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Prolonged Three-Dimensional Co-Delivery of Yamanaka Factors for Cell Reprogramming Wenwen Deng, Xia Cao, Qiang Wang, Yan Wang, Jingjing Chen, Qingtong Yu, Zhijian Zhang, Jie Zhou, Wenqian Xu, Pan Du, Jiaxin Chen, Xiangdong Gao, Jiangnan Yu, and Ximing Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05825 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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ACS Applied Materials & Interfaces

Prolonged Three-Dimensional Co-Delivery of Yamanaka Factors for Cell Reprogramming ,

,

Wenwen Deng, ‡, § Xia Cao, ‡ §Qiang Wang, ‡ §Yan Wang, §Jingjing Chen, §Qingtong Yu,§ Zhijian Zhang, † Jie Zhou, §Wenqian Xu, §Pan Du, §Jiaxin Chen, § Xiangdong Gao, ⊥Jiangnan Yu,§ and Ximing Xu*,§ ‡

These authors contributed equally to this work.

* Corresponding-Author: Prof. Ximing Xu, Tel/Fax: +86-511-85038451, Email: [email protected] §

Department of Pharmaceutics, School of Pharmacy, and Center for Drug/Gene Delivery and

Tissue Engineering, Jiangsu University, Zhenjiang 212001, P.R. China

†

Center for Drug/Gene Delivery and Tissue Engineering, and School of Medicine, Jiangsu

University, Zhenjiang 212001, P.R. China

⊥

School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009,

P.R. China

KEYWORDS: calcium phosphate, hybrid nanoparticles, induced pluripotent stem cells, polysaccharide, three-dimensional scaffolds

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ABSTRACT: Reprogramming somatic cells into a pluripotent state in two-dimensional (2D) systems has been widely investigated but not described in the more biologically faithful threedimensional (3D) scaffolds. Here, we devise a 3D porous tissue engineering scaffold that could achieve successful and efficient induction of pluripotency. To construct this 3D scaffold, non-viral hybrid nanoparticles were fabricated beforehand by employing calcium phosphate and cationized Pleurotus eryngii polysaccharide to co-deliver plasmids OCT4, SOX2, KLF4 and C-MYC (pOSKM). These hybrid nanoparticles were then loaded into a 3D porous collagen scaffold to obtain the so-called pOSKM-activated 3D scaffold. This 3D scaffold could reprogram human umbilical cord mesenchymal stem cells (HUMSCs) into a pluripotent state, generating 3D cell spheres which showed positive expression of pluripotency markers in the 3D scaffolds and tightly packed colonies when transferred to 2D feeder layers. Besides sharing similar morphology, epigenetic modification, and expression of pluripotency genes with the embryonic stem cells, the 3D system-generated colonies could also be expanded on feeder layers for more than 20 passages, indicating the successful establishment of stable iPS cell lines. Our findings represent a first employment of porous 3D scaffolds to achieve successful reprogramming via a one-time transfection, offering a safe, simple and effective alternative strategy for iPSC generation. INTRODUCTION In 2006, induced pluripotent stem cells (iPSCs) was first generated via forced overexpression of a set of transcription factors (OCT4, SOX2, KLF4 and C-MYC, OSKM) by retroviral transduction 1, marking a major breakthrough in biological research. Since then, great efforts have been devoted to developing safer alternatives to substitute the viral vectorsmediated exogenous gene delivery and designing more efficient methods to improve reprogramming efficiency, such as the utilization of proteins2, episomal vectors3, microRNAs4-5, small molecules 6and non-viral plasmids7-8.


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A previous study argued that three-dimensional (3D) systems could promote the generation of a stem cell-like niche that mimic the natural 3D tissue organization more closely than the 2D conditions 9. Recently, several research groups have investigated the beneficial effects of 3D culture conditions on promoting the self-renewal of stem cells by manipulating the 3D cell microenvironment10-11. They claimed that 3D microenvironment was favorable for iPSC colony formation while the proliferation of non-iPSCs was limited. To the best of our knowledge, all of the existing 3D systems for pluripotency promotion and maintenance are polymer hydrogel systems which are translucent, soft, wet and floppy because of the high water content 12-13, and there have been no reports exploring the induction of pluripotency in 3D porous tissue engineering scaffolds which are usually prepared by solidification treatment to acquire the porous inner structure and certain degree of mechanical strength14-15. Therefore, it is imperative to investigate whether a 3D porous scaffold could be employed for somatic cell reprogramming. To this end, we herein designed a 3D collagen scaffold in which geneladen nanoparticles were embedded. The gene-loaded nanoparticles (termed pOSKM-CPCPEPS hybrid nanoparticles) were prepared using calcium phosphate (CP) and cationized Pleurotus eryngii polysaccharide (CPEPS) to encapsulate the four plasmids (OCT4, SOX2, KLF4, C-MYC) (pOSKM). In recent years, positively charged polysaccharides have shown enormous potential in gene and drug delivery. Besides the universally investigated chitosan which naturally carries positive charges 16-17, many other natural polysaccharides have also been developed as effective gene vectors after appropriate cationic modification 18-20. In the present study, the CPEPS was produced by cationic modification of a naturally occurring polysaccharide isolated from the edible mushroom Pleurotus eryngii to acquire a positive charge which is favorable for cellular uptake 21. Previous studies have proved that CPEPS is a promising candidate for gene delivery because of its excellent capability to condense and carry DNA, good biocompatibility and biodegradability, low immunogenicity and non-toxicity 22-23. 3 ACS Paragon Plus Environment


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Another important element of this hybrid nanoparticle is CP, a commonly used biomaterial for in vitro drug/gene delivery24-25. Its excellent biocompatibility and simple applications have motivated researchers to always consider CP as the gene vector. However, its poor colloidal stability hinders extensive applications26-27. Here, for the first time, CPEPS was adopted to hybridize with CP to encapsulate pOSKM via a reverse microemulsion method, resulting in colloidally stable pOSKM-CP-CPEPS hybrid nanoparticles which possessed the joint advantages of both CP and CPEPS. Next, the hybrid nanoparticles were embedded in the 3D collagen scaffold to form the so-called pOSKM-activated 3D scaffold for virus-free iPSCs generation. The human umbilical cord mesenchymal stem cells (HUMSCs) were used as the original cells for iPSC generation because they could be easily harvested from clinical wastes after child delivery 28-29. Compared with other cell sources, collecting HUMSCs is a process of waste recycling. As a type of multipotent stromal cells, HUMSCs enjoy the capability of differentiation into a large number cell types, including fat and cartilage cells30-31, but they lack the pluripotency of differentiation into all three germ layers as ESCs and iPSCs do. Considering its easy accessibility and self-renewable property, HUMSCs would be desirable parental cells for iPSCs generation. Taken together, this pOSKM-activated 3D scaffold was expected to provide an extracellular environment mimicking in vivo conditions to achieve the induction of pluripotency. MATERIALS AND METHODS Materials. Murine type I collagen was obtained from TianXinFu Medical Appliance Co., Ltd. (Beijing, China). Poly(oxyethylene)-nonylphenyl ether (Igepal CO-520) and cyclohexane were purchased from Sigma-Aldrich (St Louis, MO, USA); disodium hydrogen phosphate, glacial acetic acid and absolute ethanol were obtained from Chemical Reagent Co., Ltd. of China National Pharmaceutical Group (Shanghai, China) and used without further purification. Silica spheres (50 µm in size, 60 Å in pore size) were purchased from Sepax Technologies 4 ACS Paragon Plus Environment

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(Newark, DE, USA). Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F12, knockout DMEM, fetal bovine serum (FBS), knockout serum replacement (KSR), bovine serum albumin, L-glutamine, penicillin, streptomycin and trypsin were obtained from Gibco BRL (Invitrogen Co., Carlsbad, CA, USA). Mitomycin C, valproic acid, collagenase IV, βmercaptoethanol, nonessential amino acids and basic fibroblast growth factor (bFGF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The four individual plasmids, each encoding one of four transcription factors (OCT4, SOX2, KLF4 and C-MYC), were purchased from GeneCopoeia, Inc. (Rockville, MD, USA) and amplified in the Escherichia coli (E. coli) host strain DH5α (Supplementary Information). Cationized Pleurotus eryngii polysaccharide (CPEPS) was prepared according to the methods described in previous studies 21. Primary HUMSCs (passage 0) were a kind gift from the Beike Jiangsu Stem Cell Bank (Taizhou, China). The use of experimental animals was adhered to the principles in the Declaration of Helsinki. The animal experimental protocol was approved by the University Ethics Committee for the Use of Experimental Animals and conformed to the Guidelines for the Care and Use of Laboratory Animals. Formation and characterization of the pOSKM-CP-CPEPS hybrid nanoparticles. The pOSKM-CP-CPEPS hybrid nanoparticles were produced by a reverse microemulsion method used in previous studies32-33, with slight modifications. Detailed procedures were presented in Supplementary Information. The resultant pOSKM-CP-CPEPS hybrid nanoparticles were observed by TEM, and the plasmid retention of the pOSKM-CP-CPEPS hybrid nanoparticles was assessed by gel electrophoresis in 1% agarose. Additionally, the size and zeta potential of the pOSKM-CPCPEPS hybrid nanoparticles were measured with a ZEN3600 Nano Series Zetasizer (Malvern Instruments, Ltd., UK). The measured scattering intensities were then analyzed using the software provided by Malvern Instruments. 5 ACS Paragon Plus Environment


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Furthermore, the amount of the encapsulated plasmids per milliliter of the pOSKM-CPCPEPS hybrid nanoparticle solution was determined. In brief, 1 mL of the prepared pOSKMCP-CPEPS hybrid nanoparticle solution was centrifuged at 50,000 rpm for 15 min at 4 °C. The resulting pellet was dissolved in 0.5 mL of sodium acetate solution (pH=3.0) to allow the release of plasmids as previously reported33. The calcium phosphate could dissolve in the mildly acidic sodium acetate solution, which led to the exposure of encapsulated pDNA. Then the total plasmid DNA was quantified by a PicoGreen dsDNA quantification kit (Invitrogen Co., Carlsbad, CA, USA) according to the instructions provided by the manufacturer. Moreover, the resultant solution of the released plasmids was subsequently subjected to the agarose gel electrophoresis as previously described 8. The cytotoxicity of the hybrid nanoparticles was also examined in the 2D culture system with MTT method as reported 5. Fabrication of the pOSKM-activated 3D scaffolds. A medical-grade type I collagen solution (2%, w/v) was prepared in a 1% (v/v) acetic acid solution at 37 °C. The collagen solution was transferred to a cuboid-shaped polytetrafluoroethylene mold (internal volume 68×38×5 mm3), which was incubated at 4 °C for 0.5 h and then rapidly transferred to -80 °C overnight before lyophilization. The lyophilized scaffold was incubated in an oven at 120 °C for 12 h to both reinforce the scaffold and evaporate the remaining acetic acid. After cooling, the scaffold was soaked in absolute ethanol for 24 h, followed by washing with 90%, 70%, 50% and 30% ethanol and sterilized double-distilled water in sequence until no ethanol odor could be detected. The wet scaffold was lyophilized again, after which the scaffold was trimmed using a cryostat microtome (Slee, Mainz, Germany) into cuboidal scaffolds with an apparent size of 5×5×3 mm3, each of which was placed in one well of a 24-well plate. After 4 h of sterilization by ultraviolet light, these scaffolds were immersed in serum-free DMEM for 0.5 h. Extra medium was removed before the hybrid nanoparticle suspension was loaded onto the scaffolds. In particular, a 100-µL aliquot of the hybrid nanoparticle suspension (containing 0.8 µg of plasmid according to the results of characterization of the pOSKM-CP-CPEPS 6 ACS Paragon Plus Environment

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hybrid nanoparticle) was added to each cuboidal scaffold. Finally, the pOSKM-activated 3D scaffolds were lyophilized under aseptic conditions and stored in a horizontal laminar-flow clean bench (Suzhou Purification Equipment Co., Ltd., Soochow, China) until use. Morphology and physical properties of the scaffolds. The morphologies of the pores in the scaffolds before and after nanoparticle loading were observed under a scanning electron microscope (SEM) (JEOL JSM5410, Japan). Furthermore, the ultrastructural features of the nanoparticle-laden scaffolds were observed by using the field emission scanning electron microscopy (FESEM) (15 kV, Hitachi, S4800, Tokyo, Japan) in combination with the energydispersive X-ray spectroscopy (EDS) (Horiba, Japan) with an accelerating voltage of 20.0 kV. The pore size was estimated based on a minimum of 30 pores from different areas along a cross-section of the scaffolds. In total, three scaffolds in each group and three different crosssections of each scaffold were used to estimate the pore size. Batches of trimmed collagen scaffolds with and without nanoparticle loading were immersed in absolute ethanol and treated with ultrasound for 5 min. The porosity was calculated using a previously reported formula34, i.e., p (%)=(Ww-Wd)/(Ww-W0)×100%, where Ww, Wd and W0 are the wet weight of the scaffold after immersion in absolute ethanol, the dry weight of the scaffold, and the weight of the scaffold in absolute ethanol, respectively. More specifically, W0 is equal to the dry weight of the scaffold with the subtraction of buoyancy. Ww–W0 is equal to the apparent volume of the scaffold, namely the total volume of the pores and the solid matrix. Additionally, another batch of scaffolds was soaked in doubledistilled water and vibrated by ultrasound for 5 min. The swelling percentage was defined as S (%) = (Ws-Wd)/WD×100%, where Ws is the wet weight of scaffold soaked in double-distilled water. In vitro release of plasmids from the pOSKM-activated 3D scaffolds. In addition, the in vitro release of plasmids from the pOSKM-activated 3D scaffolds was examined. Briefly, three dry scaffolds of equal mass were each placed in 5 mL of PBS (pH 7.4). The scaffolds 7 ACS Paragon Plus Environment


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were then placed in an orbital shaker bath at 37 °C and 120 rpm. Samples of 1 mL were removed from the release media at different time points (days 1, 3, 5, 7, 9, 12 and 15) and stored at -20 °C before plasmid DNA quantification. After each sample was collected, 1 mL of fresh PBS was added to complement the release medium. The samples were centrifuged at 50,000 rpm for 15 min at 4 °C. The resulting pellet was used to determine the DNA concentrations as described in section “Formation and characterization of the pOSKM-CPCPEPS hybrid nanoparticles”. Adhesion of HUMSCs to the scaffolds. HUMSCs, identified with flow cytometry (Figure S1), were used as the original cells for iPSC generation. The pOSKM-activated 3D scaffolds were first wetted with 200 µL of serum-free DMEM before cell seeding. A 200-µL suspension of HUMSCs was then infiltrated into the scaffolds at a density of 1× 105 cells per construct, followed by incubation in a humidified CO2 incubator for 0.5 h. Next, the cellladen scaffolds were maintained at 37 °C for 24 h in low-glucose DMEM containing 10% FBS and 100 U/mL penicillin-streptomycin. The medium was collected to determine the number of free cells using a hemocytometer. As a control, HUMSCs were seeded into the nanoparticle-free collagen scaffolds according to the same procedure. The adhesion efficiency was defined as α (%) = [(5× 104 ‒ quantity of released cells) / (5× 104)] × 100%. Preparation of the feeder cells. Mouse embryonic fibroblasts (MEFs), used as feeder cells, were purchased from the Cell Bank at the Chinese Academy of Science (Shanghai, China) and cultured in fibroblast medium (DMEM/F12 containing 10% FBS and 100 U/mL penicillin-streptomycin). The feeder cells were prepared according to the previously reported procedure 8. IPSC generation in the pOSKM-activated 3D scaffolds. HUMSCs were seeded onto the pOSKM-activated 3D scaffolds as described in section “Adhesion of HUMSCs to the scaffolds”. The scaffolds were then incubated at 37 °C in 5% CO2 in low-glucose DMEM containing 10% FBS and 100 U/mL penicillin-streptomycin. Two days later, the medium was 8 ACS Paragon Plus Environment

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changed to hESC medium, comprising knockout DMEM supplemented with 20% KSR, 2 mmol/L L-glutamine, 0.1 mmol/L β-mercaptoethanol, 1% nonessential amino acids, 4 ng/mL bFGF and 100 U/mL penicillin-streptomycin. The medium was replaced with fresh, prewarmed hESC medium every 2 days. The cells in the translucent scaffolds were observed daily by phase-contrast microscopy. Quantification of transfection efficiency. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was used to quantify transfection efficiency both in pOSKMactivated 3D scaffolds. Briefly, the HUMSCs were seeded onto the pOSKM-activated 3D scaffolds as described in section “Adhesion of HUMSCs to the scaffolds”. As control, the HUMSCs were seeded on 24-well plates at a density of 1×105 cells per well and treated with multiple transfections with the pOSKM-CP-CPEPS hybrid nanoparticles (800 ng of the plasmid mixture per well) according to the procedures in our previous work 8. After incubation for respective durations (2, 4, 6 and 8 days), cells were collected from 3D scaffolds and 2D plates for the subsequent experiments. For the QRT-PCR assay, the total RNA was extracted from the collected cells using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) following the instructions provided by the manufacturer. The RNA concentration and purity were measured using a spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA). Then, RT reaction was performed with 1.0 µg of total RNA using a RevertAid™ First Strand Synthesis Kit (Fermentas, K1622, Shenzhen, China) and random hexamer primers. The resulting complementary DNA (cDNA) was used for the subsequent PCR amplification reaction utilizing SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) according to the protocols provided by the manufacturer with the LightCycler system (Roche Molecular Biochemicals, Indianapolis, Ind.). The specific primers were listed in supplementary Table S1. The PCR conditions were as follows: 95 °C for 10 min (for initial denaturation) followed by 30 cycles of denaturation at 95 °C for 30 s, annealing for 30 s at 57 °C, and extension at 72 °C for 30 s. 9 ACS Paragon Plus Environment


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GAPDH was used as an internal standard. A no-template blank as well as a reversetranscription-negative blank were served as negative controls. The 2^(-∆∆Ct) method was used for data processing 35. The gene expression was normalized by GAPDH. All measurements were conducted in triplicates. Histological analysis. After cell seeding, several of the scaffolds were fixed in 4% paraformaldehyde at day 4, 8 and 12 for histological analysis. These scaffolds were embedded in paraffin and sliced into sections (thickness of 10 µm). Several of the resulting slides were stained using hematoxylin/eosin (HE) to reveal the morphologies of the colonies grown in the 3D scaffolds. Some of the scaffolds fixed at day 12 were subjected to immunohistochemistry to examine the expression of pluripotent markers according to an established protocol36. The primary antibodies (Abcam, Cambridge, MA, USA) used were anti-NANOG (1:100), antiOCT4 (1:100), anti-SSEA3 (1:100) and anti-TRA-1-81 (1:100). Secondary antibody (biotinconjugated goat anti-rabbit IgG) and SABC (Wuhan Boster, China) were added to the slides and incubated at 37 °C for 30 min. Finally, several slides were stained with hematoxylin and visualized with DAB (Sangon Biotech, Shanghai, China). Others were processed for alkaline phosphatase (AP) staining using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Blue) (C3206; Beyotime Institute of Biotechnology, Haimen, China) according to the instructions provided by the manufacturer. Images were then acquired using microscopy (Olympus, Tokyo, USA). Confocal laser microscopy. To better understand the process of 3D cell sphere formation, we employed confocal laser microscopy to observe the cell behavior within the scaffolds. In particular, HUMSCs were tagged with a red fluorescent dye (MINI26-1KT, PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling) from Sigma-Aldrich according to a protocol provided by the manufacturer. The detailed procedure for cell membrane labeling was in Supplementary Information. This red fluorescent dye is often used for in vivo cell tracking, labeling the cell membrane without affecting cell viability 10 ACS Paragon Plus Environment

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and functionality. The labeled cells were seeded onto the pOSKM-activated 3D scaffolds and incubated at 37 °C in 5% CO2 in low-glucose DMEM containing 10% FBS and 100 U/mL penicillin-streptomycin. Fluorescent images of the scaffolds were collected at different time points (days 4, 8 and 12) with a confocal laser scanning microscope (TCS-NT, Leica, Heidelberg, Germany). An excitation wavelength of 551 nm and an emission wavelength of 567 nm were applied to the red staining. Following the same procedure, the HUMSCs were seeded onto the nanoparticle-free 3D collagen scaffolds, acting as a negative control. Teratoma formation. All experimental procedures were performed in accordance with the standard human care guidelines of the Guide for Care and Use of Laboratory Animals. For the teratoma assay, 15 days after the seeding of HUMSCs into the pOSKM-activated 3D scaffolds, the constructs were washed twice with PBS and implanted into the flank of 4-weekold immunocompromised non-obese diabetic/severe combined immunodeficient (NODSCID) mice (Comparative Medicine Center, Yangzhou University, Yangzhou, China). Eight weeks later, the teratomas were collected and processed for Hematoxylin/Eosin (HE) staining. Reprogrammed cells release from the scaffolds. Twelve days after cell seeding, the cell-laden pOSKM-activated 3D scaffolds were enzymatically digested with type IV collagenase for 30 min at 37 °C, and then neutralized with 0.5 mL of serum-containing DMEM medium. After allowing the cell pellets that were released from the scaffolds to adhere for 8 h in a humidified CO2 incubator, the medium was replaced with fresh, prewarmed hESC medium. The cell pellets were maintained and expanded on the MEF feeder layers. Images of the colonies were acquired using a phase-contrast biological microscope. Immunofluorescence staining. Immunofluorescence staining was further conducted to examine the expression of pluripotent markers in the iPSCs after transferring onto the 2D feeder layers. Human embryonic stem cell line HN4 (obtained from Shanghai Institutes for Biological Sciences, China) was preceded as positive control. Cells were first fixed in 4% paraformaldehyde in PBS for 20 min, followed by two washes with PBS. The cells were then 11 ACS Paragon Plus Environment


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treated with 0.1% Triton X-100 for 10 min. After another washing with PBS, the cells were incubated in 4% bovine serum albumin for 1 h to block any nonspecific binding. The cells were then incubated with the primary antibodies at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. Additionally, the nuclei were counterstained with DAPI (1:2000; Sigma, St. Louis, MO, USA). The primary antibodies included anti-NANOG (1:250), anti-OCT4 (1:250), anti-SSEA3 (1:250) and anti-TRA-1-81 (1:250), which were obtained from Abcam (Cambridge, MA, USA). The secondary antibody was goat anti-mouse IgG-Cy3, purchased from Sigma (St. Louis, MO, USA), with a dilution ratio of 1:500. Fluorescence was detected using a Leica epifluorescence light microscope (Leica Microsystems, Wetzlar, Germany). Bisulfite genomic sequencing. Genomic DNA from various cell lines (iPSCs from the 3D system, HUMSCs, and HN4) was isolated using DNA extraction kit (AP-MN-MSGDNA-250, Axygen). After purification, the DNA samples (1 µg for each sample) were processed for bisulfite treatment using CpGenome Universal DNA Modification Kit (Chemicon, Temecula, CA) according to the instructions provided by the manufacturer. The promoter regions of OCT4 and NANOG were amplified by PCR suing primers listed in Supplementary Information Table S2. The resulting PCR products were cloned into pCRIITOPO vector using TOPO TA cloning kit (Invitrogen) and ten randomly selected clones were sequenced. Statistical analysis. The data were analyzed by a one-way analysis of variance (ANOVA) to determine the significance of the difference between the selected groups using the SPSS (version 14.0) statistical software (SPSS Company, USA). All data were reported as mean ± standard deviation (SD). A P < 0.05 was considered statistically significant. RESULTS AND DISCUSSION Characterization of the pOSKM-CP-CPEPS hybrid nanoparticles. The morphology of the pOSKM-CP-CPEPS hybrid nanoparticles was observed by TEM. As shown in Figure 12 ACS Paragon Plus Environment

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1a, the hybrid nanoparticles exhibited a homogenous spherical shape and uniform dispersion, with sizes ranging from 30 to 100 nm. It is well recognized that particle size and shape are very important factors that may influence cellular uptake through the pathways and efficiency of internalization37. A spherical shape has also been reported to be optimal compared with rod- and needle-like shapes 33. Additionally, nanoparticles with smaller sizes (100 nm) 38. Notably, dynamic light scattering (DLS) analysis demonstrated that the size of the pOSKM-CP-CPEPS hybrid nanoparticles was 97 nm on average (Figure 1b), which was larger than that observed with TEM. This discrepancy in particle size, as previously stated38, is because the DLS method gives the hydrodynamic diameter rather than the actual diameter of nanoparticles as the TEM does. Altogether, these findings demonstrated that the hybrid nanoparticles were able to condense plasmids into ultra-small sized particles, which is beneficial to cellular uptake. To assess the plasmid retardation effect of the pOSKM-CP-CPEPS hybrid nanoparticles, three different batches of hybrid nanoparticles were examined by agarose gel electrophoresis. The electrophoretic pattern demonstrated that all of the plasmids were completely retained in the wells (Figure 1c). This result demonstrated that the pOSKM-CP-CPEPS hybrid nanoparticles can condense plasmids and exhibit excellent plasmid retention, indicating the strong and stable binding between the plasmids and the CP and CPEPS. The surface charge of the nanoparticles is another important factor for cellular internalization. The results of the zeta potential analysis were illustrated in Figure 1d. It can be seen that the four free plasmids showed negative charges; however, when encapsulated into the hybrid nanoparticles, a significant charge reversal, from negative to positive, occurred. The resulting pOSKM-CP-CPEPS hybrid nanoparticles thus displayed a positive charge, which is favorable for cellular uptake due to the electrostatic interaction between the negatively-charged cell membrane and the positively-charged nanoparticles 39. 13 ACS Paragon Plus Environment


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Moreover, there was 0.8 µg of the total plasmids per 100-µL of the hybrid nanoparticle suspension, and the ratio of the four plasmids encapsulated in the nanoparticles was approximately 1:1:1:1 (Figure S2). The result of MTT assay showed that different batches of pOSKM-CP-CPEPS hybrid nanoparticles had negligible toxicity to cells (Figure S3), demonstrating an excellent biocompatibility of the hybrid nanoparticles. Characterization of the pOSKM-activated 3D scaffolds. One of the major efforts of the present study is to develop a non-viral gene-laden 3D system for efficient reprogramming of somatic cells into iPSCs. It would be preferable for the scaffolds to release the reprogrammed cells after the accomplishment of reprogramming. Therefore, the scaffolds should be able to naturally degrade (or degrade with mild enzyme treatment) within 2 ~ 3 weeks, without demanding for high mechanical strength. Based on this notion, herein 3D collagen scaffolds were prepared without any crosslinking agent. This would endow the scaffolds with better safety and biodegradability. The lyophilized 3D scaffolds were light yellow, soft, spongy and elastic (Figure 2a). Scanning electron microscopy (SEM) images showed that the original collagen scaffolds and pOSKM-activated 3D scaffolds both possessed a highly porous structure (Figures 2b and c). Moreover, the field emission scanning electron microscopy (FESEM) showed the magnified images of the loaded nanoparticles within the 3D scaffolds (Figures 2d and e), and the result of energy-dispersive X-ray spectroscopy (EDS) demonstrated the existence of carbon (C), nitrogen (N), oxygen (O), calcium (Ca), and phosphorus (P) (Figure 2f), confirming the attachment of the pOSKMCP-CPEPS hybrid nanoparticles on the inner surface of the scaffolds. Additionally, there was no significant change in the pore size before and after nanoparticle loading. The irregularly shaped pores exhibited pore sizes ranging from 150 to 500 µm. The highly porous structure and large pore size are suitable for cell attachment and proliferation, and the interconnected pores provide important channels for air exchange and nutrition supply.


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Moreover, the porosities of the scaffolds before and after nanoparticle loading were (87.9 ± 0.5) % and (85.1 ± 0.7) %, respectively. As we can see, there was a slight decrease in the porosity when the scaffolds were treated with the hybrid nanoparticles. A previous study reported that scaffolds are favorable for culturing cells when they exhibit a porosity of at least 85 % 40. Based on this criterion, our scaffolds are appropriate for cell culture and tissue engineering. In addition, the swelling percentages of the collagen scaffolds and pOSKMactivated 3D scaffolds were (3160± 90) % and (2970± 70) %, respectively. Overall, the physical properties of the scaffolds are suitable for cell culture. Cell adhesion. Twenty four hours after cell seeding, the percentage of cells that adhered to the scaffolds was calculated. The adhesion efficiency of the HUMSCs in the pOSKMactivated 3D scaffolds was approximately 95 %, which was higher than that of the nanoparticle-free scaffolds (approximately 91 %). The slightly increased percentage of attached cells in the pOSKM-activated 3D scaffolds was most likely due to the fact that the positively charged pOSKM-CP-CPEPS hybrid nanoparticles endowed the pOSKM-activated 3D scaffolds with a positively-charged inner surface. This result verified the previous conclusion that cells prefer to adhere to positively-charged surfaces 41. The sustained release of plasmids from 3D scaffolds and transfection efficiency. The pOSKM-activated 3D scaffolds were designed for sustained, long-lasting release of nanoparticles which further lead to a continuous, effective delivery of plasmids to HUMSCs. To address this issue, we first examined the release pattern of the total plasmids from the pOSKM-activated 3D scaffolds. As shown in Figure 3a, relatively rapid release of the plasmids was observed from the beginning to day 3, resulting in an accumulative percentage of 41 %. This relatively high initial release was able to initiate the reprogramming process. Over time, the release rate slightly decreased, and approximately 89 % of the total plasmids had been released by day 13. After that, the plasmid release reached a plateau. This result



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demonstrated the excellently sustained and relatively complete release of plasmids from the pOSKM-activated 3D scaffolds. Next, we seeded HUMSCs into the 3D scaffolds and determined the transfection efficiency. The results of PCR demonstrated robust and increasing expression of the four factors (OSKM) in the pOSKM-activated 3D scaffolds at day 2, 4 and 6, followed by continuing high level of each factor thereafter (Figure 3b). Moreover, the pOSKM-activated 3D scaffolds could generate significantly increased expression levels of exogenous OSKM in comparison with the 2D system employing pOSKM-CPNPs for gene delivery (*, p< 0.05, **, p< 0.01, Figure 3b). These data indicated that the pOSKM-activated 3D scaffolds possessed significantly higher transfection efficiency than the 2D system. It is most likely that the pOSKM-activated 3D scaffolds, acting as a reservoir for pOSKM-CP-CPEPS hybrid nanoparticles, enjoyed an efficient and lasting release of the hybrid nanoparticles, so that they could achieve continuous and effective cellular uptake of nanoparticles rather than the intermittent delivery due to the multiple transfections in the 2D system. To examine the correlation between the sustained release of plasmids from the 3D scaffolds and the transfection efficiency, a correlation curve was generated (Figure 3c). It is obvious that the transfection efficiency correlated well with the increased accumulation of released plasmids, reaching a plateau when the accumulative release percentage was over 50%. The excellent correlation between the plasmid release and transfection efficiency confirmed that pOSKM-activated 3D scaffolds were a rational design to achieve long-lasting, steady and high-level of exogenous gene expression. Formation of 3D cell spheres. The scaffolds were translucent when maintained in medium, thus cell aggregates were faintly visible. As shown in Figure 4a., single cells were uniformly distributed in the scaffolds on day 2; as time went on, small 3D spheres began to appear in the scaffolds (day 4 to 8, indicated by arrows).


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To further manifest the morphology of the spheres in the scaffolds, HE staining was performed with the cell-seeded pOSKM-activated 3D scaffolds at different time points (days 4, 8 and 12). As a result, 3D spheres (indicated by arrows) formed in the pOSKM-activated 3D scaffolds as early as on day 4, which was consistent with the results observed by phasecontrast microscopy (Figure 4b). Notably, the cell spheres in the pOSKM-activated 3D scaffolds exhibited compact spherical or ellipsoid morphologies, with clear-cut, round edges. Moreover, the cells in the colonies displayed a high nucleus/cytoplasm ratio, as the cell nuclei were stained blue by hematoxylin, and the cytoplasm was dyed light red by eosin. These are typical characteristics of human ESCs (hESCs), indicating that the cells growing in the pOSKM-activated 3D scaffolds were morphologically similar to hESCs. In contrast, the scaffold without plasmids (taken on day 12) displayed no formation of cell aggregates. To better understand the time course of colony formation, the red fluorescence-tagged cells in the pOSKM-activated 3D scaffolds were observed by confocal laser microscopy. The images obtained at different time points (days 2, 4, 6 and 8) were presented in Figure 4c (panels A-D), which demonstrated a clear process of colony formation. In contrast, the cells in the nanoparticle-free collagen scaffolds remained randomly scattered (Figure 4c, panels E-H). Compared to the previous reports which usually needed more than 7 days for the first appearance of iPSC colonies in 2D systems 7, the iPSC-like spherical colonies began to appear in the pOSKM-activated 3D scaffold 3 days earlier than in 2D systems, suggesting the accelerated reprogramming process in three dimensions. One of the possible reasons is that the pOSKM-activated 3D scaffolds could continuously release the pOSKM-CP-CPEPS hybrid nanoparticles, resulting in an efficient, steady and lasting delivery of pOSKM. Moreover, previous study claimed that 3D microenvironment could facilitate the transformation of the original cell morphology to spherical colonies; furthermore, the action of biophysical cues from the 3D microenvironment may cooperate with the exogenous



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transcription factors11. These superiorities of the pOSKM-activated 3D scaffolds greatly contributed to the accelerated cell reprogramming. Immunohistochemistry. Immunohistochemistry was conducted 12 days after cell seeding to examine the expression of pluripotent markers, including alkaline phosphate (AP), SSEA3, TRA-1-81, OCT4 and NANOG. As shown in Figure 5, the 3D cell spheres were stained in violet-blue, indicating the activation of AP (panel B). Meanwhile, those cell spheres examined for expression of SSEA3 and TRA-1-81 were positively stained in yellow on the cell membrane (panels C and D, respectively; the nuclei were stained in blue), demonstrating positive expression of these two markers. The groups stained for the examination of OCT4 and NANOG (panels E and F, respectively) showed positive in yellow inside nuclei, indicating the positive expression of the transcription factors OCT4 and NANOG, which are required for the maintenance of pluripotency. In contrast, the control group, in which HUMSCs were seeded onto nanoparticle-free scaffolds, showed no colony formation throughout the entire process (panel A). These data demonstrated that the 3D cell spheres generated in the pOSKM-activated 3D scaffolds possessed the pluripotency similar to that of the ESCs. Transferring to 2D culture conditions. The colonies were released from the scaffolds and expanded on feeder layers, which were then examined for pluripotent marker expression. An average of 306 cell spheres was released from each scaffold, making the reprogramming efficiency of 0.306 % (from the original 1×105 cells/scaffold) which was significantly higher than that of the previously reported 2D non-viral iPSC-generating system (0.128%)42. The bright field images showcased the tightly packed morphology of the colonies on the 2D feeder layers (Figure 6a). In addition, the colonies from the 3D system and the human ESCs shared similar expression of pluripotent markers, such as SSEA3, TRA-1-81, NANOG and OCT4 (Figure 6b). To establish stable iPS cell lines, these colonies were continuously cultured on MEF feeder for more than 20 passages. These colonies showed vigorous growth on the feeder 18 ACS Paragon Plus Environment

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layers (Figure 7a). Importantly, the continuously cultured colonies were positive for pluripotent markers (Figure 7b b) and several iPS cell lines have been established. These results further confirmed that the human iPSCs that were generated in the pOSKM-activated 3D scaffolds could maintain pluripotency when released into the 2D culture system. DNA methylation. To further compare the resulting iPSCs with human ESCs, epigenetic modifications were examined by analyzing the methylation states of CpG dinucleotides in the OCT4 and NANOG promoter regions. The results of the bisulfite genomic sequencing analyses showed that OCT4 and NANOG promoter regions were demethylated in comparison with the parental HUMSCs and were similar to those of human ESCs (Figure 8), which is consistent with the epigenetic remodeling during reprogramming as previously reported 43. Teratoma formation. Teratomas appeared and were performed for histological examination by HE staining. The result revealed that the teratomas could generate differentiated tissues of all three germ layers, including gland tissue (endoderm), bone and cartilage (mesoderm) and nervous tissue (ectoderm) (Figure 9). The result verified that the 3D cell spheres share similar pluripotency and multi-lineage differentiation potentials with the embryonic stem cells. CONCLUSION Here we present a pOSKM-CP-CPEPS hybrid nanoparticle-incorporating 3D collagen scaffold (pOSKM-activated 3D scaffold) for generating non-viral iPSCs. The hybrid nanoparticles possessed an ultra-small size (

Prolonged Three-Dimensional Co-Delivery of ... - ACS Publications

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