SHUMAILA USMAN - prr.hec.gov.pk

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


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

iii

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.

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.

http://frodo.wi.mit.edu/primer3/

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.

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.

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)


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


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


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.

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).


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).


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

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.


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.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Guo%20QS%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhu%20MY%5BAuthor%5D&cauthor=true&cauthor_uid=22761608


http://www.ncbi.nlm.nih.gov/pubmed/?term=Fan%20XJ%5BAuthor%5D&cauthor=true&cauthor_uid=22761608


http://www.ncbi.nlm.nih.gov/pubmed/?term=Lu%20YH%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20ZW%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhu%20SJ%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

http://www.ncbi.nlm.nih.gov/pubmed/?term=Huang%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22761608

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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118

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.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Waseem%20M%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Khan%20I%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Iqbal%20H%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Eijaz%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Usman%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Ahmed%20N%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Alam%20G%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/?term=Salim%20A%5BAuthor%5D&cauthor=true&cauthor_uid=27500307

https://www.ncbi.nlm.nih.gov/pubmed/27500307

119

PERSONAL INTRODUCTION

Shumaila Usman

[email protected]

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

122

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

124

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

125

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

127

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


Complete DMEM 8 mL

Medium 2

DMSO (filtered) 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

https://en.wikipedia.org/wiki/Laser

https://en.wikipedia.org/wiki/Chemical

https://en.wikipedia.org/wiki/Cell_sorting

https://en.wikipedia.org/wiki/Cell_counting

https://en.wikipedia.org/wiki/Protein_engineering

https://en.wikipedia.org/wiki/Biomarker

https://en.wikipedia.org/wiki/Optical_microscope

https://en.wikipedia.org/wiki/Gene

https://en.wikipedia.org/wiki/Gene_product

https://en.wikipedia.org/wiki/Protein

https://en.wikipedia.org/wiki/TRNA

https://en.wikipedia.org/wiki/SnRNA

https://en.wikipedia.org/wiki/RNA

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.

https://en.wikipedia.org/wiki/Latin_language

https://en.wikipedia.org/wiki/Research

https://en.wikipedia.org/wiki/Organism

https://en.wikipedia.org/wiki/Microorganism

https://en.wikipedia.org/wiki/Cell_(biology)

https://en.wikipedia.org/wiki/Biological_molecule

https://en.wikipedia.org/wiki/Culture_medium

https://en.wikipedia.org/wiki/Solution

javascript:glosspop('ips')

https://en.wikipedia.org/wiki/Reactive_oxygen_species

https://en.wikipedia.org/wiki/Peroxide

https://en.wikipedia.org/wiki/Free_radical


https://en.wikipedia.org/wiki/Lipid

https://en.wikipedia.org/wiki/DNA

https://en.wikipedia.org/wiki/Replicon_(genetics)

https://en.wikipedia.org/wiki/GDNA

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

https://en.wikipedia.org/wiki/Bacteria

https://en.wikipedia.org/wiki/Archaea

https://en.wikipedia.org/wiki/Eukaryote

https://en.wikipedia.org/wiki/Eukaryote

https://en.wikipedia.org/wiki/DNA_replication

https://en.wikipedia.org/wiki/DNA

https://en.wikipedia.org/wiki/DNA_sequence

https://www.nlm.nih.gov/medlineplus/ency/article/001289.htm

https://en.wikipedia.org/wiki/Oxygen

https://en.wikipedia.org/wiki/Cell_signaling

https://en.wikipedia.org/wiki/Homeostasis

https://en.wikipedia.org/wiki/Peroxide

https://en.wikipedia.org/wiki/Superoxide

https://en.wikipedia.org/wiki/Hydroxyl_radical

https://en.wikipedia.org/wiki/Singlet_oxygen

https://en.wikipedia.org/wiki/DNA_methylation

https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cells

https://en.wikipedia.org/wiki/Tissue_engineering

https://en.wikipedia.org/wiki/Molecular_biology

https://en.wikipedia.org/wiki/Molecular_biology

133

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.

https://en.wikipedia.org/wiki/Greek_language


https://en.wikipedia.org/wiki/Gamete

https://en.wikipedia.org/wiki/Germ_cell

https://en.wikipedia.org/wiki/Gametocyte

https://en.wikipedia.org/wiki/Stem_cell

https://en.wikipedia.org/wiki/Blend_word

https://en.wikipedia.org/wiki/Genetics

https://en.wikipedia.org/wiki/DNA_supercoil


https://en.wikipedia.org/wiki/Antibodies

https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell

https://en.wikipedia.org/wiki/Metaplasia

https://en.wikipedia.org/wiki/Drug_discovery

https://en.wikipedia.org/wiki/Gene_therapy

https://en.wikipedia.org/wiki/Regenerative_medicine

https://en.wikipedia.org/wiki/Diabetes_mellitus

https://en.wikipedia.org/wiki/Autoimmune

https://en.wikipedia.org/wiki/Insulin

https://en.wikipedia.org/wiki/Beta_cells

https://en.wikipedia.org/wiki/Pancreas

https://en.wikipedia.org/wiki/Polyuria

https://en.wikipedia.org/wiki/Polydipsia

https://en.wikipedia.org/wiki/Polyphagia

https://en.wikipedia.org/wiki/Genetic_material


https://en.wikipedia.org/wiki/In_vivo

https://en.wikipedia.org/wiki/Cell_culture

https://en.wikipedia.org/wiki/In_vitro

https://en.wikipedia.org/wiki/In_vitro

https://en.wikipedia.org/wiki/Virus

https://en.wikipedia.org/wiki/Genome

https://en.wikipedia.org/wiki/Gene

https://en.wikipedia.org/wiki/Transduction_(genetics)

SHUMAILA USMAN - prr.hec.gov.pk

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