Synthetic Glycopolymers for Highly Efficient Differentiation of

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Synthetic Glycopolymers for Highly Efficient Differentiation of Embryonic Stem Cells into Neurons: Lipo- or not? Qi Liu, Zhonglin Lyu, You Yu, Zhen-Ao Zhao, Shijun Hu, Lin Yuan, Gaojian Chen, and Hong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01397 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 14, 2017

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ACS Applied Materials & Interfaces

Synthetic Glycopolymers for Highly Efficient Differentiation of Embryonic Stem Cells into Neurons: Lipo- or not? Qi Liu,ab Zhonglin Lyua, You Yuc, Zhen-Ao Zhaoc, Shijun Huc, Lin Yuana, Gaojian Chen*ba and Hong Chen*a a. State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren'ai Road, Suzhou 215123, P. R. China. b. Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow University, Suzhou 215006, P. R. China. c. Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University.

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ABSTRACT

To realize the potential application of embryonic stem cells (ESCs) for the treatment of neurodegenerative diseases, it is a prerequisite to develop an effective strategy for the neural differentiation of ESCs so as to obtain adequate amount of neurons. Considering the efficacy of glycosaminoglycans (GAG) and their disadvantages (e.g. structure heterogeneity, impurity), GAG-mimicking glycopolymers (designed polymers containing functional units similar to natural GAG) with or without phospholipid groups were synthesized in the present work and their ability to promote neural differentiation of mouse ESCs (mESCs) was investigated. It was found that the lipid-anchored GAG-mimicking glycopolymers (lipo-pSGF) retained on the membrane of mESCs rather than being internalized by cells after one-hour incubation. Besides, lipo-pSGF showed better activity in promoting neural differentiation. The expression of the neural-specific maker β3-tubulin in lipo-pSGF-treated cells was ~3.8- and ~1.9-fold higher compared to natural heparin- and pSGFtreated cells at day 14. The likely mechanism involved in lipo-pSGF-mediated neural differentiation was further investigated by analyzing its effect on fibroblast growth factor 2 (FGF2)-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway which is important for neural differentiation of ESCs. Lipo-pSGF was found to efficiently bind FGF2 and enhanced the phosphorylation of ERK1/2, thus promote neural differentiation. These findings demonstrated that engineering of cell surface glycan using our synthetic lipoglycopolymer is a highly efficient approach for neural differentiation of ESCs and this strategy can be applied for the regulation of other cellular activities mediated by cell membrane receptors.

KEYWORDS: glycosaminoglycan analogues, synthetic glycopolymers, cell surface engineering, neural differentiation, mouse embryonic stem cells


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1. INTRODUCTION Embryonic stem cells (ESCs) derived from pre-implantation embryos are pluripotent cells capable of specifically differentiating into any somatic cell type.1-3 Therefore, ESCs have emerged as an almost unlimited source of any specific cell type for transplantation therapy used for the treatment of various diseases.4-9 In particular, ESCs have potential in the treatment of neurodegenerative diseases such as Alzheimer's disease (AD), multiple sclerosis (MS) and Parkinson's disease (PD). Accordingly, it is of great interest to develop a strategy for the efficient differentiation of ESCs into neural cells. In recent years, various well-established strategies have been utilized to promote the specific differentiation of ESCs into neural cells,10-13 one of which is the supplementation with exogenous biological macromolecules, the bioactive polysaccharides in particular. One important class of polysaccharides that regulate ESC differentiation are the glycosaminoglycans (GAGs); 14-16 these are highly heterogeneous extracellular polysaccharides composed of alternating sulfated glucosamine and uronic acid units.17,18 Heparin and heparan sulfate are GAGs that can form ternary complexes with fibroblast growth factor 2 (FGF2) and the corresponding FGF receptors presented on mouse ESC (mESC) membranes.19-21 Subsequently, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is phosphorylated and the activation of related signalling events indicates the differentiation of mESCs into neural cells.22,23 Nevertheless, the use of native GAGs is limited due to problems such as variation in structure and contamination.24 Many GAG analogues have been prepared in an attempt to overcome these deficiencies, while at the same time maintaining activity similar to that of the native GAG. Two general approaches for the preparation of GAG analogues have been commonly employed: (i) the direct decoration of


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natural glycans25-27 and (ii) the synthesis of new polymers from monomers with specific structures.28-30 Nevertheless, anticoagulant activity and growth factor binding have been the foci of investigation for most previous research on synthetic GAG analogues. For instance, elegant studies by Maynard and coworkers have shown that basic FGF covalently linked to GAGmimicking polymers is more stable under a variety of harsh environmental conditions than native basic FGF.30 The regulation of cell behavior using synthetic GAG analogues is less investigated, and only a few groups have made significant contributions. Among these, Ayse et al. have reported on osteo/chondrogenic differentiation of mesenchymal stem cells modulated by GAG-mimetic peptide nanofibers.31,32 In previous work, our group showed that sulfonated chitosan with building blocks similar to natural GAGs can be used to promote differentiation of mESCs into neural cells.33 However, the sulfonate groups are attached to the sugar units, leading to lack of control of the location of sulfonate sites and of the balance of sugar and sulfonate units. Recently we prepared synthetic biomimetic GAGs in which the sulfonate groups are independent of the sugar units and the degree of sulfation (DS) can be controlled.34 The promotional effect of the synthesized copolymer with a particular DS was found to be superior to that of natural heparin. This “designer polymer” approach provides a convenient means for studying the influence of defined GAGmimicking polymers on cell behavior. Considering that the FGF2-ERK1/2 pathway for neural differentiation of mESCs originates in the cell membrane, we anticipate that direct insertion of our synthetic GAG analogues into cell membrane might further enhance the promotion effect. Inserting synthetic polymers with anchoring groups into cell surface has emerged as a powerful method for cell-surface engineering. Several molecules such as cholesterol, proteins and phospholipids have been used as anchors to attach exogenous macromolecules including polymers to the cell membrane.35-38 Among these molecules, phospholipids are widely favored owing to


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their minimal impact on cell membrane structure and their superior insertion efficiency.39,40 A recent study demonstrated that passive insertion of lipid-anchored glycan mimetics with specific structure into mammary epithelial cell membrane can promote cell growth and survival.41 Hence, in the present work we attached lipid groups to our synthetic GAG-mimicking polymers and investigated the neural differentiation behavior of cell-surface glycan remodeled mESCs (Scheme 1). The promotional effect of synthetic glycopolymers without/with lipid groups for neural differentiation was compared. Furthermore, the influence of lipo-glycopolymers on FGF2 binding and the activation of ERK1/2 was investigated in order to elaborate the possible mechanism involved in lipo-glycopolymer-mediated neural differentiation.

Scheme 1. Synthesis of the specific lipid-anchored biomimetic GAGs by the post-polymerization of synthetic GAG analogues (A) and their incorporation into cell membranes for high-efficiently promoting mESC differentiation into neurons in short term (B).


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2. EXPERIMENTAL SECTION Materials and General Procedures Chemical materials and methods Chemical reagents: 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTA), sodium 4vinylbenzenesulfonate (SS), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) were purchased from Sigma-Aldrich Chemical Co.. D-(+)-Glucosamine hydrochloride was obtained from TCI Co., Ltd. (Shanghai, China). Fluorescein O-methacrylate (FluMA) was purchased from Alfa Aesar Chemical Co., Ltd. (China). 2,2’-Azoisobutyronitrile (AIBN) from Sinopharm Chemical Reagent Co. (Shanghai, China) was recrystallized from ethanol and dried under vacuum before use. 2-Methacrylamido glucopyranose (MAG) was synthesized as reported previously.42 All organic solvents were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China) and distilled prior to use. Deionized water (DIW), purified to a minimum resistivity of 18 MΩ•cm by a Millipore water purification system, was used for all experiments. Characterization: All the synthetic products were analyzed using an INOVA 400 MHz nuclear magnetic resonance spectroscopy (NMR) to verify their chemical structure and composition. Spectra was recorded in D2O solutions at 293K (for 1H NMR: D2O=4.79 ppm). The numberaverage molecular weights (Mn) and polydispersity (PDI) of the polymers were determined by gel permeation chromatography (GPC) using an Agilent PL-GPC 50 equipped with a refractive index detector, a 5 μm Guard, a 5 μm MIXED-D column with PMMA standard samples, and 0.05 mol/L LiBr solution in DMF as the eluent at a flow rate of 1 mL/min operated at 50oC. Mass spectra data was acquired on a Bruker micrOTOF-Q III instrument. Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet 6700 spectrometer with 32 scans for each sample. UV-Vis and fluorescence spectra were obtained on a Thermo Scientific Varioskan Flash instrument.


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Synthesis of pSGF and the labeled lipid-functionalized GAG analogues lipo-pSGF The fluorescent labeled glycosaminoglycan (GAG) analogs (pSGF) was synthesized via the reversible addition-fragmentation chain transfer (RAFT) polymerization of SS, MAG and FluMA. Briefly, SS (0.1547 g, 0.75 mmol), MAG (0.1853 g, 0.75 mmol), FluMA (0.0123 g, 0.031 mmol), CTA (0.0043 g, 0.015 mmol) and AIBN (0.0014 g, 0.008 mmol) were dissolved in the solution of DMF and DIW (1:1, V/V). The solution was then degassed by bubbling with nitrogen for 30 min. The polymerization was carried out at 70oC for 8 h under a nitrogen atmosphere in a glove box. When the polymerization was complete, the polymerization mixture was dialyzed for 2 days against DIW to remove the excess monomers. The copolymers formed in solution were lyophilized to give a fluffy yellow solid. Maleimide-functionalized DPPE (MAL-DPPE) was synthesized via the condensation reaction between theamino groups of DPPE and carboxyl groups in 6-maleimidohexanoic acid (MALA) as reported recently.43 The detailed improved routes are as follows: DPPE (100 mg, 0.145 mmol) was dissolved in toluene, MALA (37 mg, 0.174 mmol) and HCTU (72 mg, 0.174 mmol) were dissolved in DMF in two vials and then 72 μL DIPEA was added to the DPPE/toluene solution followed by the addition of MALA and HCTU solution. The mixture was stirred for 24 h at room temperature. After that, the appropriate volume of water was added to the final reaction solution followed by regulating pH of the aqueous phase to 4 with 10 M HCl. The reaction mixture was then extracted with ethyl acetate. The organic extract was dried with anhydrous Na2SO4 and purified via a silica column using the mixed solvent of CH2Cl2 and CH3OH (CH3OH with 0.5% CH3COOH). The solvent was removed by rotary evaporation and vacuum-dried overnight. MAL-DPPE: 1H NMR (600 MHz, CDCl3) δ 6.70 (s, 2H), 5.19 (s, 1H), 3.90 (s, 4H), 3.69-3.74 (m, J=6 Hz, 2H), 3.46-3.48 (m, 2H), 3.16-3.20 (q, J=6 Hz, 2H), 2.27-2.31 (q, J=6 Hz, 4H), 2.19-2.21 (m, 2H), 1.41-1.47 (m,


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10H), 1.24-1.30 (m, 48H), 0.86-0.88 (t, J=6 Hz, 6H). MS for C47H85N2O11P [M + H] 886.1700 (calculated); 885.9731 (found). MS for C47H85N2O11P [M + Na]

+

+

m/z:

m/z: 908.1600

(calculated); 907.5648 (found). The lipid-functionalized GAG-mimicking polymers (lipo-pSGF) were prepared via the reaction between thiol groups and maleimide functional groups. Firstly, pSGF (30 mg) was dissolved in the mixed solvent of DMF and H2O followed by the addition of excessive ethanolamine. The reaction solution was stirred for 4 h at room temperature and then dialyzed for 2 days against DIW to remove DMF and the unreacted ethanolamine. The solution was lyophilized to give a fluorescent green solid (pSGF-SH) with a yield of ~81.0%. Secondly, pSGF-SH (1 eq) was dissolved in the mixed solvent of DMF and methanol. MAL-DPPE (1.3 eq) was dissolved in the mixture of CH2Cl2 and CH3OH. Then the two solutions were mixed, bubbled with argon for 60 s and shaken in the dark for 12 h. CH2Cl2 and CH3OH were removed by rotary evaporation. Then the reaction mixture was dialyzed in water to remove DMF and lyophilized to afford a fluffy green solid with a yield of ~72.3%. The UV-Vis and fluorescence spectra are shown in Figure S6 and Figure S7, respectively. Molecular weights and compositions of lipo-pSGF are shown in Table S1. Biological reagents and general biological assays Biological reagents and methods High glucose Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Hyclone. Penicillin-streptomycin solution, non-essential Amino-acids (NEAA) and fetal bovine serum (FBS) were purchased from Gibco. PlasmocinTM was purchased from Invivogen Co., Ltd. Leukemia inhibitory factor (LIF) was obtained from Chemicon. Bovine serum albumin (BSA), paraformaldehyde, gelatin and Triton X-100 were from Sigma-Aldrich. DAPI (4’,6-diamidino-2phenylindole) was purchased from Invitrogen. Heparin sodium was obtained from Sigma-Aldrich


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Chemical Co., Ltd. Recombinant human basic FGF was obtained from Cell Signalling Technology. PCR primers were from Shanghai Sangon Biotechnology Co., Ltd. Primary antibody β3-Tubulin was obtained from Cell Signalling Technology Co., Ltd. The FITC conjugated secondary antibody was from Boster Wuhan, China. The RNA simple total RNA kit was obtained from Tiangen Biotech Co., Ltd. The RevertAid First Strand cDNA Synthesis Kit was from ThermoFisher Scientific. The Fast SYBR Green Master Mix was from Applied Biosystems. Fluorescence images were captured using an Olympus IX71 fluorescence microscopy. The images were overlapped using Image-Pro Plus 6.0 software and analyzed in ImageJ. Flow cytometry was performed on a BD FACSVerseTM and data was analyzed using FlowJo with 10, 000 events during collection. QPCR was performed in a real-time PCR machine (StepOne Plus realtime PCR system) from Applied Biosystems. Cell culture MESCs (R1/E, Stem Cell Bank, Chinese Academy of Sciences) were cultivated on a feeder layer of mouse embryonic fibroblasts (mEFs, ICR MEF, Stem Cell Bank, Chinese Academy of Sciences) inactivated with 10 μg/mL mitomycin C (Solarbio) with mESCs maintenance medium containing DMEM supplemented with 10% FBS, 100 μg/mL streptomycin, 100 U/mL penicillin, 0.1 mM NEAA, 1000 U mL-1 LIF and 0.1 mM β-mercaptoethanol (Amresco) at 37oC in a water-saturated 5% (v/v) CO2 chamber (Eppendorf Galaxy 170R). For normal passaging, cells were detached and seeded onto a T-25 plates pre-treated with a feeder layer for at least 12 h. Influence of lipid-anchored biomimetic GAG on mESCs studied by CCK-8 assay To investigate the cytocompatibility of lipo-pSGF, the classical CCK-8 test was carried out for detecting the influence on the growth of mESCs. Briefly, mESCs were seeded in the 96-well culture plates (Costar) at a density of 104 cells/well and treated with different concentrations of


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lipo-pSGF solution for 1 and 5 days, respectively. At the end of day 1 and 5, the media were replaced by 200 μL of fresh DMEM medium, followed by the addition of 20 μL CCK-8. Subsequently, the cells were incubated in a humidified 5% CO2 incubator at 37oC for 1 h. The absorbance of the resulting solutions was then recorded at 450 nm using a microplate reader. Insertion assay of synthetic polymers via microscopy After mESCs were cultured in a T-25 flask pre-treated with gelatin and passaged twice to remove mEFs, mESCs were trypsinized and seeded onto PLL coated coverslips (in a 48 well plate) at a density of 5 × 104 cells/well in mESCs maintenance medium. Following incubation overnight at 37 °C, 5% CO2, the wells were washed with 0.3 mL PBS twice and incubated with 0.5 mL of lipidcopolymer solution with different concentrations in serum-free DMEM at 37oC for 1 h. Then, 0.3 mL PBS/well was added to wash away excess polymers and fixed with 4% paraformaldehyde for 10 min. The images were captured using a fluorescence microscopy. Analysis of the insertion of lipid-copolymers into cell membranes by flow cytometry MESCs were seeded at a density of 106 cells/well into a 6-well tissue culture plate and allowed to incubate overnight at 37oC and 5% CO2 in corresponding medium. Solutions of lipid-copolymer in serum-free media with different concentrations were added to the individual well. The cells were incubated with the lipid-copolymers for 1 h at 37oC. Following incubation, the cells were washed three times with PBS, detached using trypsin and centrifuged at 1000 rpm for 5 min. Subsequently, the cells were washed twice with PBS and resuspended in 0.5 mL of PBS for flow cytometry analysis. Neural differentiation For neural differentiation, mESCs were seeded at a density of 104 cells/well into a gelatinized 48well plate in mESC growth medium. The following day, medium was replaced with neural


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induction medium (DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 0.1 mM NEAA, 0.1 mM 2-mercaptoethanol and 1.0 μM retinoic acid). Heparin and the synthetic copolymer were added to the media at final concentrations of 5 μg/mL and 1.7 μM, respectively. For neural differentiation of membrane-remodeled mESCs, cells were treated with 0.5 mL of lipidcopolymer solution in serum-free DMEM at 1.7 μM for 1 h at 37oC. After incubation, cell monolayers were washed three times with PBS, and 1 mL of fresh neural induction medium was added. Medium was replaced every two days. Immunofluorescence assay The cells in neural differentiation media after a certain period of time were washed in PBS, fixed in 4% paraformaldehyde for 10 min and rinsed three times in PBS. The cells were permeabilized with 0.1% Triton X-100 for 5 min. Following washed twice with PBS, cells were blocked using block solution (3% BSA/PBS) for 30 min. Then primary antibody β3-Tubulin was performed overnight at 4oC. After being washed three times in PBS, cells were incubated with secondary antibody at room temperature for 1 h, followed by two washes in PBS. Then, DAPI (0.5 μg mL-1) was added to stained nuclei and incubated at room temperature for 5 min. Representative images of experiments were captured using a fluorescence microscope. Herein, due to the low concentration of the polymer and the quenching of green fluorescence in the cell medium, the influence of green fluorescence of our synthetic polymers on the immunofluorescence assay can be ignored. Thus, we chose the FITC conjugated protein as the secondary antibody. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) MESCs were subjected to neuronal differentiation as mentioned above. At different time points (day 7 and day 14), total RNA was extracted from cells treated with heparin, copolymer and lipidcopolymer using the total RNA extraction kit according to the manufacturer's instructions. Then


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the RNA samples were reversely transcribed for first-strand cDNA synthesis using oligo (dT) as a reverse transcription primer. qRT-PCR was performed using a StepOnePlus real-time PCR system with primers for β-actin, OCT4 and β3-tubulin (Table S2) using a Fast SYBR Green Master Mix. Each 20 μL solution for qRT-PCR reaction was used : 2 μL of cDNA, 0.4 μL of primer mixture (20 μM forward and 20 μM reverse), 7.6 μL of ddH2O and 10 μL of SYBR Green master mix. Conditions were as follows: 95oC for 20 seconds, followed by 50 cycles of 95oC for 30 seconds, 60oC for 45 seconds, and 72oC for 45 seconds, then 95oC for 15 seconds, 60oC for 1 min and 95oC for 15 seconds. The cycle threshold (Ct) values were obtained from the amplification plots and the ∆Ct value for each sample was calculated by subtracting the Ct values of a housekeeping gene, β-actin. Then the ∆∆Ct value of the synthetic polymers samples (copolymer- and lipid-copolymer-treated samples) was normalized to the heparin-treated value at day 7 to report relative gene expression by raising 2 to the -∆∆Ct power.44 FGF2 binding test MESCs were seeded at a density of 8×105 cells/well into a 24-well tissue culture plate and allowed to incubate for 12 h at 37oC and 5% CO2 in mESCs growth medium. After that, the cells were washed twice with PBS and incubated with the synthesized lipid-anchored GAG analogue solution in DMEM and the normal medium without polymers at 37°C for 1 h. Then, the cells were washed three times with PBS to remove the unbound polymers. Following that, the cells in the plate were fixed with 4% paraformaldehyde for 10 min at room temperature and washed three times in PBS. Subsequently, cells were blocked using block solution (3% BSA/ PBS) for 1 hour at 4°C, and incubated with FGF2 solution in 3% BSA/PBS at 4°C for 1 hour. Immunostaining was carried out with rabbit anti-fibroblast growth factor as the primary antibody and AlexaFluor555 anti-rabbit


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antibody as the secondary antibody. ERK activation assays MESCs were plated in gelatinised 6-well plates at a density of 8 x 105 cells/well in mESC growth medium. MESCs were then starved overnight in DMEM without FBS. Subsequently, cells were incubated with the synthesized lipid-anchored GAG analogue solution and heparin in DMEM at 37°C for 1 h. Then, the cells were washed twice with PBS to remove the unbound polymers. After that, FGF2 with the final concentration of 10 ng/mL was added to cell monolayers for 60 minutes at 37oC, 5% CO2. Then mESCs were washed twice with PBS, followed by lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 5 nM EDTA, 0.1% SDS and complete proteinase inhibitor (Roche). After incubated on ice for 30 min, lysates were briefly sonicated and nonsoluble cell debris was removed through centrifugation at 4°C. Protein lysates in SDS loading buffer were heated for 10 minutes at 95°C, then electrophoresed in SDS-PAGE gel. Protein samples were transferred onto PVDF membranes and probed with indicated primary antibodies for 12 h at 4°C after blocked in 5% non-fat dry milk. The PVDF membrane was incubated with appropriate secondary antibodies conjugated to horseradish peroxidase for 1 h at 25oC. Finally, protein was detected using chemiluminescent HRP substrates with Molecular Imager® Gel Doc™ XR+ System (Bio-Rad). 3. RESULTS AND DISCUSSION Preparation of pSGF and the lipid-anchored GAG-mimicking polymers (lipo-pSGF) In “classical” methods to obtain polymers with lipid groups, lipid-functionalized chain transfer agents are first prepared and used for polymerization; these methods often require complex procedures including purification and de-protection.39-41 To simplify the synthesis process, postpolymerization modification (PPM)45-47 was used in the present study to prepare the lipid-anchored


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GAG analogues. Firstly, we synthesized the fluorescein-labeled biomimetic GAGs by RAFT polymerization of SS, MAG and FluMA to give pSGF. A spacer molecule consisting of maleimide-functionalized 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (MAL-DPPE) was then prepared, and attached to the copolymer via the thiol maleimide click reaction to obtain lipidderivatised polymers lipo-pSGF (Figure S1). The feed ratio of monomer SS to monomer MAG used for the preparation of lipo-pSGF was 1:1 according to our previous study.34 The 1H NMR, IR, UV-visible and fluorescence spectra (Figure S2~S5) showed that the GAGmimicking glycopolymers without lipid groups (pSGF) was obtained successfully. Then as shown in Figure 1a, the 1H NMR spectrum confirmed the expected structure of the synthetic lipo-PSGF. The characteristic peaks of MAG and benzene rings attributed to SS and FluMA can be clearly observed. The composition of the resultant lipo-pSGF was shown in Table S1 which showed that the sulfonated degree (~50.4%) similar with natural heparan sulfate was obtained. The GPC traces of pSGF-SH and lipo-pSGF were shown in Figure S8. The relative molecular weight increased after PPM of lipid groups, indicating the successful preparation of lipo-pSGF. The relative molecular weight of lipo-pSGF is ~7300 Da and the narrow polydispersity index indicated the polymerization is well controlled (Figure 1b). Furthermore, the FT-IR spectrum (Figure 1c) showed characteristic peaks of the corresponding groups in the obtained lipo-glycopolymers. The peaks at 3352 cm-1 were attributed to the O-H bonds of MAG, and the peaks at 1740 cm-1 corresponded to the ester groups from FluMA. The SO stretches from SS can be found at 1166, 1122 and 1036 cm-1., and the peaks at 2914 cm-1 and 2848 cm-1 were attributed to the methylene groups of DPPE. The sharp peak at 2848 cm-1 corresponding to methylene of DPPE was observed from IR spectra (Figure S9). The results from FT-IR spectra further confirmed that GAG analogue containing lipid end group was successfully obtained. And the 1H NMR spectrum of lipo-pSGF


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(Figure S11) showed the increase of hydrogen content from methyl andmethylene groups compared with that of pSGF (Figure S10), also indicating the successful attachment of DPPE groups. The dynamic light scattering (DLS) result (Figure S12) suggested that the polymer lipopSGF can disperse stably in aqueous solution at concentrations lower than 10 μM instead of forming micelles. The influence of lipid-anchored biomimetic GAG on the growth of live mESCs Considering specific natural glycans can manipulate cell growth, or even promote the proliferation of ESCs48-50, the growth of mESCs treated with lipo-pSGF of different concentrations was analyzed using CCK-8 assay.51-53 As shown in Figure 2, after 1 day of treatment, both of the groups treated with lipo-SGF of 0.5 and 3.4 μM showed good viability compared with the untreated group. After 3 days, the growth of cells treated with lipo-pSGF was slightly faster than the untreated control. The relative proliferation of 3.4 μM-lipo-pSGF-treated mESCs increased by ~14.5% compared to that of untreated mESCs. These results demonstrated that our synthetic lipo-GAG analogues have a good cytocompatibility, providing a good prerequisite for the subsequent biological studies. Interactions between lipo-pSGF and pSGF with mESCs Glycolipids in natural eukaryotic cell surfaces are essential complex carbohydrates, and some of them are involved in receptors-mediated signaling pathways critical for guiding cell behaviors, such as cell division, differentiation and apoptosis.54-57 We have recently achieved the insertion of synthetic lipo-glycopolymers into HeLa cell membrane.58 Here, synthetic glycopolymers was inserted into the membrane of mESCs. Before investigating its effect on cell differentiation, the retention of lipo-pSGF was firstly analyzed. MESCs were incubated in the solution of pSGF or lipo-pSGF (both containing green fluorescent groups) for 1 h at 37oC and imaged by fluorescence


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microscopy after being washed twice to remove the unbound polymers. As shown in Figure 3a, strong green fluorescence was observed in the membrane of mESC after incubation in lipo-pSGF solution. In contrast, almost no fluorescence was found in the untreated control. And cells incubating in pSGF solution showed the strongest green fluorescence intensity. Furthermore, the entire cell bodies showed strong green fluorescence for pSGF-treated cells. However, the high fluorescence contrast between internal cells and exterior of cells was observed for lipo-pSGFtreated cells, and the green fluorescence converged on cellular membranes. This should be caused by the endocytosis of pSGF which led to higher amount of fluorescent molecules in cell areas compared to lipo-pSGF-treated cells where most molecules retained on cell membrane.58 The result from flow cytometry showed that the intensity of green fluorescence was dependent on the concentration of lipo-pSGF (Figure 3b), indicating that the quantity of lipo-pSGF inserted into the cell membrane could be regulated by changing the concentration of the incubating solution. An equivalent concentration (1.7 μM) of lipo-pSGF was used for the subsequent neural differentiation experiments referring to the previous study.39 Neural differentiation of mESCs The difference in the cellular location between pSGF and lipo-pSGF enlightened us that they may show varied activity towards neural differentiation of ESCs. Thus neural differentiation of mESCs treated with pSGF and lipo-pSGF was studied. Using immunofluorescence staining, dendrites of neurons were clearly observed in the lipo-pSGF-treated group after just 7 days, and even at day 5, the similar cell phenotypes with the axonal cell bodies were observed (Figure S13), while for the lipid-derivatized polymer containing disaccharides from native heparan sulfate only differentiation to form intermediate neural rosettes occurred during the same period.39 Moreover, neurofilaments in cells treated with lipo-pSGF were observed at day 14 (Figure 4a). Quantitative polymerase


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chain reaction (qPCR) was further used to quantify the expression of neural-specific marker gene β3-tubullin.59,60 Heparin, as a kind of natural GAG, has been adopted for promoting neural differentiation of stem cells.61,62 Therefore, commercially available low-molecular-weight (3500~8000 Da) heparin that’s proven to have higher bioactivity was chosen as a positive control.63 The results showed that lipo-pSGF treated mESCs expressed the highest level of β3tubulin both at day 7 and day 14 (Figure 4b). Compared with the expression of β3-tubullin in heparin- and pSGF-treated cells, its expression in lipo-pSGF-treated cells was about 1.7 and 1.2 times higher at day 7 and 3.8 and 1.9 times higher at day 14. The expression of pluripotent gene OCT4 in mESCs treated with different molecules for 7 days was also determined (Figure S14). The expression of OCT4 in lipo-pSGF-treated cells was ~46.4% of that in untreated cells and ~35.2% of that in heparin-treated cells, demonstrating that the presence of lipo-pSGF on cell membrane accelerated the differentiation of mESCs. Taken as a whole, these results demonstrated the better efficiency of our synthetic GAG-mimicking copolymer than heparin in promoting neural differentiation of mESCs and by incorporation of GAG analogues into cell membrane, the promotion effect can be further enhanced. FGF2 binding assay In the next step, we attempted to explore the likely causes for the better activity of lipo-pSGF in promoting neural differentiation of ESCs. And the little effect of lipo-pSGF on cell viability shown in Figure 2 indicated that the promotional effect of lipo-polymers on the specific differentiation of mESCs may not be regulated by influencing cell growth. Inspired by these outcomes, we investigated the following cellular signal pathway responsible for the neural differentiation of ESCs. Many studies show that FGFs play an essential role in neuronal development.64,65 Thus, we firstly investigated the binding affinity of lipo-pSGF with FGF2 by immunostaining to look for


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possible reasons of its better promotional effect. The FGF2 binding assay was conducted following the incubation of mESCs in high glucose DMEM with lipo-pSGF for 1 h at 37oC. The results showed that mESCs remodelled with our synthesized biomimetic GAG can bind larger amount of FGF2 to cell membranes (Figure 5a) than the untreated cells. Images with multiple cells are shown in Figure S15, and indicate that a promotional effect is expected for subsequent activation of downstream events. And as shown in Figure 5b, the fluorescence intensity in lipo-pSGFremodeled group was ~2.4 times stronger compared with the untreated cells. These results suggested that incorporation of lipo-pSGF to the membrane of ESCs enhanced FGF2 binding. ERK activation assay The role of FGF2 in neural development is shown to be achieved via the ERK pathway66,67 and during neural differentiation, ERK1/2 phosphorylation is increased.22 Therefore, ERK activation assays through FGF signaling in the presence of lipo-pSGF were conducted. The expression of phosphorylated ERK1/2 was determined by western blot test.68,69 The highest level of ERK1/2 phosphorylation was found in mESCs stimulated with FGF2 for 60 min (Figure S16) and this incubation time was thereby chosen for subsequent ERK1/2 activity detection. In the presence of FGF2, lipo-pSGF-engineered mESCs exhibited a 1.8-fold increase in ERK1/2 phosphorylation compared to control cells and there is a statistical significance between lipo-pSGF- and heparin treatment (P = 0.008) (Figure 6). Based on the above results, it can be concluded that lipo-pSGFtreated mESCs have stronger binding with FGF2 and increased ERK1/2 phosphorylation which results in faster neural differentiation.


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Figure 1. (a) The 1H-NMR spectrum of lipo-pSGF in D2O. The characteristic peaks of MAG and benzene rings attributed to SS and FluMA can be clearly observed. And the peaks of the methyl andmethylene groups correspond to the polymer backbone and DPPE groups also can be observed. (b) The GPC trace of lipo-pSGF using DMF with 0.05 mol/L LiBr solution as the eluent. (c) The IR spectrum of lipo-pSGF. The appearance of characteristic peaks attributed to MAG, SS, FluMA and DPPE groups showed the successful preparation of lipo-pSGF.


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Figure 2. The growth of live cells after incubating mESCs in lipo-pSGF solutions with different concentrations for 1 and 3 days indicated the synthetic lipo-GAG analogues have a good cytocompatibility. The untreated cells cultured on normal culture plates for 1 and 3 days were used as controls and their cell viability at every time point was regarded as 100 %. Error bars represent the standard deviation of the mean (n = 3).


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Figure 3. (a) Fluorescence microscopy images of live mESCs incubated for 1 h with pSGF and lipo-pSGF (6.8 μM, shown in green), scale bars: 50 μm. The lipo-pSGF retained on the membrane of mESCs rather than being internalized by cells after one-hour incubation. (b) Analysis by flow cytometry of lipo-pSGF with different concentrations incorporated into mESC membranes. The untreated mESCs were negative control, which test mean was defined 100%. The amount of lipopSGF incorporated into mESC surfaces depends on polymer concentrations.


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Figure 4. Neural differentiation of mESCs treated with commercial heparin and synthetic glycopolymers. (a) Immunofluorescence images of mESCs (green: β3-tubulin; blue: DAPI) treated with heparin, pSGF and lipo-pSGF at day 7 and 14. Scale bar, 100 μm. More neural cells were observed in the lipo-pSGF-treated group. (b) Neuron-specific marker β3-tubulin relative expressions of differentiated cells evaluated quantitatively by qRT-PCR analysis at day 7 and 14. Error bars represent the standard deviation of the mean (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 by t-test).


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Figure 5. The synthetic lipo-pSGF significantly enhanced FGF2 binding. (a) FGF2 (red) binding assay via inverted fluorescence microscope (scale bar: 20 μm). (b) Quantitative analysis of the relative fluorescence intensity. The red fluorescence intensity of untreated cells was regarded as 100%. Error bars represent the standard deviation of the mean (n = 3, ***P < 0.001).


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Figure 6. The synthetic lipo-pSGF significantly enhanced the phosphorylation of ERK1/2. (a) Western blots and (b) subsequent quantitative analysis via densitometry. Phosphorylation levels of ERK were normalized relative to the total ERK levels for each group and compared to those of untreated mESCs. Values represent the mean ± standard error (**P < 0.01 by t-test) from two independent experiments. 4. Conclusions In summary, we have developed a simple and convenient strategy to attach phospholipid group to synthetic biomimetic GAGs that can be used to engineer stem cell membrane. Our results showed that the lipid-modified biomimetic GAG (lipo-pSGF) has a good cytocompatibility and well retained on cell membrane during one-hour incubation with mESCs. Furthermore, it accelerated the differentiation of pluripotent mESCs into neurons after only 7 days. The likely mechanisms


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involved in lipo-pSGF-mediated neural differentiation were investigated by studying FGF2 binding, and ERK1/2 phosphorylation. It was found that lipo-pSGF significantly enhanced FGF2 binding and the phosphorylation of ERK1/2. Therefore, the faster neural differentiation in lipopSGF-treated mESCs may be attributed to the more efficient binding of FGF2 on the cell membrane, which triggers the FGF2-ERK pathway. The present work provides a new strategy for modification of cell membrane using synthetic biomimetic GAGs that may be used in other biological applications that involve cell-surface signaling. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional text with details on the synthesis and characterization of pSGF and lipo-pSGF, the particle size of lipo-pSGF in water determined by DLS, the fluorescence microscopy images of FGF2 assay with multiple cells, western blot of mESCs stimulated with FGF2 for different period, q-PCR primers used in the study, immunofluorescence images of cells at day 5 and 7 and the relative expression of OCT-4 of differentiated cells at day 7.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (No. 21674074, No. 21374069, No. 21374070, No. 21334004), the Natural Science Foundation of Jiangsu Province (No.BK20161208) and Jiangsu Clinical Research Center for Cardiovascular Surgery for financial support. We thank Prof. John L. Brash and Prof. Qian Yu for the valuable advice and proofreading the manuscript. REFERENCES 1. Keller, G., Embryonic Stem Cell Differentiation: Emergence of a New Era in Biology and Medicine. Gene. Dev. 2005, 19, 1129-1155. 2. Odorico, J. S.; Kaufman, D. S.; Thomson, J. A., Multilineage Differentiation from Human Embryonic Stem Cell Lines. Stem Cells 2001, 19, 193-204. 3. Thomson, J. A.; Itskovitz-Eldor, J.; Shapiro, S. S.; Waknitz, M. A.; Swiergiel, J. J.; Marshall, V. S.; Jones, J. M., Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 1998, 282, 1145-1147. 4. Lumelsky, N.; Blondel, O.; Laeng, P.; Velasco, I.; Ravin, R.; McKay, R., Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets. Science 2001, 292, 1389-1394. 5. Sachinidis, A.; Kolossov, E.; Fleischmann, B. K.; Hescheler, J., Generation of Cardiomyocytes


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Synthetic Glycopolymers for Highly Efficient Differentiation of

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