In vitro differentiation of stem cells towards islet like cells

IN VITRO DIFFERENTIATION OF STEM CELLS TOWARDS ISLET-LIKE
CELLS

NGO KAE SIANG
(B.Sc., National University of Singapore)

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF MEDICINE
NATIONAL UNIVERSITY OF SINGAPORE
2011

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ACKNOWLEDGEMENTS
First of all, I want to thank my supervisors and advisors Professor Lee Kok Onn,
Dr. Gan Shu Uin, Professor Roy Calne, Dr. Susan Lim, Dr. Kerrie Tang, Dr. Fong Chui
Yee, and Associate Professor Phan Toan Thang. Prof. Lee has taught me the scientific
way of thinking. I am grateful for the invaluable time he spent to read and edit this thesis.
I want to thank Dr. Gan Shu Uin. It has been an honor to be her student. I appreciate all
her support, guidance and encouragement, patience and understanding during moments of
personal difficulties. She has taught me, both consciously and unconsciously, how good
experiments are done. I thank her for delivering knowledge selflessly, and spending time
to read and edit my thesis. She has been more than a supervisor. She’s been an
encouraging mentor, warm colleague, and caring friend. The enthusiasm Professor Roy
Calne has for research has always inspired me to see science with interest and curiosity. I
appreciate his contributions of ideas and funding to make my graduate experience

productive and stimulating. I would like to thank Dr. Susan Lim and Dr. Kerrie Tang for
supplying the adipose tissue derived stem cells for the experiments of this project. Dr.
Fong has generously supplied the Wharton’s Jelly derived stem cells for all the
experiments of this research project. Prof. Phan also generously supplied the cord lining
stem cells for the experiments.
In addition, I want to thank Dr. Ma FengJuan and Jayavani D/O Karuppasamy for
isolating and culturing adipose tissue derived stem cells. I would like to thank Jeyakumar
Masilamani, who did the primary isolation and culture for cord-lining cells with Prof.
Phan. I would also like to thank Arjunan Subramanian who cultured the Wharton’s Jelly

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derived stem cells. I would also like to thank Division of Regenerative Medicine, Pfizer
for help with analysis of Figure 4.13.
I would like to thank my labmates in Phoenix group: Diane Tan Ai Lin, Ooi Shu
Qin, Zhou Yue, Loke Wan Ting and Fu ZhenYing for their assistance, friendship and
happy memories in the past few years. Diane and Shu Qin have given me precious mental
support during my hard times.
I am also very grateful to my friend, Teo Peck Lian, for her love, listening ears,
support, care, and understanding.
Last but not least, I want to thank my family, for their love and encouragement,
and supporting me unconditionally.

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TABLE OF CONTENTS
TITLE PAGE...…………………………………………………...……………………....i
ACKNOWLEDGEMENTS.............................................................................................iii

TABLE OF CONTENTS..................................................................................................v
SUMMARY.......................................................................................................................xi
LIST OF FIGURES AND TABLES.............................................................................xiii
LIST OF PRESENTATIONS.........................................................................................xv
LIST OF ABBREVIATIONS........................................................................................xvi

CHAPTER 1 Introduction and Literature Review.........................................................1
1.1

Background..............................................................................................................1
1.1.1

1.2

1.3

1.4

What is diabetes?.........................................................................................1

Currently available Diabetes Treatments.................................................................3
1.2.1

Whole organ transplantation....................................................................4

1.2.2

Human pancreatic islet transplantation...................................................5

Gene therapy............................................................................................................6

1.3.1

In vitro gene therapy....................................................................................7

1.3.2

In vivo gene therapy.....................................................................................8

Stem cell therapy....................................................................................................10
1.4.1

Pancreas development................................................................................11

1.4.2

Human embryonic stem cells.....................................................................14

1.4.3

Induced pluripotent stem cells...................................................................16

1.4.4

Mesenchymal stem cells............................................................................18

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1.4.4.1 Bone marrow derived mesenchymal stem cells.............................18
1.4.4.2 Pancreatic stem cells......................................................................19

1.4.4.3 Amnion-derived stem cells............................................................21
1.4.4.4 Adipose tissue derived mesenchymal stem cells...........................22
1.4.4.5 Wharton’s jelly derived mesenchymal stem cells and cord-lining
stem cells........................................................................................24
1.5

Objectives of the present study..............................................................................26

CHAPTER 2 Materials and Methods............................................................................27
2.1

Materials................................................................................................................27
2.1.1

Primary cells and cell lines........................................................................27
2.1.1.1 Human embryonic kidney 293T cells............................................27
2.1.1.2 Cord lining stem cells....................................................................27
2.1.1.3 Wharton’s jelly derived mesenchymal stem cells………………..27
2.1.1.4 Adipose tissue derived mesenchymal stem cells……………….28

2.2

2.1.2

RT-PCR primers........................................................................................29

2.1.3

Antibodies and assay kits...........................................................................30

Methods..................................................................................................................31
2.2.1

In vitro differentiation towards insulin-producing cells............................31
2.2.1.1 3-step differentiation protocols............…………………………..31
2.2.1.2 1-step 7-day differentiation protocols...............………………….31

2.2.2

Reverse transcription-polymerase chain reaction (RT-PCR)....................32

2.2.3

Agarose gel electrophoresis.......................................................................33

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2.2.4

Flow Cytometry.........................................................................................33

2.2.5

Enzyme-linked immunosorbent assay (ELISA)........................................33

2.2.6

Cytospin.....................................................................................................34

2.2.7

Immunocytochemistry...............................................................................34

2.2.8

Dithizone Staining.....................................................................................34

2.2.9

Microscopy………………………………………………………......…..35

CHAPTER 3 Characterization of Cord Lining Stem Cells (CLSCs), Wharton’s
Jelly Derived Mesenchymal stem cells (WJSCs), and Adipose Tissue Derived
Mesenchymal Stem Cells (ADSCs).................................................................................36
3.1

Introduction............................................................................................................36

3.2

Results....................................................................................................................37

3.3

3.2.1

Morphology of CLSCs...............................................................................37

3.2.2

Morphology of WJSCs..............................................................................38

3.2.3

Morphology of ADSCs..............................................................................39

3.2.4

Fluorescence-activated cell sorting (FACS) analysis for MSC markers...40

3.2.5

Sox17 expression of WJSCs......................................................................45

3.2.6

Nestin expression of CLSCs, WJSCs, and ADSCs...................................48

3.2.7

Nanog, Oct4, SOX2 expression of CLSCs, WJSCs, and ADSCs.............50

Summary................................................................................................................53

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CHAPTER 4 In Vitro Differentiation of Stem Cells towards Islet-like Cells using the
3-step differentiation protocol........................................................................................54

4.1

Introduction............................................................................................................54

4.2

Results....................................................................................................................56
4.2.1

Differentiation of CLSCs into islet-like cells............................................56
4.2.1.1 3-step differentiation protocol…………………………....………56
4.2.1.2 Comparison of pancreatic lineage markers of CLSCS before and
after differentiation........................................................................58
4.2.1.3 Comparison of pancreatic lineage markers of differentiated CLSCs
derived from different donors........................................................60

4.2.2

Differentiation of ADSCs into islet-like cells............................................62
4.2.2.1 3-step differentiation protocol……………………………………62
4.2.2.2 Comparison of pancreatic lineage markers of ADSCs before and
after differentiation........................................................................64
4.2.2.3 Comparison of pancreatic lineage markers of differentiated
ADSCs derived from different donors...........................................66

4.2.3

Differentiation of WJSCs into islet-like cells............................................68
4.2.3.1 3-step differentiation protocol………………………………….68
4.2.3.2 Comparison of pancreatic lineage markers of WJSCs before and

after differentiation........................................................................70
4.2.3.3 Comparison of pancreatic lineage markers of differentiated WJSCs
derived from different donors………............................................72

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4.2.3.4 Reproducibility of differentiation of the WJSCs from the same
donors……….................................................................................74
4.2.4

Expression of PC1/3, PC2, glucagon and somatostatin of CLSCs, ADSCs,
and WJSCs after differentiation.................................................................76

4.2.5

Expression of Glut2, PC1/3, PC2, glucagon and somatostatin at different
stages along the 3-step differentiation pathway.........................................78

4.2.6

Real-time PCR analysis of expression of glucagon and PC1/3 of
differentiated WJSCs……………………….……………………………80

4.2.7

Dithizone staining of differentiated islet-like clusters...............................82

4.2.8

C-peptide release of differentiated CLSCs, WJSCs, and ADSCs……….84

4.2.9

GLP-1 expression of differentiated WJSCs...............................................86

4.2.10 Glucagon immunostaining of uninduced and differentiated WJSCs.........87
4.3

Summary...................................................................................................89

CHAPTER 5 In Vitro Differentiation of Stem Cells Towards Islet-like Cells using 1step 7-day differentiation protocol................................................................................91
5.1

Introduction............................................................................................................91

5.2

Results...................................................................................................................92
5.2.1

Islet-like cells differentiation of WJSCs, CLSCs, and ADSCs using
1-step 7-day differentiation protocol........................................................92

5.2.2

PC1/3, PC2, glucagon, and somatostatin expression of uninduced and
differentiated CLSCs, ADSCs, and WJSCs..............................................94

5.3

Summary...............................................................................................................96

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CHAPTER 6 Discussion and Conclusion.....................................................................97
6.1

Characterization of WJSCs, CLSCs, and ADSCs................................................97

6.2

In vitro differentiation of CLSCs, ADSCs, and WJSCs towards islet-like cells
using 3-step differentiation protocol....................................................................100

6.3

In vitro differentiation of CLSCs, ADSCs, and WJSCs towards islet-like cells
using 1-step differentiation protocol....................................................................104

6.4

Conclusion...........................................................................................................104

6.5

Future work..........................................................................................................105

REFERENCES...............................................................................................................106

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SUMMARY
Mesenchymal stem cells of different origins were shown to differentiate into
insulin producing cells under appropriate conditions. This study assessed the potential of
umbilical cord lining stem cells (CLSCs), Wharton’s jelly derived mesenchymal stem
cells (WJSCs), and adipose tissue derived mesenchymal stem cells (ADSCs) to
differentiate into islet-like cells.
The basic morphology, pluripotency markers, immunophenotyping surface
markers of mesenchymal stem cells, and possible marker of pancreatic progenitor cells,
nestin, and sox17 of stem cells derived from umbilical cord, Wharton’s jelly, and adipose
tissue were defined and described. We investigated the potential of WJSCs ,CLSCs and
ADSCs to differentiate into islet-like cells in vitro using a 3 stage differentiation protocol
which had successfully differentiated bone marrow derived mesenchymal stem cells into
glucose responsive insulin secreting pancreatic islet-like clusters as described by Sun et al.
(Sun, Chen et al. 2007).
Transcripts of glucagon, proprotein convertase 1/3, ISL-1, and Nkx6-1 were
consistently upregulated at the end of differentiation in CLSCs, WJSCs, and ADSCs.
Other markers such as insulin, proprotein convertase 2, somatostatin, Glut2 transporter,
glucokinase, pdx1, pax4, pax6, mafA, neuroD1, neurogenin3 were upregulated at
transcript level at the end of differentiation, but not in a consistent manner. The
differentiated islet-like clusters which were shown to express insulin at transcript level
also stained positively with dithizone, a zinc chelating agent that selectively stains insulin
producing cells. C-peptide was also detected by ELISA in one of the experiments.

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Furthermore, the protein expression of glucagon, another hormone known to be produced
by islets of Langerhans, was also verified by immunostaining.
ADSCs, WJSCs and CLSCs were subjected to the 7-day differentiation protocol
described by Chiou et al. to determine if a shorter period of differentiation could be
achieved (Chiou, Chen et al. 2011). The 7-day differentiation protocol contained defined
factors which were similar to the combination of factors of stage 2 and stage 3 of the 3stage differentiation protocol but the time required for differentiation was shorter.
CLSCs and ADSCs could not tolerate this recipe and the cells started to die.
Furthermore, the pancreatic lineage profile of these differentiated ADSCs and CLSCs
was not comparable to the differentiated clusters underwent 3-stage differentiation
protocol. Upregulation of expression of glucagon, somatostatin were not detected in
ADSCs and upregulation of expression of PC2, and somatostatin were not detected in
CLSCs that underwent 7-day differentiation protocol. WJSCs tolerated the protocol but
insulin expression was not detected at the end of differentiation.
In conclusion, Wharton’s Jelly derived-stem cells, cord lining stem cells and
adipose tissue derived stem cells have potential to differentiate into islet-like insulin
expressing cells in response to defined culture conditions. However, further optimization
is required to obtain consistent glucose responsive insulin secretion from these
differentiated cells.

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LIST OF FIGURES AND TABLES
Figure 1

A schematic overview of the cell lineage determination during pancreas
development...............................................................................................14

Figure 3.1

Morphology of CLSCs...............................................................................37

Figure 3.2

Morphology of WJSCs..............................................................................38

Figure 3.3

Morphology of ADSCs..............................................................................39

Figure 3.4

Expression of mesench ymal markers in WJSCs, CLSCs, and
ADSCs.......................................................................................................41

Figure 3.5

Sox17 expression of WJSCs…………………………………………......46

Figure 3.6

Nestin expression of WJSCs, CLSCs, and ADSCs...................................49

Figure 3.7

Nanog, Oct4, and Sox2 expression of WJSCs, CLSCs, and ADSCs........52

Figure 4.1

Morphology of CLSCs at different stages of differentiation…………..57

Figure 4.2

Expression of pancreatic genes by uninduced and differentiated CLSCs
....................................................................................................................59

Figure 4.3

Expression of pancreatic genes by CLSCs derived from different
donors.........................................................................................................61

Figure 4.4

Morphology of ADSCs at different stages of differentiation....................63

Figure 4.5

Expression of pancreatic genes by uninduced and differentiated
ADSCs…...................................................................................................65

Figure 4.6

Expression of pancreatic genes by ADSCs derived from different
donors.........................................................................................................67

Figure 4.7

Morphology of WJSCs at different stages of differentiation.....................69

Figure 4.8

Expression of pancreatic genes by uninduced and differentiated
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WJSCs.................................................................................................…...71
Figure 4.9

Expression of pancreatic genes by WJSCs derived from different
donors….....................................................................................................73

Figure 4.10

Expression of pancreatic genes by differentiated
WJSC011m……............................................................................………75

Figure 4.11

PC1/3, PC2, glucagon, and somatostatin expression of uninduced and
differentiated CLSCs, ADSCs, and WJSCs…………………………….77

Figure 4.12

PC1/3, PC2, glucagon, and somatostatin, Glut2, and nestin expression of
WJSCs at different stages of differentiation…………………………….79

Figure 4.13

Glucagon and PC1/3 expression of differentiated WJSCs…………….81

Figure 4.14

Dithizone staining of differentiated WJSCs and CLSCs...........................83

Figure 4.15

Glucagon immunostaining of uninduced and differentiated WJSCs.........88

Figure 5.1

Morphology of CLSCs, WJSCs, and ADSCs a 1-step
differentiation.............................................................................................94

Figure 5.2

PC1/3, PC2, glucagon and somatostatin expression of differentiated
WJSCs, ADSCs, and CLSCs……………...…………………….............96

Table 1

Primer sets used in RT-PCR......................................................................29

Table 2

C-peptide secretion of differentiated CLSCs, ADSCs, and WJSCs into
culture medium..........................................................................................85

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LIST of PRESENTATIONS
1.

Pancreatic islet lineage differentiation from human umbilical cord-lining stem
cells and Wharton’s Jelly derived mesenchymal stem cells.
Ngo K S, Fong C Y, Phan T T, BISWAS A, BONGSO A, CALNE R, LEE K O,
Gan S U (2010)
Oral presentation at Proceedings of a Colloquium/Workshop: Stem cells and gene
therapy strategies to treat diabetes 2010

2.

Pancreatic islet lineage differentiation from human umbilical cord derived stem
cells
Ngo K S, Fong C Y, Phan T T, BISWAS A, BONGSO A, CALNE R, LEE K O,
Gan S U (2010)
Abstract presented at Singapore Stem Cell Consortium (SSCC) 2010

3.

Differentiation of glucagon producing cells from Wharton’s Jelly derived
mesenchymal stem cells
Ngo K S, Fong C Y, Phan T T, BISWAS A, BONGSO A, CALNE R, LEE K O,
Gan S U (Manuscript in preparation)

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LIST of ABBREVIATIONS
ABCG2

ATP-binding cassette sub-family G member 2

ADSCs

adipose-tissue derived mesenchymal stem cells

bp

base pairs

CD4

cluster of differentiation 4

CD8

cluster of differentiation 8

cDNA

complementary deoxyribonucleic acid

CER

cerberus

CK19

cytokeratin 19

CLSCs

cord-lining stem cells

c-myc

V-myc myelocytomatosis viral oncogene homolog (avian)

CXCR4

C-X-C chemokine receptor type 4

DAPT

N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester

DMEM

Dulbecco’s modified Eagle’s medium

DTZ

dithizone

EDTA

ethylenediaminetetraacetic acid

EGF

epidermal growth factor

ELISA

enzyme-linked immunosorbent assay

ES cells

embryonic stem cells

FACS

fluorescence-activated cell sorting

FBS

fetal bovine serum

FGF

fibroblast growth factor

FITC

Fluorescein isothiocyanate
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FoxA2

Forkhead box A2

GADA

glutamic acid decarboxylase

GATA4/6

GATA binding protein 4/6

GDM

gestational diabetes mellitus

GLP-1

glucagon like peptide 1

GLP-2

glucagon like peptide 2

Glut-2

glucose transporter 2

GVHD

graft-versus-host disease

HEK293T

human embryonic kidney 293T

HGF

hepatocyte growth factor

HLA

human leukocyte antigen

HNF1B

hepatocyte nuclear factor 1 homeobox B

HNF4A

hepatocyte nuclear factor 4 homeobox A

HNF6

hepatocyte nuclear factor 6

HRP

horseradish peroxidase

IAA

insulin autoantibodies

ICA

islet cell antibodies

IGF-1

insulin like growth factor 1

IgG

immunoglobulin G

IL-6

interleukin 6

iPS cells

induced pluripotent stem cells

ISL-1

ISL1 transcription factor, LIM/homeodomain

ITS

insulin–transferrin−selenium

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KAAD

3-Keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl

Klf4

Kruppel-like factor 4

mafA

v-maf musculoaponeurotic fibrosarcoma oncogene homolog A

mafB

v-maf musculoaponeurotic fibrosarcoma oncogene homolog B

MHC

major histocompatibility complex

Mnx1

motor neuron and pancreas homeobox 1

mRNA

messenger ribonucleic acid

NeuroD1

neurogenic differentiation 1

Ngn3

neurogenin 3

Nkx2-2

NK2 transcription factor related, locus 2 (Drosophila)

Nkx6-1

NK6 homeobox 1

NOD

non-obese diabetic

Oct4

octamer-binding transcription factor 4

Pax4

paired box 4

Pax6

paired box 6

PC1/3

prohormone convertase 1/3

PC2

prohormone convertase 2

Pdx-1

pancreatic and duodenal homeobox 1

PFA

paraformaldehyde

PGP9.5

protein gene product 9.5

PLA

processed lipoaspirate

PP cells

F cells

Rfx-6

regulatory factor 6

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RPE

R-phycoerythrin

RT-PCR

reverse transcription-polymerase chain reaction

SCF

stem cell factor

SLC30A8

Solute carrier family 30 (zinc transporter), member 8

SVF

stromal vascular fraction

Sox17

SRY (sex determining region Y)-box 17

Sox2

SRY (sex determining region Y)-box 2

SSEA4

stage specific embryonic antigen 4

STZ

streptozotocin

T1DM

type 1 diabetes mellitus

T2DM

type 2 diabetes mellitus

TAE

tris-acetate-EDTA

TGF

transforming growth factor

Thy1

thymus cell antigen 1, theta

TRA1-60

tumor rejection antigen 1-60

Tra-1-81

tumor rejection antigen 1-81

VEGF

vascular endothelial growth factor

WJSCs

Wharton’s jelly derived stem cells

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Chapter 1

Introduction and Literature Review

Chapter 1 Introduction and Literature Review
1.1

Background

1.1.1

What is diabetes?
Diabetes is a metabolic disorder which results in abnormally high blood glucose

levels and impaired glucose tolerance. Based on report of International Diabetes
Federation in 2010, South East Asia region has the highest number of deaths due to
diabetes of all the regions in the world. An estimated 1.1 million adults are expected to
die from the diabetes-related causes, accounting for 14.3% of all deaths. Diabetes can
lead to serious complications like heart attack, stroke, high blood pressure, blindness,
kidney disease, neuropathy, amputation and premature death. However, it can be
controlled by monitoring of the blood glucose, blood pressure, and blood lipids of
individual.
Classification of diabetes often depends on the circumstances present at the time
of diagnosis, and many diabetic individuals do not easily fit into a single class.
(American_Diabetes_Association, 2004a). For example, a person with gestational
diabetes mellitus (GDM) may continue to be hyperglycemic after delivery and may be
determined to have Type 2 diabetes. Therefore, for clinician and patient, it is less
important to label the particular type of diabetes than it is to understand the pathogenesis
of the hyperglycemia and to treat it effectively. Type 1 diabetes mellitus (T1DM) is the
autoimmune destruction of insulin producing beta cells of islet of Langerhans in pancreas
(Devendra, Liu et al. 2004). It will eventually lead to absolute insulin deficiency and
increased blood and urine glucose. Type 1 diabetes accounts for about 5-10% of those
with diabetes and develops most often in children and young adults but can appear at any

1


age (American_Diabetes_Association, 2002). The symptoms of type 1 diabetes include
polyuria (frequent urination), polyphagia (increased hunger), polydipsia (increased thirst),
and weight loss (Cooke and Plotnick 2008).
T1DM commonly results from the autoimmune destruction of pancreatic beta
cells which involve the autoreactive T cells, CD4+ and CD 8+ T cells. The appearance of
a series of autoantibodies including islet cell antibodies (ICA), insulin autoantibodies
(IAA), auto-antibodies to the 65kD isoform of glutamic acid decarboxylase (GADA), the
protein tyrosine phosphotase-related IA-2 molecule (IA-2A) (Knip 2002). The zinc
transporter Slc30A8 residing in the insulin secretory granule of the beta cells (Wenzlau,
Juhl et al. 2007) was shown to be the first detectable sign of emerging beta-cell
autoimmunity. T1DM also has strong HLA associations, with linkage to the DQA and
DQB genes, and it is influenced by the DRB genes (Todd, Walker et al. 2007). These
HLA-DR/DQ alleles can be either predisposing or protective.
Type 2 diabetes is characterized by a relative insulin deficiency, reduced insulin
action, and insulin resistance of glucose transport in skeletal muscle and adipose tissue. It
is the most common form of the disease, accounting for 85-95% of all cases worldwide.
The risk of developing Type 2 diabetes increases with obesity, age, and lack of physical
activity. It occurs more frequently in women with prior gestational diabetes mellitus and
in individuals with hypertension. Most patients with Type 2 diabetes are obese, and
obesity itself is able to cause insulin resistance. They may have insulin levels that appear
normal or elevated, however, high blood glucose may lead to higher insulin levels which
overwhelm the secretory machinery of normal functional beta cells. Thus, insulin
secretion is defective and insufficient to compensate for insulin resistance. Insulin

2

resistance may improve with weight reduction and drug treatment of hyperglycemia but it
is seldom restored to normal.
1.2

Currently available Diabetes Treatments
Patients with Type 1 Diabetes Mellitus (T1DM) are insulin dependent as a result

of autoimmune destruction of insulin producing beta cells of islet of Langerhans in
pancreas. Therefore, exogenous insulin therapy is required to improve the blood glucose
levels.
There are two types of insulin analogues whose structure has been altered to have
different pharmacokinetics properties to when compared to natural insulin available for
glycaemic control of diabetic patients. Rapid-acting insulin analogues are readily
absorbed from the injection site and thus act faster than natural insulin to supply the
insulin required after a meal. Long-acting insulin analogues are those released slowly
over a period of 8 to 24 hours, to supply the basal level of insulin for the day.
Multiple insulin injection (three or more injections per day) and continuous
subcutaneous insulin infusion are commonly used by diabetic patients for blood glucose
control to prevent long-term diabetes complications. Continuous subcutaneous insulin
infusion is used when patients fail to achieve good glycaemic control with conventional
injection regimes. It also led to the risk of more frequent and rapid onset ketotic episodes
whenever insulin delivery is interrupted. Besides, infections and inflammation at the
needle site may also affect the therapy (American_Diabetes_Association 2004b).
Other than the conventional subcutaneous and intravenous injection of insulin,
inhalation of insulin was also suggested to be a route for glycaemic control of diabetic
patients after meal. Inhalable insulin was available from September 2006 to October 2007
3