Transcript
Studies on Transdifferentiation of Mature Cell Type
into Insulin Producing β-Cells
A DISSERATION SUBMITTED FOR THE PARTIAL
FULFILLMENT OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
BY
SHUMAILA USMAN
Dr. Panjwani Center for Molecular Medicine and Drug Research
(International Center for Chemical and Biological Sciences)
University of Karachi, Karachi-75270, Pakistan
2016
Dedication
I would like to dedicate my doctoral dissertation to my
dearest mother, “ Rehana Perveen”
&
dearest father,” Muhammad Usman”
for their incredible love, prayers, encouragement and endless support which give me strength to chase my
dreams.
LIST OF CONTENTS
ACKNOWLEDGMENTS……………………………………………………………….I
LIST OF FIGURES……………………………………………………………………III
LIST OF TABLES…………………………………………………………………......VI
LIST OF ABBREVIATIONS…………………………………………………..…....VII
SUMMARY………………………………………………………………………..…….X
KHULASA…………………………………………………………………………...…XI
1. INTRODUCTION
_____________________________________________________________
1.1 Diabetes Mellitus……………………………………………………..….1
1.2 Current Treatments Used for Type 1 Diabetes Mellitus……………...1
1.2.1 Medications Maintaining Blood Glucose Level………………..3
1.2.2 Whole Pancreas Transplant………………………………….…3
1.2.3 Isolated Pancreatic Islet Cell Transplantation………………...3
1.2.4 Cellular Therapy and Regenerative Medicine………………...4
1.3 Cellular Therapeutic Strategies for β-Cells Regeneration……………4
1.3.1 Use of Small Molecules …………..…………………………….4
1.3.2 Differentiation of Stem Cells………………………………...….6
1.3.3 Transdifferentiation of Somatic Cells………………………...12
1.4 Environmental Factors Affecting Differentiation of β-Cells ………..15
1.4.1 Oxidative Stress ………………………………………………..15
1.4.2 Pancreatic Tissue Environment……………………………….16
1.5 Objectives of the Present Study………………………………….....…17
2. MATERIAL AND METHODS
_____________________________________________________________
2.1 Reagents and Cell Lines…………………………………………….….18
2.2 Gene Expression Analysis of Pancreatic Tissue……………………...18
2.2.1 Pancreatic Tissue……………………………………………….18
2.3 Culturing of NIH3T3 Cells………………………………………….…21
2.3.1 Cell Thawing and Expansion………………………………….21
2.3.2 Sub Culturing of NIH3T3 cells…………….…………………23
2.3.3 Cryopreservation…………………………………………….…23
2.4 Analysis of Protein Expression in NIH3T3 Cells…………………….23
2.4.1 Immunocytochemistry…………………………………………23
2.4.2 Flow Cytometry……………………………………………...…24
2.5 Analysis of Genes and Proteins Expression in NIH3T3 Cells
Transfected with Nrp1, MafA, Nrp1/MafA…………………………..24
2.5.1 Culturing of Bacteria…………………………………………..25
2.5.2 Plasmid Isolation by Maxiprep………………………………..26
2.5.3 Plasmid Quantification……………………………………...…26
2.5.4 Culturing of Packaging Cell Line………………………….….27
2.5.5 Adenovirus Production……………………………………...…27
2.5.6 Transfection of NIH3T3 Cells with MafA, Nrp1, and
MafA/Nrp1 ……………………………………………………..28
2.5.7 Analysis of Pancreatic Genes after Transfection…………….29
2.5.8 Analysis of Pancreatic Proteins after Transfection………….29
2.6 Preconditioning of NIH3T3 Cells……………………………………..30
2.6.1 Experimental Groups…………………………………….….....30
2.6.2 Pancreatic Extract……………………………………………...30
2.6.3 Dexamethasone Treatment………………………….…………30
2.6.4 Combination of Pancreatic Extract and Dexamethasone……31
2.6.5 Morphological Examination of Preconditioned Cells………..32
2.6.6 Analysis of Pancreatic Genes in of Preconditioned Cells …...32
2.6.7 Analysis of Pancreatic Proteins in of Preconditioned Cells....32
2.7 Analysis of Pancreatic Gene Expression in Response to 2,4-
Dinitrophenol (DNP)……………………………………………...……32
2.7.1 DNP Treatment……………………………………...…………32
2.7.2 Morphological Examination of DNP Treated Cells………….33
2.7.3 Analysis of Pancreatic Genes in DNP Treated Cells..………..33
2.7.4 Analysis of Pancreatic Proteins in DNP Treated Cells………33
2.8.1 Statistical Analysis………………………………………...…...33
3. RESULTS
_____________________________________________________________
3.1 Propagation and Characterization of Mouse Embryonic Fibroblasts
(NIH3T3 Cells)………………………………………………………....34
3.1.1 Morphological Characteristics…………………………..….…34
3.1.2 Gene Expression Analysis by RT-PCR…………………….…34
3.1.3 Protein Expression Analysis by Immunocytochemistry……..34
3.2 Transfection of NIH3T3 Cells with Nrp1………………………...…...41
3.2.1 Morphological Characteristics…………………………..….…41
3.2.2 Gene Expression Analysis…………………………………..…41
3.3 Transfection of NIH3T3 Cells with MafA……………..……………..41
3.3.1 Morphological Characteristics…………………….………….41
3.4 Co-transfection of NIH3T3 Cells with MafA and Nrp1……………..47
3.4.1 Morphological Characteristics……………………………..….47
3.4.2 Expression of Pancreatic Genes in MafA, Nrp1 and Co-
transfected Cells…………………………………………….….47
3.4.3 Expression of Pancreatic Proteins in MafA, Nrp1 and Co-
transfected Cells……………………………………………..…47
3.5 Preconditioning of NIH3T3 Cells with Dexamethasone and
Pancreatic Extract………………………………………………......…61
3.5.1 Cytotoxic Effect of Dexamethasone Quantified by JC-1
Mitochondrial Membrane Potential Assay……………...……61
3.5.2 Oxidative Stress Induction by Dexamethasone: Effect on
Reactive Oxygen Species (ROS) Level Quantified by 3’-(p-
hydroxyphenyl) Fluorescein (HPF)……………...……………61
3.5.3 Morphological Characteristics………………………………...62
3.5.4 Pancreatic Gene Expression after Preconditioning of NIH3T4
Cells with Dexamethasone and Pancreatic Extract………….62
3.5.5 Pancreatic Protein Expression after Preconditioning of
NIH3T3 Cells with Dexamethasone and Pancreatic
Extract…………………………………………………………..68
3.6 Preconditioning of NIH3T3 Cells by 2,4- Dinitrophenol (DNP)…….86
3.6.1 Optimization: Morphological Characteristics………………..86
3.6.2 Pancreatic Gene Expression after Preconditioning of NIH3T3
cells with DNP……………………………………………….….86
3.6.3 Pancreatic Protein Expression after Preconditioning of
NIH3T3 cells with DNP…………………………………….….86
4. DISCUSSION AND CONCLUSION
_____________________________________________________________
DISCUSSION………………………………………………………….….........98
CONCLUSION……………………………………………………..…………104
REFERENCES……………………………………………………………..…105
LIST OF PUBLICATIONS…………………………………………………..118
PERSONAL INTRODUCTION ………………………………………….…119
APPENDIX-I……………………………………………………………….…121
APPENDIX-II………………………………………………………………....125
GLOSSARY………………………………………………………………..….130
i
ACKNOWLEDGMENT
Alhmadulillah! Above all, my first and most profound thanks to Almighty ALLAH (the
Most Merciful and Most Beneficent) for his countless blessings, for guiding me at each
and every path of life. I also pay highest respect to Holy Prophet Muhammad (Peace
be upon him) for enlightening our souls with Allah’s message.
I am grateful to my mentor Dr. Asmat Salim with the core of my heart, for her
motivation, and support during hurdles at any time. Her generosity and dedication helped
me in building up my confidence. I am thankful for her interminable guidance and
support.
Moreover, this research could not have been performed without the state of the art infra-
structure which is indeed the vision of Prof. Dr. Atta-ur-Rahman (F.R.S., N.I., S.I., T.I.) and
its appreciation by Mrs. Nadira Panjwani (H.I) which has nurtured under the visionary,
academic and administrative leadership of Prof. Dr. M. Iqbal Choudhary (H.I., S.I., T.I.). I
thank these three personalities from the core of my heart, who toiled their sleep for
meritorious students of Pakistan, despite having chances and every material luxury of
life.
My affectionate gratitude to my beloved teacher Dr. Siddiqa Jamall (belated), the
driving force in research. She has always inspired me not only in my studies but also in
other aspects of life. I am very thankful for her help, leadership and tremendous
contribution in the accomplishment of my goal.
My intense gratitude to my senior lab colleague, Dr. Irfan Khan and Dr. Nadia Naeem
for tutoring me all the molecular biology techniques and guiding me throughout my
research work. My immense thanks to Dr. Kanwal Haneef for her moral support,
kindness and positive attitude throughout my studies. My enormous thanks to all stem
cell laboratory members, Dr. Nazia Ahmed, Dr. Sumreen Begum, Dr. Hana’a Iqbal,
Dr. Sana Ejaz, Dr. Rakhshinda Habib, Dr. Uzma Jabeen, Ms. Sehrish Usman, Ms.
Aneesa Gul, Ms. Masooma Batool, Ms. Ramla Sana Khalid, Ms. Rida-e-Maria
Qazi, Ms. Tuba Mustafa, Ms. Hiba Warraich, Ms. Midhat Batool, Ms. Tuba Mallik,
Dr. Anwar Ali, Dr. Aleem Akhtar, Dr. Muhammad Waseem and Mr. Gulzar Alam.
ii
All of them were very supportive and contributed with professional and have contributed
with professional and constructive discussions throughout the project. I would also like
to thank our laboratory assistant Mr. Shahid Shakoor for his assistance.
I would also like to thank my friends and colleagues from different laboratories for being
very kind and supportive throughout the study. My friends Ms. Areeba Anwar, Ms.
Aisha Kamal, Ms. Aneesa Gul, Ms. Masooma Batool, Ms. Sana Sharif and Ms.
Syeda Ayesha Naveed for their support, encouragement, care and ecstatic company.
During the entire research period my batch mates have played a great role. They are the
most helping and generous people I have ever met. I feel honoured to be a part of batch
2011. I want to express my deepest appreciation to all of them for their enduring support
and encouragement.
I would like to convey my deepest gratitude to my beloved mother Mrs. Rehana
Perveen and my father Mr. Muhammad Usman, for their unconditional love, prayers,
continuous and perpetual support. Their constant support and believe facilitate me to
achieve my goal. My most loving and affectionate thanks to my cherished siblings
Muhammad Faizan, Rimsha Usman, Shalina Usman and Muhammad Sameer for
their awful care, love and innocent prayers.
I would also like to extend an immense heartfelt gratitude to my husband Mr.
Muhammad Tahir Yaqoob and my in-laws and for their encouragement, love,
kindness, care and prayers.
Special thanks to my sister Ms. Sehrish Usman and my best friend Mrs. Shahgul Saad
for their boundless love, moral support, admiration, motivation and prayers.
Shumaila Usman
Stem Cell Laboratory
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LIST OF FIGURES
Figure
No.
Title
Page
No.
1.1
Central objective of diabetes therapy
2
1.2
Three routes of cellular regeneration
5
1.3
Embryonic / induced pluripotent stem cells differentiation into
pancreatic β-Cells
7
1.4
The iPSC approach to therapy for type 1 diabetes
10
1.5
Insulin producing cells (IPCs) from different cell sources
14
3.1
Morphology of mouse embryonic fibroblasts (NIH3T3)
35
3.2
Expression of pancreatic genes NIH3T3 cells and pancreatic
tissue
36
3.3
Graphical representation of pancreatic genes expression in
NIH3T3 cells and pancreatic tissue
38
iv
3.4
Immunocytochemical analysis of pancreatic proteins in NIH3T3
cells
39
3.5
Morphology of NIH3T3 cells after transfection with Neuropilin-1
42
3.6
Relative insulin expression in NIH3T3 after transfection with
Neuropilin-1 at different time intervals
44
3.7
Morphology of packaging cell line HEK-293
45
3.8
Morphology of NIH3T3 cells after transfection with MafA
46
3.9
Morphology of NIH3T3 cells after transfection with MafA and
Nrp1
48
3.10
Analysis of pancreatic genes in transfected NIH3T3 cells by RT-
PCR
49
3.11
Analysis of pancreatic proteins in transfected NIH3T3 cells
53
3.12
Graphical representation of islet proteins expression in transfected
NIH3T3 cells
57
v
3.13
Cytotoxic measurement of NIH3T3 after dexamethasone
treatment
63
3.14
ROS measurement after dexamethasone treatment
66
3.15
Morphology of NIH3T3 cells after pre-conditioning with
dexamethasone and pancreatic extract
69
3.16
RT-PCR analysis of pancreatic genes in NIH3T3 cells
preconditioned with dexamethasone and pancreatic extract
72
3.17
Analysis of pancreatic proteins in NIH3T3 cells preconditioned
with dexamethasone and pancreatic extract
75
3.18
Analysis of pancreatic proteins in NIH3T3 cells preconditioned
with dexamethasone and pancreatic extract by
Immunocytochemistry
82
3.19
Morphology of NIH3T3 cells after DNP treatment
87
3.20
RT-PCR analysis of pancreatic genes in NIH3T3 cells
preconditioned with DNP
90
3.21
Flowcytometric analysis of pancreatic proteins in NIH3T3 cells
preconditioned with DNP
97
vi
LIST OF TABLES
Table
No.
Title
Page
No.
1.
Forward and Reverse primer sequences, accession number,
annealing temperature and expected product sizes for PCR
analysis
22
vii
LIST OF ABBREVIATIONS
µg Microgram
mL Millilitre
mM Millimolar
ANOVA Analysis of variance
ASCs Adipose derived stem cells
Ad-MSCs Adipose derived mesenchymal stem cells
bp Base pair
BM Bone marrow
BM-MSCs Bone marrow derived mesenchymal stem cells
BSA Bovine serum albumin
cDNA Complementary deoxyribonucleic acid
CO2 Carbon dioxide
DAPI 4’, 6-Diamidino-2-phenylindole
DEPC Diethylpyrocarbonate
DMEM Dulbecco’s modified eagle’s medium
DNA Deoxyribonucleic acid
dNTPs Deoxyribonucleotide triphosphate
DNP 2, 4-Dinitrophenol
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
ESCs Embryonic stem cells
FACS Fluorescence activated cell sorting
FBS Fetal bovine serum
FGF Fibroblast growth factor
FTIC Fluorescein isothiocyanate
Fig Figure
FSC Forward scatter
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
viii
GFP Green fluorescent protein
GLP-1 Glucagon like peptide-1
GSK3β Glycogen synthase kinase 3 beta
hADSCs Human adipose-derived stem cells
HIF-1α Hypoxia inducible factor-1
hiPSC Human induced pleuripotent stem cell
HNF3B Hepatocyte nuclear factor 3b
ICM Inner cell mass
IDE-1 Inducer of definitive endoderm-1
IDE-2 Inducer of definitive endoderm-2
IGF-1 Insulin like growth factor -1
iPCs Insulin producing cells
iPSC Induced pleuripotent stem cell
Klf4 Kruppel- like factor 4
JC-1 5,5’ , 6,6’- tetrachloro-1,1’ ,3,3’ tetra ethylbenzimi-dazolyl
carbocyanin iodide
LB Luria bertani
MafA v-maf musculoaponeurotic fibrosarcoma oncogene homolog A
mE-ASCs Murine epididymal adipose stem cells
mRNA Messenger RNA
MSCs Mesenchymal stem cells
NeuroD1 Neurogenic differentiation 1
Ngn3 Neurogenin 3
Nkx2.2 NK2 homeobox 2
Nkx6.1 NK6 homeobox 1
Nrp1 Neuropilin-1
Oct4 Octamer-binding transcription factor 1
Pax4 Paired box gene 4
PBS Phosphate buffer saline
Pdx1 Pancreatic and duodenal homeobox 1
ix
PFA Paraformaldehyde
RA Retinoic acid
RNA Ribonucleic acid
Rpm Revolution per minute
PI3K Phosphoinositol 3-phosphate
RT-PCR Reverse transcriptase polymerase chain reaction
Sca1 Stem cell antigen 1
SD rats Sprague Dawley rats
Sox-2 SRY (sex determining region Y)- box 2
STZ Streptozotocin
T1D Type 1 Diabetes
TBE Tris/ Borate/ EDTA
TE Tris- EDTA
TGF-β Transforming growth factor beta
TSA Trichostatin
VEGF Vascular endothelial growth factor
Wnt-4 Wingless-type MMTV integration site family, member 4
x
SUMMARY
The present study evaluated different therapeutic strategies to trans-differentiate mouse
embryonic fibroblasts (NIH3T3 cells) into insulin producing cells (IPCs) with respect to
changes in morphology and pancreatic genes and proteins expression in the treated cells.
In the first part of the study, NIH3T3 cells were transfected with MafA (transcription
factor) and Neuropilin-1 (angiogenic factor), either alone or in combination. It was
observed that the genetic manipulation of NIH3T3 cells enhanced the expression of
pancreatic genes as well as proteins, particularly insulin and Ngn3.
In the second part of the study, preconditioning of NIH3T3 cells was done with
dexamethasone (Dx), either alone or in combination with pancreatic extract at two
different protein concentrations (0.05 and 0.4 mg/mL). Pancreatic extract which contains
different pancreatic proteins and growth factors, provide specific microenvironment to
the cells so that their differential potential can be enhanced. Addition of pancreatic
extract enhanced both insulin gene and protein expression levels. Optimal concentration
of Dx (5µM) was selected on the basis of having least cytotoxicity and significant
increase in the ROS production. Preconditioning with Dx showed increase in the insulin
gene expression, while there is a possible inhibitory effect on insulin translation.
NIH3T3 cells were also preconditioned with 2, 4-dinitrophenol (DNP). DNP is a
lipophilic weak acid that uncouples the oxidative phosphorylation by decreasing ATP
production. An optimized concentration (0.1 mM) of DNP was selected on the basis of
having less number of apoptotic cells. After 48 hours re-oxygenation, expression of
almost all pancreatic genes (Ngn3, Nkx6.1, insulin, and glucagon) and proteins (insulin,
MafA, glucagon, Pdx1 and Ngn3) were significantly increased in the treated NIH3T3
cells.
The strategies to induce efficient trans-differentiation of NIH3T3 cells into IPCs have
shown enhanced but variable expression pattern of endocrine markers, specifically β-cell
specific transcription factors demonstrating their successful regeneration. The study
could further be evaluated for their in vivo effect and serve as an improved and effective
cellular therapeutic option for type 1 diabetes.
xi
CHAPTER 1 introduction
1
1.1 Diabetes Mellitus
Diabetes Mellitus (DM) is the most common life-threatening metabolic disease, caused
either by autoimmune destruction of pancreatic β-cells or by insulin resistance in the
peripheral tissues (Akinci, Banga, Tungatt, Segal, Eberhard, Dutton, and Slack, 2013;
Bhonde, Sheshadri, Sharma, and Kumar, 2014). Latest survey reveals that about 347
million people worldwide are suffering from DM (Masuda, Wu, Hishida, Pandian,
Sugiyama, and Belmonte, 2013). This pandemic disease is growing exponentially during
the last few decades, exerting a huge economic burden on individuals and government
(Bluestone, Herold, and Eisenbarth, 2010). By 2030, an estimated 440 million adults will
be suffering from diabetes (Shaw, Sicree, and Zimmet, 2010; Terzic and Waldman,
2011). This chronic disease is categorized into three major types. Type 1 diabetes
(T1DM), a well-known childhood disease, prompting substantial morbidity and
mortality, is the outcome of β- cells destruction. (Borowiak and Melton, 2009; Vija,
Farge, Gautier, Vexiau, Dumitrache, Bourgarit, Verrecchia, and Larghero, 2009;
Borowiak, 2010). The main challenges in developing a treatment of T1DM are
autoimmunity and paucity of insulin producing cells (Borowiak, 2010). The non insulin-
dependent Diabetes Mellitus (Type II DM) is an age dependent metabolic disease,
identified by insulin resistance and dysfunctional pancreatic β cells (Gillies, Abrams,
Lambert, Cooper, Sutton, Hsu, and Khunti, 2007). Gestational DM is another major form
of this disease affecting about 3 -10% of pregnancies, which in severe cases can lead to
neonatal and intrauterine foetal mortality (Pandian, Taniguchi, and Sugiyama, 2014).
1.2 Current Treatments Used for Type I Diabetes Mellitus
T1D patients are unable to conserve normoglycemia which eventually leads to a number
of pathological outcomes like cardiovascular complications, nephropathy, neuropathy,
retinopathy and often death (Zhao, Jiang, Zhao, Ye, Hu, Yin, Li, Zhang, Diao, and Li,
2012). The main objective of the treatment is to maintain insulin demand of the body.
Various treatment options are available to regulate proper blood glucose level, which
largely on the lifestyle changes, particularly dietary restrictions (Fig. 1.1) (Pandian,
Taniguchi, and Sugiyama, 2014).
2
Fig. 1.1: Central objective of diabetes therapy: In hyperglycemia (e.g. diabetes
mellitus), there is excessive blood glucose in the body that is usually caused by low
insulin levels. Diabetes therapies tend to increase the insulin supply more than what is
needed. Maintaining insulin demand and production is the essential objective of the
diabetes therapy. Different strategies including pharmacological approaches and
regenerative strategies have been developed ranging from islet transplantation to stem
cell-based therapy targeted to restore insulin homeostasis and normoglycemia.
Abbreviation: GLP-1, glucagon-like peptide-1 (Holditch, Terzic, and Ikeda, 2014).
3
1.2.1 Medications Maintaining Blood Glucose Level
Major drugs used for the treatment of diabetes mellitus include insulin, metformin,
sulfonylureas, glucagon-like peptide 1 agonists, thiazolidinediones, α-glucosidase
inhibitors, and dipeptidyl peptidase-4 inhibitors (Pandian, Taniguchi, and Sugiyama,
2014). Since the discovery of insulin, exogenous insulin administration is the principal
source to maintain normoglycemia, but it cannot prevent the long-term complications
which include cardiovascular disorders, diabetic retinopathy, and nephropathies (Kelly,
Flatt, and McClenaghan, 2011; Efrat and Russ, 2012). Continuous administration of anti-
diabetic drugs leads to several pathological outcomes including hypoglycemic episodes,
ketoacidosis as well as micro and major complications affecting nervous, retinal, renal,
cerebrovascular, and cardiovascular systems (Pandian, Taniguchi, and Sugiyama, 2014).
1.2.2 Whole Pancreas Transplant
The easiest solution to replace the damaged islet β-cells in T1D, is to implant a whole
pancreatic organ harvested from cadaveric donors (Chhoun, Voltzke, and Firpo, 2012).
Whole pancreatic grafts generally result in rapid control of hyperglycemia, with
substantial termination of exogenous insulin supplementation. However, there are two
main drawbacks, i.e. significant morbidity of the recipient; and consistent and imminent
severe undesirable effects, make the need of strict and life-long immuno-suppression
crucial (Calafiore and Basta, 2015).
1.2.3 Isolated Pancreatic Islet Cell Transplantation
Islet transplantation is another method to replace damaged β-cells. They occupy an
incomparably smaller volume than whole pancreas with minimal invasive procedures.
However, islets disengage from their native ECM upon recovery from donor pancreas,
and are therefore, more difficult to engraft in a different organ. Isolated islets like whole
pancreas also cause immune rejection upon allograft, therefore, also demands strict
recipient’s general immuno-suppression (Pandian, Taniguchi, and Sugiyama, 2014;
Calafiore and Basta, 2015).
4
1.2.4 Cellular Therapy and Regenerative Medicine
Regenerative medicine is defined as the restoration of degenerated or injured tissues by
stimulation of endogenous cells, or by cellular transplantation (Allsopp, Bunnage, and
Fish, 2010). It aims at understanding the tissue development, expansion, homeostasis,
and discovering of novel therapies that restore the function of the damaged or injured
tissue (Allsopp, Bunnage, and Fish, 2010; Green and Lee, 2013). The ultimate goal can
be accomplished via different cellular reprogramming strategies including
dedifferentiation, transdifferentiation and reprogramming with the help of different
transcription factors; and proteins and small molecules involved in the production,
maintenance, and differentiation of pluripotent and somatic cells, as well as in their
target tissue incorporation (Fig. 1.2) (Jopling, Boue, and Belmonte, 2011; Green and
Lee, 2013).
Most favourable therapy for the treatment of T1D is the restoration of a functional β-
cells to regulate blood glucose level (Zhou, Brown, Kanarek, Rajagopal, and Melton,
2008; Melton, 2011). Cellular therapies evade the exogenous insulin dependence or the
modern pump technology and are able to manage hyperglycemia (Zhou, Brown,
Kanarek, Rajagopal, and Melton, 2008). Numerous promising methodologies have been
recommended for β-cells restoration including β-cells stimulation, reprogramming of non
β-cells into β-cells, and regeneration of insulin producing β-cells by direct differentiation
of stem cells or somatic cells (Borowiak, 2010; Baiu, Merriam, and Odorico, 2011;
Melton, 2011; Efrat and Russ, 2012). Cellular regeneration of the lost cell type (β-cells)
is the best approach to target T1D (Borowiak, 2010).
1.3 Cellular Therapeutic Strategies for β-Cells Regeneration
1.3.1 Use of Small Molecules
Small molecules play a significant role in the modulation of stem cell behaviour.
Moreover, this transgene free reprogramming with small molecules is non immunogenic
and more conventional approach (Masuda, Wu, Hishida, Pandian, Sugiyama, and
Belmonte, 2013). Small molecules, like the epigenetic enzyme inhibitors and signalling
5
Fig. 1.2: Three routes of cellular regeneration: Dedifferentiation, Transdifferentiation,
and Reprogramming (Jopling, Boue, and Belmonte, 2011).
6
pathway factors, promote the direct or indirect differentiation to insulin producing cells
(IPCs) either from stem cells or somatic cells by inducing key transcription factors
(Pandian, Taniguchi, and Sugiyama, 2014). For example, 5-aza-2′-deoxycytidine (5-
AZA), a DNA methyl transferase inhibitor, triggers Ngn3 expression and promotes
endocrine cell differentiation in the human pancreatic ductal cell line (PANC-1)
(Lefebvre, Belaich, Longue, Vandewalle, Oberholzer, Gmyr, Pattou, and Kerr-Conte,
2010). Definitive endoderm inducers, IDE-1 and IDE-2, along with indolactam V
generate pancreatic progenitors from mouse ESCs (Borowiak, Maehr, Chen, Chen,
Tang, Fox, Schreiber, and Melton, 2009). Retinoic acid is important for early embryonic
pancreas development, specifically for differentiating mouse and human ESCs into
Pdx1-expressing cells (Ostrom, Loffler, Edfalk, Selander, Dahl, Ricordi, Jeon, Correa-
Medina, Diez, and Edlund, 2008). MSCs were also differentiated recently with XW4.4,
an aminopyrole derivative, into IPCs via HNF3b (hepatocyte nuclear factor 3b)
induction. IPCs generated with XW4.4 have shown cluster formation, insulin secretion
and positive expression of pancreatic endocrine cell markers (Ouyang, Huang, Yu,
Xiong, Mula, Zou, and Yu, 2014).
1.3.2 Differentiation of Stem Cells
Stem and progenitor cells with pluripotent characteristic, possess a promising use in
cellular therapies for several degenerative diseases (Baiu, Merriam, and Odorico, 2011;
Bernardo, Hay, and Docherty, 2008). A remarkable progress has been made in the in
vitro generation of IPCs from embryonic and induced pluripotent stem cells by using
cytokines, hormones, and small molecules (Borowiak and Melton, 2009; Borowiak,
2010). These chemical inducers (small molecules) direct step wise differentiation of ES
cells and iPSC first into definitive endoderm, then into gut tube cells, pancreatic
endoderm, endocrine pancreatic progenitors, and finally into hormone expressing β-
pancreatic cells (Bernardo, Hay, and Docherty, 2008; Allsopp, Bunnage, and Fish, 2010)
(Fig. 1.3).
7
Fig. 1.3: Embryonic / induced pluripotent stem cells differentiation into pancreatic
β-Cells: Step wise differentiation of ESCs or iPSCs into insulin producing β-cells after
sequential treatment with different compounds (Borowiak, 2010).
8
1.3.2.1 Sources of Stem Cells for β-cell Regeneration
(i) Embryonic Stem Cells
At the early embryo stage, inner cell mass (ICM) of the blastocyst is the main source of
embryonic stem cells (ESCs), with the highest differential potential and unlimited self-
renewal capacity to generate a variety of cells for therapeutic purposes (Calafiore and
Basta, 2015). In a study, mouse ESCs were differentiated into IPCs that were found to
stabilize blood glucose level in streptozotocin-induced diabetic mice (Soria, Roche,
Berna, León-Quinto, Reig, and Martín, 2000). Pdx1 positive cells were also generated
from ESCs with Indolactam V (Chen, Borowiak, Fox, Maehr, Osafune, Davidow, Lam,
Peng, Schreiber, and Rubin, 2009). ESCs were successfully differentiated to IPCs by
using five-stage pancreatic differentiation protocol (D'Amour, Bang, Eliazer, Kelly,
Agulnick, Smart, Moorman, Kroon, Carpenter, and Baetge, 2006). The protocol was
further modified to four developmental stages to increase the number of IPCs (Kroon,
Martinson, Kadoya, Bang, Kelly, Eliazer, Young, Richardson, Smart, and Cunningham,
2008). In another study, mouse ESCs has also been differentiated to IPCs via nestin over
expression (Lumelsky, Blondel, Laeng, Velasco, Ravin, and McKay, 2001). However,
with different modified approaches, the number of IPCs obtained is still very low and
hence, could not meet the required amount of insulin produced by these cells to regulate
blood glucose level as compared to the inherent β-cells (Zhang, Jiang, Liu, Sui, Yin,
Chen, Shi, and Deng, 2009). Apart from the ethical and religious concerns, further
limitations of using undifferentiated ESCs involve the rapid proliferation, self-renewal
tendency, lack of contact inhibition, telomerase activity and a potency of post-
transplantation teratoma formation, which are the molecular sources of the iPSCs
tumorigenicity (Kooreman and Wu, 2010).
(ii) Induced Pluripotent Stem Cells (iPSCs)
Despite remarkable progress in cell replacement using human ESCs, it has major ethical
concerns. The use of allogeneic ESC-derived cells is also accompanied with
immunological mismatch. Nuclear reprogramming technology, which allows generation
of induced pluripotent stem cells (iPSCs) from adult somatic cells, has opened a new
path for generating patient-specific pluripotent stem cells (Robinton and Daley, 2012;
Takahashi and Yamanaka, 2013). The recent advent of iPSCs has elevated extensive
9
enthusiasm in the field of regenerative medicine, including interventional therapies for
diabetes. Upon genetic introduction of selected pluripotency-associated factors in adult
somatic cell, a cell is reprogrammed and dedifferentiated into a pluripotent stem cell
(Takahashi, Tanabe, Ohnuki, Narita, Ichisaka, Tomoda, and Yamanaka, 2007; Yu, Hu,
Smuga-Otto, Tian, Stewart, Slukvin, and Thomson, 2009). iPSCs hold incredible
promise as they are pluripotent in nature (Robinton and Daley, 2012). Derived iPSCs
have been shown the characteristics similar to human ESCs; including morphology, gene
expression profiles, elongated telomeres, and the tendency to differentiate into all three
germ layers (Ohmine, Dietz, Deeds, Hartjes, Miller, Thatava, Sakuma, Kudva, and
Ikeda, 2011; Thatava, Armstrong, De Lamo, Edukulla, Khan, Sakuma, Ohmine,
Sundsbak, Harris, and Kudva, 2011). These characteristics of iPSCs give scientists a new
promising opportunity to generate autologous insulin-producing cells (IPCs) to replace
pancreatic β-cells. Patient specific somatic cells can be reprogrammed into iPSCs. These
iPSCs after further conversion into IPCs could be transplanted back to the patient for
effective regulation of blood glucose levels (Manzar, Kim, Rotti, and Zavazava, 2014)
(Fig. 1.4).
One of the examples is the retroviral expression of pluripotency associated factors in
human skin fibroblast derived iPSCs that resulted in insulin-producing islet-like cell
clusters (ILCCs) formation (Tateishi, He, Taranova, Liang, D'Alessio, and Zhang, 2008).
iPSCs have also been produced by lentiviral transduction of SOX2, OCT4, and KLF4
into human fibroblasts. These factors, that share many target genes in embryonic stem
cells, were shown to have remarkable potential for the induction of pluripotency in
mature cells. The iPSCs can be differentiated into insulin producing cells (IPCs) by using
specific ingredients in the culture medium in a stepwise protocol. IPCs generated by this
strategy were shown to express most of the essential β-cells specific transcription factors
(Zhang, Jiang, Liu, Sui, Yin, Chen, Shi, and Deng, 2009). iPSCs treated with GSK3β
inhibitor and activin A and then with a combination of BMP/TGF-β inhibitor and
retinoic acid (RA) showed improved endodermal differentiation into pancreatic
progenitor cells. Additionally, dexamethasone, forksolin, and a TGF-β inhibitor
treatment of pancreatic progenitor cells also resulted in IPCs generation (Kunisada,
Tsubooka-Yamazoe, Shoji, and Hosoya, 2012).
10
Fig. 1.4: The iPSC approach to therapy for type 1 diabetes: Fibroblast of the diabetic
patient reprogrammed into self-renewing iPSCs further differentiated to IPCs, and then
could be transplanted back into the patient to maintain normoglycemia (Manzar, Kim,
Rotti, and Zavazava, 2014).
11
(iii) Bone Marrow derived Mesenchymal Stem Cells (MSCs)
MSCs isolated from different sources; including adipose tissue, bone marrow, umbilical
cord blood etc, have been studied for their differentiation potential into IPCs (Chen,
Jiang, and Yang, 2004; Zhang, Jiang, Liu, Sui, Yin, Chen, Shi, and Deng, 2009). In an
approach, MSCs isolated from bone marrow were differentiated into pancreatic
endocrine cells in the presence of conophylline, betacellulin and activin A. Differentiated
cells secreted insulin and upon transplantation to diabetic mice resulted in the reduction
of blood glucose level (Hisanaga, Park, Yamada, Hashimoto, Takeuchi, Mori, Seno,
Umezawa, Takei, and Kojima, 2008). In another study, BM-MSCs derived from a human
trimester abortus were differentiated into pancreatic islet-like cells and following a four-
step induction protocol, expressed pancreatic islet β-cells specific markers (Ngn3, Pdx1,
MafA, NeuroD1, insulin). Upon transplantation to diabetic mice, the differentiated cells
released insulin and maintained normal blood glucose concentration (Zhang, Shen, Hua,
Lei, Lv, Wang, Yang, Gao, and Dou, 2010).
(iv) Adipose derived Mesenchymal Stem cells
Mesenchymal stem cells can also be isolated from a rich and easily available source,
adipose tissue (Rodriguez, Elabd, Amri, Ailhaud, and Dani, 2005). IPCs differentiated
from human adipose-derived stem cells (hADSCs) has shown up regulation of pancreatic
transcription factors and islet hormones (Timper, Seboek, Eberhardt, Linscheid, Christ-
Crain, Keller, Müller, and Zulewski, 2006). Murine epididymal adipose stem cells (mE-
ASCs) have also been differentiated into insulin producing cells (IPCs) by step-wise
induction protocol. Differentiated cells effectively expressed pancreatic markers; insulin,
glucagon, C-peptide, Pdx1, somatostatin, Glut-2 and pancreatic polypeptide (Chandra,
Phadnis, Nair, and Bhonde, 2009). Similarly, hADSCs were also differentiated into IPCs
(Chandra, Swetha, Muthyala, Jaiswal, Bellare, Nair, and Bhonde, 2011). Forced
expression of Pdx1 by retrovirus mediated transduction into ADSCs successfully
differentiate them to iPCs (Kajiyama, Hamazaki, Tokuhara, Masui, Okabayashi,
Ohnuma, Yabe, Yasuda, Ishiura, and Okochi, 2010).
12
(v) MSCs from Miscellaneous Sources
Human umbilical cord-derived MSCs have been differentiated into islet-like clusters,
which showed increased over expression of pancreatic β-cell related genes (insulin,
Nkx2.2, Pdx1, Glut-2 and Nkx6.1) (Chao, Chao, Fu, and Liu, 2008). Recently, insulin
producing cells have been generated from periosteum-derived MSCs and showed genes
related to β-cell development (Kim, Choi, Ko, Lim, Lee, and Kim, 2012). Endometrial
MSCs and pancreas-derived MSCs have also been differentiated into functional IPCs
(Li, Chen, Chen, Kao, Tseng, Lo, Chang, Yang, Ku, and Twu, 2010). Role of
extracellular matrix (ECM) proteins have also been validated by different studies in IPCs
differentiation, proliferation, and insulin secretion.
1.3.3 Transdifferentiation of Somatic Cells
An alternative strategy is the cellular reprogramming of somatic cells with different
transcription factors (Pandian, Taniguchi, and Sugiyama, 2014). The inter conversion of
one cell type into another can serve as a promising approach for a number of biomedical
applications. Genetic modification of somatic cells has generated different cell types;
including cardiomyocytes, neurons, and β-cells, for use in cellular therapy of
degenerative disorders (Huang, He, Ji, Sun, Xiang, Liu, Hu, Wang, and Hui, 2011;
Vierbuchen, Ostermeier, Pang, Kokubu, Südhof, and Wernig, 2010). Insulin producing
cells have been produced by transdifferentiation of a wide range of cells (Pandian,
Taniguchi, and Sugiyama, 2014). Pancreatic exocrine cells have also been successfully
differentiated into insulin-producing cells after reprogramming with three transcription
factors (Pdx1, Ngn3 and MafA). Glucagon producing cells have also been differentiated
into insulin producing cells (Melton, 2011). Virus mediated gene transfer of exogenous
transcription factors, Pdx1; Pdx1/VP16 (fusion protein) + NeuroD1; Pdx1/VP16 (fusion
protein) + Ngn3, reprogrammed hepatocytes into insulin producing cells (IPCs). IPCs
have also been generated by the forced expression of Ngn3 + Pdx1 + MafA and Pax4,
into non-β cells such as acinar cells and α-cells respectively (Collombat, Xu, Ravassard,
Sosa-Pineda, Dussaud, Billestrup, Madsen, Serup, Heimberg, and Mansouri, 2009).
Transduction of a single transcription factor, Pdx1, results in the differentiation of
13
adipose-derived stem cells into functional IPCs (Fig.1.5) (Kajiyama, Hamazaki,
Tokuhara, Masui, Okabayashi, Ohnuma, Yabe, Yasuda, Ishiura, and Okochi, 2010).
The choice of defined transcription factors hold key to efficient transdifferentiation.
Some of these factors play important roles in pancreatic beta cell development. MafA is
a basic leucine zipper, the homologue of v-Maf oncoprotein and belongs to
musculoaponeurotic fibrosarcoma oncogene family. It is expressed in the initial stages of
β-cell production and involved in insulin gene expression. It is the principle transcription
factor for β-cell development, maturation, reprogramming, production and maintenance
of insulin producing cells. MafA is considered a potent transactivator of insulin gene
(Matsuoka, Zhao, Artner, Jarrett, Friedman, Means and Stein, 2003; Matsuoka, Kaneto,
Stein, Miyatsuka, Kawanori, Henderson, Kojima, Matsuhisa, Hori, and Yamasaki, 2007).
During pancreas development, MafA expression is first detected at the beginning of the
principal phase of insulin-producing cell production. Neurogenin3 (Ngn3) belongs to
basic helix loop helix transcription factor family, and is known to play an important role
in pancreatic development and endocrine differentiation (Gu, Dubauskaite and Melton,
2002; Dominguez-Bendala, Klein, Ribeiro, Ricordi, Inverardi, Pastori, and Edlund,
2005). Ngn3 is also involved in the regulation of a variety of pancreatic transcription
factors such as NeuroD, Pax4 and Nkx2.2 (Watada, Mirmira, Leung and German, 2000;
Watada, Scheel, Leung and German, 2003). Early pancreatic marker, Sca-1 is co-
expressed with Pdx-1 and Ngn3 (Ma, Chen, Chi, Yang, Lu and Han, 2012). Sca-1 is
expressed especially in islets and ductal cells (Seaberg, Smukler, Kieffer, Enikolopov,
Asghar, Wheeler, Korbutt and van der Kooy, 2004). Other transcription factors or factors
that enhance the differntiation potential towards the beta cell lineage can also be used for
the efficient differntiation. One of these factors could be Neuropilin-1 (Nrp1) which is a
transmembrane glycoprotein. It also functions as a co-receptor for VEGF165 in
endothelial cells (Hasan, Kendrick, Druckenbrod, Huelsmeyer, Warner, and MacDonald,
2010). VEGF plays an important role in pancreatic islet cell proliferation (Lammert, Gu,
McLaughlin, Brown, Brekken, Murtaugh, Gerber, Ferrara, and Melton, 2003).
A transgene-free cellular reprogramming showed that it is possible to generate a desired
cell type by using small molecules. Combination of small molecules can be used to
induce pluripotency in the somatic cells that are non-immunogenic, and strategy is
relatively easier than the reprogramming approach (Pandian, Taniguchi, and Sugiyama,
14
Fig. 1.5: Insulin producing cells (IPCs) from different cell sources: Over expression
of the defined exogenous transcription factors (Pdx1+ NeuroD + Ngn3) in liver cells
generate IPCs (A), IPCs could also be generated from pancreatic non-β cells and α-cells
by forced expression of Ngn3 + Pdx1 + MafA and Pax4, respectively (B), Pdx1
transduction into adipose tissue-derived mesenchymal stem cells resulted in IPCs
generation (C) (Pandian, Taniguchi, and Sugiyama, 2014).
15
2014). Several small molecules that activate or inhibit the epigenetic enzymes could
enhance the differentiation potential of stem cells or somatic cells into insulin producing
β-cells. Combined treatment of selenite, 5-AZA, RA and Trichostatin A (TSA),
chromatin remodelling regulator proteins like insulin and transferrin resulted in direct
differentiation of insulin producing cells from rat liver epithelial stem-like WB-F344
cells (WB cells) (Liu, Liu, Wang, Hao, Han, Shen, Shi, Li, Mu, and Han, 2013). NIH3T3
cells were also differentiated into islet-like clusters with Swertisin, a compound isolated
from a perennial herb (Enicostemma littorale). Furthermore, these differentiated cells
have also shown promising results in maintaining normal blood glucose level upon
transplantation (Dadheech, Soni, Srivastava, Dadheech, Gupta, Gopurappilly, Bhonde,
and Gupta, 2013).
1.4 Environmental Factors Affecting Differentiation of β-
Cells
1.4.1 Oxidative Stress
Beyond its role in aerobic respiration, oxygen plays a crucial role in many development
events and cellular homeostasis (Fraker, Ricordi, Inverardi, and Domínguez‐Bendala,
2009). It has been shown to regulate stem cell functions and embryonic development of
several organs, including pancreas (Heinis, Simon, Ilc, Mazure, Pouysségur,
Scharfmann, and Duvillié, 2010; Shah, Esni, Jakub, Paredes, Lath, Malek, Potoka,
Prasadan, Mastroberardino, and Shiota, 2011). The deficiency of oxygen in normal cells
contributes to the cell death, while in stem cells, it controls stem cell self-renewal and
pluripotency by stimulating specific signalling pathways and the expression of
transcriptional factors (Hakim, Kaitsuka, Raeed, Wei, Shiraki, Akagi, Yokota, Kume,
and Tomizawa, 2014). It is known to control cell differentiation in various tissues,
including pancreatic endocrine cell type (Fraker, Alvarez, Papadopoulos, Giraldo, Gu,
Ricordi, Inverardi, and Domínguez‐Bendala, 2007). A high O2 condition during the early
stage of differentiation is reported to increase the percentage of Ngn3-expressing
endocrine progenitor and insulin positive cells in both mESC and hiPSC at the terminus
of differentiation via HIF-1α inhibition and stimulation of Ngn3 gene expression
16
(Hakim, Kaitsuka, Raeed, Wei, Shiraki, Akagi, Yokota, Kume, and Tomizawa, 2014).
HIF-1α is reported to activate Notch signalling in stem cells and embryonic pancreatic
cells (Heinis, Simon, Ilc, Mazure, Pouysségur, Scharfmann, and Duvillié, 2010). Down
regulation of Notch signalling give rise to Ngn3 expressing cells (Hakim, Kaitsuka,
Raeed, Wei, Shiraki, Akagi, Yokota, Kume, and Tomizawa, 2014). Ngn3 gene
expression and pancreatic endocrine development are tightly regulated by Hes1 which is
an inhibitory bHLH factor activated by Notch signalling. The high oxygen condition
inhibits HIF-1α signalling which might lead to Hes1 repression and induction of Ngn3
expression (Hakim, Kaitsuka, Raeed, Wei, Shiraki, Akagi, Yokota, Kume, and
Tomizawa, 2014). Furthermore, high O2 concentration induces Wnt signalling activation.
The Wnt/beta-catenin pathway is involved in the regulation of pluripotency,
differentiation, and pancreatic development (McLin, Rankin, and Zorn, 2007).
1.4.2 Pancreatic Tissue Environment
Micro-environment, pancreatic developmental signal control, and gene expression are
the essential parameters for an efficient in vitro induction of pancreatic β-cells (Parnaud,
Bosco, Berney, Pattou, Kerr-Conte, Donath, Bruun, Mandrup-Poulsen, Billestrup, and
Halban, 2008). The proficiency and extent of differentiation depends on both the gene
expression pattern and the external micro-environment that play an important role in
stem cell survival and differentiation (Kim, Choi, Ko, Lim, Lee, and Kim, 2012). In
response to damage, large amount of β-cell regeneration and stem cell differentiation
factors, transcription proteins, and pancreatic development-related cytokines are released
from the pancreatic tissue (Xu, Chen, Hou, Lin, Sun, Sun, Dong, Liu, and Fu, 2009).
Thus, it could play a vital part in promoting stem cell differentiation, β-cell proliferation,
and insulin secretion. Proteins present in the pancreatic extract could therefore, able to
promote pancreatic islet regeneration and MSCs differentiation (Xie, Wang, Zhang, Qi,
Zhou, and Li, 2013). Conditioned medium supplemented with the pancreatic tissue
extract has been shown to differentiate IPCs from rat bone marrow mesenchymal stem
cells (BM-MSCs). Co-culture studies of islets and pancreatic stem cells with the
conditioned medium have also been reported in the production of mature β-cells
(Parnaud, Bosco, Berney, Pattou, Kerr-Conte, Donath, Bruun, Mandrup-Poulsen,
Billestrup, and Halban, 2008).
17
1.5 Objectives of the Present Study
The study was designed to evaluate the effect of different cellular therapeutic strategies
on the trans-differentiation of mouse embryonic fibroblasts into insulin producing β-cells
in an attempt to improve treatment options for type I diabetes.
The objectives of the proposed study include:
1- differentiation of mature cell type (NIH3T3 cells) into pancreatic β-cells
2- study of the role of various transcription factors in the trans-differentiation of
NIH3T3 cells into pancreatic β-cells
3- study of the role of various preconditioned media (dexamethasone, pancreatic
extract, DNP) in the trans-differentiation of NIH3T3 cells into pancreatic β-cells
by analyzing the expression of pancreatic genes and proteins.
CHAPTER 2 Materials and Methods
18
2.1 Reagents and Cell Lines
List of all chemicals, reagents, consumables, plasmids, kits and antibodies used in this
study are outlined in Appendix I and details of reagent preparations are given in
Appendix II. All cell lines used in this study were purchased from the American Type
Culture Collection (ATCC) by the Biobank facility of Dr. Panjwani Center for Molecular
Medicine and Drug Research (PCMD).
2.2 Gene Expression Analysis of Pancreatic Tissue
2.2.1 Pancreatic Tissue
Pancreas was isolated from 3-4 months old SD rats. Animals were anesthetized, pancreas
was isolated and the blood was removed by washing the tissue with sterile PBS. Tissue
was stored in RNA Later solution at -20 ˚C for later use.
(a) RNA Isolation
Two methods for RNA isolation were followed:
(i) Trizol Method
Prior to RNA isolation, pipettes, glassware and bench top were sanitized by RNAse
Erase spray. Pancreatic tissue (~20 mg) was homogenized immediately after
resuspending in 1 mL Trizol reagent. The mixture was incubated at 25 °C for 15 minutes.
Chloroform (200 μL per mL Trizol) was then added to the mixture and incubated at 25
˚C for 15-20 minutes. Phase separation was performed by centrifuging the mixture at
11,000 X g for 30 minutes. Two phases appeared; lower organic phase and upper
transparent aqueous phase and in between these two phases, a pellet of protein and DNA
was present. Upper aqueous phase was proceeded for RNA extraction by transferring to
a sterile DEPC treated centrifuge tube. RNA was then precipitated by chilled isopropanol
with centrifugation at 6,000 X g for 10 minutes at 4 ˚C. RNA was re-hydrated by 75%
ethanol and pelleted at 6,500 X g for 15 minutes followed by air drying. The dried pellet
19
was then re-suspended in 40 µL sterile, nuclease free water and stored at -80 ˚C till
further use.
(ii) Spin Method
SV total RNA system kit (Promega, USA) was used for RNA isolation. Briefly, 80-90%
confluent cells were harvested by trypsinization. Trypsin action was inhibited after
complete detachment of cells by adding 7-8 mL of complete DMEM. The dissociated
cell suspension after centrifugation was resuspended in 175 μL RNA lysis buffer and β
mercaptoethanol (10 µL per mL buffer RLT). DNA dilution buffer (350 μL) was added
to the lysate, vortexed for 15 seconds and heated at 70 °C for 3 minutes. The mixture was
then immediately centrifuged at 12000 rpm for 10 minutes at 4 °C. The supernatant was
transferred to a fresh DEPC treated microcentrifuge tube. 200 μL pre-chilled 70%
ethanol was added to the lysate, mixed well and applied to the spin column in a 2 mL
collection tube, and centrifuged for 2 minutes at the same speed. Column was washed
with 600 μL RNA wash buffer (RWB) and centrifuged at 12000 rpm for 2 minutes
before DNase solution was applied to the column and incubated for 15 minutes at RT.
DNase stop solution (200 μL) was applied to the column to stop the reaction and
centrifuged at 12000 rpm for 1 minute. Washing of the column was done twice with
RWB followed by centrifugation at the same speed for 2 minutes. Column was shifted to
the collection tube and RNA was eluted by applying 50 μL nuclease free water to
column membrane, followed by centrifugation at 12000 rpm for 3 minutes. The isolated
RNA was stored at -80 oC till further use.
(b) RNA Quantification
1: 200 dilution of the eluted RNA was prepared to determine the concentration, by
measuring absorbance at 260 nm in a UV visible spectrophotometer (UV-1700,
Shimadzu, Japan). The ratio A260/280 was used for purity. Formula for concentration
determination is listed in Appendix II.
(c) cDNA Synthesis
RNA was subjected to cDNA synthesis by using SuperScript III first-strand synthesis kit
20
(Invitrogen life technology, USA) according to the manufacturer's instructions. Amount
of RNA equivalent to 1 μg was taken and RNA/ primer mixture was prepared in an
RNAse / DNase free PCR tube. Reaction mixture contained 1 μg RNA, 1 μL random
hexamer and the volume was made up to 12 µL with DEPC treated sterile water. This
mixture was first incubated at 70 oC for 5 minutes, and then placed on ice for 1 minute. 4
µL 5X reaction buffer, 2 µL dNTPs and 1µL RNase out (20u/µL) were added in the tube
and incubated at 25 °C for 5 minutes. After incubation, 1 µL of Reverse Transcriptase
enzyme (Superscript, TM-III RT) was added, microfuged and then incubated at 25 °C for
10 minutes followed by two other incubations, first at 42 °C for 60 minutes and second
at 70 °C for 10 minutes. After incubation, the cDNA mixture tubes were incubated at 37
oC for 10 minutes, chilled on ice and were either used immediately for PCR or stored at -
20 oC till further use.
(d) Gene Amplification
RT-PCR of NIH3T3 cells and pancreatic tissue were performed for the analysis of
expression of islet specific genes. Islet specific primers used in this study include
glucagon, insulin, somatostatin, and Musculoaponeuroticfibrosarcoma oncogene family
protein A (MafA) (Table 1). Primers for each gene were designed using
the primer3 design program at http://frodo.wi.mit.edu/primer3/, and purchased from
Integrated DNA technologies (IDT, USA). The primers were reconstituted in 10 mM
Tris-HCl/EDTA (TE) buffer (pH 8). 100 μM primer stock was prepared from master
primer vials and was further diluted to 10 μM in TE buffer, pH 8.0. Formula for
annealing temperature (Tm) calculation is listed in Appendix II. Sequence, annealing
temperature, and product sizes of each primer are enlisted in Table 1.
For 25 µL PCR reaction, 1μg of cDNA was amplified by using Go Taq® Green Master
Mix 2X (Promega, USA) according to manufacturer’s instructions. The reaction mixture
contained 12.5 µL Master mix, 0.5 μL of each primer, and 1 μg of cDNA. Reaction
volume (25 µL) was maintained by adding sterile, nuclease free water. The mixture was
subjected to centrifugation and then placed in thermal cycler.
21
PCR reaction was carried out in Master cycler, 5531, Eppendorf, Germany. GAPDH was
used as internal standard in all experiments. Reaction started with the initial denaturation
at 95 °C for 2 minutes, followed by 35 cycles of denaturation, annealing, and extension
at 95 °C, 58-64 °C and 72 °C, respectively and final extension at 72 °C for 10 minutes.
The amplified PCR products were stored at -20 °C.
(e) Analysis of Gene Expression by Agarose Gel Electrophoresis
10 µL of PCR product was then electrophoretically resolved on 1% agarose gel, prepared
in freshly made 1X TBE buffer containing 0.3 μg/mL ethidium bromide. Gel was
polymerized in the gel casting unit (Sub-Cell GT Agarose Electrophoresis Systems, Bio-
Rad, USA) and then placed in the horizontal gel apparatus. 1X TBE buffer was used as
the running buffer. 6 μL DNA ladder (100 to 1000 bp) and 10 µL PCR products were
loaded into the wells and electrophoresis was carried out at 70 volts for 70 minutes. Gel
documentation system (Alpha Innotech, AlphaEAse FC imaging system, FluorChemTM,
USA) was used to analyze the gel. Relative gene expression was calculated by
normalization of expressed gene density with the corresponding GAPDH band density
and compared with the control group.
2.3 Culturing of NIH3T3 Cells
2.3.1 Cell Thawing and Expansion
Mouse embryonic fibroblast cells, NIH3T3 (ATCC® CRL-1658TM) were stored in liquid
nitrogen at -196 °C. Just before the start of the experiments, cryovial was removed from
the liquid nitrogen tank, immediately thawed in pre-heated warm water at 37 °C and then
sterilized with 70% ethanol. Thawed cells along with the freezing medium were
transferred to a 15 mL falcon tube having 9 mL complete Dulbecco’s modified Eagle’s
medium/F12 (DMEM/F12). Cells were centrifuged at 1000 rpm for 8 minutes and
seeded in tissue culture treated flask containing high glucose DMEM/F12. Flask was
then incubated in CO2 incubator (NU5500E, NuAire, USA) at 37 °C for proper cell
proliferation.
22
23
2.3.2 Sub Culturing of NIH3T3 Cells
When the cell confluency reached 80%, they were sub-cultured into two T75 cm2 flasks.
Cells were washed twice with 1X PBS after medium aspiration. 2-3 mL of 0.1% trypsin
was added and the cells were incubated at 37 °C for 3 minutes. Trypsin reaction was
terminated with complete medium and the suspended cells were then transferred into 15
mL falcon tube, centrifuged at 300 X g for 8 minutes and seeded in sterile tissue culture
treated flasks.
2.3.3 Cryopreservation
All the consumables used in the process of cryopreservation were autoclaved. Two types
of freezing media were prepared. Medium A contains 20% FBS and 80% high glucose
DMEM/F12, while medium B was composed of 10% FBS, 10% DMSO and 80%
DMEM/F12. The media were prepared just before the start of the experiment and chilled
on ice. In order to freeze the cells for long term storage, the cells were trypisinized using
the same protocol as described in Section 2.3.2. Following centrifugation, medium was
discarded and the cells were resuspended in Medium A (500 µL) in the pre-chilled and
labelled cryovial. 500 µL Medium B was added drop wise along the side of the vial.
From one T75 flask, 5 cryovials were prepared having cells in 1 mL of freezing medium
in each vial. Vials were first stored at -20 °C for 2 hours, then transferred to -80 °C for
24 hours and for long term storage finally placed in liquid nitrogen tank.
2.4 Analysis of Protein Expression in NIH3T3 Cells
2.4.1 Immunocytochemistry
NIH3T3 cells (~10,000 cells) were cultured in chambered glass slides (5712-002.PS/
Glass, IWAKI, Japan) by adding 1 mL of cell suspension (1 X 103 cells) to each well.
After proper cell attachment, medium was aspirated and the cells were gently rinsed
twice with 1X PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1%
Triton X-100 in PBS for 15 minutes at RT. The cells were rinsed 2-3 times with 1X PBS.
24
The cells were then incubated in blocking solution overnight at 4 °C with primary
antibodies at 1:100 dilution against actin, insulin, glucagon, MafA, Ngn3 and Pdx1.
After incubation, the antibody solution was removed and cells were rinsed with PBS and
incubated for 1 hour at RT with Alexa fluor 546 goat anti mouse secondary antibody (for
insulin, glucagon, Ngn3 and actin) and Alexa fluor 546 goat anti rabbit secondary
antibody (for MafA and Pdx1) at a dilution of 1:200. This was followed by washing with
1X PBS. Nuclei were counterstained with 0.5 µg/mL 4', 6-Diamidino-2-phenylindole
(DAPI). Finally, cells were rinsed 2-3 times with PBS, mounted with mounting medium
and observed under inverted fluorescent microscope (Eclipse TE 2000-S, Nikon, Japan).
2.4.2 Flow Cytometry
NIH3T3 cells were dissociated with cell dissociation buffer. Supernatant was discarded
and pellet was washed twice with 1X PBS. Blocking solution (5 µL) was added to the
pellet, mixed well and incubated for 2 minutes at RT. Cells were incubated in dark for 3
minutes with primary antibodies ( MafA, Pdx1, insulin, Ngn3 and Sca1 ) diluted at a
ratio of 1:40 with cold FACS solution. Cell suspension was washed twice with cold
FACS solution and centrifuged at 400 x g for 8 minutes at 4 °C. The cells were then
incubated with either Alexa Fluor 546 goat anti mouse or anti rabbit secondary
antibodies at 1:500 dilution, mixed well and incubated in dark at 4 °C. Cells were
washed twice with 2 mL FACS solution and centrifuged at the same speed. 500 µL of
FACS solution was added to the pellet, vortexed and analyzed through flow cytometer.
Unlabelled cells or cells labelled with secondary antibody were used as controls.
Labelled cells were observed in FL-2 filter.
2.5 Analysis of Genes and Proteins Expression in NIH3T3
Cells Transfected with Nrp1, MafA and Nrp1/MafA
The plasmids of Nrp1 (pCherry-mNrp1) and MafA (pAd-MafA-I-nGFP) gifted by Guido
Serini (Addgene plasmid # 21934) and Douglas Melton (Addgene plasmid # 19412),
respectively, were obtained in the form of inserts in E. coli stab culture from Addgene
(www.addgene.org). Following steps were performed for transfection experiment.
25
2.5.1 Culturing of Bacteria
E. coli from the stab cultures were grown in LB agar medium. The bacterial culture
medium composed of agar (3.75 gm), yeast extract (1.25 gm), peptone (2.5 gm), and
sodium chloride (2.5 gm). All the contents were dissolved in 100 mL of autoclaved
distilled water in a clean conical flask. Once all contents were dissolved, the final
volume was made up to 250 mL with distilled water, divided equally into two clean
conical flasks for processing of Nrp1 and MafA plasmids and autoclaved. When the
medium cooled down to 37°C, antibiotics in each medium were added accordingly. 50
µL of kenamycin from stock of 100 mg/mL was added to the culture medium to be used
for Nrp1 plasmid, while same concentration of ampicillin was used in case of MafA
plasmid. 20-25 mL LB agar medium was then poured into sterile petri dishes in sterile
condition and kept in the Laminar Flow Cabinet (EN 1822.1 ESCO, USA) to solidify.
After 2 hours, the bacteria were inoculated with sterile wire loop on the surface of the
agar. Petri dishes were labelled properly and incubated at 37 oC for 16 hours.
(a) Bacterial Stock Culture
The bacterial colonies were further processed in Luria broth (LB). 250 mL of LB was
prepared by dissolving yeast extract (1.25 gm), sodium chloride (2.5 gm) and peptone
(2.5 gm) in distilled water and then autoclaved. When the medium was cooled down to
37 °C, 50 µL of antibiotics were added in the medium accordingly from the stock of 100
µg/mL and mixed well to distribute homogeneously. 1 mL LB medium was transferred
into 2 mL sterile microcentrifuge tubes. A single colony was picked up with 10 µL
sterile pipette tip and placed in the microcentrifuge tube containing the broth. The tubes
were then incubated at 37 oC in the shaking incubator (Incubator Shaker Series 126, New
Brunswick Scientific, USA) for 16 hours. After 16 hours incubation, the turbid medium
was stored at 4 oC to cease the bacterial growth.
(b) Bacterial Glycerol Stock
To make the glycerol stock of the plasmids, 250 µL deionized autoclaved water was
added in 250 µL 99.6% glycerol. To this 50% glycerol solution, 500 µL of LB bacterial
culture was added, mixed well and stored at -80 °C for long term storage.
26
(c) Bacterial Culture for Plasmid DNA Isolation
For plasmid DNA isolation, 500 µL of bacterial culture stock was added to 250 mL Luria
broth in a sterile conical flask. The flask was covered with aluminium foil and incubated
in the shaking incubator at 37 °C for 16 hours. After incubation, the medium appeared
turbid confirming the bacterial growth. The flask was taken out and the medium was
processed for plasmid DNA isolation by maxiprep kit.
2.5.2 Plasmid Isolation by Maxiprep
WizardRPlusMaxiprep DNA purification kit (Promega, USA) was used to purify plasmid
DNA. Bacterial culture was transferred to sterile 50 mL falcon tubes and centrifuged at
3220 X g for 20 minutes. Supernatant was poured off and pellet was resuspended in 15
mL of Cell Resuspension Solution followed by addition of 15 mL of Cell Lysis Solution.
This mixture was inverted for 20 minutes in order to achieve complete lysis. Cell lysis
was complete when the solution became clear and viscous. 15 mL of Neutralizing
Solution was added and mixed by gently inverting the falcon tubes. The tubes were
centrifuged at 3220 X g for 10 minutes. The upper pellet was dissolved by mixing and
the mixture was then again centrifuged at the same speed for 40 minutes. Supernatant
was filtered in a sterile 50 mL falcon tube by using Whatman filter paper. 0.5 volume of
isopropanol was added to the mixture and incubated for 15 minutes at RT. The mixture
was centrifuged again for 40 minutes. Supernatant was discarded and the plasmid DNA
pellet was resuspended in 1 mL RNase free water. The walls of the falcon tubes were
washed thoroughly with RNase free water to recover the DNA. The plasmid DNA was
transferred to a sterile microcentrifuge tube labelled and stored at -20 ˚C.
2.5.3 Plasmid Quantification
Plasmid DNA was quantified at 260 nm and concentration was calculated with the
formula mentioned in Appendix II.
27
2.5.4 Culturing of Packaging Cell Line
Adenovirus packaging cell line, HEK-293 (ATCC® CRL-1573TM) was stored in liquid
nitrogen until further use. Just before the start of the experiment, the vial was taken out
from the liquid nitrogen and immediately thawed at 37 °C. The vial was decontaminated
with 70% ethanol and the cells were transferred to a 15 mL falcon tube containing 9 mL
complete medium and centrifuged at 120 X g for 8 minutes. Supernatant was aspirated
and 2 mL of complete DMEM was added to resuspend the cells thoroughly. Finally, the
cell suspension was transferred to 75 cm2 flask containing 8 mL of complete DMEM and
incubated at 37 °C in a humidified chamber with 5% CO2. When the cells achieved 80-
90% confluency, they were subcultured as described in Section 2.3.2.
2.5.5 Adenovirus Production
HEK-293 cells were seeded in 75 cm2 flask in DMEM supplemented with 10% FBS
prior to transfection. When cells reached 50-60% confluency, they were transfected with
lipofectamine2000. 10µL of lipofectamine was diluted in 490 µL of serum and antibiotic
free medium (Solution A). In a separate tube, volume equivalent to 10 µg of plasmid was
diluted in serum and antibiotic free medium to make the total volume upto 500 µL
(Solution B). The microcentrifuge tubes were incubated for 5-10 minutes at room
temperature. Both solutions were mixed carefully and incubated for 30 minutes at room
temperature. Complete medium was aspirated with serological pipette and the cells were
washed thoroughly with serum free medium 2-3 times. After 30 minutes of incubation,
the mixture was added to the flask and mixed gently in order to spread the mixture
homogeneously. Serum and antibiotic free DMEM was added to the flask and incubated
in a humidified chamber for 20-22 hours. At the end of the incubation, medium was
collected in a sterile 15 mL falcon tube and cells were trypsinized with 0.25% trypsin.
The typsinized cells were transferred to 15 mL falcon tube and centrifuged at 4000rpm
for 30 minutes at 4 °C. The supernatant was filtered with 0.22 µM filter and stored at -80
°C.
28
2.5.6 Transfection of NIH3T3 Cells with MafA, Nrp1, and MafA/Nrp1
(a) Transfection with MafA
Adenovirus was collected by the method described in Section 2.5.5. Different ratios (1:1,
1:2, 1:3, 2:1) of MafA adenovirus and serum free media respectively was used to
transfect the NIH3T3 cells. The cells were incubated at 37 °C for 22-24 hours. After
incubation, the medium was aspirated and cells were washed twice with serum free
medium. Cells were further processed for RNA isolation after 3-4 days.
(b) Transfection with Nrp1
NIH3T3 transfection with Nrp1 plasmid DNA was done by using lipofectamine2000. 10
µg Nrp1 plasmid was diluted in 500 µL incomplete medium (Solution A). In a separate
eppendorf tube, 10 µL lipofectmine was added in 490 µL incomplete medium (Solution
B), mixed well and incubate for 10 minutes at room temperature. After incubation, both
the solutions (1 mL) were mixed and incubated for 30 minutes at room temperature.
Before transfection, NIH3T3 cells were washed with incomplete medium. Solution
containing Nrp1 plasmid, lipofectamine and incomplete medium was spread on the cells
and incubated for 3-4 minutes. 9 mL incomplete medium was added in the flask and
incubated in CO2 incubator for 20-22 hours. After 20 hours of transfection, medium was
removed and cells were washed with 1X PBS. Fresh complete medium was added and
cells were analyzed for gene and protein expression after 3-4 days.
(c) Co-Transfection with MafA/Nrp1
Combined transfection of MafA/Nrp1 was carried out by using lipofectamine. Complete
medium was removed and the cells were first washed gently with serum free medium.
Transfection with Nrp1 was carried out in the same way as described in Section 2.5.6.
After 20 hours of transfection, medium was removed and cells were transfected with
MafA adenovirus in a ratio of 1:2 as described in Section 2.5.5. The concentration of the
MafA was chosen after checking the viability of the cells. At the end of the transfection,
transfected medium was aspirated and fresh complete medium was added in the flask.
29
Cells were incubated in CO2 incubator for 3-4 days and then analyzed for gene
expression studies.
2.5.7 Analysis of Pancreatic Genes after Transfection
(a) RNA Isolation and Quantification
Following transfection with Nrp1, MafA and MafA/Nrp1, RNA was isolated at different
time intervals i.e. 0, 24, 48, 72 and 96 hours. Medium was removed and cells were
washed with 1X PBS twice. The remaining protocol is same as described in Section
2.2.1a.
(b) cDNA Synthesis
cDNA synthesis was carried out by using Super Script III first-strand synthesis kit as
described in Section 2.2.1c.
(c) Gene Amplification
RT-PCR of normal and non-transfected NIH3T3 cells was performed for the analysis of
expression of insulin, MafA, Ngn3, Nkx6.1 and somatostatin genes. Glyceraldehyde 3-
phoshate dehydrogenase (GAPDH) was used as internal standard. The results of gene
expression after transfection with Nrp1, MafA and the combination of these plasmids
were quantified by densitometry. Sequence, annealing temperature, and product sizes of
each primer are enlisted in Table 1.
2.5.8 Analysis of Pancreatic Proteins after Transfection
After 4 days of transfection, cells were dissociated with cell dissociation buffer and
protein expression of insulin, MafA, Ngn3, glucagon and Sca1 was determined with flow
cytometer by using the same protocol as mentioned in Section 2.4.2.
30
2.6 Preconditioning of NIH3T3 Cells
2.6.1 Experimental Groups
Cells were divided into six groups on the basis of different treatments:
Group 1: No treatment (Control)
Group 2: Dexamethasone (5 µM)
Group 3: Pancreatic extract (0.05 mg/mL protein)
Group 4: Pancreatic extract (0.4 mg/mL protein)
Group 5: Dexamethasone (5 µM) and pancreatic extract (0.05 mg/mL protein)
Group 6: Dexamethasone (5 µM) and pancreatic extract (0.4 mg/mL protein)
2.6.2 Pancreatic Extract
Pancreas was processed according to a reported protocol (Xie, Wang, Zhang, Qi, Zhou,
and Li, 2013). Pancreas isolated from SD rats was rinsed with sterile 1X PBS and
transferred in a sterile petri dish having 4-5 mL 1X PBS. The pancreas was chopped with
the help of scissors, homogenized and centrifuged at 3000 rpm for 10 minutes at 4 °C.
Supernatant was further centrifuged at 12000 rpm for 20 minutes at 4 °C and then
filtered with 0.22 µM syringe filter. Protein concentration of the pancreatic extract was
determined by using Nanodrop. The pancreatic extract was stored at -80 °C for further
use. Two different concentrations of the proteins (i.e. 0.05 mg/mL and 0.4 mg/mL) were
used to induce differentiation.
2.6.3 Dexamethasone Treatment
NIH3T3 cells were treated with different concentrations (5, 10, 15, and 20µM) of
dexamethasone when they reached 40-50% confluence. After 4 days, the cells were
analyzed for cytotoxicity, reactive oxygen species (ROS) production and gene expression
studies.
31
(a) Analysis of Cytotoxic Effect of Dexamethasone by JC1 Mitochondrial
Membrane Potential Assay
NIH3T3 cells were cultured at a density of 1x106 cells/mL. When cells reach 40-50%
confluence, dexamethasone was added at different concentrations for 4 days. After 4
days, cells were trypsinized. Supernatant was removed and the cell pellet was
resuspended in 0.5 mL PBS. Following centrifugation at 400 x g for 5 minutes, pellet
was incubated with 500 µL JC1 stain (Cayman, USA) at a working concentration of 10
μg/mL at 37 °C in humidified CO2 incubator for 15 minutes and centrifuged at the same
speed. Cells were washed twice with PBS. After final washing, pellet was resuspended in
500 µL PBS. Number of apoptotic cells was analyzed through flow cytometer.
(b) Analysis of the Effect of Dexamethasone on Reactive Oxygen Species (ROS)
Production by 3'-(p-hydroxyphenyl) Fluorescein (HPF)
Cells were grown to 70-80% confluence and treated with different concentration of
dexamethasone for 4 days. Following treatment, cells were trypsinized with 0.25%
trypsin and washed with PBS. 500 μL of 3′-(phydroxyphenyl) fluorescein (HPF), an
ROS indicator (Invitrogen, USA), at a working concentration of 5 µM was added to the
pellet, mixed well and incubated at 37 °C for 15 minutes. The cells were then centrifuged
at 180 X g for 8 minutes. Pellet was resuspended in 500 μL of 1X PBS and analyzed in
flow cytometer. Unlabelled cells were used as colour compensatory control and
untreated HPF labelled cells were used as control. Oxidative stress induced in cells was
observed in FL-1 filter (excitation 488 nm; emission 530 nm) and data was evaluated
using BD Cell Quest pro software.
2.6.4 Combination of Pancreatic Extract and Dexamethasone
NIH3T3 cells were preconditioned using dexamethasone and pancreatic extract.
Different conditioned media were used; (1) CMa and (2) CMb in which cells were grown
separately in media containing only pancreatic extract at either of the two concentrations
i.e. 0.05 mg/mL and 0.4 mg/mL respectively; and mixture of both dexamethasone and
pancreatic extract, (3) MXa in which cells were treated with 5 µM dexamethasone and
32
grown in medium containing 0.05 mg/mL of pancreatic extract and (4) MXb in which
cells were treated with 5 µM dexamethasone and grown in medium containing 0.4
mg/mL of pancreatic extract. After different treatments, cells were propagated for 4 days.
2.6.5 Morphological Examination of Preconditioned Cells
At the end of each treatment, preconditioned NIHT3T3 cells were analyzed for
morphological changes and compared with that of untreated control.
2.6.6 Analysis of Pancreatic Genes in Preconditioned Cells
RNA isolation, and cDNA synthesis, was performed as described in Section 2.2.1 and
2.2.3. Gene expression analysis of islet specific genes, insulin, somatostatin, Ngn3, Nkx
6.1 and MafA was performed by RT-PCR as described in Section 2.2.4. GAPDH was used
as positive control (Table 1). Gene amplification program and analysis are described in
Section 2.2.1d and e.
2.6.7 Analysis of Pancreatic Proteins in Preconditioned Cells
Cells were dissociated with cell dissociation solution and analyzed for the expression of
insulin, glucagon, MafA, Ngn3, Pdx1, and Sca1 by flow cytometry using the protocol
described in Section 2.4.2. Expression of insulin, Ngn3 and Sca1 were also analyzed with
immunocytochemistry using the protocol described in Section 2.4.1.
2.7 Analysis of Pancreatic Gene Expression in Response to
2, 4 Dintrophenol (DNP)
2.7.1 DNP Treatment
70% confluent NIH3T3 cells were treated with 2, 4 dinitrophenol (DNP). To find
optimal concentration, cells were treated with different concentrations of DNP (0.025- 2
mM) for 10 and 20 minutes. The optimal dose of 0.1 mM for 20 minutes was selected on
the basis that cells at this concentration only experienced shock but did not die. Initially,
33
medium was aspirated and cells were washed twice with incomplete medium. 0.1 mM
DNP with FBS free medium was added for 20 minutes and cells were incubated at 37
°C. After 20 minutes, medium was aspirated and cells were washed with incomplete
medium. Finally, cells were reperfused in the presence of complete medium for 48 hours
in CO2 incubator.
2.7.2 Morphological Examination of DNP Treated Cells
After DNP treatment and 48 hours of reperfusion, cells were analyzed for morphological
changes and compared with that of untreated control.
2.7.3 Analysis of Pancreatic Genes in DNP Treated Cells
RNA isolation, and cDNA synthesis were performed as described in Section 2.2.1 and
2.2.3. Gene expression analysis of islet specific genes, insulin, somatostatin,
Neurogenin3 (Ngn3), glucagon, NK6 homeobox 1 (Nkx 6.1) and MafA was performed
by RT-PCR as described in Section 2.2.4. GAPDH was used as positive control (Table
1). Gene amplification program and analysis are described in Section 2.2.1d and e.
2.7.4 Analysis of Pancreatic Proteins in DNP Treated Cells
Cells were dissociated with cell dissociation solution and analyzed for the expression of
insulin, MafA, Ngn3, Pdx1 glucagon and Sca1 by flow cytometry using the protocol
described in Section 2.4.2.
2.8 Statistical Analysis
Significance of difference among the groups was analyzed by using one-way ANOVA
followed by Bonferroni post hoc tests for comparison between groups. The results were
illustrated as mean ± S.E.M. P-value < 0.05 was considered statistically significant.
Analysis was done by using SPSS program (version 13, SPSS Inc, Chicago, IL, USA).
CHAPTER 3 results
34
3.1 Propagation and Characterization of Mouse Embryonic
Fibroblasts (NIH3T3 Cells)
3.1.1 Morphological Characteristics
Mouse embryonic fibroblasts (NIH3T3) grown in DMEM/F12 showed spindle shaped
morphology. Cells were adherent and have high proliferation rate (Fig. 3.1).
3.1.2 Gene Expression Analysis by RT-PCR
Expression of pancreatic genes, MafA, insulin, glucagon, and somatostatin was analyzed
in NIH3T3 cells. GAPDH gene was used as internal standard and pancreatic tissue was
taken as positive control. There was a basal level expression of insulin and MafA in
NIH3T3 cells. Glucagon and somatostatin expressions were found in the pancreas tissue
while they show no expression in NIH3T3 cells (Figs. 3.2 - 3.3).
3.1.3 Protein Expression Analysis by Immunocytochemistry
Basal levels of MafA, Pdx1, glucagon, insulin and Ngn3 proteins were analyzed in the
NIH3T3 cells by direct immunofluorescence. The cells showed low or no expression of
these proteins in the NIH3T3 cells (Fig. 3.4).
35
Fig. 3.1: Morphology of mouse embryonic fibroblasts (NIH3T3): Cells show
adherent spindle shaped morphology under inverted phase contrast microscope at 10X
(a) and 20X (b) magnifications.
36
37
Fig. 3.2: Expression of pancreatic genes in NIH3T3 cells and pancreatic tissue: Gene
expression of glucagon (a), insulin (b), MafA (c), and somatostatin (d) were analyzed in
NIH3T3 cells to see their basal expression. The same genes were also analyzed in
pancreatic tissue for comparison.
38
Fig. 3.3: Graphical representation of pancreatic genes expression in NIH3T3 cells
and pancreatic tissue: Graphical representation of glucagon, insulin, MafA, and
somatostatin in NIH3T3 cells and pancreatic tissue. Significantly increased expression of
somatostatin (p <0.001), glucagon (p <0.001), MafA (p<0.05) and insulin (p<0.01) was
observed in pancreatic tissue as compared to NIH3T3 cells. Data is presented as mean ±
S.E.M.; level of significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * =
p < 0.05).
39
(a)
(b)
(c)
40
(d)
(e)
(f)
Fig. 3.4: Immunocytochemical analysis of pancreatic proteins in NIH3T3 cells:
NIH3T3 cells were shown to be positive for actin (a), MafA (b) and Pdx1 (c) and
negative for insulin (d), glucagon (e) and Ngn3 (f). Alexa Fluor 546 goat anti mouse and
anti rabbit secondary antibodies were used for detection. Nuclei were stained with DAPI.
10X magnification images were captured by inverted fluorescent microscope.
41
3.2 Transfection of NIH3T3 Cells with Neuropilin-1 (Nrp1)
3.2.1 Morphological Characteristics
pCherry-Neuropilin-1 transfected NIH3T3 cells have shown elongation, cluster
formation and increased number of projections. Fig. 3.5 shows the morphological
changes in NIH3T3 cells after transfection with pCherry-Neuropilin-1 at different time
intervals. Zero (0) hour corresponds to the time just after the removal of transfection
medium while 24, 48, 72, and 96 hours correspond to the time after removal of
transfection medium plus incubation times.
3.2.2 Gene Expression Analysis
Changes in the insulin expression levels after transfection at different time intervals were
analyzed by RT-PCR. Significant increase in insulin expression was observed after
Neuropilin-1 transfection (Fig. 3.6).
3.3 Transfection of NIH3T3 Cells with MafA
3.3.1 Morphological Characteristics
Human embryonic kidney (HEK-293) packaging cell line showed adherent, fibroblast
like morphology in high glucose supplemented medium before transfection (Fig. 3.7a).
MafA plasmid construct was added to HEK-293 cells using lipofectamine according to
manufacturer’s instructions. After 18 hours, the cells swell up and burst indicating cell
lysis and virus production (Fig. 3.7b). NIH3T3 cells prior to transfection appeared
homogenous and spindle in shape (Fig. 3.8a). After 18 hours of transfection with MafA
adenovirus at different media and virus ratios, transfected cells became round (Fig. 3.8b-
d).
42
(a)
(b)
(c)
43
(d)
(e)
(f)
Fig. 3.5: Morphology of NIH3T3 cells after transfection with Neuropilin-1: Changes
in NIH3T3 cells after transfection with pCherry-Neuropilin-1 after 0 (b), 24 (c), 48 (d),
72 (e), and 96 (f) hours and in control cells (a). Cells became elongated and showed
cluster formation after transfection as compared to control having non transfected cells.
Images were taken at 10X and 20 X magnifications under inverted phase contrast
microscope.
44
Fig. 3.6: Relative insulin expression in NIH3T3 after transfection with Neuropilin-1
at different time intervals: Significant increase in insulin expression was observed at 0
hour (p<0.001), 48 hours (p<0.01), 72 hours (p<0.001) and 96 hours (p<0.001) of
transfection compared to non transfected control. Data is presented as mean ± S.E.M.;
level of significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * = p <
0.05).
45
(a)
(b)
Fig. 3.7: Morphology of packaging cell line HEK-293: HEK-293 cells at passage 2
before (a) and after 20 hours (b) of MafA plasmid delivery. Images were taken at 10X
magnification under inverted phase contrast microscope.
46
(a) (b)
(c) (d)
Fig. 3.8: Morphology of NIH3T3 cells after transfection with MafA: NIH3T3 cells
before (a) and after transfection at ratios of 1:1 (b), 1:2 (c), and 2:1 (d). Transfected cells
appeared round in shape. Images were taken at 10X magnification under inverted phase
contrast microscope.
47
3.4 Co-Transfection of NIH3T3 Cells with MafA and Nrp1
3.4.1 Morphological Characteristics
MafA and Nrp1 transfected NIH3T3 cells have shown round shaped morphology. Fig.
3.9 shows the morphological changes in NIH3T3 cells after co-transfection of MafA and
Nrp1.
3.4.2 Expression of Pancreatic Genes in MafA, Nrp1 and Co-
Transfected Cells
Pancreatic genes, insulin, Ngn3, MafA, Nkx 6.1 and somatostatin were analyzed by RT-
PCR after 4 days of transfection with MafA, Nrp1 and combined transfection. Relative
gene expression as normalized with GAPDH after MafA transfection at the ratio of 2:1
(virus: medium), Nrp1 transfection and combined transfection of MafA (1:2) and Nrp1
showed increase in insulin, MafA and Ngn3 gene expressions while somatostatin
expression was found to be decreased (Fig. 3.10).
3.4.3 Expression of Pancreatic Proteins in MafA, Nrp1 and Co-
Transfected Cells
After 4 days of transfection, NIH3T3 cells were analyzed for insulin, MafA, Ngn3,
glucagon and somatostatin expression by flow cytometry. MafA, Ngn3 and Sca1
expressions were significantly increased (p<0.001) after Nrp1, MafA and co-
transfection. Insulin expression was found to be significantly increased (p<0.001) in
Nrp1 and co-transfected cells while glucagon expression was significantly increased
(p<0.001) in MafA and co-transfected cells (Figs. 3.11 - 3.12).
48
(a) (b)
Fig. 3.9: Morphology of NIH3T3 cells after co-transfection with MafA and Nrp1:
NIH3T3 cells before (a) and after transfection (b). Transfected cells appeared round in
shape. Images were taken at 10X magnification under inverted phase contrast
microscope.
49
50
51
52
(g)
Fig. 3.10: Analysis of pancreatic genes in transfected NIH3T3 cells by RT-PCR:
Relative gene expressions of GAPDH (a), insulin (b), MafA (c), Ngn3 (d), Nkx6.1 (e)
and somatostatin (f) in non-transfected control, and MafA, Nrp-1 and co-transfected
NIH3T3 cells are shown. Insulin and Ngn3 expressions were found to be significantly
increased (p < 0.01 and p < 0.001 respectively), while no significant change was
observed in MafA, Nkx 6.1 and somatostatin expressions after transfection. Combined
graphical representation of pancreatic gene expression in control and transfected groups
is also shown (g). Data is presented as mean ± S.E.M; level of significance is p < 0.05;
(where *** = p < 0.001, ** = p < 0.01, and * = p < 0.05).
53
(a)
Insulin
Glucagon
MafA
Ngn3
Sca1
58.21%
1.89%
1.51%
1.68%
3.98%
54
(b)
Insulin
Glucagon
MafA
Ngn3
Sca1
12.03%
1.90%
5.15%
6.39%
76.05%
55
(c)
Insulin
Glucagon
MafA
Ngn3
Sca1
3.86%
3.32%
5.13%
6.29%
82.81%
56
(d)
Fig. 3.11: Analysis of pancreatic proteins in transfected NIH3T3 cells: Pancreatic
proteins, insulin, glucagon, MafA, Ngn3 and Sca1 were analyzed in Nrp1 (b), MafA (c),
and co-transfected (d) NIH3T3 cells. Alexa Fluor 546 goat anti mouse or anti rabbit
secondary antibodies were used as controls (a). Number of positive cells is shown as
percentage of non-transfected labelled cells.
Insulin
Glucagon
MafA
Ngn3
Sca1
14.39%
6.10%
5.68%
5.14%
64%
57
(a)
(b)
58
(c)
(d)
59
(e)
60
(f)
Fig 3.12: Graphical representation of pancreatic proteins expression in transfected
NIH3T3 cells: Protein expressions of MafA (a), insulin (b), Ngn3 (c), Sca1 (d) and
glucagon (e) in non transfected control, and MafA transfected, Nrp-1 transfected and co-
transfected NIH3T3 cells are shown. Significant increase in MafA, Ngn3 and Sca1
expressions (p<0.001) after Nrp1, MafA and co-transfection was observed in NIH3T3
cells. Insulin expression was found to be significantly increased (p<0.001) in Nrp1 and
co-transfected cells while glucagon expression was significantly increased (p<0.001) in
MafA and co-transfected cells. Combined graphical representation of pancreatic proteins
expression in control and transfected groups is also shown (f). Data is presented as mean
± S.E.M; level of significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * =
p < 0.05).
61
3.5 Preconditioning of NIH3T3 Cells with Dexamethasone
and Pancreatic Extract
3.5.1 Cytotoxic Effect of Dexamethasone Quantified by JC-1
Mitochondrial Membrane Potential Assay
During apoptosis, several key events occur in mitochondria including changes in electron
transport chain (ETC), caspase activators release and loss in mitochondrial
transmembrane potential (Δψm). Therefore, mitochondrial function and cell death could
be analyzed with the change in mitochondria membrane potential. JC1 staining was used
to quantify the apoptosis induced by dexamethasone at different concentrations. JC-1 is a
lipophilic dye that can selectively penetrate into mitochondria. Color of the dye
reversibly changes from green to red with the increase in the membrane potential.
Healthy cells with high mitochondrial membrane potential give intense red fluorescence
of J-aggregates whereas, monomeric form of JC-1 in apoptotic or unhealthy cells give
green fluorescence with low mitochondrial membrane potential.
NIH3T3 cells after dexamethasone treatment at different concentrations did not show
increase in the dead cells as compared to untreated control. The percentages of apoptotic
cells were 2.62 ± 0.03, 2.6 ± 0.33, 3.19 ± 0.19, and 2.61 ± 0.26 for untreated control and
treated cells at 5 µM, 10 µM and 15 µM concentrations of dexamethasone, respectively
(Fig. 3.13).
3.5.2 Oxidative Stress Induction by Dexamethasone: Effect on Reactive
Oxygen Species (ROS) Level Quantified by 3’-(p- hydroxyphenyl)
Fluorescein (HPF)
3’-(p-hydroxyphenyl) Fluorescein (HPF) is a ROS indicator. ROS increase after
oxidative stress induction was quantified by HPF staining using flow cytometry. Upon
reaction with the ROS, HPF oxidizes and exhibits bright green fluorescence that can be
acquired on FL-1 filter.
62
ROS level after dexamethasone treatment at different concentrations was found to be
significantly increased as compared to untreated control. Mean percentage of untreated
control and dexamethasone treated cells at 5µM, 10µM, and 15µM were 14.46 ± 0.82,
19.47 ± 2.0, 17.98 ± 0.18 and 20.95 ± 0.62, respectively (Fig. 3.14).
3.5.3 Morphological Characteristics
NIH3T3 cells were preconditioned using dexamethasone and pancreatic extract.
Different combination of treatments showed different morphology. Cells showed
flattened morphology with extended cytoplasmic processes and cluster formation after
dexamethasone (Fig. 3.15 b) as well as combined treatment of MXa (5 µM
dexamethasone and 0.05 mg/mL of pancreatic extract) and MXb (5 µM dexamethasone
and 0.4 mg/mL of pancreatic extract) (Fig. 3.15 e, f) . Cells grown in CMa (0.05 mg/mL
of pancreatic extract) and CMb (0.4 mg/mL of pancreatic extract) appeared small and
round (Fig. 3.15 c, d).
3.5.4 Pancreatic Gene Expression after Preconditioning of NIH3T3
Cells with Dexamethasone and Pancreatic Extract
To check the transdifferentiation of NIH3T3 cells, pancreatic transcription factors
(MafA, Ngn3) and pancreatic genes (insulin, somatostatin) were analyzed at mRNA
level by RT-PCR. Insulin expression was significantly increased (p<0.01) after
dexamethasone treatment, and when grown in conditioned media, CMa, CMb (p<0.01),
MXa and MXb (p<0.001) respectively; MafA and Ngn3 expression levels were
increased but this increase is non-significant while somatostatin was down regulated
(p<0.001) in all groups after treatment as compared to untreated control (Fig. 3.16).
63
(a)
(b)
(c)
0.07%
2.62%
2.60%
64
(d)
(e)
3.19%
2.61%
65
(f)
Fig. 3.13: Cytotoxic measurement of NIH3T3 after dexamethasone treatment:
Dexamethasone treated NIH3T3 cells labelled with JC-1 dye were analyzed by flow
cytometry. Panels show untreated unlabelled cells (a), JC-1 labelled cells (b), and
dexamethasone treated cells having concentrations of 5 µM (c), 10 µM (d) and 15 µM
(e). Graphical representation of dexamethasone treated NIH3T3 cells labelled with JC-1
stain showed no significant apoptotic cells at all concentrations as compared to untreated
labelled control (f). Data is presented as mean ± S.E.M; level of significance is p < 0.05.
66
(a)
(b)
(c)
(d)
67
(e)
(f)
Fig. 3.14: ROS measurement after dexamethasone treatment: NIH3T3 cells treated
with dexamethasone were analyzed for ROS level. Panels show untreated unlabelled
cells (a), untreated labelled cells (b), and dexamethasone treated cells having
concentrations of 5 µM (c), 10 µM (d) and 15 µM (e). Treated cells labelled with HPF
showed increase in ROS level by the shift in FL-1 filter. Overlay diagram shows peaks
of dexamethasone treated cells at 5 µM (pink), 10 µM (blue), and 15 µM (orange)
concentrations and untreated unlabelled (red) and untreated labelled (green) cells.
Graphical representation of ROS level in NIH3T3 cells after dexamethasone treatment at
5 µM and 15 µM concentrations showed a significant (p < 0.01 and p < 0.05
respectively) increase in ROS levels (f). Data is presented as mean ± S.E.M; level of
significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * = p < 0.05).
68
3.5.5 Pancreatic Protein Expression by Flowcytometry after
Preconditioning of NIH3T3 Cells with Dexamethasone and
Pancreatic Extract
To evaluate the effect of dexamethasone and pancreatic extract on the differentiation of
NIH3T3 cells, expression of pancreatic proteins, insulin, glucagon, MafA, Ngn3, Pdx-1,
and Sca1 were analyzed by flow cytometry. Insulin, Sca1, and Pdx1 expressions were
significantly decreased (p<0.001) in dexamethasone treated cells, while glucagon, Ngn3
and MafA had no significant effect. Cells grown in CMa have shown significant increase
(p<0.001) in insulin, glucagon and Sca1 expressions. Conditioned medium with
increased concentration of pancreatic extract (CMb) has shown significant increase
(p<0.001) in insulin, glucagon, MafA, Ngn3, Pdx1 and Sca1 expressions. In case of MXa
and MXb, significant increase (p<0.001) was observed in MafA, Pdx1, glucagon and
Sca1 expressions while no significant change was seen on insulin expression (Fig. 3.17).
Protein expression of insulin, Ngn3, and Sca1 were also analyzed by
immunocytochemistry. The results showed positive expression of these proteins in all
groups (Fig. 3.18).
69
(a)
(b)
(c)
70
(d)
(e)
(f)
71
Fig. 3.15: Morphology of NIH3T3 cells after preconditioning with dexamethasone
and pancreatic extract: NIH3T3 cells grown in the presence of 5 µM dexamethasone
(b), MXa (5 µM dexamethasone and 0.05 mg/mL pancreatic extract) (c) and MXb (5 µM
dexamethasone and 0.4 mg/mL pancreatic extract) (d) showed flattened, elongated and
clustered morphology while in case of CMa (pancreatic extract; 0.05 mg/mL) (e) and
CMb (pancreatic extract; 0.4 mg/mL) appeared round and small (f) as compared to
untreated control cells (a). Images were taken at 10X magnification under inverted phase
contrast microscope.
72
(a)
GAPDH
73
74
(f)
Fig. 3.16: RT-PCR analysis of pancreatic genes in NIH3T3 cells preconditioned
with dexamethasone and pancreatic extract: Relative gene expression of GAPDH (a),
insulin (b), somatostatin (c), MafA (d), and Ngn3 (e) in NIH3T3 cells before and after
preconditioning with 5 µM dexamethasone, CMa (pancreatic extract 0.05mg/mL), CMb
(pancreatic extract 0.4mg/mL), MXa (5 µM dexamethasone and 0.05mg/mL pancreatic
extract) and MXb (5 µM dexamethasone and 0.4mg/mL pancreatic extract). Insulin
expression was significantly increased in dexamethasone (p < 0.01), CMa (p < 0.01),
CMb (p < 0.01), MXa (p < 0.001), and MXb groups (p < 0.001). Somatostatin
expression was significantly decreased (p<0.001) after all treatments. Ngn3 was found to
be decreased (p< 0.01) after dexamethasone treatment only. No significant change was
observed in MafA expression. Combined graphical representation of pancreatic gene
expression in untreated and treatment groups is also shown (f). Data is presented as mean
± S.E.M; level of significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * =
p < 0.05).
.
75
(a)
76
(b)
77
(c)
78
(d)
79
(e)
80
(f)
81
(g)
Fig 3.17: Analysis of pancreatic proteins in NIH3T3 cells preconditioned with
dexamethasone and pancreatic extract: Protein expressions of MafA (a), insulin (b),
glucagon (c), Pdx1, (d) Ngn3 (e) and Sca1 (f) in NIH3T3 cells before and after
preconditioning with 5 µM dexamethasone, CMa (pancreatic extract 0.05 mg/mL), CMb
(pancreatic extract 0.4 mg/mL), MXa (5 µM dexamethasone and 0.05 mg/mL pancreatic
extract) and MXb (5 µM dexamethasone and 0.4mg/mL pancreatic extract) are shown.
Insulin, Sca1 and Pdx1 expressions were significantly decreased (p<0.001) in
dexamethasone treated cells. Cells grown in the CMa and CMb have shown significant
increase (p<0.001) in insulin, glucagon and Sca1 expressions. MafA. Ngn3 and Pdx1
were also increased (p<0.001) in MXb group. Cells in MXa and MXb have shown
significant increase (p<0.001) in MafA, Pdx1, glucagon and Sca1 expressions. No effect
on insulin expression was observed in both groups. Combined graphical representation
of pancreatic proteins expression in untreated and treatment groups is also shown (g).
Data is presented as mean ± S.E.M; level of significance is p < 0.05; (where *** = p <
0.001, ** = p < 0.01, and * = p < 0.05).
82
(a)
83
(b)
84
(c)
85
Fig 3.18: Analysis of pancreatic proteins in NIH3T3 cells preconditioned with
dexamethasone and pancreatic extract by immunocytochemistry: Protein expression
of insulin (a), Ngn3 (b) and Sca1 (c) in normal NIH3T3 cells and after preconditioning
with 5 µM dexamethasone, CMa (pancreatic extract 0.05 mg/mL), CMb (pancreatic
extract 0.4 mg/mL), MXa (5 µM dexamethasone and 0.05 mg/mL pancreatic extract) and
MXb (5 µM dexamethasone and 0.4mg/mL pancreatic extract) are shown. Insulin, Ngn3
and Sca1 expressions were positive in all treated groups. Alexa Fluor 546 goat anti
mouse or anti rabbit antibody was used for detection.
86
3.6 Preconditioning of NIH3T3 Cells with 2, 4 Dinitrophenol
(DNP)
3.6.1 Optimization: Morphological Characteristics
NIH3T3 cells treated with different concentrations of DNP (0.025 mM – 2 mM) for 20
minutes showed round and contracted morphology (Fig: 3.19a) as compared to the
untreated control. After 48 hours of reoxygenation, cells regained normal morphology at
all concentrations except at 1 and 2 mM at which most of the cells died (Fig: 3.19b). We
selected 0.1 mM concentration of DNP for 20 minutes for all subsequent experiments as
cells were only shrunk and their normal morphology was restored after reoxygenation.
3.6.2 Pancreatic Gene Expression after Preconditioning of NIH3T3
Cells with DNP
To see the effect of DNP on transdifferentiation NIH3T3 cells, pancreatic transcription
factors (MafA, Ngn3, Nkx 6.1) and genes (insulin, glucagon somatostatin) were analyzed
by RT-PCR. Treated cells expressed insulin, MafA, and Ngn3 whereas somatostatin
expression was down regulated (Fig. 3.20). Significant increase in the expression of
insulin, glucagon (p< 0.05), Ngn3 and Nkx6.1 (p<0.01), while significant decrease (p<
0.05) in the somatostatin expression was observed after DNP treatment. No significant
change was observed in MafA levels as compared to untreated control (Fig. 3.20).
3.6.3 Pancreatic Proteins Expression after Preconditioning of NIH3T3
Cells with DNP
To evaluate the effect of DNP on the differentiation of NIH3T3 cells, expression of
pancreatic proteins, insulin, glucagon, MafA, Ngn3, Pdx-1, and Sca1 were analyzed by
flow cytometry. Treated NIH3T3 cells showed significant increase (p <0.001) in insulin,
MafA, Pdx1, glucagon and Ngn3 expressions as compared to untreated control (Fig.
3.21).
87
NIHT3T3 Cells
(After DNP Treatment)
NIHT3T3 Cells
(After Reoxygenation)
Control
DNP (0.025 mM)
DNP (0.05 mM)
88
DNP (0.1 mM)
DNP (0.25 mM)
DNP (0.5 mM)
89
DNP (1 mM)
DNP (2 mM)
Fig. 3.19: Morphology of NIH3T3 cells after DNP treatment: NIH3T3 cells treated
with different concentrations of DNP (0.025 mM – 2 mM) for 20 minutes were round
and shrunken (left panel). After 48 hours of reoxygenation (right panel), cells regained
normal morphology except in case of 1 mM and 2 mM concentrations in which most of
the cells died. Images were taken at 10X magnification under inverted phase contrast
microscope.
90
(a)
(b)
91
(c)
(d)
92
(e)
(f)
93
(g)
94
(h)
Fig. 3.20: RT-PCR analysis of pancreatic genes in NIH3T3 cells preconditioned
with DNP: Expression levels of GAPDH (a), insulin (b), Ngn3 (c), MafA (d), Nkx6.1 (e)
and somatostatin (f) in NIH3T3 cells before and after preconditioning with DNP.
Graphical representation is also shown. Ngn3, Nkx 6.1, insulin, and glucagon expression
was significantly increased (p < 0.01, p < 0.01, p < 0.05 and p < 0.05 respectively) while
that of somatostatin was significantly decreased (p<0.001) after DNP treatment. No
significant change was observed in MafA expression after DNP treatment. Combined
graphical representation of pancreatic gene expression in untreated and DNP treated
groups is also shown (h). Data is presented as mean ± S.E.M; level of significance is p <
0.05; (where *** = p < 0.001, ** = p < 0.01, and * = p < 0.05).
95
(a)
Insulin Glucagon
Ngn3 Pdx1
Sca1 MafA
3.76% 1.83%
1.91% 19.32%
56.42% 9.22%
96
(b)
Insulin Glucagon
Ngn3 Pdx1
Sca1 MafA
23.76% 8.75%
35.79% 26.06%
43.6% 36.2%
97
(c)
Fig. 3.21: Flow cytometric analysis of pancreatic proteins in NIH3T3 cells
preconditioned with DNP: Pancreatic proteins, MafA, Pdx1, insulin, glucagon, Ngn3
and Sca1 were analyzed in NIH3T3 cells before (a) and after treatment with DNP (b).
Cells labelled with Alexa Fluor 546 goat anti mouse or anti rabbit secondary antibodies
were used as controls. Number of positive cells is shown as percentage of untreated cells.
Also shown is the graphical representation of proteins expression of MafA, Pdx1,
insulin, glucagon, Ngn3 and Sca1 in NIH3T3 cells before and after DNP treatment (c).
Significant increase (p<0.001) in insulin, glucagon, MafA, Ngn3 and Pdx1 expressions
was observed after DNP treatment. Data is presented as mean ± S.E.M; level of
significance is p < 0.05; (where *** = p < 0.001, ** = p < 0.01, and * = p < 0.05).
CHAPTER 4 discussion
98
Type 1 diabetes (T1D) generally results from the autoimmune destruction of pancreatic
β-cells, resulting in the elevated blood glucose levels (Atkinson, Eisenbarth, and
Michels, 2014; Gerace, Martiniello-Wilks, O'Brien, and Simpson, 2014). To maintain
normal blood glucose concentration, the lost β-cells must be replenished. Since the
discovery of insulin, exogenous insulin administration has been the only effective
treatment to maintain the proper blood glucose levels. However, insulin therapy comes
along with the hypoglycemic episodes and does not eliminate the chronic complications
associated with diabetes (Gerace, Martiniello-Wilks, O'Brien, and Simpson, 2014).
Pancreas and islet transplantation are the current effective methods for the treatment of
T1D, however, this treatment is hampered because of limited cadaveric donors and the
use of lifetime immuno-suppressants (Johannesson, Sui, Freytes, Creusot, and Egli,
2015). The cell replacement therapy constitutes one of the best approaches to target T1D,
as it is caused by the lack of a single, well-defined cell type, the β-cell (Borowiak, 2010).
In the last few years, several promising approaches have been suggested for β-cells
regeneration (Borowiak, 2010), including reprogramming of non β-cells, direct
differentiation of stem cells or trans-differentiation of mature somatic cells like
pancreatic duct cells and hepatocytes into pancreatic β-like cells, thus giving new insight
to cure T1D (Lemper, Leuckx, Heremans, German, Heimberg, Bouwens, and Baeyens,
2014).
Somatic cells can be differentiated into a number of different cell types (cardiomyocytes,
neurons, pancreatic β-cells) by genetic modification, in order to be used in cellular
therapy of degenerative disorders (Vierbuchen, Ostermeier, Pang, Kokubu, Südhof, and
Wernig, 2010; Huang, He, Ji, Sun, Xiang, Liu, Hu, Wang, and Hui, 2011). Cellular
reprogramming with transcription factors also suggest a possibility to generate insulin
producing cells by trans-differentiation of a wide range of cells (Pandian, Taniguchi, and
Sugiyama, 2014). Transcription factors bind to the specific DNA sequences within the
region of enhancer, promoter or repressors and control different biological processes
including proliferation, differentiation and apoptosis. A number of transcription factors
have been identified which are involved in β-cell development (Guo, Zhu, Wang, Fan,
Lu, Wang, Zhu, Wang, and Huang, 2012; Hang, Yamamoto, Benninger, Brissova, Guo,
Bush, Piston, Powers, Magnuson, and Thurmond, 2014). These transcription factors can
therefore be used to induce the differentiation into insulin producing β-cells.
99
Islet of Langerhans is composed of different types of exocrine and endocrine cells that
release different hormones having a wide range of functions. Pancreatic β-cells are
specifically responsible for insulin release (Zhao, Jiang, Zhao, Ye, Hu, Yin, Li, Zhang,
Diao, and Li, 2012). The specificity for islet cell development comes from the unique
combination of transcription factors (MafA, Pdx1, Pax6 and Beta2) that bind to the
conserved enhancer regions and stimulate insulin expression (Zhao, Jiang, Zhao, Ye, Hu,
Yin, Li, Zhang, Diao, and Li, 2012). Combined action of the exclusive transcription
factors is capable of reprogramming one cell type into another. Pooled transfection of
three important factors, Pdx1 (pancreatic and duodenal homeobox-1), MafA (V-
mafmusculoaponeuroticfibrosarcoma oncogene homolog A) and Ngn3 (neurogenin 3) in
pancreatic acinar cells resulted in the formation of insulin producing cells (IPCs) (Hang,
Yamamoto, Benninger, Brissova, Guo, Bush, Piston, Powers, Magnuson, and Thurmond,
2014). Similarly, transfection of MafA, NeuroD1 (neurogenic differentiation-1) and
Pdx1 in bone marrow derived mesenchymal stem cells (BM-MSCs) result in their
differentiation into IPCs (Guo, Zhu, Wang, Fan, Lu, Wang, Zhu, Wang, and Huang,
2012). Genetic profiling revealed that MafA plays key role in insulin production and
secretion pathways as well as in maintaining genes involved in β-cell function (Zhang,
Moriguchi, Kajihara, Esaki, Harada, Shimohata, Oishi, Hamada, Morito, and Hasegawa,
2005; Wang, Brun, Kataoka, Sharma, and Wollheim, 2007; Hang, Yamamoto,
Benninger, Brissova, Guo, Bush, Piston, Powers, Magnuson, and Thurmond, 2014).
In our study, we used MafA (transcription factor) and Neuropilin-1 (angiogenic factor)
transfection, either alone or in combination, for the trans-differentiation of NIH3T3 cells
into insulin producing cells (IPCs). NIH3T3 cells transfected with Neuropilin-1 showed
morphological changes as compared to non transfected cells. Transfected cells appeared
elongated with increased number of projections and cluster formation. Significant
increase (p<0.01 and p<0.001) in insulin mRNA transcription from 24 to 96 hours of
transfection was also observed. Cells transfected with MafA have shown small and round
morphology as compared to untreated control and increase in insulin gene expression
(p<0.01) while combined transfection with Neuropilin-1 and MafA resulted in more
enhanced expression (p<0.001). These results suggest an important role of these
transcription factors in coordinating and controlling the insulin gene expression levels.
Moreover, Neuropilin-1 transfected cells alone and in combination with MafA
100
transfected cells have shown significant increase in insulin protein expression (p<0.001)
as compared to untreated control and MafA transfection alone, thus, signifying the role
of Neuropilin-1 in insulin protein expression. A significant increase in Neurogenin3
(Ngn3) expression was also observed after transfection with Neuropilin-1 (p<0.001),
MafA (p<0.01) and their co-transfection (p<0.001).
The transcription factor, MafA, used in our study for transdifferentiation of somatic cells
is known to play important role in pancreatic development, maturation, reprogramming,
production and maintainance of insulin producing cells. The other molecule i.e.
neuropilin-1 (Nrp1) is a transmembrane glycoprotein, that functions as a co-receptor for
VEGF in endothelial cells (Hasan, Kendrick, Druckenbrod, Huelsmeyer, Warner, and
MacDonald, 2010). VEGF is present in islet cells increases insulin content, and plays an
important role in pancreatic islet cell proliferation. VEGF signalling is also involved in
pancreatic islet neogenesis (Lammert, Gu, McLaughlin, Brown, Brekken, Murtaugh,
Gerber, Ferrara, and Melton, 2003). Previous research suggests that Neuropilin-1
peptides are confined to the islets and two SNPs in the intron 9 of the Neuropilin-1 gene
are found to be associated with type 1 diabetes (T1D) (Hasan, Kendrick, Druckenbrod,
Huelsmeyer, Warner, and MacDonald, 2010). Ngn3 is a basic helix-loop-helix
transcription factor, expressed in the pancreatic endocrine progenitor cells (Gradwohl,
Dierich, LeMeur, and Guillemot, 2000).
Cell’s regeneration potential can be enhanced by different preconditioning strategies.
Microenvironment plays an important role in the cell specification, differentiation and
development. Bone marrow isolated mesenchymal stem cells have been shown to
successfully differentiate into insulin producing β-cells in the presence of the injured
pancreatic tissue extract (Xie, Wang, Zhang, Qi, Zhou, and Li, 2013). Oxygen also
functions as a vital component of cellular differentiation, endocrine cell specification,
homeostasis, and many developmental events besides its role in aerobic respiration
(Fraker, Ricordi, Inverardi, and Dominguez-Bendala, 2009). Increased oxygen delivery
to the developing pancreas has shown significant up regulation of endocrine
differentiation markers (Fraker, Alvarez, Papadopoulos, Giraldo, Gu, Ricordi, Inverardi,
and Dominguez-Bendala, 2007). Increase in oxygen tension inhibits hypoxia inducible
101
factor-1α (HIF-1α) mediated activation of Notch signalling and inhibition of β-catenin
directed Wnt signalling activation. Down regulation of Notch yields cells that express
Ngn3 which is a pro-endocrine marker leading to further differentiation into all
endocrine cell types (Fraker, Ricordi, Inverardi, and Dominguez-Bendala, 2009).
One of the preconditioning strategies used in this study for the transdifferentiation of
NIH3T3 cells into insulin producing β-cells, is the use of dexamethasone (Dx), either
alone or in combination with the pancreatic extract at two different protein
concentrations, 0.05 and 0.4 mg/mL. Pancreatic extract provide the microenvironment to
the cells, as it is rich in pancreatic proteins and growth factors. Dexamethasone is a
glucocorticoid; glucocorticoids are involved in late phases of differentiation when
endocrine and exocrine cell differentiation occurs during pancreas development
(Dumortier, Theys, Ahn, Remacle, and Reusens, 2011). Previously, dexamethasone has
been used for different trans-differentiation studies and shown to differentiate pancreatic
AR42J cells to exocrine cells (Mashima, Yamada, Tajima, Seno, Yamada, Takeda, and
Kojima, 1999) and pancreatic exocrine cells to hepatocytes (Shen, Seckl, Slack, and
Tosh, 2003). In the present study, dexamethasone concentration was selected on the basis
of least cytotoxicity and significant increase in the ROS production after treatment of
NIH3T3 cells with different concentrations of dexamethasone (5 µM, 10 µM and 15
µM). Highest increase in the ROS level (p<0.01) was observed at 5µM concentration
after 4 days of treatment with no substantial increase in the apoptotic cells at all
concentrations. This optimized dose (5µM) was therefore selected for all experiments.
After treatment, cells appeared flattened and hexagonal in shape, and cluster formation
was also observed in some areas. Dexamethasone increased (p<0.01) insulin gene
expression while the expressions of Pdx1, Sca1, MafA, glucagon, Ngn3 and somatostatin
were either decreased or no change was observed both at the gene or protein levels. Cells
grown in the presence of pancreatic extract at both concentrations (0.05 and 0.4 mg/mL)
appeared round and small in shape and showed increase (p<0.01) in the insulin gene
expression and decrease (p<0.001) in somatostatin expression while protein expression
of insulin, glucagon, Sca1, Pdx1 and Ngn3 have also showed significant change
(p<0.001). This increase in insulin expression can be attributed to the presence of β-cell
specific cytokines and growth factors that help in the transdifferentiation of NIH3T3
cells into insulin producing β-cells.
102
Combined treatment with dexamethasone and pancreatic extract have shown a
significant increase (p<0.001) in insulin gene expression; however, it did not enhance the
insulin protein expression. Moreover, it has shown a significant increase (p<0.001) in
MafA, Pdx1 and glucagon protein expressions. This suggests that dexamethasone may
have an inhibitory effect on insulin translation. In earlier studies, insulin DNA content
was found to be increased in the islet-like cell clusters after dexamethasone treatment;
however, the cells were unable to control blood glucose levels when injected in diabetic
animals (Korsgren, Andersson, and Sandier, 1993). We have found similar effect of
dexamethasone after treatment of NIH3T3 cells for four days either alone or in the
presence of pancreatic extract as it is only increasing the transcription of insulin gene.
Moreover, preconditioning with dexamethasone, pancreatic extract alone and in
combination resulted in increased (p<0.001) glucagon protein expression signifying the
differentiation of NIH3T3 cells into glucagon producing α-cells.
The other preconditioning strategy used in this study is the treatment with 2, 4-
dinitrophenol (DNP). In our recent study, we have observed that a number of genes
involved in the cell survival, growth, differentiation and homing have been upregulated
after preconditioning of mesenchymal stem cells with DNP (Ali, Akhter, Haneef, Khan,
Naeem, Habib, Kabir, and Salim, 2015). DNP is a lipophilic weak acid that uncouple the
oxidative phosphorylation by decreasing the ATP production (Shavell, Fletcher, Jiang,
Saed, and Diamond, 2012). DNP treatment has been shown to decrease fasting and
random blood glucose levels and improve glucose homeostasis (Goldgof, Xiao,
Chanturiya, Jou, Gavrilova, and Reitman, 2014). We optimized DNP dose by treating
cells with different concentrations (0.025 – 2 mM) followed by 48 hours of reperfusion.
Cells after DNP treatment appeared small and got shrunk displaying stress condition.
However, it attains normal morphology after reoxygenation. The concentration of 0.1
mM was selected on the basis of having lesser number of apoptotic cells. After
preconditioning, expression of almost all pancreatic genes was upregulated in NIH3T3
cells. Ngn3, Nkx6.1, insulin, and glucagon expressions were significantly increased
(p<0.01, p<0.01, p<0.05 and p<0.05 respectively). MafA expression was also increased
but the change was not significant. In addition, somatostatin was significantly decreased
(p<0.001) after DNP treatment. Also, protein concentrations of insulin, MafA, glucagon,
Pdx1 and Ngn3 were significantly increased (p<0.001) after DNP treatment.
103
The enhanced expression of endocrine markers, specifically β-cell specific transcription
factors demonstrate the successful regeneration of insulin producing β-cells.
Furthermore, significant change in glucagon expression suggests that the cells are also
differentiating to glucagon producing α-cells. DNP was used to give oxidative stress and
it has been shown to enhance the differential potential of NIH3T3 cells by significantly
increasing the endocrine marker (Ngn3). A high O2 condition during the early stage of
differentiation is reported to increase the percentage of Ngn3-expressing endocrine
progenitor and insulin positive cells in both mESC and hiPSC at the terminus of
differentiation via HIF-1α inhibition and stimulation of Ngn3 gene expression (Hakim,
Kaitsuka, Raeed, Wei, Shiraki, Akagi, Yokota, Kume, and Tomizawa, 2014). However,
during hypoxia, HIF-1α expression was enhanced which results in the activation of
Notch signalling. Activation of Notch results in the inhibition of β-cell differentiation by
inhibiting Ngn3 expression. DNP is also used to give chemical hypoxia to the cells as it
depletes ATP production (Jovanović, Sukhodub, and Jovanović, 2009). However, DNP
treatment is reported to suppress HIF-1α expression and enhance Ngn3 expression, as
well as it is reported to improve Nrp1 expression (Ali, Akhter, Haneef, Khan, Naeem,
Habib, Kabir, and Salim, 2015). This increase in Nrp1 expression or enhanced Ngn3
expression via HIF-1α suppression may lead to increase in the insulin level in treated
NIH3T3 cells.
In our study, the strategies to induce efficient trans-differentiation of NIH3T3 cells into
IPCs have shown enhanced but variable expression pattern of endocrine markers,
specifically β-cell specific transcription factors demonstrating their successful
regeneration. The study could further be evaluated for their in vivo effect and serve as an
improved and effective cellular therapeutic option for type 1 diabetes.
CONCLUSION
104
We used genetic modification as well as different preconditioning strategies to
differentiate mature fibroblasts into insulin producing β-cells. Our study concluded, that
genetic manipulation of mature fibroblasts (NIH3T3 cells) with MafA, and Nrp1 genes
enhanced the expression of pancreatic genes, Ngn3, and insulin, as well as increased the
protein expression of MafA, Ngn3, insulin, glucagon, and Sca1. Preconditioning with
dexamethasone showed increase in the insulin gene expression. Microenvironment in the
form of pancreatic extract also enhanced insulin gene and protein expression levels.
Dexamethasone shows a possible inhibitory effect on insulin translation when used in
combination with the pancreatic extract. Glucagon was also found to be upregulated after
preconditioning with dexamethasone, pancreatic extract as well as after combined
treatment, showing that these cells may also be differentiating towards alpha cell lineage.
The best preconditioning effect was observed with 2, 4-dinitrophenol (DNP) as it
enhanced the gene, as well as protein expressions of almost all pancreatic genes. The
present study is yet another attempt to induce efficient transdifferentitaion of mature cell
type into insulin producing β-cells by using different strategies that could serve as a
therapeutic approach for the treatment of diabetes mellitus in future.
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LIST OF PUBLICATIONS
1. Usman S, Khan I, Naeem N, Iqbal H, Ali A, Usman S, and Salim A. (2016).
Conditioned Media Trans-differentiate Mature Fibroblasts into Pancreatic
Beta like Cells. Life Sci., 164, 52-59.
2. Waseem M, Khan I, Iqbal H, Eijaz S, Usman S, Ahmed N, Alam G, and Salim A.
(2016). Hypoxic preconditioning Improves the Therapeutic Potential of
Aging Bone Marrow Mesenchymal Stem Cells in Streptozotocin- Induced
Type-1 Diabetic Mice. Cell Reprogram., 18, 344-355.
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PERSONAL INTRODUCTION
Shumaila Usman
shumaila_iccs@yahoo.com
I am Shumaila Usman, born and raised in Karachi. I received my schooling from
Shaheen Public School and Jasmine Care Primary Secondary School with 1st position in
each class. Secured 85.4% in secondary school exams in 2004. My higher secondary
studies were from Govt. Degree College (Malir Cantt.). Unlike others which had their
primary priority for medical schooling, my self-motivation encouraged me to carry on
my education in basic biological sciences, thus, I opted botany as my major subject in the
undergraduate studies from University of Karachi. During under graduate studies, I got
acquainted with Biochemistry as subsidiary course and this was the turning point in my
academic career which later turned over as a transition from Botany to Biochemistry as a
graduate subject. During undergraduate studies, exposure to several workshops and
scientific conferences aroused my interest towards scientific research. The thirst to gain
research exposure motivated me to secure an internship at the Stem Cell laboratory of
International Center for Chemical and Biological Sciences (ICCBS), University of
Karachi. Inspirations from graduate studies and a decent exposure at stem cell research
laboratory harnessed my intent to join ICCBS as research student.
Immediately after passing the graduate exam in 2010, I was fortunate to secure a position
of Junior Research Fellow at ICCBS in July 2011 under the admirable guidance of Dr.
Asmat Salim. Joining ICCBS as research scholar opened new horizons of research for me
and raged my thirst to obtain a doctorate. After completion of courses with distinction,
passed GRE type exam with the percentile of 93, I wasted no time on working out my
doctoral dissertation on “Transdifferentiation of Mature Cell type into Insulin producing
β-Cells”.
120
During this time period not only I learned different molecular biology techniques
including animal cell and tissue culture, flow cytometry, immunocytochemistry, PCR etc
but it also gave me confidence in interpretation and analysis of data. It should also be
mentioned that my research advisor, Dr. Asmat Salim groomed my personality and
trimmed my academic as well as professional temperament.
My stay at the PCMD not only helped me building up my career but also taught me to
deal with interactive issues and professional pressures. It also helped me to identify my
leadership capacity within. The training here kindled the essence of working and
volunteering in various extra-curricular activities. Moreover, I was able to pivot
numerous conferences, workshops and scientific sessions at the institute.
I would like to acquire more skills in the ocean of knowledge with new-fangled research
goals ahead in future and I hope that this study and training contribute further in future
for betterment and welfare of society and mankind.
“Think big, think fast and think ahead. Ideas are no-one’s monopoly.” (Mr D. H.
Ambani)
Shumaila Usman
Research Fellow (Molecular Medicine)
PCMD, ICCBS- Batch 2011.
APPENDICES
121
APPENDIX-I
Reagents and Chemicals Company Name Catalog Number
Agarose Sigma, Germany A-9539
Ampicillin Serva 13398.02
Bovine Serum Albumin MP Biomedical Inc, USA 151429
Cell Dissociation Solution Gico, USA 13150-016
Chloroform Scharlau CL 0205
Dexamethasone Serva electrophoresis 18660
DNA Ladder (100bp) Thermo Scientific, USA R0611
DNA Loading Dye Bioron, Germany 11400
Diethylpyrucarbonate (DEPC) Serva 18835
Dulbecco’s modified Eagle’s
medium (DMEM) high glucose
Gibco, life technologies, USA 11995-065
Dimethylsufoxide (DMSO) Fisher Scientific, USA D4121/PB08
EDTA Fisher Scientific, USA 60-009-4
Ethanol Fisher Scientific, USA 64-17-5
Ethidium Bromide MP Biomedical Inc, USA 04802511
Fetal bovine serum (FBS) PAA, Austria A11-104
Formaldehyde Sigma, USA F1268
Go Taq® Green Master Mix Promega, USA M7122
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Glycerol Fisher Scientific 56-81-5
Goat Serum Jackson Immuno Research
Lab, USA
005-000-121
Isopropanolol Scharlau AL03232500
KCl Merck TA200635
KH2PO4 Acrose, USA 205925000
L-glutamine Gibco, Boston, USA 25030
Lipofectamine Invitrogen, life technologies,
USA
11668-019
Mercaptoethanol MP Biomedical, Inc 190242
Methanol Fisher Scientific, USA 67-56-1
Mounting Medium Merck UN1307
Na2HPO4 Merck 1.06586.0500
Paraformaldehyde Riedel-dehaen 16005
RNA erase MP Biomedical Inc, USA 821682
Sodium Azide Serva 30175
Sodium Choride (NaCl) Serva 30183
Sodium Pyruvate Gibco, USA 11360
Sodium Phosphate dibasic dehydrate Sigma, USA 30435
Streptomycin and Penicillin Gibco, Boston, USA 15140
Tris base Promega H5131
123
Taq Polymerase Invitrogen, USA 11615-010
Triton X 100 Sigma, USA T8787
Trizole Invitrogen, USA 1559026
Trypsin EDTA (0.5 %) Gibco, life technologies,
Canada
15090-046
Tween-20 MP Biomedical, Inc 195532
Tryptone Acumedia 7351A
Xylene Scharlau 100572500
Yeast Extract Lab M limited MC-1
Kits
JC-1 Mitochondrial Membrane
Potential Assay Kit
Cayman Chemical Company,
USA
10009172
HPF staining Invitrogen, Molecular Probes,
USA
H36004
SV Total RNA isolation System Promega, USA Z3100
SuperScriptIII first strand
synthesis
Invitrogen life technologies,
USA
18080-051
Plasmid Isolation Kit Real Genomics Y PD 100/Y PD
300
Antibodies
Alexa fluor 546 goat anti mouse
secondary antibodies
Invitrogen, USA A-11001
Alexa fluor 546 goat anti rabbit Invitrogen, USA A-11010
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secondary antibodies
Insulin Millipore 05-1066
Glucagon Santa Cruz, Inc, USA Sc-57171
MafA Santa Cruz, Inc, USA Sc-66958
Neurogenin3 Santa Cruz, Inc, USA Sc-136002
Pdx-1 Santa Cruz, Inc, USA Sc-25403
Sca1 Santa Cruz, Inc, USA Sc-24758
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APPENDIX- II
FORMULA
RNA Concentration:
Primer Calculations:
Plasmid Concentration:
126
Solution Preparations for Immunocytochemistry
Solution Preparations for Flowcytometry
1X PBS (1000 mL)
NaCl 8 grams
KCl 0.2 grams
Na2HPO4 1.15 grams
KH2PO4 17.6 grams
4% Paraformaldehyde
Paraformaldehyde 4 grams
1 X PBS 100 mL
0.1% Triton X-100
Triton X-100 10 µL
Milli Q water 9.99 mL
Blocking Solution
Bovine Serum
Albumin
2 %
Goat Serum 2 %
Tween 10 0.1 %
FACS solution
Bovine Serum
Albumin
1 %
EDTA 1 mM
Sodium Azide 0.1 %
PBS 100 mL
Blocking Solution
Bovine Serum
Albumin
1 %
PBS 1 mL
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Buffer Preparations
TE Buffer for Primers
Stock Solution 1M Tris-HCl/EDTA (TE) buffer pH 8.0
Tris 3.02 grams
H2O 20 mL
EDTA 0.292 grams
Adjust pH, mixed well and stored at 4 ºC
Working Solution 1 X TE buffer
Tris-HCl/EDTA buffer 0.01 mL
DEPC treated water 1 mL
TBE Buffer for Gel Electrophoresis
Stock Solution 5 X TBE
Tris Base 54 grams
Boric Acid 55 grams
EDTA 1.86 grams
Add 1000 mL distilled water, mixed well and stored at 4 ºC
Working Solution 1 X TBE
5 X TBE 200 mL
Milli Q water 800 mL
Mixed well and store at room temperature
Agarose Gel (1 %) for Electrophoresis
Agarose 1 gram
1 X TBE 100 mL
Ethidium Bromide 1.5 µL
128
Medium Preparations
Preperation of Complete Medium
Fetal Bovine Serum 58 mL
L-glutamine (200 mM) 11.5 mL
Penicillin / Streptomycin 6 mL
Sodium Pyruvate (100 mM) 6 mL
DMEM 500 mL
Mixed all the ingredients, filtered, sealed and stored at 4 ºC
Preparation for Cell line Freezing Medium
Medium 1
Fetal Bovine Serum 2 mL
Complete DMEM 8 mL
Medium 2
DMSO (filtered) 1 mL
Fetal Bovine Serum 1 mL
Complete DMEM 8 mL
Incomplete Medium for Transfection
L-glutamine (200 mM) 11.5 mL
Sodium Pyruvate (100 mM) 6 mL
DMEM 500 mL
Mixed all the ingredients, filtered, sealed and stored at 4 ºC
129
Preparation of 0.1% Diethylpyrocarbonate (DEPC) treated water
DEPC 1 mL
milli Q water 99 mL
Mixed well and incubated at 37 ºC for 12 hours. Autoclaved and stored at 4 ºC.
Sterilization with DEPC water
1.5-2 mL eppendorf tubes were soaked completely in 0.1 % DEPC treated water
(unsterile) and incubated at 37 ºC for 2 hours. In the laminar flow hood, with the help of
a forcep, eppendorfs were rinsed two to three times with DEPC treated water (sterile)
and placed in an empty beaker for autoclave.
Preparation of Dexamethasone
Chemical Formula C22H29FO5
Molecular weight 392.46 grams
Stock Solution (50 mM)
Dexamethasone 9.811 grams
Methanol 500 µL
Working solution was made from 50 mM stock solution with complete medium
according to the requirement
GLOSSARY
130
GLOSSARY
Apoptosis
It is defined as a natural process of programmed cell death generally characterized by
distinct morphology and sequential biochemical events that results in the eradication of
the cell without inflammation.
Cellular Differentiation
It is a process of cell changing from a less specialized type to a more specialized type.
Electrophoresis
Electrophoresis is a technique used to separate macromolecules (proteins, DNA, RNA)
on the basis of their size. Separation of the charged molecules was carried under the
influence of uniform electric field.
Fluorescence Activated Cell Sorting
Flow cytometry is a laser-based, biophysical technology which detects the cells in a
stream of fluid and allow multi parametric analysis of the physical
and chemical characteristics of up to thousands of particles per second. It is employed
in cell sorting, cell counting, protein engineering and biomarker detection.
Fluorescence Microscope
A fluorescence microscope is an optical microscope that uses fluorescence to generate an
image.
Gene Expression
Gene expression is the process by which information from a gene is used in the synthesis
of a functional gene product. These products are often proteins, but in non-protein coding
genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product
is a functional RNA. This process is used by prokaryotes, eukaryotes as well as to virus
in order to generate the macromolecular machinery for life.
Immunocytochemistry
Immunocytochemistry (ICC) is a technique used to visualize proteins and peptides in
cells by using biomolecules capable of binding the protein of interest. Usually the
131
biomolecule is an antibody that is linked to a reporter, e.g. a fluorophore, fluorescent
dye, or enzyme. The reporter will give rise to a signal, e.g. fluorescence or color from an
enzyme reaction, which is then detectable by a microscope.
In Vitro Study
In vitro (Latin: in glass) refers to the studies that are conducted using components of
an organism, isolated from their usual biological surroundings, such
as microorganisms, cells or biological molecules. For example, microrganisms or cells
can be studied in artificial culture medium, proteins can be examined in solutions.
Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells to an
embryonic stem cell–like state by forced expression of genes and factors important for
maintaining the defining properties of embryonic stem cells. IPSCs demonstrate
important characteristics of pluripotent stem cells, including expressing stem cell
markers, forming tumors containing cells from all three germ layers, and being able to
contribute to many different tissues when injected into mouse embryos at a very early
stage in development.
Melting Temperature
Meting temperature (Tm) is defined as the temperature at which 50% of all molecules of
a given DNA sequence are hybridized into a double strand, and 50% are present as single
strands. It simply referes to the dissociation of half of the two molecules of the DNA
double helix.
Oxidative Stress
Oxidative stress refers to the imbalance between the reactive oxygen species generation
and a biological system's ability to readily detoxify the reactive intermediates. Imbalance
of the redox state of cell results in the production of peroxides and free radicals that
cause toxic effects and damage to all components of the cell, including proteins, lipids,
and DNA.
Plasmid DNA
Plasmids or replicons, is a small unit of DNA molecule that is physically separated from
a chromosomal DNA and capable of replicating autonomously within a suitable host.
132
They are most commonly found in bacteria as small, circular, double-stranded DNA
molecules; however, plasmids are sometimes present in archaea and eukaryotic
organisms.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a technology used to amplify a single copy or a
few copies of a piece of DNA across several orders of magnitude, generating thousands
to millions of copies of a particular DNA sequence.
Proliferation
Proliferation is the growth of tissue cells. In many diseases, it is abnormal. Cancer cells
are very prolific, they have high rates of cell division and growth.
Protein Expression
Protein expression refers to the way in which proteins are synthesized, modified
and regulated in living organisms
Reactive Oxygen Species
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen,
formed as a natural by-product of the normal metabolism of oxygen and have important
roles in cell signalling and homeostasis. Examples include peroxides, superoxide,
hydroxyl radical, and singlet oxygen.
Reprogramming
Reprogramming refers to remodelling of epigenetic marks, such as DNA methylation by
using different transcription factors and small molecules. It is mainly used in the creation
of induced pluripotent stem cells from mature cell types by transfecting the mature cell
type with stem cell associated genes.
Regenerative Medicine
Regenerative medicine is an emerging branch of tissue engineering and molecular
biology which involves the study of different processes of cell/ tissue replacement, tissue
engineering or restoration of injured tissues or organs to repair or establish normal
function, by stimulating the body's own repair mechanisms.
Somatic Cells
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The word "somatic" is derived from the Greek word sōma, meaning "body". A somatic
cell is any biological cell forming the body of an organism; like bones, blood, skin,
connective tissue or any cell other than a gamete, germ cell, gametocyte or
undifferentiated stem cell. 220 types of somatic cells are present in the human body.
Transfection
The word transfection is a blend of trans- and infection. In this process genetic material
(such as supercoiled plasmid DNA ), or even proteins such as antibodies, may be
transfected into cells. The term is often used for non-viral methods in eukaryotic cells
and can be carried out by using calcium phosphate, electroporation, and cationic lipids
etc which allow the cargo delivery by fusion of the cell membrane.
Transdifferentiation
Transdifferentiation, also known as lineage reprogramming, is a process of
transformation of one mature somatic cell into another mature somatic cell without
undergoing an intermediate pluripotent state. It is a type of metaplasia, which includes all
cell fate switches, including the interconversion of stem cells. Current uses of
transdifferentiation include disease modelling and drug discovery and in the future may
include gene therapy and regenerative medicine.
Type I Diabetes
Type 1 Diabetes Mellitus (also known as type 1 diabetes, or T1D; formerly insulin-
dependent diabetes or juvenile diabetes) is a form of diabetes mellitus that results from
the autoimmune destruction of the insulin-producing beta cells in the pancreas. The
subsequent lack of insulin leads to increased blood and urine glucose. The classical
symptoms are polyuria (frequent urination), polydipsia (increased
thirst), polyphagia (increased hunger) and weight loss.
Viral Vector
Viral vectors are common tools used to deliver genetic material into cells. This process
can be performed inside a living organism (in vivo) or in cell culture (in
vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport
their genomes inside the cells they infect. Delivery of genes by a virus is termed
transduction and the infected cells are described as transduced.
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