{'fraction_non_alphanumeric': 0.050913447139862233, 'fraction_numerical': 0.026954177897574125, 'mean_word_length': 4.528973509933775, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Engineering hydrogels with homogeneous mechanical properties for controlling stem cell lineage specification

Significance Hydrogels are extensively used for cell culture, tissue engineering, and flexible electronics. In all of these applications, mechanical properties of hydrogels play an important role. Although tremendous studies have been devoted to optimizing the stiffness, strain, toughness, and dynamic mechanical response, the mechanical homogeneity of hydrogels has rarely been considered. By developing a general strategy to control the mechanical homogeneity of hydrogels, here we show that nanoscale variation in matrix stiffness can considerably affect the lineage specification of human embryonic stem cells. Inhomogeneous hydrogels suppress mechanotransduction and facilitate stemness maintenance, while homogenous hydrogels promote mechanotransduction and osteogenic differentiation. Therefore, engineering hydrogels with controllable and well-defined nanoscale homogeneity may have considerable implications in stem cell culture and regenerative medicine.

The compression Young's modulus was the approximate linear fitting value of the stress-strain curves in the strain range of ~20%.
Swelling and porosity measurements. In a typical swelling experiment, the volume of the initial hydrogel was recorded as V1. Then, the hydrogel was immersed in 50 times deionized water for 24 hours at room temperature to reach swelling equilibrium, and the volume was recorded as V2. The swelling ratio (Ɛ) was calculated by the equation Ɛ =V2/V1. The porosity of the hydrogel was calculated as the sol/gel fraction of the hydrogel. The hydrogels were first dialyzed in deionized water for another 24 hours. The hydrogel samples were weighed, and the wet weight was recorded as W1. Then, the hydrogel samples were lyophilized, and the weight was recorded again as W2. The porosity () was calculated as  =(1-W2/W1)×100%.
Scanning electron microscopy (SEM). SEM images were obtained using a Quanta scanning electron microscope (Quanta 200,FEI) at 20 kV. The samples were lyophilized prior to the measurement.

Thiol detection using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB).
PEG-SH/PEG-Mal/β-CD hydrogels were prepared as described above and soaked in deionized water for 24 hours. The hydrogels were soaked in the solution containing DTNB for 5 hours, and the concentration of the reaction product of thiol and DTNB, 3-carboxy-4-nitrophenyl disulfide, was monitored by monitoring the UV absorbance at 412 nm of the solutions.

Analysis of the RGD distribution in the hydrogels.
The thiol-containing fluorescent dye (Cy5-PEG-SH, 2 kDa) was used to mimic the RGD ligand in the hydrogel preparation process. Then, the Cy5-PEG-SH containing hydrogels were placed in ddH2O 50 times of the volume of the hydrogels for 24 h to remove the trapped unreacted fluorescent dyes. This process was repeated for 3 times to make sure the unlabeled dyes were completely removed. As the hydrogels were immersed in ddH2O for sufficient long time, the volume of the hydrogels remained the same after each equilibration step in ddH2O (~2.9 times of the original hydrogels). Then the distribution of Cy5-PEG-SH in hydrogels was investigated using a laser confocal fluorescence microscopy (Olympus FV3000, Japan).

Cell culture and viability.
For all the hydrogels used for cell culture, 0.2 mg mL -1 cRGD (Cyclo(Arg-Gly-Asp-D-Phe-Cys)) was added to the hydrogel mixture during preparation. SMCC-7721 cells and HK-2 cells were cultivated in RPMI-1640 medium (Gibco, USA) and DMEM (Gibco, USA), respectively. Ten percent foetal bovine serum and 1% penicillin & streptomycin were added to the medium. All the cells used in cell viability testing were used before 5 passages. Typically, 100 µL of hydrogel was prepared in each well of opaque-walled multiwall plates before collecting cells, and cells were digested from culture dishes. Then, the cells were seeded into wells containing hydrogel at a density of 2×10 4 per well. Cells were incubated at 37 °C and 5% CO2. After 24 hours, the plates were equilibrated to room temperature for approximately half an hour. Fifty microlitres of CellTiter-Glo Reagent (Promega, USA) was added to each well. After mixing, the intensities of the luminescence signals were recorded using a SpectraMax M5e Multi-Mode Microplate Reader (Molecular Devices, USA).
Additionally, calcein AM and propidium iodide (PI) staining was used to evaluate the cell viability of cells living on gel. The hydrogels were prepared, and cells were seeded into the wells as described above. After incubation for 24 hours, each well of a 96-well plate was washed twice with PBS. Then, calcein AM and propidium iodide (PI) dye solution were mixed and added to each well. The plate was incubated at 37 °C for 30 min. Then, whole-cell images were obtained using an OLYMPUS-IX73 fluorescence microscope (OLYMPUS, USA).          S9. Young's modulus distributions of surfaces determined by IT-AFM for hydrogels at the PEG concentration of 52.5 mg mL -1 (CPEG-Mal =CPEG-SH=52.5 mg mL -1 ). (A) Two-dimensional Young's modulus distributions of surfaces determined by IT-AFM for hydrogels at PEG concentration of 52.5 mg mL -1 (CPEG-Mal =CPEG-SH=52.5 mg mL -1 ). The molar ratios of β-CD and 4armed PEG-Mal were 0:1 (left) and 1:1 (right). The scale bar is 1.0 μm. (B) Histograms of Young's modulus quantified by IT-AFM for PEG-SH/PEG-Mal/β-CD hydrogels at PEG concentration of 52.5 mg mL -1 (CPEG-Mal =CPEG-SH=52.5 mg mL -1 ). The Young's moduli of the hydrogels are 58.4±10.9 kPa and 60.9±6.9 kPa at β-CD and 4-armed PEG-Mal molar ratios of 0:1 (left) and 1:1 (right), respectively, in which the error bars indicate the half-peak breadth of the Gaussian fitting. (C) Scatter diagram of the Young's modulus of the PEG-SH/PEG-Mal/β-CD hydrogels at PEG concentration of 52.5 mg mL -1 (CPEG-Mal =CPEG-SH=52.5 mg mL -1 ). The molar ratios of β-CD and 4-armed PEG-Mal were 0:1 (left) and 1:1 (right).

Fig. S16. Cell viabilities of HK-2 and SMCC-7721 cells cultured on I-gels and H-gels. (A)
Viabilities determined by luminescence of HK-2 (A) and SMCC-7721 (B) cells on I-gels and Hgels after being cultured for 24 hours. The β-CD and PEG-Mal ratios used for the preparation of H-gels were 0:1, 1:1, 2:1 and 4:1. (C) Images of stained live cells on I-gels and H-gels. The β-CD and PEG-Mal ratio used for the preparation of H-gels was 1:1. Green represents live cells with high enzymatic activity, indicated by calcein AM. The red colour of PI shows dead cells with compromised membranes. Additionally, the cellular morphology was checked by bright-field (BF) microscopy. Scale bar = 200 µm.