Pleurotus eryngii Polysaccharide Promotes Pluripotent

Jan 26, 2016 - ... with calcium phosphate (CP), was used to codeliver plasmids (Oct4, Sox2, Klf4, c-Myc) for generating induced pluripotent stem cells...
0 downloads 0 Views 4MB Size
Subscriber access provided by KUNGL TEKNISKA HOGSKOLAN

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

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.

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

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

2

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

6

the hybrid nanoparticles could significantly enhance the process and efficiency of

7

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

8

facilitated epigenetic modification during the reprogramming. Moreover, these hybrid

9

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

10

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

11

findings therefore present a novel application of Pleurotus eryngii polysaccharide in

12

pluripotent reprogramming via active epigenetic modification.

13

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

14

non-viral, induced pluripotent stem cells

15

2 ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

Journal of Agricultural and Food Chemistry

16 17

INTRODUCTION

18

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

19

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

20

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

21

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

22

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

23

circumventing the ethical issues and complications emanating from immune rejection

24

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

25

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

26

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

27

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

28

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

29

much attention although with lower reprogramming efficiency.

30

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

31

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

32

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

33

various chemical modifications, and exhibit excellent biocompatibility and low

34

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

35

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

36

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

37

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

38

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

39

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

40

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

Journal of Agricultural and Food Chemistry

41

characterization and bioactive effects .25 The Pleurotus eryngii polysaccharide has

42

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

43

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

44

polysaccharide (CPS) with significantly enhanced transfection efficiency than the

45

positive transfection reagent (Lipofectamine2000).

46

The outstanding gene delivery and biocompatibility of CPS could promote its

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

yield hybrid nanoparticles for effective gene transfection because of their multiple

56

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

57

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

58

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

59

preliminarily investigate the possible mechanisms involved in the reprogramming. .

60

MATERIALS AND METHODS

61

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

62

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

63

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

64

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

65

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

Page 4 of 38

Page 5 of 38

Journal of Agricultural and Food Chemistry

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

USA).

81

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

82

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

83

Laboratory Animals.

84

Preparation and characterization of CPS. The crude polysaccharide was extracted

85

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

86

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

87

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

88

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

89

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

90

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

91

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

Journal of Agricultural and Food Chemistry

92

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

93

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

94

described .17

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

4 °C for subsequent use.

6 ACS Paragon Plus Environment

Page 6 of 38

Page 7 of 38

Journal of Agricultural and Food Chemistry

116

Characterization of pOSKM-encapsulated CPS-CPNPs. Transmission electron

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

UK).

126

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

127

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

128

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

129

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

130 131 132

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

133

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

134

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

135

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

136

reported. 29

137

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

138

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

139

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

140

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

141

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

Journal of Agricultural and Food Chemistry

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

efficiency of the reprogramming as previously reported.29

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

Page 8 of 38

Page 9 of 38

Journal of Agricultural and Food Chemistry

168

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

169

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

170

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

171

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

172

follows: GAPDH forward, CGGAGTCAACGGATTTGGTCGTAT; GAPDH

173

reverse,AGCCTTCTCCATGGTGGTGAAGAC; Sox2

174

forward,GCCCTGCAGTACAACTCCAT; Sox2 reverse, GACTTGACCACCGAACCCAT;

175

Oct4 forward, ATGTGGTCCGAGTGTGGTTC; Oct4 reverse,

176

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

177

reverse, GCTGGTGCATTTTCGGTTGT; Klf4 forward, GGAAGTCGCTTCATGTGGGA;

178

and Klf4 reverse, GGAAGTCGCTTCATGTGGGA.

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

CPNPs) to track the pathways of cellular uptake.

187

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

188

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

189

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

190

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

191

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

192

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

193

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

Journal of Agricultural and Food Chemistry

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

(Leica Microsystems, Wetzlar, Germany).

211

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

212

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

213

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

214

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

215

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

216

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

217

Teratoma formation. All experimental procedures were performed in accordance with

218

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

219

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

Page 10 of 38

Page 11 of 38

Journal of Agricultural and Food Chemistry

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

229

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

230

hematoxylin/eosin according to the standard procedures.

231

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

232

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

233

determine the significance of differences (significance was accepted at p