem cells (MSCs) with stress resistance by blocking the activity of apoptotic cytokines including the binding of tumor necrosis factor α (TNFα) to tumor necrosis factor receptor, which in turn maintains mitochondrial integrity, function and protects MSCs from TNFα‐induces apoptosis. The administration of surface engineered MSCs to hearts results in significant improvements in engraftment, cardiac function, infarct size, and vascularity compared with using uncoated MSCs in treating myocardial infarction. The surface‐anchored, biocompatible cell surface engineering with nanogel armor provides a new way to produce robust therapeutic stem cells and may explore immense potentials in cell‐based therapy.

6 w/v in NaHCO 3 buffer, pH = 9.0); then, the mixture was stirred at 150 rpm and 37 °C for 6~8 h and dialyzed against deionized water for 2 days. The water was replenished every 4-6 h; then, the sample was lyophilized. All procedures were conducted in the dark, and the lyophilized product was dissolved in PBS (8%, w/v), filtered, and stored at 4 °C.
Sulfo-cyanine 7 (300 mg) was dissolved in DMSO (300 mg/mL) and added to 30 mL gelatin solution (5% w/v in Na 2 CO 3 buffer, pH = 8.4); then, the mixture was stirred at 150 rpm and 20 °C for 5 h and dialyzed against PBS overnight. Then the mixture was dialyzed in pure water for 4 h and lyophilized finally. All procedures were conducted in darkness, and the lyophilized product was dissolved in PBS (8%, w/v), filtered, and stored at -20 °C.
MSC isolation and culture conditions. The tibias of 2-month-old male Sprague-Dawley (SD) rats (Slac Laboratory Animal, Shanghai, China) were rinsed with Dulbecco's modified eagle medium (DMEM, Thermo) to collect the bone-marrow cells; then, the cell mixture was centrifuged, the supernatant was discarded, and the cells were resuspended and cultured in MSC culture medium consisting of low glucose DMEM (90% v/v), FBS (10% v/v), and penicillin-streptomycin (1% v/v). Two or three days later, the medium was changed, the non-MSC population (i.e., the non-adherent cells) was removed, and the adherent cells were 7 cultured until a purified population of MSCs was obtained. MSCs were characterized by flow cytometry using specific cell surface antigens [S1] .
Cells were maintained under standard culture conditions in low glucose DMEM and 10% fetal bovine serum under 21% oxygen and 5% carbon dioxide. Hypoxia was induced by replacing the standard medium with glucose-and serum-free medium (except as indicated) and incubating the cells for 24 hours in a ProOx-C-chamber system (Biospherix) under 0.5% O 2 , 5% CO 2 , and N 2 . For TNFα treatment, the standard medium was replaced with optimal medium (Gibco) for 24 hours, and then with 100 ng/mL TNF-α in optimal medium for another 24 hours.
Nanogel-coated MSC generation. MSCs were cultured on a cell culture dish and trypsinized; then, 3.0-6.0×10 5 cells were suspended in 200 μL PBS containing PAAM-mTG (50-100 μM), cultured for 10-15 min at 37 °C, and centrifuged (1000 rpm, 3 min). The supernatant was removed; then, the MSCs were soaked in gelatin solution (8%, w/v) for 30 min at 37 °C, centrifuged (4000 rpm, 4 min) to remove excess gelatin solution, and rinsed with PBS. Formation of the nanogel coating was confirmed by laser scanning confocal microscopy (Nikon A1 Ti) of MSCs that had been coated with the FITC-conjugated gelatin 8 solution, and the encapsulation rate was determined by staining the cells with Hoechst 33342 (Aladdin) and evaluating FITC fluorescence through flow-cytometry analysis performed with a Beckman FC500 MPL instrument (Beckman). Subsequently, a series of ethanol gradient solutions with concentrations (v/v) of 30%, 50%, 70%, 80%, 90%, and 95% was used to dehydrate the fixed cells (15 min each) sequentially.

Scanning electron microscopy (SEM
Finally, the resulted cell samples were further dehydrated twice in 100% ethanol (20 min each). After that, the samples were dried and observed with a scanning electron microscope (Phenom Pro, Phenom-World, The Netherlands) with an accelerating voltage of 10 kV.
Size analysis of nanogel-coated MSCs. The size of nanogel-coated MSCs is measured by Mastersizer 3000 laser particle size analyzer (Malvern Instruments Ltd., Malvern Worcestershire, UK). The conditions of determination were as follows: 2.0 -6.0 × 10 5 in 250 9 ml PBS, the pump set at 1000 rpm, and laser obscuration was sat at 10%. The sample was injected when the shading degree is between 0.1-15%.
Distribution and thickness of gelatin on the MSC surface. The MSCs were resuspended with electro-transfected buffer. Then mCherry plasmid (expressed in the cytoplasm) was added into the buffer and then the mixture was transferred into micropulser. The indicated program was used to transfer the plasmid into cells and these MSCs were plated on a dish for nanogel coating with FITC-labeled gelatin. After 8 hours, the distribution of gelatin in the MSC surface was observed via SIM microscopy (Nikon, Japan), then the image was processed by Imaris (Batch) 9.3.1. And the thickness of the coated nanogel was calculated by the homemade code with Image J software.
The pore size of gelatin hydrogel. PAAM-mTG (10 μM) was added into 20 mL gelatin solution (8%), then, the mixture was stirred at 150 rpm and 37 °C for 10 min and centrifuged at 3000 g to remove air bubbles. Pour mixture into 6 cm cell culture dish at 37°C. After 20 min, the gelatin was crosslinked, then the crosslinked gelatin was quickly frozen in liquid nitrogen for 10 min and then sublimated at room temperature for 15 s to sublimate all water inside the crosslinked gelatin. The freeze-dried hydrogel samples were then cut, fixed on aluminum stubs, and coated with gold for 10 seconds for interior morphology observation with Cryo-scanning electron microscope (FIB Company, USA). The pore size distribution was determined by randomly selecting 300 pores from the FIB-SEM images.
Particle size distributions. The particle size distributions of isolated exosomes suspended in PBS was determined using ZetaView (Particle Metrix, Germany).

MSC mechanical properties.
The mechanical properties of the cells were characterized through micropipette aspiration as described previously [S2] . Briefly, micropipettes with an inner diameter of 8-10 μm were created from borosilicate glass capillary tubes with a Flaming/Brown micropipette puller (P-1000, Sutter Instrument) and a microforge (Narishige), and the cells were trypsinized and suspended in warmed PBS. Suction pressure was applied through the capillary to the surface of the cell with a water negative pressure control system, and the equilibrium pressure (i.e., when the cell was neither aspirated nor pushed away from the micropipette) was recorded; then, the pressure was increased until the diameter of the cell was equal to the capillary diameter [S2a] , and the pressure, the micropipette diameter, and the diameter of the portion of the cell that remained outside the pipette were recorded by the system software. Measurements were completed no more than 2 h after trypsinization, and 10 11 cells were measured for each experimental group or condition. Young's modulus and cortical tension were calculated according to the following equations (Tc: cortical tension; Pp: pipette pressure; Rp: pipette radius; Rc: cell radius; E: Young's modulus).
( ) Binding force. The MSCs were suspended in DMEM buffer with 1% BSA, and red blood cells (RBCs) were fused to TNFα-biotin using a biotin protein labeling kit (Elabscience).
Single MSCs and RBCs were gently aspirated with micropipettes and brought into contact with each other for 2 s; then, the cells were pulled apart with a pipette connected to a computer-controlled piezoelectric actuator. In the absence of adhesion, the RBC immediately separated from the MSC and returned to its original spherical shape, while in the presence of adhesion, the RBC remained bound to the MSC and was stretched into an elongated shape until separation. Eight to 12 pairs of cells were tested, and the cycle was repeated 50 times for each pair of cells.
Transmission electron microscopy (TEM). Cells (1×10 6 ) were suspended in doubledistilled water, and the sample solution was dipped with a copper mesh supporting membrane.
The membrane surface liquid was absorbed with filter paper; then, the membrane was dried slightly, and 2% acetic acid uranyl uranium dye solution was added dropwise. The excess staining solution was removed with filter paper; then, the samples were dried and observed with a JEM 1200-EX transmission electron microscope (JEOL).
TEM was used to detect the mitochondrial length. In brief, 1×10 6 cellswerefixed with 2.5% glutaraldehyde for 12 h. After washing three times with phosphate-buffered saline, the cells were post-fixed with 1% OsO 4 for 1-2 h. Next, the specimens were dehydrated by an ethanol gradient, followed by acetone for overnight infiltration. Furthermore, the specimens were embedded in Spurr resin and sectioned in Leica EM UC7 Manufacturer (Leica, Wetzlar, Germany). The sections were stained with uranyl acetate and alkaline lead citrate, and the images were obtained by Hitachi Model H-7650 TEMat6,800 × and 30,000 × magnification.
Cell apoptosis. Cells were cultured in 24-well plates (1×10 5 cells/well) under normal or hypoxic conditions for 24 hours, and then apoptotic cells were identified via deoxynucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL) with an In-Situ Cell Death Detection Kit (TMR red; Roche Applied Science, Indianapolis).
Tube formation. Tube formation was evaluated as described previously [S3] . Briefly, cells were plated in 24-well or 96-well plates (1×10 5 or 2×10 4 cells/well) that had been pre-coated 13 with growth factor-reduced Matrigel (BD) and incubated with the indicated medium for 4-6 h; then, tube length was quantified with Image-Pro Plus 6.0 software (Media Cybernetics). Five fields (100 × magnification) were counted in each well and3 wells in each group from three independent experiments were evaluated for each experimental group or condition.
Cell migration. Gel-coated and uncoated MSCs migration was performed in Transwell chemotaxis 24 well chamber (Corning). Cells (2×10 4 cells/well) in DMEM with 1% FBS were plated in the upper chamber, the lower chamber was filled with DMEM with 10% FBS.
After 12 h, non-migrating cells were removed and washing with PBS. The migrated cells were fixed with 10% formaldehyde and stained with 0.1% crystal violet. Six fields were counted for each well (200 × magnification) and 3 wells in each group from three independent experiments, the migrated cells were counted by Image-pro plus 6.0 (Media Cybernetics).

Distribution of gelatin in MSC division. The FITC-labeled gelatin coated MSCs cultured
incomplete medium with 10% FBS. Then, during MSCs was proliferation, the nanogel-coated MSC was observed every 10 min via laser scanning confocal microscopy (Leica, Wetzlar, Germany) to show the distribution of gelatin after cell division.
Proliferation. Cells were cultured in standard medium on 96-well plates at a density of 2×10 4 cells/well; then, the medium was replaced with 0.1 mL of DMEM containing 10% FBS, and the cells were placed in a humidified incubator maintained at 5% CO 2 /95% room air and 37 °C. Cell proliferation was measured 24, 48, and 72 h later with a Universal Microplate Spectrophotometer (MD-SpectraMax M5) and a CCK-8 Kit as directed by the manufacturer's instructions.
MSC differentiation. Cells were seeded in a 6-well plate (1×10 5 cells/well) and differentiated into osteocytes, chondrocytes, and adipocytes via the protocols as described previously [S4] . Cellular ATP production. Cellular ATP content was measured by using an ATP determination kit (Beyotime Biotechnology) as directed by the manufacturer's instructions.
Cytokine array. Cells (1×10 6 ) were cultured for 48 h with 10 mL of phenol red-free DMEM plus 10% FBS, and centrifuged for 3 min at 2500 rpm to remove cell debris; then, the proteins in the supernatants were quantified with the Rat Cytokine Array (RayBio, GSR-CAA-67), which can detect 67 proteins, via recommended protocols (Wayen Biotechnologies, Shanghai, China). Briefly, antibodies were printed onto slides to capture the corresponding cytokines, incubated with a mixture of biotinylated secondary antibodies, and labeled with Cy3conjugated streptavidin; then, Cy3 fluorescence was detected with a GenePix 4000B Microarray Scanner (Axon), and the signal was digitized with GenePix Pro 6.0 software. Western blot. Cells were rinsed with cold PBS, lysed in 2.5×sodium dodecyl sulfate (SDS) gel loading buffer (30 mM Tris-HCl, pH 6.8, 1% SDS, 0.05% bromphenol blue, 12.5% glycerol, and 2.5% mercaptoethanol) and boiled for 30 min; then, the proteins in the lysate were separated on 12% SDS polyacrylamide gels, electro-transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Boston, MA), and stained with the Luciferase transfection. Cells (1×10 6 ) were infected with luciferase-encoding recombinant lentiviruses (Genechem Company) in 10 μg/mL polybrene (Millipore) for 12 h; then, the growth medium was replaced, and luciferase activity was detected by using an X Pack CMV-XP-Luciferase-EF1-Puro Expression Lentivector Kit (System bioscier) 48 h later.
Generation of conditioned medium. Cells were seeded in 6-well plates (1×10 5 cells/well) and cultured for 24 h; then, the medium was replaced with 2 mL of DMEM plus 10% FBS, and the cells were cultured for an additional 48 h. The conditioned medium was collected and centrifuged for 3 min at 2500 rpm to remove cell debris before use in subsequent experiments.
Isolation of neonatal rat cardiomyocytes. Hearts were extracted from 1-to 3-day-old neonatal rats (purchased from Zhejiang Chinese Medical University) and transferred into PBS. The tip of heart was cut into 1-mm 3 pieces, digested with 0.25% trypsin (Genom), and centrifuged for 10 min at 100 rpm; then, the supernatant was collected, and digestion/centrifugation was repeated until the tissue was fully digested. The collected supernatants were added to DMEM with 10% FBS, centrifuged for 10 min at 1500 rpm, and incubated for 1.5 h at 37 °C; then, the adherent cells (mostly fibroblasts) were discarded and the cardiomyocytes in the supernatant were collected.

MI model and treatment. Experiments involving live animals were performed in accordance
with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Zhejiang University. Male Sprague-Dawley 18 (SD) rats (250 g) were purchased from Zhejiang Chinese Medical University. The animals were fed a standard laboratory diet with free access to food and water and housed under a controlled temperature (22 ± 1°C) and humidity (65-70%) with a 12:12 h light : dark cycle.
For MI induction, the animals were anesthetized via intraperitoneal injection of pentobarbital sodium (50 mg/kg) and ventilated via tracheal intubation and a rodent ventilator; then, the left anterior descending coronary artery was ligated with an 8-0 nylon suture. Treatments (1 × 10 6 MSCs or nanogel-coated MSCs in 100 μL PBS, or PBS alone) were administered 30 minutes later to five sites at the border of the infarction.
Echocardiography. Rats (number of animals in each group ≥ 8) were anaesthetized (3% sevoflurane mixed with 97% O 2 ) in an induction chamber, and transthoracic echocardiography was performed with a Vevo 2100Imaging System (Visual Sonics Inc). Left ventricular ejection fraction (EF) and fractional shortening (FS) were calculated from guided M-mode recordings as described previously [S6] .
Bioluminescence. Animals (number of animals in each group ≥ 4) were anesthetized with pentobarbital sodium (i.p., 50 mg/kg) and intraperitoneally injected with D-luciferin (150 19 mg/kg in PBS, R&D); then, bioluminescence images were acquired over a 5-min period with an In vivo Imaging System (PerkinElmer).
Histological staining. Hearts were dehydrated in 30% sucrose solution, embedded in Tissue- Tek OCT compound, snap frozen in liquid nitrogen, and cut into 5-μm sections; then, the Masson's trichrome staining was performed to evaluate infract area by Masson's trichrome staining kit (Solaribio, Beijing, china). The images were measured using the Image-Pro Plus RNA sequencing. Total RNA was isolated from three replicates per experimental group or condition with a RNeasy Mini Kit (Qiagen) and sent to GENOME (Beijing, China) for preparation, sequencing, and mapping. The data were evaluated via hierarchical clustering analysis, pathway analysis, and cluster analysis.
Conditioned medium was collected from MSC and Gel-MSC and analyzed according to the manufacturer's instruction.
Statistical analysis. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using one tailed t-tests for comparisons between two groups and one-way analysis of variance (ANOVA) and Tukey correction for comparisons among more than two groups. Analyses were performed with GraphPad Prism software (version 5.0), and p < 0.05 was considered statistically significant.