Pleurotus eryngii Polysaccharide Promotes Pluripotent

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Polysaccharide from Pleurotus eryngii promotes pluripotent reprogramming Wenwen Deng, Xia Cao, Yan Wang, Qingtong Yu, Zhijian Zhang, Rui Qu, Jingjing Chen, Genbao Shao, Xiangdong Gao, Ximing Xu, and Jiangnan Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05661 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Journal of Agricultural and Food Chemistry

Pleurotus eryngii polysaccharide promotes pluripotent

reprogramming via facilitating epigenetic modification Wenwen Deng, §, ‡ Xia Cao, §, ‡ Yan Wang, §, ‡ Qingtong Yu, Zhijian Zhang, † Rui Qu, ‡ ⊥

⊥

Jingjing Chen, ‡ Genbao Shao, † Xiangdong Gao, Ximing Xu,*, ‡ and Jiangnan Yu*, ‡ §

These authors contributed equally to this work.

[*]

Corresponding-Authors: Prof. Ximing Xu and Prof. Jiangnan Yu

Department of Pharmaceutics and Tissue Engineering, School of Pharmacy, Jiangsu University, Zhenjiang 212001, P.R. China. Tel/Fax: +86-511-85038451 Email: [email protected]; [email protected] ‡

Department of Pharmaceutics and Tissue Engineering, School of Pharmacy, Jiangsu

University, Zhenjiang 212001, P.R. China. †

Center for Drug/Gene Delivery and Tissue Engineering, and School of Medical Science and

Laboratory Medicine, Jiangsu University, Zhenjiang 212001, P.R. China. ⊥

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

P.R. China.

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ABSTRACT: Pleurotus eryngii is a medicinal/edible mushroom with great nutritional value

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and bioactivity. Its polysaccharide has recently been developed into an effective gene vector

3

via cationic modification. In the present study, cationized Pleurotus eryngii polysaccharide

4

(CPS), hybridized with calcium phosphate (CP), was used to co-deliver plasmids (Oct4, Sox2,

5

Klf4, c-Myc) for generating induced pluripotent stem cells (iPSCs). The results revealed that

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the hybrid nanoparticles could significantly enhance the process and efficiency of

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reprogramming (1.6-fold increase) compared with the CP nanoparticles. The hybrid CPS also

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facilitated epigenetic modification during the reprogramming. Moreover, these hybrid

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nanoparticles exhibited multiple pathways (both caveolae- and clathrin-mediated endocytosis)

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in their cellular internalization, which accounted for the improved iPSCs generation. These

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findings therefore present a novel application of Pleurotus eryngii polysaccharide in

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pluripotent reprogramming via active epigenetic modification.

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KEYWORDS: Pleurotus eryngii polysaccharide, calcium phosphate, hybrid nanoparticles,

14

non-viral, induced pluripotent stem cells

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INTRODUCTION

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Induced pluripotent stem cells (iPSCs) has drawn wide attention from the public,

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clinicians, and scientists since their discovery in 2006.1 The iPSCs represent a very

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promising cell source due to their close resemblance to embryonic stem cells (ESCs) in

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morphology, gene expression profile and the propensity to differentiate into three germ

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lineages (in vitro and in vivo). Thus, they provide an ideal substitute for ESCs while

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circumventing the ethical issues and complications emanating from immune rejection

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after transplantation.2, 3 Therefore, the iPSCs has become a promising candidate for

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regenerative medicine, disease modelling and treatment, as well as drug screening.4-7

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It is widely known that viral transduction often carries the risks of insertional

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mutagenesis and tumor formation,8-10 thus hampering the therapeutic applications of

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iPSCs. Therefore, non-viral strategies for delivering transcription factors have gained

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much attention although with lower reprogramming efficiency.

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Currently, naturally occurring polysaccharides have taken the centre stage of several

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studies due to their great potential in the field of non-viral gene delivery.11-14 These

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natural products enjoy abundant sources (especially in plants and fungi),15-17 allow

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various chemical modifications, and exhibit excellent biocompatibility and low

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immunogenicity. Mushrooms, especially the edible/medicinal types with high

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nutritional values, are sustainable source of high-quality natural polysaccharides.18, 19

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Pleurotus eryngii, one of such mushrooms, is also rich in dietary fibres, proteins and

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polysaccharides. This mushroom has been developed as a functional food due to its

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immunoregulatory effect, antioxidant, anti-fatigue, anti-viral and anti-tumour

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functions.20-23 The polysaccharides of Pleurotus eryngii, enriched in the matured

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fruiting bodies,24 has also been mostly investigated in the area of isolation, purification, 3 ACS Paragon Plus Environment


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characterization and bioactive effects .25 The Pleurotus eryngii polysaccharide has

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further been explored as an effective non-viral gene vector via appropriate cationic

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modification in previous reports.,17 which resulted in cationic Pleurotus eryngii

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polysaccharide (CPS) with significantly enhanced transfection efficiency than the

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positive transfection reagent (Lipofectamine2000).

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The outstanding gene delivery and biocompatibility of CPS could promote its

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potential application in highly appreciated field of iPSC technology; however, such

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investigations are yet to be reported. In this regard, the current study looks at the

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possibility of using CPS as an efficient gene vector for generating iPSCs. Previous

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pilot project used CPS to condense and incorporate four plasmids (each encoding one

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of the four transcription factors: Oct4, Sox2, Klf4 and c-Myc, also abbreviated as

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pOSKM) without any highly significant iPSCs generation. In an attempt to change the

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status quo, calcium phosphate (CP), a commonly used inorganic material, was adopted

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to hybridize the CPS. The CP can successfully form a complex with the organic CPS to

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yield hybrid nanoparticles for effective gene transfection because of their multiple

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advantages such as non-toxic nature, easy preparation, biocompatibility and

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biodegradability..26-28 Here, the present work aims at generating iPSCs via hybrid

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nanoparticles using CPS and CP to co-incorporate plasmid mixture (pOSKM), and

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preliminarily investigate the possible mechanisms involved in the reprogramming. .

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MATERIALS AND METHODS

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Materials. DEAE-52 cellulose resin was purchased from Whatman, UK. SephadexG-

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100 was provided by Shanghai RiChu Bioscience Co., Ltd, Shanghai, China.

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Poly(oxyethylene)-nonylphenyl ether (Igepal CO-520) and cyclohexane were purchased from

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Sigma-Aldrich (St Louis, MO, USA). Disodium hydrogen phosphate, glacial acetic acid and

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absolute ethanol were obtained from Chemical Reagent Co., Ltd. of China National 4 ACS Paragon Plus Environment

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Pharmaceutical Group (Shanghai, China), and used without any further purification. Silica

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spheres (50 µm in size, 60 Å in pore size) were purchased from Sepax Technologies (Newark,

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DE, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM),

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DMEM/F12, knockout DMEM, knockout serum replacement (KSR), bovine serum albumin,

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L-glutamine, penicillin, streptomycin and trypsin were obtained from Gibco BRL (Invitrogen

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Co., Carlsbad, CA, USA). Mitomycin C, valproic acid (VPA), collagenase IV, β-

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mercaptoethanol, non-essential amino acids and basic fibroblast growth factor (bFGF) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Human Oct4, Sox2, Klf4 and c-Myc

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ELISA kits were purchased from Yantai Science and Biotechnology Co., Ltd. (Shandong,

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China). Four non-viral plasmids containing the CMV promoter were purchased from

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GeneCopoeia, Inc. (Rockville, MD, USA), and routinely amplified as previously reported.29

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NH4Cl, filipin Ш, sodium orthovanadate (SOV), glucose, 5-(N, N-dimethyl)-amiloride

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(DMA), chlorpromazine hydrochloride (CPZ) and genistein were obtained from Sigma-

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Aldrich (St. Louis, MO, USA). YOYO-1 was purchased from Invitrogen (Carlsbad, CA,

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USA).

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The animal experiment was approved by Jiangsu University Ethics Committee for the

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Use of Experimental Animals and conformed to the Guidelines for the Care and Use of

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Laboratory Animals.

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Preparation and characterization of CPS. The crude polysaccharide was extracted

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from fresh fruiting bodies of Pleurotus eryngii (Zhenjiang edible mushroom growth base,

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Zhenjiang, China), followed by purification with anion-exchange chromatography and gel

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chromatography according to other previous report.17 Similar process was also employed to

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obtain cationized Pleurotus eryngii polysaccharide (CPS). After that, gel permeation

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chromatography (GPC) was used to determine molecular mass. Other investigations included

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the use of trinitrobenzene sulfonic acid method (Total nitrogen of CPS), Fourier transform

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infrared (FT-IR, structural information) and thin layer chromatography (Monosaccharide 5 ACS Paragon Plus Environment


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composition). Zeta potential analysis and gel retardation assay were further conducted to

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assess the positively charged nanoparticles and their potential to carry genes as previously

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described .17

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Synthesis of pOSKM -encapsulated CPS-CP hybridized nanoparticles (pOSKM-

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encapsulated CPS-CPNPs). The pOSKM-encapsulated CPS-CPNPs were prepared by

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reverse microemulsion method according to previous studies with some modifications.30, 31

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Briefly, Igepal CO-520 was dissolved in cyclohexane to form a mixture of Igepal CO-

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520/cyclohexane (29 %, v/v). The nanoparticles were prepared by mixing two pre-fabricated

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microemulsions (A and B). For microemulsion A, a calcium chloride solution (650 µL, 0.01

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M) and CPS (0.8 µg) were added to 25 mL Igepal CO-520/cyclohexane mixture, followed by

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continuous stirring for 2 min to form the microemulsion. Similarly, a disodium hydrogen

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phosphate (650 µL, 0.06 M) and a mixture of four plasmids (10 µg) encoding Oct4, Sox2,

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Klf4 and c-Myc (2.5 µg of each plasmid) were added to the Igepal CO-520/cyclohexane

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mixture (25 mL). After 2 min of continuous agitation, microemulsion B was obtained. The

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microemulsion B was then added dropwise to the microemulsion A with continuous magnetic

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stirring at 4 °C until the entire system was completely translucent. The prepared plasmid-

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encapsulated CPS-CPNPs-doped microemulsion was diluted with pH-adjusted absolute

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ethanol (pH 7.0), followed by isolation using van der Waals chromatography laundering as

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described previously30. The fraction obtained from the silica column was concentrated using a

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vacuum rotary evaporator at 37 °C to yield virtually organic solvent-free solution (1 mL). The

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product was dialyzed with saline at 4 °C for 3 days using a dialysis bag (cutoff molecular

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mass of 500 Da, Sigma, St. Louis, MO, USA) to remove NaCl. The pOSKM-encapsulated

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CPS-CPNPs solution was further filtered through 0.22 µm membrane pore size and stored at

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4 °C for subsequent use.


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Characterization of pOSKM-encapsulated CPS-CPNPs. Transmission electron

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microscopy (TEM) using a JEM-2100 instrument (JEOL, Tokyo, Japan) was used to observe

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the morphology of pOSKM-encapsulated CPS-CPNPs as previously reported.32

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The particle size of pOSKM-encapsulated CPS-CPNPs was determined using a dynamic

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light scattering technique, performed at 25 °C with a Brookhaven BI-90 plus instrument

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(Brookhaven Instrument Corporation, Holtsville, NY, USA). The scattering intensities were

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analyzed using software provided by Brookhaven (Holtsville, NY, USA).

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The zeta potential of pOSKM-encapsulated CPS-CPNPs was measured with a Malvern

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Instruments ZEN3600 Nano Series Zetasizer (Malvern Instruments, Ltd., Worcestershire,

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UK).

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The sample solution (20 ml) was lyophilized to obtain a dried powder (1.0 ± 0.1 mg).

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The product was mixed with KBr (approximately 1.0 g) to yield KBr pellets, which were

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dried under an infrared lamp. The infrared spectra were recorded on Nicolet FT-IR-170SX

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spectrophotometer (Nicolet Instruments Corporation, Madison, Wisconsin, USA).

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The plasmid DNA retardation effect of pOSKM-encapsulated CPS-CPNPs was analyzed using gel electrophoresis (1% agarose gel) as reported in other studies .32 Generation of induced pluripotent stem cells. Primary human umbilical cord

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mesenchymal stem cells (HUMSCs) were generously provided by Beike Jiangsu Stem Cell

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Bank (Taizhou, Jiangsu, China). The HUMSCs culture and preparation of feeder cells

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(mitomycin C-treated primary mouse embryonic fibroblasts) were conducted as previously

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reported. 29

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The cytotoxicity of pOSKM-encapsulated CPS-CPNPs was evaluated with 3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay 32 prior to

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transfection. The HUMSCs were seeded in a 6-well plate at a density of 2×105 cells per well

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and cultured in DMEM (containing 10% FBS and 100 U/mL penicillin-streptomycin) at

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37 °C in 5% CO2. Following an 80~90% confluence, the medium was replaced with a mixture 7 ACS Paragon Plus Environment


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of serum-free DMEM (1.8 mL) and pOSKM-encapsulated CPS-CPNP solution (200 µL),

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containing 4 µg of total plasmids per well. After 4 h, the medium was replaced with low-

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glucose DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin.

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Another three consecutive transfections (4 h each) were carried out using pOSKM-

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encapsulated CPS-CPNPs at days 2, 4 and 6 under the same conditions. For the respective

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days, valproic acid (VPA) was added to the medium at a final concentration of 0.5 mM. The

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hESC-like iPSC colonies appeared a day after the last transfection (day 7). The medium was

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then replaced with hESC medium, which consisted of knockout DMEM supplemented with

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20% knockout serum replacement (KSR), L-glutamine (2 mM), β-mercaptoethanol (0.1 mM),

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1% non-essential amino acids, basic fibroblast growth factor (bFGF) (4 ng/mL) and

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penicillin-streptomycin (100 U/mL). In establishing the iPSC lines, the colonies were

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mechanically picked and maintained on MEF feeder layers in hESC medium without VPA.

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Twenty three days later, alkaline phosphatase (AP) staining was performed to determine the

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efficiency of the reprogramming as previously reported.29

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Enzyme-linked immunoabsorbent assay (ELISA). Three groups of different samples

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were prepared for the ELISA test, namely free plasmid group, pOSKM-encapsulated calcium

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phosphate nanoparticles (CPNPs) group and a pOSKM-encapsulated CPS-CPNPs group.

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After the second, third and fourth transfections, the total proteins of the cells were extracted

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respectively using a RIPA Lysis Kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu,

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China) according to the manufacturer’s protocol. Following complete lysis, the reaction

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solution was centrifuged at 10,000 g for 5 min. The supernatant was analyzed using ELISA to

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determine the level of ectopic expression of the four factors Oct4, Sox2, Klf4 and c-Myc.

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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The

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qRT-PCR was used to further examine the transfection efficiency of pOSKM-encapsulated

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CPS-CPNPs. . The pOSKM-CPNPs and Lipofectamine2000 were employed as positive

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control standards . The total RNA was extracted using TRIzol reagent (Invitrogen Co., 8 ACS Paragon Plus Environment

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Carlsbad, CA, USA) as directed by the manufacturer. The quantitative PCR was also

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conducted using SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) according to the protocol

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provided by the manufacture with the LightCycler system (Roche Molecular Biochemicals,

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Indianapolis, IN, USA). GAPDH was used as an internal standard. Primer sequences were as

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follows: GAPDH forward, CGGAGTCAACGGATTTGGTCGTAT; GAPDH

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reverse,AGCCTTCTCCATGGTGGTGAAGAC; Sox2

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forward,GCCCTGCAGTACAACTCCAT; Sox2 reverse, GACTTGACCACCGAACCCAT;

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Oct4 forward, ATGTGGTCCGAGTGTGGTTC; Oct4 reverse,

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AAACCCTGGCACAAACTCCA; c-Myc forward, CGTCCTCGGATTCTCTGCTC; c-Myc

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reverse, GCTGGTGCATTTTCGGTTGT; Klf4 forward, GGAAGTCGCTTCATGTGGGA;

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and Klf4 reverse, GGAAGTCGCTTCATGTGGGA.

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Cellular uptake pathways. The underlying mechanisms of cellular uptake of both

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pOSKM-CPNPs and pOSKM-encapsulated CPS-CPNPs were investigated using

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internalization inhibition tests. Seven inhibitors including NH4Cl (10 mM), filipin Ш (1

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µg/mL), sodium orthovanadate (SOV) (5 µM), glucose (0.45 M), 5-(N,N-dimethyl)-amiloride

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(DMA) (10 µM), chlorpromazine hydrochloride (CPZ) (10.0 µg/mL) and genistein (50 µM)

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were used for the evaluation. YOYO-1, a fluorescent probe (green fluorescence), was used to

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tag the plasmid DNAs in the nanoparticles (pOSKM-CPNPs and pOSKM-encapsulated CPS-

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CPNPs) to track the pathways of cellular uptake.

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In brief, each inhibitor was diluted to an appropriate concentration of 500 µL using

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serum-free DMEM/F12 medium. Then the medium in each well was replaced with their

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respective inhibitors. Thirty minutes later, the mixture of YOYO-1 and pOSKM-CPNPs or

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pOSKM-encapsulated CPS-CPNPs was added to each well. After incubation at 37 °C for 2 h,

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the culture medium in each well was replaced by 10% serum-containing DMEM/F12 medium.

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After 24 h incubation, the cells were observed under ﬂuorescence microscope (Leica, DMI

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6000B, Wetzlar, Germany). 9 ACS Paragon Plus Environment


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Immunofluorescence staining. The cells were fixed in 4% paraformaldehyde, followed

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by immunofluorescence staining according to previous procedures.29 The primary antibodies

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used for detecting the expression of the four transcription factors were anti-Oct-4 (1:500),

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anti-Sox2 (1:500), anti-Klf4 (1:250) and anti-c-Myc (1:250), whereas that of pluripotency

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markers included anti-Oct-4 (1:500), anti-SSEA3 (1:500), anti-SSEA4 (1:500), anti-Tra-1-

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81(1:250) and anti-Nanog (1:250). All the antibodies were obtained from Abcam (Cambridge,

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MA, USA). Fluorescently labelled sheep anti-mouse IgG-Cy3 was used as secondary

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antibody, and it was obtained from Sigma (St. Louis, MO, USA). The nuclei were

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counterstained using DAPI (1:2000; Sigma, St. Louis, MO, USA).

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Determination of histone H3-lysine 4 (H3K4) methylation and acetylation. Histone

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H3K4 di-methylation (H3K4me2), tri-methylation (H3K4me3) and acetylation were

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evaluated via immunostaining. The cells were immunostained with antibodies against di- and

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tri-methyl-lysine 4 of histone H3, and acetyl-lysine 4 of histone H3 (Santa Cruz

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Biotechnology, Santa Cruz, CA, USA). The samples were then probed with Cy3-conjugated

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goat anti-mouse IgG secondary antibody (Sigma, St. Louis, MO, USA). Counterstaining was

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performed using DAPI . Images were obtained using a Leica epifluorescence light microscope

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(Leica Microsystems, Wetzlar, Germany).

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In vitro differentiation. For three-germ layer differentiation, the process was conducted

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as previously presented. 29 Four weeks later, the differentiated cells were processed for

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immunofluorescence. The primary antibodies used included anti-βIII tubulin (Tuj1, ectoderm

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marker, 1:500; ab18207, Abcam), anti-α-fetoprotein (AFP, endoderm marker, 1:250;

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SAB3500533, Sigma, St. Louis, MO, USA) and anti-collagen II (mesoderm marker, 1:500;

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SAB4500366, Sigma, St. Louis, MO, USA).

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Teratoma formation. All experimental procedures were performed in accordance with

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the standard human care guidelines of the Guide for Care and Use of Laboratory Animals. In

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the teratoma assay, human iPSCs were washed with PBS and treated with collagenase IV for 10 ACS Paragon Plus Environment

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30 min at 37 °C. The resulting cells were collected using centrifugation. The cells were

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resuspended in hESC medium at a density of 1×107 cells/mL. The cell suspension (100 µL)

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was subcutaneously injected into 4-week old immunocompromised non-obese diabetic-severe

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combined immunodeficient (NOD-SCID) mice (Comparative Medicine Center, Yangzhou

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University, Yangzhou, Jiangsu, China). After 8 weeks of cell injection, the teratomas were

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collected and processed for hematoxylin/eosin (HE) staining. Briefly, the tumors were fixed

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in 4% paraformaldehyde for 24 h and dehydrated using ethanol with increasing concentrations,

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followed by treatment with dimethylbenzene and embedment in paraffin. Afterwards, serial

228

sections (5 µm in thickness) were prepared, and the slices were dried in an oven at 65 °C for 6

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h. The samples were gradually subject to dewaxing and hydration, followed by staining with

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hematoxylin/eosin according to the standard procedures.

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Statistical analysis. The data were analyzed using Student’s t-test, one-way analysis of

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variance (ANOVA) and Fisher’s protected least squares differences (FLSD) post-hoc tests to

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determine the significance of differences (significance was accepted at p

Pleurotus eryngii Polysaccharide Promotes Pluripotent

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