Directing Induced Pluripotent Stem Cell Derived Neural Stem Cell

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Biological and Medical Applications of Materials and Interfaces

Directing IPSC-derived Neural Stem Cell Fate with 3D Biomimetic Hydrogel for Spinal Cord Injury Repair Lei Fan, Can Liu, Xiuxing Chen, Yan Zou, Zhengnan Zhou, Chengkai Lin, Guoxin Tan, Lei Zhou, Chengyun Ning, and Qiyou Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05293 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Directing IPSC-derived Neural Stem Cell Fate with 3D Biomimetic Hydrogel for Spinal Cord Injury Repair Lei Fan1, 2, *, Can Liu1, *, Xiuxing Chen3, *, Yan Zou6, Zhengnan Zhou4, Chenkai Lin5, Guoxin Tan4, Lei Zhou2, #, Chenyun Ning2, #, Qiyou Wang1, # 1

Department of Spine Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou

510630, Guangdong Province, China. 2

College of Materials Science and Technology, South China University of Technology, Guangzhou,


State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou


Institute of Chemical Engineering and Light Industry, Guangdong University of Technology,

Guangzhou 510006, Guangdong Province, China. 5

Department of Orthopedics, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen,


Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou

510630, Guangdong Province, China. * The first three authors contributed equally to this work and should be considered as co-first authors. # To whom correspondence may be addressed. Email Address: [email protected] (L. Zhou), [email protected] (C. Ning) and [email protected] (Q. Wang)

Key words: gelatin methacrylate hydrogel scaffold; 3D culture; induced pluripotent stem cells; spinal cord injury; neuroregeneration

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Abstract Current treatment approaches for spinal cord injuries (SCIs) are mainly based on cellular transplantation. Induced pluripotent stem cells (iPSCs) without supply constraints and ethical concerns have emerged as a viable treatment option for repairing neurological disorders. However, the primarily limitations in the neuroregeneration field are uncontrolled cell differentiation, and low cell viability caused by the ischemic environment. The mechanical property of three-dimensional (3D) hydrogel can be easily controlled and shared similar characteristics with nerve tissue, thus promoting cell survival and controlled cell differentiation. We propose the combination of a 3D gelatin methacrylate (GelMA) hydrogel with iPSC-derived NSCs (iNSCs) to promote regeneration after SCI. In vitro, the iNSCs photo-encapsulated in the 3D GelMA hydrogel survived and differentiated well, especially in lower-moduli hydrogels. More robust neurite outgrowth and more neuronal differentiation were detected in the soft hydrogel group. To further evaluate the in vivo neuronal regeneration effect of the GelMA hydrogels, a mouse spinal cord transection model was generated. We found that GelMA/iNSC implants significantly promoted functional recovery. Further histological analysis showed that the cavity areas were significantly reduced, and less collagen was deposited in the GelMA/iNSC group. Furthermore, the GelMA and iNSC combined transplantation decreased inflammation by reducing activated macrophages/microglia (CD68-positive cells). Additionally, GelMA/iNSCs implantation showed striking therapeutic effects of inhibiting GFAP-positive cells and glial scar formation while simultaneously promoting axonal regeneration. Undoubtedly, use of this 3D hydrogel stem cell-loaded system is a promising therapeutic strategy for SCI repair.

Key words: gelatin methacrylate hydrogel scaffold; 3D culture; induced pluripotent stem cells; spinal cord injury; neuroregeneration

1 Introduction Traumatic injury or disease may result in spinal cord injury (SCI). Generally, SCI have devastating condition that can leads to permanent sensory and motor dysfunction below the injury site, mainly due to the appearance of a harsh microenvironment around the damage site, which impair neuroregeneration and functional recovery after SCI. Because of the severity of SCI, no effective treatment has yet to be developed [1-3].Recent advances in stem cell transplantation provide a potentially effective method to repair damaged spinal cord. Induced pluripotent stem cells (iPSCs) without supply constraints and ethical concerns have been implanted into models of SCI and have achieved promising results, and iPSC-based treatments have emerged as a powerful approach for neurological regeneration [4-6]. With the advent of iPSC technology, iPSCs have become a promising cell type for use in therapeutic approaches to treat patients with SCI. However, various limitations still need to be overcome to use direct cell injections as a therapeutic strategy in SCI. 1) Direct


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injections inevitably cause the cell loss in the injury site [7]. 2) Dead or dying cells caused by ischemia and the adverse environment at the injured site can worsen outcomes [8]. 3) The differentiation of transplanted stem cells to their mature phenotypes including neurons, astrocytes, and oligodendrocytes was uncontrolled [9, 10]. The cells in traditional two-dimensional (2D) systems are very different from those cultured in 3D environments regarding their adhesion, spreading, polarity, and migration [11, 12]. A promising three-dimensional (3D) biomimetic platform that mimics the unique physiological microenvironment of native CNS is needed to deliver stem cells and promote cell survival and neural differentiation. The aim of 3D cell culture systems is to simulate the in situ functions of living tissue by rebuilding organizational structure and providing a favorable microenvironment for cell survival and proliferation [13]. In particular, to study the CNS, given the poor representation of soft tissue provided by 2D cultures, 3D cultures of neural lineage cells (neurons and glial cells) are being exploited to more accurately rebuild the complex function and structure of the human brain [13, 14]. Solid porous scaffolds or other fibrous 3D scaffolds with cells seeded on the surface of the scaffold do not well imitate the natural structure of the CNS. For example, the direction of NSC differentiation can be well controlled by the mechanical properties of the material, which cannot be simulated by these 3D scaffolds. In our study, hydrogels composed of the collagen degradation product gelatin combined with methacrylate (GelMA) were used to photo-encapsulate iNSCs and provided an excellent scaffold material for SCI repair. The GelMA 3D hydrogel system shared similar characteristics with nerve tissue, including high permeability for oxygen and nutrients, a high water-content matrix and moderate mechanical properties, which together provide an excellent environment for cell growth [10]. Promoting and controlling the differentiation of iPSC-derived neural stem cells (iNSCs) into neurons is a major aim in transplantation studies. Biophysical aspects of the biomaterials, especially elasticity, play an important role in cellular behaviours containing stem cell expansion, migration and differentiation [15]. The mechanical properties of the hydrogel have been reported to have profound influence on NSC differentiation [16]. The fates of NSCs can be modulated by the mechanical properties of the hydrogel. NSCs can not survive well in materials which possess very low (100kPa), and it is more likely to neuronal differentiation in soft scaffolds (≈0.1−1 kPa), otherwise, they are prone to astrocyte differentiation in slightly stiffer materials ( ≈ 7−10 kPa) [17, 18]. Thus,the proliferation and fate of NSCs could be manipulated by just regulating the mechanical properties of hydrogels. Importantly, the phenotype and migration of neural cells play important roles in neuronal maturation. Furthermore, whether the mechanical properties of GelMA hydrogels also have profound influences on the survival, migration, and differentiation of iNSCs has not been addressed in previous research [19]. In addition, the GelMA hydrogel is rich in the cell-adhesive Arg-Gly-Asp (RGD) peptide, which increases the adhesion and differentiation of encapsulated stem cells [10, 20]. Several groups have reported the application of this ECM-like GelMA



scaffold for skin tissue engineering, bone repair, treatment of cardiovascular diseases, and so on [21-25]. However, its potential for applications in neural tissue engineering has not been widely investigated. We hypothesized that photo-encapsulated iNSCs in a tunable mechanical GelMA hydrogel 3D culture system that mimics both the structure and mechanical properties of nervous tissue would promote iNSC neuronal differentiation in vitro and in vivo. In the vivo study, hydrogels were used to fill the lesion site of spinal cord in adult male mice to evaluate their neuroregenerative ability at time points up to 6 weeks. A GelMA hydrogel based a biomaterial was used to efficiently deliver iNSCs. Hydrogels are particularly appealing for use as the vehicle for cell transplantation because they not only can promote cell survival but also inhibit inflammation and promote neuroregeneration. To our knowledge, this is the first report to evaluate the effects of GelMA scaffold mechanical properties on the differentiation of iNSCs and to combine the good biocompatibility of GelMA scaffolds with iNSCs to treat SCI model mice. 2. Materials and Methods 2.1 Fabrication and Characterization of GelMA Hydrogels The synthesis process of GelMA have been reported in previously work [26, 27]. 1H NMR spectra of GelMA were obtained according to the literature [28]. At room temperature, a Bruker Avance 300 MHz instrument was used to determinate the methacrylation degree of GelMA at 1 H resonance frequency of 400 MHz, 16 scans after dissolved in D2O (30 mg/mL). Hydrogels were prepared from GelMA solution (3% w/v) with 0.5% photoinitiator, 2-hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959; Sigma) under UV irradiation (6.9 mW/cm2, 360-480nm). Varying UV exposure time (15s, 25 s and 40s) yielded three hydrogels of variable stiffness. The internal microstructures of GelMA hydrogels with different stiffness were observed under a scanning electron microscope (SEM) as previously described [27]. Then the software (Image J) was used to calculate the pore sizes. The compressive modulus of GelMA hydrogels were characterized on a Bose ELF 3200 universal testing system (Bose Corp., Eden Prairie, MN, USA). Briefly, aftert a 6 cm in diameter, and 800µm in thickness disc were removed from DPBS and the residual liquid was removed by blotting with kimwipes, then the hydrogel disc was tested at a rate of 20 % strain/min. From the linear region of the stress–strain, the compressive modulus can be calculated. 2.2 In Vitro Study 2.2.1 Isolation of Murine Embryonic Fibroblasts (MEF) and IPS Generation The episomal vectors pEP4-103EO2S-ET2K (7 µg) (Addgene plasmid 20927), which encoded three transcription factors, Oct4, Sox2, and Klf4, and pCEP4-miR302-367


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clusters (5 µg) were electroporated into 2×106 MEFs, which were obtained from mouse embryos at approximately two weeks according to previously published protocols [29]. Plasmids carrying the reprogramming factors were transfected by using a Nucleofector 2b Device (Lonza, Basel, Switzerland) according to the instructions. After transfection, 1×105 cells were cultured in MEF medium (DMEM+10% FBS (GIBCO)) for 48 h. Then, these cells were collected and cultured with an MEF feeder in iPSC induction medium (DMEM supplemented with 15% KSR (GIBCO), 1 mM L- glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), 0.1 mM nonessential amino acids (NEAA, GIBCO), 1× penicillin/streptomycin, 0.1 mM β-mercaptoethanol (GIBCO) and 1000 units of recombinant leukemia inhibitory factor (LIF, Millipore)); the medium was changed every two days. (Fig. 1). 2.2.2 Differentiation of IPSCs into INSCs After dissociating with 0.125% trypsin-EDTA, iPSCs were transferred to agarose G-10-coated (BIOWEST) tissue culture dishes (low-attachment dishes) and cultured in iPSC medium without LIF to promote embryoid body (EB) formation. For differentiation toward a neuronal lineage, the EBs were treated with 1 µM all-trans retinoic acid (RA; Sigma) for 4 days and then transferred to 0.01% poly-L-lysine (PLL, Sigma)-coated tissue culture dishes and maintained with NSC proliferation medium. The medium was changed every two days. Seven days later, NSC-like cells were trypsinized, transferred to low-attachment dishes and cultured with NSC medium. 2.2.3 INSC Photoencapsulation and 3D Culture Cell photoencapsulation was following previously published protocols [27]. Briefly, iNSC were pelleted and resuspended in pure GelMA solution containing 0.5 % (w/v) photoinitiator, Irgacure 2959 at a concentration of 1×107 cells/mL. Microgel units with entrapped cells were fabricated in a 6 mm in diameter and 2 mm in height PDMS mold following exposure to 6.9 mW/cm2 UV light (360–480 nm) for 15s, 25s and 40s, respectively. Then the crosslinking hydrogels were immediately transferred into NSC differentiation medium (1:1mix of Neurobasal Medium (NB, GIBCO) and DMEM/F12 supplemented with N2, B27, as well as 1×Glutamax and Penicillin/Streptomycin) and cultured at 37°C with 5% CO2. Half amount of media was changed every other days. 2.2.4 Live-dead Assays and Cell Proliferation within GelMA Hydrogels Encapsulation iNSCs were washed by phosphate buffered saline (PBS) and then incubated with Calcein-AM/ethidium (Calcein-AM/PI Invitrogen) homodimer fluorescence for 20 min at 37°C, 5% CO2. After washed by PBS for two times, the samples were visualized using an inverted fluorescence microscope (Olympus IX71, Olympus Co. Tokyo, Japan). A Cell Counting Kit-8 (CCK-8, Dojindo, Japan) test was



used to measure cell proliferation. In brief, the proliferation of encapsulated cells were measured at 1day, 3days and 7days respectively. After 3 hours incubated in the medium containing CCK8 (1:10), 100ul supernatant were transferred into 96-well culture plates. The absorbance of the solution was measured on microplate reader (EON, Gene Company Limited) at 450 nm. 2.2.5 Gene Expression GelMA hydrogels were degraded in collagenase type II solution at 100 U/mL concentration (Sigma) at 37°C for 1 h to release entrapped cells. Next, The process of reverse transcription quantitative polymerase chain reaction (RT-qPCR) were performed as described previously [29]. The primer information is provided in Table 1. All experiments were performed in triplicate. Gene expression was calculated using the 2-∆∆Ct method. 2.2.6 Immunofluorescence After the hydrogels were blocked with 4% paraformaldehyde at 4°C for 20 min, they were permeabilized with a mixture of 6% fetal bovine serum (BSA Sigma) and 0.2% Triton X-100 (Sigma) for 1 h at room temperature. They were then incubated with primary antibodies (Table 2) overnight. After collecting the primary antibody, the secondary antibody was added, and cells were further incubated for 1 h. Finally, cells were stained with Hoechst 33342 (Sigma) in PBS for 5 min. Between each step, PBS were used to wash for three times. 2.3 In Vivo Testing 2.3.1 Ethics Statement All experimental protocols and animal handling procedures were conducted according to National Institutes of Health Guide for the Care and Use of Laboratory Animal, which was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. 2.3.2 Experimental Groups and Spinal Cord Injury Surgery Twenty-four 6- to 8-week-old C57BL/6N male mice were randomly divided into three equal groups as follows: spinal cord transection with saline injection (SCI group), spinal cord transection with simple cell treatment (iNSCs group), iNSC-laden GelMA hydrogel treatment (GelMA/iNSCs group). The spinal cord was transected at the level of T9-10 in adult male C57BL/6N mice as the method described previously [30, 31]. Briefly, mice were anesthetized by injection of a mixture of 70 mg/kg ketamine and 5 mg/kg xylazine via intraperitoneal. After corneal reflexes disappearing, surgical shaved the fur from the surgical site and


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disinfected the skin with Iodine tincture and ethanol 70%. Then layered cut the skin and paravertebral muscles in the back of the mice.. The spinal cord was transected and a 2mm spinal cord segment completely removed after laminectomy at T9-10 level. After the respective treatment; the paravertebral muscles and skin were closed in layers, then disinfected with ethanol 70% again. The mice were allowed to recover in a warmed cage and free access to food and water. 2.3.3 Motor Behavior and Footprint Analysis The motor behavior of the hindlimbs was assessed by using BMS scores weekly [32]. Hind limb movement of all three group mice were observed in a grid (80 cm side length) at one to six week postoperatively. The locomotion of hindlimb were assessed with BMS scores ranging from 0 (no ankle movement) to 9 (complete functional recovery) points. In addition, Mice were excluded from future evaluation if their BMS score were higher than 3 point 1day after injury. Body weight support and limb coordination ability were assessed by footprint analysis. Briefly, the fore and hind limbs of the mouse were pressed down onto blue and red ink, respectively. After that, animals were allowed to walk on white paper (1 m in length and 7 cm in width) without constraints. The distance between the left and right hindpaws was determined as the base of support. Stride length which assessed limb coordination ability was characterized as the distance between the center pads of the forelimb and hind limb perpendicular. The angle of rotation (AR) was defined as the angle formed by two lines connecting the third toe and the stride line at the center of hindpaw. The outcomes was determined by average of five sequential steps [33, 34]. 2.3.4 Histological Analysis Mice were euthanized by deeply anesthetized using 10g/0.1ml 0.6% sodium pentobarbital (merck) at six weeks post-SCI. Then they were perfused with 20ml 0.9% NaCl through the Left ventricle and followed by 50ml 4% paraformaldehyde. A dissection of spinal cord containing the lesion site was performed, and the tissues were fixed in 4% paraformaldehyde 24 hours prior to embedding in paraffin. Ten thickness longitudinal slice were performed using a Leica RM2245 Electric slicer. Hematoxylin and eosin (H&E) staining was performed to general review the cellular and extracellular matrix features. Adjacent tissue sections were stained with for observation of, Masson's trichrome was used to measure the collagenous tissue deposition within the injury site. 2.3.5 Immunohistochemistry (IHC) For IHC, paraffin-embedded spinal cord tissues were dewaxed and rehydrated, and then, antigens were retrieved by incubating slides in 2% ethylene diamine tetraacetic acid (EDTA) for 5 min at 95°C and for 1 hour at room temperature. After incubation with 6% hydrogen peroxide for 10 min, slides were blocked for 1 h at room



temperature in goat serum. Then the slides were incubated with primary antibody overnight at 4°C. Secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG polymer (ZSGB-BIO, China) was then added for 60 min at 37°C. The slides were developed with 3,3’-diaminobenzidine (DAB) for 3 min at room temperature and counterstained with Hematoxylin for 1 min. Finally, the samples were dehydrated by ethanol and mounted with coverslips. 2.3.6 Western Blot (WB) Analysis Forty-five days after surgery, the injured spinal cord of each mouse was extracted using liquid nitrogen. The spinal cord tissues were homogenized in RIPA lysis buffer (CWBIO, China) and extraction buffer including 20 mM Tris-HCl (pH 7.4) and lysed on ice for 1 hour. After the sample centrifuged at 12,000 g for 30 min, the tissue lysates were obtained from the supernatant. Then a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA) was used to quantitatively measure. SDS-PAGE Protein Loading Buffer (5X) was added to the samples, and the samples were heated at 100°C for 10 min. Equal amounts (20 µg) of protein suspension were loaded on 6% (for NF protein) or 8% (for Tuj-1, GFAP and CD68 protein) polyacrylamide gels. The gel electrophoresis was used to separate proteins and the samples were then transferred onto polyvinylidenefuloride (PVDF) membranes; 5% skimmed milk was added to the PVDF membranes to block for one hour. Next, the membranes were incubated with the primary antibodies listed in Table 2 at 4°C overnight. Other day, horseradish peroxidase-conjugated secondary antibody (ZSGB Bio, Beijing, China) was performed to incubate for 2 h. Immunoblots were visualized by using an enhanced chemiluminescence (ECL) kit and chemiluminescence were detected by Mini Chemiluminescence Imager. The density of each protein band was calculated by using ImageJ software. 2.4 Statistical Analyses The statistical software SPSS13.0 were used to perform all statistical analyses. All data are presented as means ± standard deviations (SD). Multiple group comparisons were tested via one-way analysis of variance (ANOVA) with an LSD-t (equal variance assumed) or Bonferroni was performed. A statistically significant difference was admitted at p< 0.05. 3 Results 3.1 Characterization of Hydrogels As the proton NMR of gelatin showed, the peak was appeared at 7.4 ppm confirmed the existence of the aromatic amino acid and the peaks at 5.5 ppm and 5.7 ppm verified the double bonds of the methacrylate groups (Figure S1.). The soft, medium and stiff hydrogels all possessed a transparent porous structure that


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provided adequate space for cell adherence and proliferation (Fig. 2A1-A3). The average pore sizes of the soft, medium and stiff hydrogels were 183.5 µm, 141.2 µm and 86.7 µm, respectively (Fig. 2B). The pore size of the hydrogels significantly decreased as the modulus of the hydrogels increased. All three GelMA hydrogels displayed a typical linear elastic behavior region and a non-linear region at low and higher stress, respectively. As shown in Fig. 2C, as the time of UV radiation increased, the stiffness of stress-strain curves increased. Young’s modulus was lowest for the soft hydrogel at 0.68 ± 0.02 kPa and incrementally increased to 1.23 ± 0.11 kPa and 2.03 ± 0.09 kPa for medium and stiff hydrogels, respectively (Fig. 2D). The modulus of soft hydrogel was significantly lower than the moduli of medium and stiff hydrogels (p

Directing Induced Pluripotent Stem Cell Derived Neural Stem Cell

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