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
