୯ҥѠεᏢғڮࣽᏢଣғϯࣽמᏢس!
ᅺγፕЎ!
Department of Biological Science and Technology College of Life Science
National Taiwan University Master Thesis
ว҅ႝ܄ڼԯಈηբࣁ!Sox2, Klf4, Oct4!Ϸ!d-Myc!
ೈқ፦ၩᡏᇙΓᜪᇨᏤӭૈ༸ಒझϐࣴز!
Development of cationic nanoparticles with Sox2, Klf4, Oct4 and c-Myc proteins in induced pluripotent stem cells’
generation
হྷඳ!
Jui-Ching Hsia
ࡰᏤ௲Ǻഋࡏᄪ!റγ!
AdvisorǺYen-Rong Chen, Ph.D.
ύ҇୯!103 ԃ!7!Д!
July 2014
α
α၂ہቩۓਜ
Acknowledgement
In the beginning, I would like to thank my research advisor, Dr. Yen-Rong Chen for his assistant and support in this study. Besides, advice and materials given by Dr. Yi-You Huang and Dr. Ming-Ju Chou have been a great help in my research. Equipment and materials provided by Dr. Hung-Yuan Chi and Dr. Kung-Ta Lee was also greatly appreciated. I would like to offer my sincerely thanks to my thesis committee: Dr. Yi-You Huang and Dr. Hong-Ren Jiang for their advice and comments. Also, I wish to acknowledge the help provided by all the members in Technology Commons, College of Life Science, NTU, for their technical supports.
On the other hand, my special thanks are extended to all my colleagues in niChe lab for their help and encouragement in my research, especially Min-Hao Chiang, Fang-Ting Kuo, Woan-Ing Twu and Chih-hsun Liao for their assistance in plasmid construction and overexpression system preparation.
Finally, I want to give my special thanks to my parents and my girlfriend Fang-Yu Huang for their encouragement and comments during my academy life. This work would not accomplish smoothly without their support.
ύ
ύЎᄔा
ΓᜪᇨᏤӭૈ܄༸ಒझ (induced pluripotent stem cells, iPS cells) ࢂᅿஒᡏ ಒझӆำׇϯࡕౢғޑΓ༸ಒझǴځᗺӧܭஒςϩϯޑಒझଏϩϯӣڀԖϩ ϯૈΚޑ༸ಒझǴׯᡂΑၸ۳ᇡࣁϩϯѝૈࢂൂՉၰޑཷۺǶҞനЬाޑΓᜪ
ᇨᏤӭૈ܄༸ಒझᇙБݤࢂξύ՜ᔆറγܭ 2006 ԃ܌วǴճҔԖѤӢη
(Klf4, Sox2, Oct4 аϷ c-Myc) ޑϸᙯᒵੰࢥགࢉᡏಒझӆำׇϯԶԋǶԾ၀ࣴ
زϐࡕǴӭᙖҗ DNA ൪ΕஎЬ୷Ӣᡏٰ߄ѤӢηǴஒᡏಒझӆำׇ
ϯԋࣁΓᜪᇨᏤӭૈ܄༸ಒझޑБԄഌഌុុวрٰǴ೭٤а DNA բࣁӢ
ηၮଌၩᡏޑΓᜪᇨᏤӭૈ܄༸ಒझΨᆀࣁಃжΓᜪᇨᏤӭૈ܄༸ಒझǶ
ฅԶǴᗨฅа DNA ࣁѤӢηၮଌၩᡏځӆำׇϯޑਏКځдБݤٰޑ
ଯǴԜᅿБݤޑനεલᗺӧܭၩᡏ܌ឫޑѤӢηࢂᒿᐒ൪ΕಒझࢉՅᡏύǴஎ Ь୷ӢँᡂޑᅪቾᏤठΑಃжΓᜪᇨᏤӭૈ܄༸ಒझӧᙴᕍޑวεڙज़
ڋǶࣁΑှ،୷Ӣ൪ΕޑୢᚒǴճҔߚ DNA ޑၩᡏٰᇙΓᜪᇨᏤӭૈ܄༸ಒ
झԋࣁ߈යࣴزख़ЈǶ೭٤ߚ DNA ޑၩᡏхࡴೈқ፦ǵߞ٬ RNA аϷ༾λ
RNA (MicroRNA) ǴځӅ೯ᗺӧܭӆำׇϯޑၸำύόሡჹஎЬ୷ӢᡏҺ Ֆ׳ǴځᇨᏤрٰޑΓᜪᇨᏤӭૈ܄༸ಒझڀԖ׳٫ޑᙴᕍᔈҔඳǶ
୷ܭॊҗǴӧԜࣴزύך׆ఈճҔೈқ፦բࣁѤӢηޑၩᡏଌΕΓᜪх
Ҝᠼᆢ҆ಒझ (HS68) ύٰᇨᏤΓᜪᇨᏤӭૈ܄༸ಒझޑౢғǶೈқ፦ڀԖܰܭ
εໆ߄ǵપϯаϷᓯӸޑᓬᗺǴځբࣁၩᡏޑЬाምᛖӧܭೈқ፦คݤЬऀ
ၸಒझጢǴሡᙖҗڼԯಈη܈ऀጢอ两ޑᔅշωૈճΕಒझϣǹӢԜǴךճ Ҕᙴπ܌໒วޑ gelatin-polyethyleneimaine (gelatin-PEI) ڼԯಈηٰхᇗᆘՅᑻӀ
ೈқ (բࣁኳԄೈқ) аϷѤӢη,ᙖԜஒ೭٤ೈқ፦ଌΕΓᜪᠼᆢ҆ಒझύǴ٠
ӧտӞࡕᙖҗڼԯಈηޑભᆶΒભբࣁ፦ηੇᆟ (proton sponge) ڐշೈ
ӧڼԯಈηޑտӞਏෳ၂ύǴёـᐚࡋගϲډ 50ug/ml ਔǴHS68 ૈ
տӞஒ߈ΐԋޑڼԯಈηǹԶӧಒझࢥ܄ෳ၂ύǴHS68 ᗨӧᛰࡕ෧ϿऊΟ ԋޑಒझǴࠅૈӧΟϺޑᎦࡕӣൺډচԖኧໆǴᡉҢ҂ٰӆำׇϯਔϸᙟᛰ
ࢂёՉޑǶ
Զӧೈқ፦߄ޑ่݀ύёـǴόፕࢂѤӢη܈ࢂᆘՅᑻӀೈқૈԋфޑ ӧεဉఎ߄سύ߄ǴӕਔԜϖᅿೈқ፦ΨૈճҔᙻᒃӝᆅࢊٰՉપ
ϯǶӧપϯࡕޑᆘՅᑻӀೈқௗڙ gelatin-PEI ڼԯಈηхᇗࡕǴךஒٿޣޑష
ӝނуΕ HS68 ޑᎦ୷ύǴวᆘՅᑻӀೈқаϷڼԯಈηૈճಒझտ
ӞǴЪᆘՅᑻӀೈқ٩ᙑૈౢғᑻӀǶᡉҢճҔ೭ኬޑڼԯಈηѐхᇗೈқ፦ٰ
ଌΕಒझࢂёՉޑǴჹܭೈқ፦ޑཞΨࢂёௗڙޑǶ
аࣴز่݀ᡉҢ҂ٰճҔ gelatin-PEI ڼԯಈηٰхᇗѤӢηೈқǴᙖԜ
Չ ᡏಒझӆำׇϯᇙΓᜪᇨᏤӭૈ܄༸ಒझࢂॶள၂ޑ,҂ٰ೭ኬޑБݤ ёૈԋࣁ׳ӼӄޑಒझӆำׇϯБԄǶ
Abstract
Induced pluripotent stem cells (iPS cells) are artificial stem cells, generated by
somatic cell reprogramming. In 2006, Shinya Yamanaka and Kazutoshi Takahashi
investigated 24 candidates that are specifically expressed in embryonic stem cells (ES
cells) as pluripotent-correlated genes, they finally found out that Klf4, Sox2, Oct4 and
c-Myc, which are known as Yamanaka factors, are able to derive iPS cells from adult
fibroblasts. Since then many DNA-dependent reprogramming methods have been
developed, and these methods have the same problem, which hinder the clinical
application of this type of iPS cells. The problem of DNA-dependent methods is
uncontrollable genome integration during reprogramming process, so the following
researcher focused on DNA-free reprogramming vectors, such as proteins, microRNA
and mRNA. Those DNA-free methods won’t modify host genome and therefore those
methods are more promising in regenerative medicine area.
Based on these reasons, I used proteins as DNA-free reprogramming vectors to
generate iPS cells. Proteins are easy to overexpress, purify and store up, but without
specific peptide sequence or protein carrier, most proteins are unable to cross cell
membrane, and hence I cooperated with Dr. Yi-You Huang and Dr. Ming-Ju Chou using
their gelatin–polyethyleneimine (gelatin-PEI) nanoparticles as my protein carrier. On
the other hand, gelatin-PEI nanoparticles have many primary and secondary amines
work as proton sponge, which provide high efficiency of endosomal escape.
In my research, the uptake efficiency experiment performed by flow cytometry
showed that HS68 cells are able to uptake almost 90% of gelatin-PEI nanoparticle when
particle concentration reach 50ug/ml. Besides, cell viability assay showed that although
HS68 population will decrease after 24 hours particle application, HS68 cells will
repopulate after three days, indicating that repeating protein delivery is possible.
On the other hand, I also showed that both enhanced green fluorescent proteins
(eGFPs) and Yamanaka four factors can be overexpressed in Escherichia coli
expression system, and these proteins were able to be purified by Ni-NTA affinity
columns. Furthermore, after I mixed gelatin-PEI nanoparticles and eGFPs with HS68
cells, these particles are able to transport our model protein eGFPs into HS68 cell line,
and transfection process won’t cause severe cell death.
Overall, these data showed that gelatin-PEI nanoparticles are able to carry our
model proteins, eGFPs, into human foreskin fibroblasts. I suppose that we can combine
proteins and nanoparticles as reprogramming vectors, and this might be a new method
to generate iPS cells
Table of contents
α၂ہቩۓਜ... i
Acknowledgement ... ii
ύЎᄔा... iii
Abstract... v
Table of contents... vii
List of figures... ix
List of tables ... x
Chapter one Introduction ... 1
1.1 Induced pluripotent stem cells: History, characteristics and further applications ... 1
1.2 Factor delivery: First step in iPS cells generation ... 4
1.3 Yamanaka factors: Sox2, Klf4, Oct4 and c-Myc ... 6
1.5 Gelatin- Polyethyleneimaine (Gelatin-PEI) nanoparticles ... 8
1.6 Research aims...11
Chapter two Materials and methods ... 13
2.1 Cell culture ... 13
2.1.1 Cell line... 13
2.1.2 Cell culture condition ... 13
2.2 Plasmid construction... 13
2.2.1 Plasmid ... 13
2.2.2 Polymerase chain reaction (PCR) condition... 14
2.2.3 Gel filtration ... 14
2.2.5 Competent cell preparation... 15
2.2.6 Ligation and transformation ... 16
2.3 Plasmid mini-preparation ... 17
2.4 Plasmid midi preparation... 17
2.5 Total protein extraction... 18
2.6 SDS-PAGE and CBR staining. ... 19
2.7 Western analysis ... 20
2.8 Affinity chromatography... 21
2.9 Protein concentration determination... 22
2.10 Cell viability analysis ... 22
2.11 Cellular uptake analysis ... 23
2.10 Intracellular protein delivery of gelatin-PEI nanoparticles ... 24
Chapter three Result ... 26
3.1 Identification of His-eGFP and His-Yamanaka factors overexpressed by BL21 and Rosetta™ competent cells... 26
3.1.1 Total protein analysis by coomassie brilliant blue and Western blotting26 3.1.2 Induction time optimization of His-eGFP and His-SKOM ... 27
3.2 Purification of His-eGFP and His-Yamanaka factors by affinity columns... 28
3.3 Cellular uptake of gelatin-PEI nanoparticles in HS68 cell line... 29
3.4 Cytotoxicity analysis of gelatin-PEI nanoparticles in HS68 cell line ... 29
3.5 His-eGFP delivery by gelatin-PEI nanoparticles in HS68 cell line... 30
Chapter four Discussion ... 32
Chapter five Conclusion ... 35
Reference ... 55
List of figures
Figure 1-1. eGFP Overexpression Verification by CBR Staining. ... 38
Figure 1-2. eGFP Overexpression Verification by Western Blot Analysis. ... 39
Figure 1-3. Sox2, Klf4, Oct4 and c-Myc Overexpression Verification by CBR Staining. ... 40
Figure 1-4. Sox2, Klf4, Oct4 and c-Myc Overexpression Verification by Western Blotting Analysis. ... 41
Figure 2-1. Induction Time Optimization of His-eGFP... 42
Figure 2-2. Quantification of His-eGFP Overexpression Level with Different IPTG Induction Period. ... 43
Figure 2-3. Induction Time Optimization of His-Yamanaka Factors. ... 45
Figure 2-4. Quantification of His-Yamanaka Factors Overexpression Level with Different IPTG Induction Period. ... 47
Figure 3-1. His-eGFP Purification was Verified by CBR Staining. ... 48
Figure 3-2. His-eGFP Purification was Verified by Western Blotting... 49
Figure 3-3. His-Yamanaka Factors Purification was Verified by CBR Staining. ... 50
Figure 3-4. His-Yamanaka Factors Purification was Verified by Western Blotting. ... 51
Figure 4. In vitro Protein Delivery Efficiency of GR-PEI Nanoparticles in Treating Hs68 Cell Line. ... 52
Figure 5. In vitro Cytotoxicity Analysis of Gelatin-PEI Nanoparticles in Treating HS68 Cell Line. ... 53
Figure 6. In vitro His-eGFP Delivery by Gelatin-PEI Nanoparticles in Treating HS68 cell line. ... 54
List of tables
Table 1. Primer List of Yamanaka Factors, eGFP and Adaptor for Yamanaka Factors. . 37
Chapter one Introduction
1.1 Induced pluripotent stem cells: History, characteristics and further
applications
Stem cells are cells that are able to renew themselves through mitotic cell division
and differentiate into a diverse range of specialized cell types. Therefore, stem cells can
be used in developmental research, therapies and regenerative medicine, such as cardiac
cell therapy (1) and islet-beta cell generation (2). Also, stem cells are classified into
several classes, including embryonic stem cells, fetal stem cells, perinatal stem cells,
adult stem cells and induced pluripotent stem cells based on their origin (3). The self-
maintenance and differentiation of stem cells are controlled not only by some specific
genes but also affected by “stem cell niche”, the microenvironment surrounding stem
cells in vivo (4).
Before reprogramming techniques were available, it was believed that adult
somatic cells would not switch fates automatically after committed to specific cell type.
In 1952, Briggs and King (5) generated tadpoles from blastocyst nucleus with
enucleated oocyte by somatic cell nuclei transfer (SCNT) technique. Later on, Gurdon
(6) combined mature somatic cell’s nucleus with enucleated oocyte by using same
technique to generate adult frog. With SCNT, reprogramming is even possible for fetal
and adult mammalian cells (7).
On the other hand, cell-cell fusion is another method to generate dedifferentiated
somatic cell before induced pluripotent stem cells were available. In 1976, Miller and
Ruddle (8) mixed pluripotent PCC4aza1 embryonal carcinoma cells with thymocytes
from young adult mice, and these mixed-up cells were able to generate tumors which
contained differentiated tissues after transplanted into nude mice. After Miller and
Ruddels’ research, both Tada M et al. (9) and Cowan CA et al. (10) have successfully
reprogrammed somatic cells with embryonic stem cells using cell-cell fusion technique.
These experiments together indicated that unfertilized eggs and ES cells contain factors
that can confer totipotency or pluripotency to somatic cells (11).
Based on this idea, Yamanaka and Takahashi have scanned 24 factors which play
pivotal roles in the maintenance of ES cell identity (11). Eventually, they found out that
by delivering some specific factors, such as Sox2, Klf4, Oct4 and c-Myc, murine
fibroblasts were able to be reprogrammed back into embryonic-stem-cell-like state, and
these cells are what we called iPS cells today. Compared with human embryonic stem
(hES) cells, iPS cells possessed some advantages, such as ethical-issues-free, easier to
obtain, and immunologically compatible when applying to disease modeling (12), cell
therapy (13) and organ reconstruction (14).
Considering pluripotency and the ability of differentiation, ES cells are the best
choice of all. But due to the ethical problem, ES cells are not convenient to obtain. From
previous researches, iPS cells’ genome-wide expression patterns and histone
modifications have high similarity with ES cells, and they have similar differentiation
behavior (15). In addition, the problem of epigenetic memory due to reprogramming of
iPS cells generation is not as serious as SCNT and cell-cell fusion techniques (16).
Although iPS cells generation is a well-known techniques these days, the
mechanism behind those transcription factors (that is to say, Sox2, Klf4, Oct4 and c-
Myc) is still a mystery. In the experiments executed by Soufi A et al., they found out
that c-Myc plays an important role in OSK chromatin engagement, and low
reprogramming efficiency of iPS cells might due to massive H3K9me3 in many genes
required for pluripotency (17). Besides, p53 (18) and Mbd3 (19) were both correlated to
iPS cells’ reprogramming process, p53 knockout or Mbd3 knockout cells can both
achieved higher reprogramming efficiency. Moreover, the process of reprogramming
can be further divided into different stages (20), and either epithelial-to-mesenchymal
transition (EMT) or mesenchymal-to-epithelial transition (MET) is required for
reprogramming (21). These experiments indicated that reprogramming might not be a
one-step process, but a series of epigenetic modification, gene activation and
inactivation processes.
In addition to the mechanism of iPS cells’ reprogramming, the efficiency of factor
delivery and the elimination of iPS cells’ tumor-genic potential are also important
during iPS cells generation, and these problems are associated with the methods used to
generate iPS cells.
1.2 Factor delivery: First step in iPS cells generation
The factor delivery methods can be generally classified into two groups: DNA-
dependent and DNA-free methods. With former can be further subdivided into two
major categories: Integrating and non-integrating vectors (11, 22). From lentiviral
vectors (11) to retroviral vectors (23), non-specific genome mutation caused by these
integrating vectors is the major problem during reprogramming process. Although using
integrating vector is a prevalent way during the iPS cell generation, but it is still not
proper when applying to clinical use (24).
For non-integrating vectors like piggyBac transposition (25) or episomal vectors
(22), the efficiencies of iPS cells generation by both methods are much more higher
than general DNA-free methods, but whether any piece of vector sequence will remain
in the host genome or not is still a possible problem. Besides, transposon needs a
specific integration site, but it is still unclear whether this method would induce
nonspecific genome alteration in iPS cells (25). As a result, the DNA-free method might
be a safer way to generate iPS cells.
Since 2006, DNA-free methods including microRNA (26), mRNA (24), small
molecules (27) and proteins (28) were developed rapidly. Messenger RNA or
microRNA were both able to avoid genome mutation and gene insertion during
reprogramming process. Nevertheless, the complex technique and the instability of
RNA may limit its universalization and reprogramming efficiency. Also, even though
iPS cells generated by small molecules provide promising reprogramming efficiency
(about 0.2%), but the interaction between different small molecules and the
reprogrammed cells is still unclear, and the reprogramming process is much more
complicated than usual methods. Therefore, proteins were chosen to be the
reprogramming vector in this research.
In previous researches, Hongyan Zhou et al. combined Yamanaka factors with
poly-arginine, which was also known as cell-penetrating peptide, to deliver bare protein
into the cells. These recombinant proteins were able to transform human fibroblast into
ES cells-like cells (28). Compared with other delivering means, its efficiency is
particularly low even with the addition of deacetylase (HDAC) inhibitor valproic acid
(VPA) (28). On the other hand, Dohoon Kim et al. also combined Yamanaka factors
with poly-arginine tag, and successfully deliver these proteins into human newborn
fibroblast (HNF). The iPS cells which them generated from these recombinant proteins
were able to maintain for more than 35 passages and exhibit morphology similar to that
of hES cells (29). However, the reprogramming efficiency of both experiment were
extremely low, this phenomena indicates that there is still room for improvement,
especially for protein delivery and protein protection.
Overall, DNA-free methods are able to substitute for viral vectors and become the
new delivering way in the future. Nevertheless, the obstacles, such as the
reprogramming efficiency, of the iPS cell generation process still need to be eliminated.
1.3 Yamanaka factors: Sox2, Klf4, Oct4 and c-Myc
SRY-related HMG box (Sox) 2 is a member of the Sox gene family with a single
HMG DNA-binding domain. In 2000, Zappone et al. found out that Sox2 expression is
correlated with uncommitted dividing stem and precursor cells of the developing central
nervous system (30). Also, Avilion et al. showed that Sox2 marks the pluripotent lineage
of the early mouse embryo. In contrast, Sox2 was down-regulated in development
committed cells (31). These results indicated that Sox2 might play an important role in
pluripotency maintenance.
Krüppel-like factors (Klfs) are transcription factors that bind to specific DNA
sequences and regulate gene transcription. Klf4 is highly expressed in epithelial tissues,
especially in postmitotic epithelial cells of the intestinal mucosa. This characteristic
suggests that klf4 may be link to cell grow arrest (32). During iPS cells generation, klf4
binds to the Oct3/4-Sox2 complex to co-regulate the expression of Nanog, which is the
pluripotency-defining protein (33), with homeobox protein PBX1.
OCT4, a POU domain protein also known as Oct3, is expressed in blastomeres,
pluripotent early embryo cells and later in germ cells (34). In 1998, Jennifer Nichols et
al. discovered that Oct4-deficient embryos developed to the blastocyst stage, but the
inner cell mass cells were not pluripotent. Furthermore, the trophoblastic proliferation
of Oct4 knockout embryos was restricted (35). That is to say, Oct4 plays a vital role in
embryonic development and pluipotency maintenance.
c-Myc, a cellular homolog of the retroviral v-Myc oncogene (36), belongs to a
family of helix-loop-helix/leucine zipper transcription factors. In several animal and
human tumors, c-Myc proto-oncogene was found to be activated (37). c-Myc mutation
is lethal in homozygotes between 9.5 and 10.5 days of gestation, and the embryos are
smaller and retarded in development than normal ones (38). In 2004, Peter Cartwright et
al. reveled that Myc expression alone can render self-renewal and maintenance of
pluripotency, even in the absence of LIF (39).
Overall, these Yamanaka factors are either ES cells proliferation correlated or
pluripotent correlated, and participated in several different early developmental stages.
These facts may be the clues that why iPS cell can be generated by applying four
Yamanaka factors to adult somatic cells.
1.5 Gelatin- Polyethyleneimaine (Gelatin-PEI) nanoparticles!!
Biomaterials have been use in several bioengineering areas, including tissue
engineering, tissue regeneration and drug delivery. By using biocompatible materials as
bio-macromolecules carriers, the tolerability and bioavailability of bio-macromolecules
can be significantly elevated. Those biocompatible carriers including nanoparticles,
nanocapsules, micellar systems, and conjugates (40). The nanoparticle-based carriers
possessed several advantages, such as high uptake efficiency due to sub-micron particle
size, longer retention time, good surface-to-volume ratio and acceptable drug-release
control (41). In fact, those advantages are the most important factors which can be
manipulated by specific process during nanoparticles construction, including size,
encapsulation efficiency, surface charge (zeta potential) and release characteristics.
In past decades, the most widely-used polymers were poly lactic acid (PLA), poly
glycolic acid (PGA), and poly lactide-co-glycolide (PLGA). The polymers described
above are known for their biocompatibility and plasticity, and these polymers were all
approved by Food and Drug Administration (FDA). On the other hand, natural polymers
such as gelatin (42), chitosan (43), collagen (44) and silk fibroin (45) were also possible
to be the ingredients of nanoparticles. These natural polymers have two major
advantages, one is the various functional groups on natural polymers makes then easy to
be modified depending on the needs. The second is the natural derivatives after polymer
degradation are mostly amino acids and saccharides, which can be easily cleared after
medical applications.
Gelatin is a natural, biocompatible (46) and biodegradable (47) polymer, which
derived from animal skin white connective tissue, bones and collagen . It is wildly used
in pharmaceutical products, medical products and, most important of all, as a carrier
matrix due to its low cell toxicity (48) and capability of preserving the bioactivity of
encapsulated agent in vivo (49).
Gelatin is obtained from partial hydrolysis of collagen, and the charge it possesses
depends on the methods that were used in collagen pre-treatment. Due to the proportion
of the carbonyl groups in gelatin, the polymer is positively charged in acidic solutions
and negatively charged in alkaline solutions (50). Also, the isoeletric point (IEP) is
ranging from 4.8 to 9.4 depends on the pH value of the pre-treatment (51). By adjusting
the charge and the IEP value, gelatin polymer will be able to attract either positively or
negatively charged proteins due to ionic strength. In fact, gelatin and the albumin were
the first nanoparticles for pharmaceutical applications (52, 53).
Polyethyleneimaine (PEI) is a synthetic cationic polymer, which is regarded as the
most effective DNA carrier since 1995 (54). The numerous amine groups within PEI
provide PEI ability to interact with negatively charged drugs and biomolecules,
including DNA and proteins (55). Besides, these amine groups provide several reactive
sites, and therefore a wide range of chemical modifications are possible. During
biomolecules delivery, amine groups were believed to act as proton sponge which can
help biomolecules escape from endosomes, and cationic polymer may induce nanoscale
holes on cell membrane which increase the permeability of the membrane (56). The
amount of primary and secondary amines was indicated to be the most important factor
for transfection efficiency and cytotoxicity (57), so it is important to find the balance
between these two.
In this research, I cooperated with Dr. Yi-You Huang and his lab by using their
gelatin-PEI nanoparticles as protein carriers. In 2010, Wei-Ti Kuo et al. developed a
gelatin (1.8kDa)-PEI nanocarries with high positive ζ potential and buffering effect.
And with specific nanoparticles-to-proteins ratio (30:1), gelatin-PEI nanoparticles were
able to deliver 2.12 × 104RLU/μg protein with acceptable cell viability (58). Based on
this research, gelatin-PEI nanoparticles were used as protein carriers in my study to see
whether it can deliver Yamanaka factors into human fibroblasts or not, and if so, target
cells might be able to be reprogrammed into iPS cells.
1.6 Research aims
The first generation of iPS cells were generated by using constitutively active viral
vectors that can integrated into the host genome, but gene insertion might cause genome
instability, which limited the possibility of iPS cells in clinical usage. Also, iPS cells
were able to be derived via transposon, transient plasmid, episomal or adenovirus, but
the problem of insertion mutagenesis and the complex operation, such as sequential
selection and vector construction during generation process remains to be solved. (25,
59-62)
To solve these problems, I try to deliver proteins of four Yamanaka factors (Sox2,
Klf4, Oct4 and c-Myc) into somatic cells with gelatin-PEI nanoparticles which
generated by Dr. Yi-You Huang and his group. Dr. Yi-You Huang and his research group
mainly focused on tissue reconstruction and drug delivery with several different
nanoparticles, such as liposome and polymersome in the past. I wanted to combine their
techniques in biomolecule delivery with iPS cells generation, hoping to overcome the
disadvantage of DNA-dependent methods we mentioned above. It is possible that by
using proteins with nanoparticles as delivery vectors, the genome integrity and cell
uptake efficiency would be improved largely compared with DNA-dependent methods.
And, application of this technique to tissue reconstruction might provide a possible
therapeutic use in the future.
Chapter two Materials and methods
2.1 Cell culture
2.1.1 Cell line
HS68 (ATCC®CRL-1635)
HS68 cell line is a kind of human foreskin fibroblast cell line, and HS68 cell line’s
donor was possibly suffered Canavan disease, a deficiency in aspartoacyclase. In
this research, HS68 cell line was bought from Bioresource Collection and Research
Center, and this cell line is going to be reprogrammed by Yamanaka factors.
2.1.2 Cell culture condition
HS68 cells were cultured and transfected in Dulbecco’s modified eagle medium
(Life technologe) with 10% fetal bovine serum (Hyclone) and 3.7 g/l Sodium
hydrogen carbonate. HS68 cells were culture in 10 cm cell dish (Thermo) at 37°D.
2.2 Plasmid construction
2.2.1 Plasmid
The following plasmids were used in protein overexpression in E.coli strain BL21:
pETDUET-1-Sox2, pETDUET-1-Klf4, pETDUET-1-Oct4, pETDUET-1-c-Myc and
pETDUET-1-eGFP. Due to frame shift, adaptors were design for Sox2, Klf4, Oct4 and
c-Myc as Table.1.
Sox2, Klf4, Oct4 and c-Myc sequences were amplified from pCAG2LMKOSimO,
and eGFP was amplified from pEGFP-C1 (Clontech) by LA taqTMDNA polymerase
(Takara). pETDUET-1 (Novagen) was obtained from Institute of Biochemical Sciences,
National Taiwan University. Cloning primers were designed as Table.1. Sox2, Klf4,
Oct4 and c-Myc were inserted by restriction site EcoRI (C-terminal) and NotI (N-
terminal). eGFP was inserted by restriction site NotI on both ends.
2.2.2 Polymerase chain reaction (PCR) condition
Polymerase chain reaction was performed with following condition: LA TaqTM
DNA Polymerase (Takara, 1%) was mixed with 10x LA PCR buffer (10%), dNTP mix
(16%), Mg2+(10%), template DNA (4%), each primer (2%) and ddH2O up to total value
as 50μl.
2.2.3 Gel filtration
After PCR, DNA product was analyzed by 1% TAE agarose gel. DNA was further
purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid) following by kit’s
protocol. In brief, 500 μl DF buffer was added into each tubes which contained gel less
than 300 mg, then incubated at 60°C for 15 minutes. Then DF column was placed in a
new collection tube. The sample was transferred to the DF column 800μl each time,
then centrifuged at 15000 rpm for 30 seconds. Discard the solution in the collection
tube. Added 400 μl W1 buffer into DF column and centrifuged with same condition
again. Discard the solution in the collection tube then add 600 μl wash buffer. The DF
column was centrifuged at same condition and discarded the solution in the collection
tube again. The empty column was centrifuged at 15000 rpm for 3 minutes. The DF
column was moved to a new tube, then added 30 μl ddH2O and stood at room
temperature for 2 minutes. And the tube was centrifuged at 15000 rpm for 2 minutes.
The DNA sample was preserved at 4°C.
2.2.4 Restriction enzyme digestion
Purified DNA inserts and pETDUET-1 were cleavage with EcoRI-HF® (New
England Biolabs) and NotI-HF® (New England Biolabs) for 2 hours at 37°C. Reaction
mixture contained NEBuffer (10%), restriction enzyme (1%), template (up to 5 μg) and
ddH2O. After restriction process, the reaction mixture was placed in dry bath at 65°C for
30 minutes to eliminate enzyme activity.
2.2.5 Competent cell preparation
For plasmid preparation and protein overexpression, DH5α and BL21 competent
cells were prepared according to the Molecular Cloning (Sambrook, 2001). The E. coli
cultured from a single colony were incubated at 18°C for about 40 hours to reach
absorbance of 0.4-0.8 at O.D. 600 nm. Let the culture medium stand on ice for 10
minutes. Medium was centrifuged at 3600 rpm under 4°C for 10 minutes. Added 1/3
total volume transformation buffer (10mM PIPES, 15mM CaCl2Ƿ2H2O, 250mM KCl,
55mM MnCl2Ƿ4H2O, pH 6.7) then let the tube stand on ice for another 10 minutes.
Centrifuged at 3600 rpm under 4°C for 10 minutes. Added transformation buffer (8%
total medium volume) and DMSO (Bioman, 7 % transformation buffer volume) into the
pellet and resuspended. Then let liquid nitrogen freeze the sample and stored in -80°C.
2.2.6 Ligation and transformation
The insert and pETDUET-1 were mixed with 2 μl ligation high ver.2 (Toyobo) at
16°C for 30 minutes, eGFP and pETDUET should both be treated with shrimp alkaline
phosphatase (sAP) first to prevent self-ligation. After ligation, I added 50 μl DH5α
competent cell into the ligation solution and put the mixture on ice stood for 10 minutes.
Then the tube was incubated at 37°C for 3 minutes. Let it stood on ice for another 2
minutes. 50 μl SOB (2% bacto tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5mM KCl,
10mM Mg2+added after autoclaved) was added into the tube and the transformed cells
were recovered for 30 minutes at 37°C. Transformed cells were further spread out on
the LB plate with ampicillin (5% tryptone, 2.5% yeast extract, 5% NaCl, 2% 1N NaOH,
1.5% agarose, 100 μg/ml ampicillin) and cultured at 37°C for 14~16 hours.
2.3 Plasmid mini-preparation
The proper colony was picked up and cultured in up to 2 ml Luria-Bertani (LB)
broth (10 g tryptone, 5 g yeast extract and 5 g sodium chloride in 1 liter dH2O) with
ampicillin at 37°C for 14~16 hours. Then the medium and the cells were centrifuged at
15000 rpm for 5 minutes. After the supernatant was discarded, 150 μl MP I solution (25
mM Tris-HCl, 10 mM EDTA, pH 8.0, 50 mM glucose, RNase μg/ml) was added to
resuspend cell pellet. Then 150 μl MP II (0.2N NaOH, 1% SDS, freshly prepared)
solution was added to lysis pellet and immediately followed by adding 150 μl MP III
(3M potassium acetate solution, pH 5.2) solution III to neutralize the solution. The
mixture was centrifuged at 15000 rpm for 5 minutes and transferred the supernatant to
the new tube twice. Isopropanol (KANTO KAGAKU, 0.7x total volume) was added
then the mixture was centrifuged at 15000 rpm for 15 minutes. The supernatant was
discarded then 250 μl 80% alcohol was added to wash the DNA pellet. The mixture was
centrifuged at 15000 rpm for 5 minutes and the supernatant was discarded again. 250 μl
80% alcohol was added to wash again and the mixture was centrifuged again. The DNA
sample was dissolved in 20 μl ddH2O and stored at -20°C.
2.4 Plasmid midi preparation
The proper colony was picked up and cultured in 50 ml LB broth with ampicillin at
37°C for 14~16 hours. The cultured medium was transferred into the 50 ml centrifuge
tube and centrifuged at 3000 rpm for 10 minutes under 4°C, then the supernatant was
discarded. Preparation procedures were referred to HiPure Plasmid Midiprep Kit’s
protocol (Invitrogen). The sample was then added Suspension Buffer (RNase added),
Lysis Buffer, and chilled Neutralization Buffer, 4 ml each. The flow-through was then
transferred into the column which was equilibrated by 10 ml Equilibrium Buffer.
Discard flow-through and washed the column by 10 ml Wash Buffer twice.
Then the column was put onto a new 15 ml centrifuge tube after washing. 5 ml
elution Buffer was added into the column to elute the plasmid, and the plasmid was
precipitated by adding 3.5 ml isopropanol into the centrifuge tube. The tube was then
centrifuged at 9000 rpm for 30 minutes under 4°C, and the supernatant was further
discarded. DNA pellet was washed by 1 ml chilled 70% ethanol, then the sample was
centrifuged at 12000 rpm for 10 minutes under 4°C.The sample was later transferred
into a new microcentrifuge tube and the washing process should be repeated at least two
times. Let the plasmid pellet stood for 30 minutes before dissolved by 30 μl TE-8.0 (10
mM Tris-HCl, 1 mM EDTA, pH 8.0).
2.5 Total protein extraction.
Transformed BL21 and Rosetta™ competent cells (Novagen) were cultured in 50
ml LB broth with ampicillin under 37°C until O.D. 600 reached 0.4~0.6. Isopropyl β-D-
1-thiogalactopyranoside (IPTG) was later added into the culture to a final concentration
of 1mM and cells were cultured at 27°C for several hours (depends on the experiment).
The medium was later transferred into a 15ml centrifuge tube and spun at 6000 rpm for
10 minutes under 4°C. Then we discarded the supernatant and added 5 ml lysis buffer
(50 mM Na3PO4Ƿ12H2O, pH 7.0, 0.1 M NaCl, 0.1 mM EDTA, 0.2% Triton X-100,
added and 20 μg/ml Roche cOmplete protease inhibitor cocktail before use) to
resuspend the pellet. Cells were destructed by sonication (Misonix Sonicator 3000).
During the whole sonication process, the sample should be placed on ice. The lysate
was later transferred into microcentrifuge tubes and spun at 12000 rpm for 20 minutes
under 4°C. Collect both supernatant and pellet separately, then the supernatant should
be filtrated with 0.45 μm PVDF filter (Millipore) before SDS-PAGE and affinity
chromatography.
2.6 SDS-PAGE and CBR staining.
The protein samples were mixed with 5x sample buffer (250 mM Tris-HCl, pH 6.8,
10% SDS, 0.5% (w/v) bromophenol, 50% glycerol, 5% β-mercaptoethanol) and placed
on dry bath incubator (Major Science) under 100°Cġfor 10 minutes. The SDS-
polyacrylamide gel (10% separation gel and 4% stacking gel) was placed in the tank
filled with TGS buffer (50 mM Tris-HCl, pH 8.3, 380 mM Glycine, 0.1% SDS). The gel
was further stained by CBR solution (1 g coomassie brilliant blue R-250 [Sigma B-
0149] dissolved in 10% glacial acetic acid and 20% methanol) for 30 minutes and the
stained gel was destained by destain buffer (10% glacial acetic acid, 20% methanol).
2.7 Western analysis
PVDF membrane (Roche) and filter papers (Blot Absorbent Filter Paper [extra
thick], Bio-Rad) were rinsed with western transfer buffer (25 mM Tris-HCl, 192 mM
Glycine, pH 8.3). The membranes and gel should be placed on platinum anode of the
cell (Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell, Bio-Rad) in following
arrangement: filter paper, PVDF membrane, SDS-PAGE gel, and filter paper. The
current should be set at 0.06~0.18 A (3 mA/cm2, depends on the membrane size) during
the transferring and the membrane should be transferred for 1 hour.
After protein transfer, TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1%
Tween 20) was used to wash the membrane twice. The membrane would later be
blocked with blocking buffer (5% steam milk dissolved in TBST) for 1 hour. The
membrane should be washed with TBST thrice (10 minutes each) after blocking, then
the membrane was soaked in 1stantibody solution, which would be anti-His mouse
monoclonal antibody (Santa Cruz) or anti-eGFP rabbit polyclonal antibody (Bioman), at
room temperature for an hours. Membrane was washed with TBST three times after 1st
antibody application, and the membrane was soaked in 2ndantibody solution (Anti-
mouse goat antibody conjugated with horseradish peroxidase [Jackson 115-035-075] or
anti-rabbit goat antibody conjugated with horseradish peroxidase [Jackson 111-035-
003]) for an hour. The membrane should be washed with TBST three times after 2nd
antibody application and then rinsed with HRP substrate (Millipore WBLUC0500) for 1
minute. Use ddH2O to wash the membrane and then the membrane was imaged by
BioSpectrum 2D Imaging System (UVP BioSpectrum 800).
2.8 Affinity chromatography.
Ni-NTA spin column (Qiagen 31314) and His-Trap gravity column (GE) were used
to purify 6x His fusion proteins, and the experiments were performed according to each
column’s protocol. For Ni-NTA spin columns, the Ni-NTA spin column was equilibrated
with 600 μl NPI-10 buffer (50 mM Na2H2PO4, 300 mM NaCl, 10 mM imidazole, pH =
8.0). Centrifuge for 2 minutes at 890 g. Load up to 600 μl of the cleared sample onto the
pre-equilibrated Ni-NTA spin column. Centrifuge for 5 minutes at 270 g, and collect the
flow-through. Wash the Ni-NTA spin column twice with 600 μl NPI-20 buffer (50 mM
Na2H2PO4, 300 mM NaCl, 20 mM imidazole, pH = 8.0). Centrifuge for 2 minutes at
890 g. Elute the protein twice with 300 μl buffer NPI-500 (50 mM Na2H2PO4, 300 mM
NaCl, 500 mM imidazole, pH = 8.0). Centrifuge for 2 minutes at 890 g, and collect the
flow through.
For Ni-NTA gravity columns, the His-Trap gravity columns were equilibrated with
10 ml binding buffer (50 mM Na2H2PO4, 300 mM NaCl, 10 mM imidazole, pH = 8.0).
Load up to 3 ml of the cleared sample into the pre-equilibrated column and collect the
flow-through. Wash the Ni-NTA spin column with 10 ml binding buffer and collect the
flow through. Elute the protein with 5 ml elution buffer (50 mM Na2H2PO4, 300 mM
NaCl, 250 mM imidazole, pH = 8.0) and collect the flow through.
2.9 Protein concentration determination
Different concentration of bovine serum standards (100, 200, 300, 400 and 500
μg/ml) were prepared. Standards and protein samples were added into 96-well
microtiter plate in duplicate. The Dye-Reagent (Bio-Rad 500-0006, diluted 5 folds with
dH2O) was later added to each samples and standards. Samples and dye were mixed and
placed at room temperature for 3 minutes before measured the O.D. 595 nm (Thermo
Multiskan FC).
2.10 Cell viability analysis
Cell viability was measured by MTT (Sigma M5655) assay. MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a water soluble tetrazolium
salt (yellow), and the concentration of MTT stock solution was 5 mg/ml . According to
MTT product applications, after MTT was cleft by active mitochondrial dehydrogenases
of living cells, the soluble yellow dye will be converted to the insoluble purple
formazan. The amount of formazan can be measured by dissolving formazan in 0.08 N
HCl in isopropanol, and the absorbance of converted dye was measured at a wavelength
of 570 nm by Microplate Reader (Beckman DTX 880).
In this experiment, 3 x 104HS68 cells were seeded on 96 well plates (BD) 12
hours before adding different amount of gelatin-PEI nanoparticles (Obtained from Dr.
Yi-You Huang and Dr. Min-Ju Chou). Serum-free DMEM should be used during
gelatin-PEI nanoparticles application. Wash with PBS three times after 2 hours
nanoparticles application. HS68 cells were further cultured in 10% FBS DMEM with
24, 48 or 72 hours. Each well was treated with 10 μl MTT stock solution for 3 hours.
Remove the medium and 0.08 N HCl in isopropanol was added. Measure the
wavelength of 570 nm by Microplate Reader (Beckman DTX 880).
2.11 Cellular uptake analysis
Gelatin-PEI nanoparticles (conjugated with rhodamine isothiocyanate, RITC)
uptake efficiency was determined by the RITC-positive cells detected by flow
cytometry (BD FACSCanto II). In brief, HS68 cells were cultured in 6 well plate
(Corning) for 12 hours. Gelatin-PEI nanoparticles were added into each well with
different concentration (0, 5, 10 and 50 μg/ml). Serum-free DMEM should be used
during gelatin-PEI nanoparticles application. Wash with PBS three times after 2 hours
nanoparticles application. HS68 cells were further cultured in 10% FBS DMEM with 22
hours. Use 0.05% trypsin to collect HS68 cells and fix them with 4 % paraformaldehyde
(Sigma P6148). Wash the cells two times with PBS between previous steps. Cells were
filtrated with .40 nm mesh filter (BD) before flow cytometry analysis. Flow data was
analyzed by FlowJo 7.6.1.
2.10 Intracellular protein delivery of gelatin-PEI nanoparticles
Purified eGFP and gelatin-PEI nanoparticles were mixed with weight ratio as 1:2
(final eGFP concentration = 10 μg/ml). The mixed solution was kept in dark and stood
at room temperature for an hours. HS68 cells were cultured in 24 well plate (Corning)
with cover glass (Deckgläser 18 mm) for 12 hours. Gelatin-PEI nanoparticles and eGFP
mixture were added into each well with different concentration (0 and 10 μg/ml eGFP).
Serum-free DMEM should be used during gelatin-PEI nanoparticles application. Wash
with PBS three times after 2 hours nanoparticles application. HS68 cells were further
cultured in 10% FBS DMEM with 4 hours. Discard culture medium and fix HS68 cells
with 4 % paraformaldehyde (Sigma P6148). Fix cover glass with mounting medium on
the micro slide glass (MATSUNAMI pro-01). Red fluorescence and green fluorescence
were observed and recorded by confocal microscopy (Zeiss LSM 780 Confocal).
Chapter three Result
3.1 Identification of His-eGFP and His-Yamanaka factors overexpressed by BL21
and Rosetta™ competent cells
3.1.1 Total protein analysis by coomassie brilliant blue and Western blotting
After IPTG induction, total protein extract from empty pETDUET-1 and target
genes were collected after sonication and centrifugation. Protein concentration after
extraction was determined by Bradford method.
In total protein SDS-PAGE, total proteins were stained by coomassie brilliant blue-
R for 5 minutes. Total protein analysis (figure 1-1) showed that eGFP was
overexpressed only in eGFP-pETDUET-1 sample, but not in empty pETDUET control
group. Western blot analysis also showed the same result obviously (figure 1-2). Due to
high expression level of his-eGFP, only BL21 was used in eGFP overexpression
experiments.
For Yamanaka four factors, all factors were first overexpressed by BL21, but the
expression level were not obvious. So Rosetta™ competent cells were used to improve
His-Yamanaka factors’ expression level.
Total protein analysis (figure 1-3) showed that only Oct4 in BL21, Sox2 in both
BL21 and Rosetta™ and c-Myc in Rosetta™ had clear overexpression pattern, but other
groups had no significant change. In contrast, Western blot analysis (figure 1-4) showed
that all four factors were successfully overexpressed after IPTG induction, including
Klf4 and Oct4, indicated that overexpression level or the solubility are quite different
between all four factors and different competent cells.
Overall, His-eGFP and all Yamanaka factors were successfully overexpressed by
these two competent cells, and except for His-eGFP, all other factors needs further
condition modification to elevate the expression amount of these fusion proteins.
3.1.2 Induction time optimization of His-eGFP and His-SKOM
In order to optimize the best IPTG induction time for His-eGFP fusion proteins, the
inducted cells (three replicates, Group A, B and C) was collected every 3 hours after
IPTG application. After only 3 hours IPTG induction, His-eGFP was able to be
overexpressed by BL21 (figure 2-1), and after 9 to 15 hours IPTG induction, expression
level of His-eGFP would reach the highest point, than gradually decreased according to
Western blot quantification by ImageJ (figure 2-2).
These results indicated that His-eGFP should be inducted between 9~15 hours to
reach the highest expression level, and longer induction would not be suggested.
On the other hand, all His-Yamanaka factors were able to be detected after 4 hours
IPTG induction (figure 2-3), but no specific overexpression pattern could be observed in
His-Sox2 group, and 4 hour IPTG induction is enough for all His-Sox2 groups (figure
2-4). For His-Klf4 and His-Oct4, highest expression level appeared after 4 hour IPTG
induction, but no significant expression could be seen after 12 hours (figure 2-4).
Besides, the highest expression level of His-Myc appeared after 16 hour IPTG
induction, but 8 hour induction might be enough (figure 2-4). These result indicated that
except for c-Myc, 4 hours IPTG induction is enough to obtain sufficient amount of His-
SKO, longer induction culture is unnecessary.
3.2 Purification of His-eGFP and His-Yamanaka factors by affinity columns
After protein extraction, His-eGFP and His-Yamanaka factors were purified by Ni-
NTA spin columns and HisTrap gravity columns. Total protein analysis of His-eGFP
(figure 3-1) and Western blot analysis (figure 3-2) both showed that most of the His-
eGFP could be purified by both spin and gravity columns, and purified extracts
contained only a small portion of proteins other than His-eGFP, especially in spin
column group. According to this data, His-SKOM were further purified by spin columns
only to obtain purer samples.
For His-Yamanaka factors, total protein analysis (figure 3-3) showed that purified
extracts contained less proteins that were not His-Yamanaka factors, but His-Yamanaka
factors had no significant change. Western blot analysis (figure 3-4) showed that after
purification, all four fusion proteins could be successfully purified even though protein
amount of all factors were less than total extracts.
3.3 Cellular uptake of gelatin-PEI nanoparticles in HS68 cell line
To evaluate the uptake efficiency of gelatin-PEI nanoparticles in HS68 cell line,
gelatin-PEI nanoparticles were added into culture medium with different concentration
(0,5,10,50 μg/ml separately) and analyzed by flow cytometry.
Histogram analysis by FlowJo 7.6.1 (figure 4) showed that when nanoparticle’s
concentration was below 50 μg/ml, no significant peak shift could be observed, but
almost 90 % cells were RITC-positive when nanoparticle’s concentration reach 50
μg/ml. Further experiments are needed to determine uptake efficiency when
concentration over 50 μg/ml.
3.4 Cytotoxicity analysis of gelatin-PEI nanoparticles in HS68 cell line
Nanoparticle cytotoxicity at different concentration (10, 20, 30, 40 and 50 μg/ml)
was measured by MTT assay. The result of MTT assay (figure 5) indicated that after
nanoparticle application, cell viability would decrease to about 70% for all groups
except 0 μg/ml control after 24 hours culture. On the other hand, cells would recover to
more than 90% of original population after 72 hours culture compared with control
group.
To successfully generate iPS cells with proteins, nanoparticles and proteins should
be apply to cells several times. Therefore, according to the result of MTT assay, it is
possible to use HS68 cell line as reprogramming target due to low cytotoxicity of the
nanoparticles and good recovery rate of HS68 cells.
3.5 His-eGFP delivery by gelatin-PEI nanoparticles in HS68 cell line
Gelatin-PEI and His-eGFP were applied to HS68 cells after 2 hours mix with 2:1
weight ratio (30 μg/ml: 15 μg/ml), and the delivery results were taken by confocal
microscopy. Green fluorescence (His-eGFP) and red fluorescence (RITC tag on
nanoparticles) were both detectable after 24 hours application, and co-localization was
observed (figure 6). Even though there were a lot of nanoparticles that were not
successfully entering the cells can be observed, some of them truly penetrated into the
cells and remain in the cells can be detected by Z-stack analysis by confocal
microscopy.
Overall, gelatin-PEI nanoparticles were able to deliver His-eGFP into HS68 cells
without destroy eGFP’s function. This result indicated that His-Yamanaka factor may be
transported into the cells with same method, but further tests are still needed.
Chapter four Discussion
iPS cells are considered to be the future star in regenerative medicine due to its
ethical- and transplant rejection-free characteristics. But the reprogramming vectors of
iPS cells, especially viral systems, limit the possibility of iPS cells in clinical use. In
order to solve this problem, several DNA-free methods have been developed. However,
almost every method has its limitation and advantages, and these methods are not as
user-friendly as traditional viral vectors. Therefore, in this research proteins were
combined with nanoparticles as a new reprogramming vectors, hope to find out a
balance between potential risk of genome instability and transfection efficiency.
In this study, although no primary reprogramming colony has been observed yet,
proteins have been shown to be a possible reprogramming vectors in previous research
with extremely low reprogramming efficiency (28) (29). In 2013, Majad Khan et al.
developed a cationic bolaamphiphile combine with purchased Yamanaka factors as
reprogramming vectors (63). These researches indicated that using proteins as
reprogramming vectors is possible, but none of them can avoid co-treatment with
specific chemical compounds or even culture transformed cells on Matrigels during
reprogramming process. It suggested that during protein transduction process, the
amount of proteins might be not enough to sustain the reprogramming condition.
Protein loss during reprogramming and delivery may due to several reasons,
including encapsulation waste, endosome escaping failure and protein degradation
within the host cells. By using cationic nanoparticles, the protein escaping percentage
may evaluate due to massive proton acceptors in the polymers, and the encapsulation
waste may be reduced due to free of organic solvent during protein encapsulation.
Besides, with different cationic polymers and target proteins, the protein-
nanoparticle binding efficiency and surface conformation after binding would be
different, so it is important to find out a proper cationic polymer which can match the
need in different delivery aims. Although Majad Khan et al. successfully generate iPS
cells by this method, more cationic nanoparticles need to be test in order to increase the
protein binding efficiency, and further increase the amount of proteins that can be
successfully delivered into the host cells.
In addition to cationic polymer optimization, there are several important factors
need to be examined in future experiments. One is the cytotoxicity of protein samples,
especially overexpressed by E.coli. The endotoxins generated by bacteria should be
further removed before reprogramming experiments, and test whether endotoxin
removal can enhance the cell viability or not. Another problem is the proper amount of
proteins that need to be loaded during every reprogramming cycle, and find out the
balance between reprogramming efficiency and the toxicity of nanoparticles. Also, the
expression system of Yamanaka factors can be substituted for mammalian expression
system, such as CHO cells to overcome post-translation modification and folding
problems.
Overall, nanoparticles combine with proteins as delivery vectors may have wide
applications besides reprogramming, if the safety and stability of this method can be
proved by further experiments, proteins and cationic nanoparticles together may have
promising future in medical and reprogramming area.
Chapter five Conclusion
iPS cells are artificial stem cells originated from reprogrammed somatic cells by
specific transcription factors, such as Sox2 and Oct4. In order to develop a novel DNA-
free method for the generation of iPS cells, proteins were considered to be the proper
reprogramming vectors combine with cationic gelatin-PEI nanoparticles. Proteins are
able to modulate the gene expression of the host cells, easier to obtain than other DNA-
free vectors, and can be simply modified by plasmid construction. Also, proteins are
overall negatively charged, so cationic nanoparticles may able to interact with them and
carry them into the cells as well as DNA.
In this study, eGFP and Yamanaka factors were fused with 6 x His-tag and
successfully overexpressed by E.coli overexpression system. 6 x His-tag was added
because it provides an affinity tag to purify those target proteins. Also, the proper
induction time, which should be between 9 to 12 hours, was found as the result
indicated. Although His-Yamanaka factors were not expressed as many as His-eGFP,
these factors were still possible to be obtained by E.coli overexpression system.
Furthermore, His-eGFP and all His-Yamanaka factors were able to be purified by Ni-
NTA affinity columns for nanoparticle encapsulation.
On the other hand, cell viability assay showed that the cytotoxicity of gelatin-PEI
nanoparticles was durable with HS68 cell line, and these cells could repopulate after
three days application, which indicated that repeated cycles during iPS cells generation
are possible. Also, cellular uptake efficiency would reach almost 90% when
nanoparticle concentration is 50 μg/ml, and it is enough for further applications.
At the end of the study, the protein delivery result showed that eGFP were able to
cross the membrane with the presence of gelatin-PEI nanoparticles. Although it is
unclear whether these proteins were in the endosomes or not, the result still indicated
that using gelatin-PEI to deliver Yamanaka factors is possible in the future.
In conclusion, gelatin-PEI nanoparticles were able to carry proteins, such as eGFP
in this research, into human fibroblast cell line. Combining cationic nanoparticles and
proteins is a high potential way to become a stable and integration-free reprogramming
method in the future. For further study, repeat applications of Yamanaka factors
combine with gelatin-PEI nanoparticles should be performed and the reprogramming
efficiency should be measured and compared with other methods.
Name Sequence (5’- 3’) Adaptor forward
Adaptor reverse Sox2 forward Sox2 reverse Klf4 forward Klf4 reverse Oct4 forward Oct4 reverse c-Myc forward c-Myc reverse eGFP forward eGFP reverse
Table 1. Primer List of Yamanaka Factors, eGFP and Adaptor for
Yamanaka Factors.
Figure 1-1. eGFP Overexpression Verification by CBR Staining.
Control
Figure 1-2. eGFP Overexpression Verification by Western Blot Analysis.
Control
Figure 1-3. Sox2, Klf4, Oct4 and c-Myc Overexpression Verification by CBR Staining.
Control R
R B
Figure 1-4. Sox2, Klf4, Oct4 and c-Myc Overexpression Verification by Western Blotting Analysis.
Control RR
B
Figure 2-1. Induction Time Optimization of His-eGFP.
Control
μμ
Figure 2-2. Quantification of His-eGFP Overexpression Level with Different IPTG Induction Period.
Control
μμ
Figure 2-3. Induction Time Optimization of His-Yamanaka Factors.
Control
μμ
Figure 2-4. Quantification of His-Yamanaka Factors Overexpression Level with Different IPTG Induction Period.
μμ
Figure 3-1. His-eGFP Purification was Verified by CBR Staining.
Control
μμ
Figure 3-2. His-eGFP Purification was Verified by Western Blotting.
Control
μμ
Figure 3-3. His-Yamanaka Factors Purification was Verified by CBR Staining.
Control
B P
μμ
Figure 3-4. His-Yamanaka Factors Purification was Verified by Western Blotting.
Control
B P
μμ
Figure 4. In vitro Protein Delivery Efficiency of GR-PEI Nanoparticles in Treating Hs68 Cell Line.
Figure 5. In vitro Cytotoxicity Analysis of Gelatin-PEI Nanoparticles in Treating HS68 Cell Line.
Figure 6. In vitro His-eGFP Delivery by Gelatin-PEI Nanoparticles in Treating HS68 cell line.
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