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1 Suppression of Epithelial to Mesenchymal Transitioning (EMT) Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue towards Functional Insulin Producing β-Like Cells* 1 ǂMaria João Lima, 1 ǂKenneth R Muir, 1 Hilary M Docherty 2 Robert Drummond, 3 Neil WA McGowan, 4 Shareen Forbes, 5 Yves Heremans, 5 Isabelle Houbracken, 2 James A Ross, 6 Stuart J. Forbes, 7 Philippe Ravassard, 5 Harry Heimberg, 3 John Casey, and 1 Kevin Docherty 1 School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, UK., 2 MRC Centre for Regenerative Medicine, Tissue Injury and Repair Group, University of Edinburgh, Chancellor's Building, Edinburgh, EH16 4SB, UK., 3 Department of Surgery, University of Edinburgh, Edinburgh Royal Infirmary, Edinburgh, EH16 4SU, 4 Endocrinology Unit, University/BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, EH16 4TJ, 5 Diabetes Research Center, Vrije Universiteit Brussel, B1090 Brussels, Belgium, 6 MRC Centre for Regenerative Medicine, SCRM Building, The University of Edinburgh, 5 Little France Drive Edinburgh, EH16 4UU and 7 Biotechnology and Biotherapy Laboratory, CNRS UMR 7225, INSERM 975 and University Pierre and Marie Curie, Hôpital Pitié Salpêtrière, Paris, 75013, France *Running title: Reprogramming of pancreatic exocrine tissue ǂ Joint first authors. To whom correspondence should be addressed: Dr K Docherty, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, UK. Tel. 44 1224 437359. Fax 44 1224 437388. Email: [email protected] Page 1 of 54 Diabetes Diabetes Publish Ahead of Print, published online April 22, 2013

Transcript of 1ǂKenneth R Muir, 1Hilary M Docherty 2Robert Drummond, · PDF fileeffective and viable...

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Suppression of Epithelial to Mesenchymal Transitioning (EMT) Enhances Ex Vivo

Reprogramming of Human Exocrine Pancreatic Tissue

towards Functional Insulin Producing β-Like Cells*

1ǂMaria João Lima,

1ǂKenneth R Muir,

1Hilary M Docherty

2Robert Drummond,

3Neil WA McGowan,

4Shareen Forbes,

5Yves Heremans,

5Isabelle Houbracken,

2James

A Ross, 6Stuart J. Forbes,

7Philippe Ravassard,

5Harry Heimberg,

3John Casey, and

1Kevin Docherty

1School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences,

Foresterhill, Aberdeen, AB25 2ZD, UK., 2MRC Centre for Regenerative Medicine, Tissue

Injury and Repair Group, University of Edinburgh, Chancellor's Building, Edinburgh, EH16

4SB, UK., 3

Department of Surgery, University of Edinburgh, Edinburgh Royal Infirmary,

Edinburgh, EH16 4SU, 4Endocrinology Unit, University/BHF Centre for Cardiovascular

Science, Queen’s Medical Research Institute, University of Edinburgh, EH16 4TJ, 5Diabetes

Research Center, Vrije Universiteit Brussel, B1090 Brussels, Belgium, 6MRC Centre for

Regenerative Medicine, SCRM Building, The University of Edinburgh, 5 Little France Drive

Edinburgh, EH16 4UU and 7Biotechnology and Biotherapy Laboratory, CNRS UMR 7225,

INSERM 975 and University Pierre and Marie Curie, Hôpital Pitié Salpêtrière, Paris, 75013,

France

*Running title: Reprogramming of pancreatic exocrine tissue ǂ Joint first authors.

To whom correspondence should be addressed: Dr K Docherty, School of Medical Sciences,

University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD,

UK. Tel. 44 1224 437359. Fax 44 1224 437388. Email: [email protected]

Page 1 of 54 Diabetes

Diabetes Publish Ahead of Print, published online April 22, 2013

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ABSTRACT

Due to the lack of tissue available for islet transplantation, new sources of β-cells have been

sought for the treatment of type 1 diabetes. The aim of this study was to determine whether

the human exocrine enriched fraction from the islet isolation procedure could be

reprogrammed to provide additional islet tissue for transplantation. The exocrine enriched

cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic

lineage tracing confirmed that these mesenchymal cells arose in part through a process of

epithelial to mesenchymal transitioning (EMT). A protocol was developed whereby

transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA

and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like

cells. Transdifferentiation or reprogramming towards insulin secreting β-cells was enhanced,

however, when using unpassaged cells in combination with inhibition of EMT by inclusion of

ROCK and TGF-β1 inhibitors. Resultant cells were able to secrete insulin in response to

glucose and on transplantation, to normalise blood glucose levels in streptozotocin diabetic

NOD/Scid mice. In conclusion, reprogramming of human exocrine enriched tissue can best

be achieved using fresh material under conditions whereby EMT is inhibited. .rather than

allowing the culture to expand as a mesenchymal monolayer.

Keywords: islet transplantation, stem cells, pancreatic transcription factors, gene therapy

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INTRODUCTION

Since the establishment of the Edmonton protocol, islet transplantation has become an

effective and viable therapeutic option for Type 1 diabetes, however it typically requires

multiple donors to achieve insulin independence (1). The lack of donor material is a

significant problem and is fuelling the drive towards new sources of insulin-producing cells

(2). Several potential strategies exist for developing a replenishable supply of β-cells. One of

these is through directed differentiation of human embryonic stem cells (hESCs) or induced

pluripotent stem cells (iPSCs) towards a β-cell lineage, through an attempt to mimic the

signalling pathways that are triggered during pancreatic development (3-13). Another

strategy involves transdifferentiating or reprogramming one fully differentiated adult cell

type to another (14). Thus, insulin-producing cells can be generated from liver (15-17), bone

marrow (18) adipose tissue (19) and cells derived from the umbilical cord (20). Of particular

relevance is the finding that murine pancreatic exocrine cells can be reprogrammed (21) in

vivo and in vitro towards insulin-producing cells that are phenotypically similar to β-cells.

Most of the strategies applied to murine models involved the exogenous expression of

pancreatic transcription factors that are important for normal endocrine pancreatic

development (22, 23). Although expression of the three transcription factors Pdx1, Ngn3 and

MafA in exocrine cells of murine pancreas resulted in transdifferentiation of these cells

towards the β-cell lineage in vivo (24), the same transcription factors were unable to generate

functional β-cells in vitro (23) and further studies have shown that additional transcription

factors such as Nkx6.1, Pax4 or IA-1 (21-23) and growth factors such as betacellulin, TGF-β

and EGF (25, 26) may be important for generating functional transdifferentiated β-cells in

vitro.

The successful reprogramming of murine exocrine cells has driven further studies

aimed at the reprogramming of human pancreatic tissue. Implementation of the Edmonton

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protocol facilitated access to human cadaveric tissue that results as a by-product of the islet

isolation procedure. When placed in culture, this exocrine enriched fraction rapidly

dedifferentiates to form a mesenchymal monolayer that can be expanded through 20 or more

passages (27). Several studies have attempted to expand β-cell numbers through

redifferentiation of these human exocrine or islet derived mesenchymal cells (28-32). Despite

some success in generating glucose-responsive insulin producing cells from both islet and

exocrine cell sources, the ability of the transdifferentiated cells to rescue diabetes in an

animal model is still unclear.

Here we describe how cells of the adult human exocrine pancreas obtained from the

islet isolation procedure can be reprogrammed towards functional β-like cells in vitro. When

placed in culture the acinar cells undergo EMT, as demonstrated by genetic lineage tracing, to

form a monolayer of mesenchymal cells. Efficient reprogramming was achieved using forced

expression of four pancreatic transcription factors (Pdx1, Ngn3, Pax4 and MafA) in

combination with the growth factors betacellulin, exendin-4, the vitamin nicotinamide and

small molecules that facilitate DNA binding of transcription factors. We show that this

protocol generates predominantly glucagon positive cells, which respond to glucose in a

manner similar to that of pancreatic α-cells in vitro and in vivo. Importantly, our studies

demonstrate that reprogramming of pancreatic exocrine cells towards functional insulin

producing cells could be further enhanced by suppressing EMT using inhibitors of TGF-β1

and Rho-kinase signalling pathways. The resultant cells secreted insulin in response to

glucose and successfully prevented the onset of diabetes when grafted in a streptozotocin

diabetic mouse model.

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RESEARCH DESIGN AND METHODS

Culture of human exocrine pancreatic fractions. All human tissue was procured with

appropriate ethical consent. Human pancreata (n=16) were isolated from brain-dead adult

donors in the Scottish Islet Isolation Laboratory (SNBTS, Edinburgh, UK). The mean donor

age was 39.4 years (range 23-61 years) and BMI 27.2kg/m2 (range 22-36.5kg/m

2).

Following islet isolation for clinical application the low purity exocrine fractions were

transported to Aberdeen where the cells were immediately plated at a density of 300,000

exocrine clusters on 75 cm2 tissue culture flask (Greiner, Stonehouse, UK) and cultured in

serum complete medium (SCM) prepared using RPMI 1640 (Gibco, Life Technologies,

Paisley, UK) supplemented with 10% foetal bovine serum (FBS), 10 mM HEPES, 1 mM

sodium pyruvate (all from Gibco) and 75 µM β-mercaptoethanol (Sigma Aldrich, Dorset,

UK). Human exocrine pancreatic cells were passaged every 7 days with a solution of Trypsin

(0.05%)-EDTA (0.02%, Gibco). Serum free medium (SFM) was prepared using RPMI 1640

supplemented with 1% bovine serum albumin (BSA, Sigma), 10 µg/ml insulin and 5.5 µg/ml

transferrin (both from Roche Diagnostics, West Sussex, UK).

Flow cytometry. Cells were incubated for 30 min with 10 µl of each primary antibody

(Suppl. Table 1), washed twice with phosphate buffered saline (PBS), and incubated for a

further 30 min with secondary antibodies (anti-Mouse Ig, Silenus, Victoria, Australia). They

were then washed twice in PBS and resuspended in 1% formaldehyde/PBS prior to analysis

using a Beckman Coulter EPICS XL-MCL Flow Cytometer.

Differentiation towards adipocytes, osteoblasts and chondrocytes. For differentiation,

cells were trypsinised and resuspended in NH Adipodiff Medium, NH Osteodiff Medium or

NH Chondrodiff Medium (all from Milentyi Biotec, Surrey, UK) and processed as per the

manufacturer’s instructions.

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Genetic lineage tracing

The adenoviral vectors expressing Cre recombinase under the control of the amylase

promoter and the two lentiviral vectors expressing Cre recombinase under the insulin

promoter and a floxed STOP dsRED promoter under control of the CMV promoter have been

previously described (33, 34). The lentiviral floxed STOP dsRED was generated by ligating

dsRED into the BamHI/XhoI digested pTrip-CMV-Lox-STOP-Lox-EGFP-deltaU3 to replace

EGFP.

Preparation of adenoviruses. Recombinant adenoviruses encoding the mouse sequences of

Pdx1, MafA, Ngn3 and Pax4 (35) were prepared using the Ad-EasyTM

system (Agilent

Technologies, Edinburgh, UK). The adenoviruses containing Pdx1 and Ngn3 also expressed

GFP through a downstream CMV promoter. Viral transduction was performed in SFM for

4h at a multiplicity of infection (MOI) of 100 for each virus.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). QRT/PCR

was performed as previously described (22). Data were analysed using the 2-∆∆CT

method.

Statistical analysis was performed using PRISM software and the student’s t-test or one-way

ANOVA followed by the Dunnet’s post-hoc test, as appropriate. The list of TaqMan probes

are listed in Suppl. Table 3.

Immunocytochemistry and immunohistochemistry. Immunocytochemistry and

immunohistochemistry were performed as previously described (13, 22), using the antibodies

listed in Suppl. Table 3.

C-Peptide and glucagon release studies. Glucagon or C-peptide levels were measured

using a human glucagon Quantikine ELISA kit (R&D Systems, Abingdon, UK) or a human

C-peptide ELISA kit (Millipore, Livingston, UK).

Insulin content. Cells were harvested in Trizol® reagent (Invitrogen). DNA and protein

were extracted according to the manufacturer’s instructions. Insulin was measured using an

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insulin ELISA kit (Millipore) and protein and DNA as previously described (22). Animal

Studies. All animal experiments were performed under UK Home Office regulations and

with the approval of the University of Aberdeen Ethics Committee. Male 8-10 week old

NOD/SCID mice (NOD.CB17-Prkdcscid/NCrHsd) were obtained from Harlan Laboratories

(Blackthorn, UK) and maintained on a 12h light/dark cycle with ad libitum access to food.

Mice were rendered diabetic by one single intraperitoneal (i.p.) injection of streptozotocin

(STZ) (150 mg/kg), and transdifferentiated human cells were grafted under the left kidney

capsule as previously described (13).

RESULTS

Human exocrine enriched tissue dedifferentiates in culture to form a mesenchymal

monolayer. The material left over from the islet isolation procedure was mainly composed of

aggregates of exocrine cells, which were epithelial in nature. There was some islet debris that

could be detected with dithizone or anti-insulin antibodies (Suppl Fig. 1A and 1B). The

amount of insulin positive material varied between preparations but was never more than 2%

of the total tissue. When these exocrine enriched pancreatic fractions were placed in culture

the cells readily attached to the dish and expanded into a monolayer of proliferative cells with

a morphology that resembled that of mesenchymal stromal cells (MSCs) , which could be

further expanded in vitro for at least 20 passages (Fig. 1A). This change in morphology was

associated with a loss of expression of exocrine (amylase and CK19), endocrine (insulin) and

epithelial (E-cadherin and EpCAM) markers with a concomitant increase in the mesenchymal

markers Snai2 and α-smooth muscle actin (Fig. 1B). Immunocytochemistry confirmed the

presence of amylase and CK19 positive exocrine cells as well as vimentin positive cells at

Day 2, the earliest time that could be measured after plating (Fig. 2). The presence of

vimentin RNA (Fig. 1B) probably indicated the presence of endogenous stromal cells in the

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exocrine fraction. The number of amylase and CK19 positive cells rapidly decreased, while

the vimentin positive cells increased towards Day 18. Interestingly, at Day 4 and Day 10

there was evidence for cells that co-expressed amylase and vimentin or CK19 and vimentin,

suggesting that the exocrine cells were undergoing dedifferentiation towards a mesenchymal

phenotype. There were also cells that co-expressed amylase and CK19 confirming a previous

report of human acinar cell plasticity (34). Flow cytometry analysis showed that >90% of the

passaged cells were positive for the main characteristic mesenchymal stem cell surface

markers, while endothelial and hematopoietic cells were very rare in these cultures (Suppl.

Fig. 1B). In keeping with the characteristics of MSCs the pancreatic derived mesenchymal

cultures were able to differentiate towards cells that stained positive for markers of adipose

tissue (Oil Red O), bone (alkaline phosphatase) and cartilage (aggregan) (suppl. Fig. 1D).To

map the origins of the MSC population genetic lineage tracing was undertaken using an

adenovirus containing Cre-recombinase under the control of the amylase promoter and a

lentiviral vector containing a CMV driven dsRed reporter preceded by a floxed stop cassette

blocking its expression (Fig. 3A). In acinar cells transduced by both viruses dsRed is

permanently expressed, permitting continuous tracking of acinar cell fate even after amylase

expression has stopped (34). The function of this lineage tracing system was validated using

AR42J-B13 exocrine pancreatic cells (Suppl. Fig. 2A). After transduction of the human

exocrine enriched cells at Day1, dsRed expressing cells were present at Day 4 and time points

thereafter (Fig. 3A). There was a massive increase in the number of dsRed positive cells

between Day 4 and Day 10, associated with a change from a rounded morphology towards a

more elongated stellate shape. By day 10 the dsRed positive cells constituted ∼30% of the

mesenchymal population. Immunocytochemistry showed that already by Day 3 the dsRed

positive cells were positive for amylase, CK19, vimentin and Ki67, a marker for cell

proliferation (Fig. 3C). By Day 10, the dsRed cells were all positive for vimentin and Ki67,

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and negative for amylase and CK19. These results demonstrated that the vimentin-positive

mesenchymal monolayer was derived by a process of EMT, although this does not rule out

the presence of stromal cells within the original exocrine-enriched fractions.

The fate of β-cells was also mapped using a lentivirus containing Cre-recombinase under

control of the rat insulin promoter in combination with the floxed stop dsRed lentivirus

described above. The function of this system was evaluated in Min-6 β-cells (Suppl Fig. 2B).

Using an islet enriched fraction we were able to confirm the previous finding (37) that

genetically labelled β-cells could transdifferentiate via a process of EMT into vimentin

positive mesenchymal cells (Suppl Fig. 3). It was therefore possible that any residual islet

debris (or ductal cells) in the exocrine enriched fraction could also contribute to the MSC

population that expanded in culture.

Human exocrine MSCs can be reprogrammed towards functional α-like cells.

Having characterised in detail the source of the pancreatic mesenchymal monolayer we next

set out to reprogramme these cells towards functional β-cells. Ectopic expression of

pancreatic transcription factors (TFs) in cultured islet derived pancreatic MSCs has

previously been accomplished with infection with adenoviral vectors (38). In the present

study a similar strategy was used to induce reprogramming of the human exocrine derived

MSCs by introducing TFs that control key stages in the developing pancreas, namely Pdx1,

Ngn3, MafA and Pax4 (4TF). The individual adenoviruses transduced the pancreatic MSCs

with similar efficiency as evidenced by immunocytochemistry (Suppl Fig. 4) Individually the

TFs had no effect on the expression of glucagon, insulin or somatostatin (Fig. 4A). However,

in combination the TFs significantly increased expression of glucagon with a maximal effect

seen with the 4TFs (Fig. 4A). Under the same conditions the TFs, either individually or in

combination, had no effect on the extremely low levels of expression of insulin or

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somatostatin). Immunocytochemistry confirmed the expression of glucagon in 40 ± 4.9% of

the cells (n= 400 cells per donor, from 4 donor preparations, (Fig. 4B) and the absence of

insulin (data not shown). A control adenovirus containing a GFP reporter had no effect on the

expression of insulin, C-peptide or any other marker (Suppl Fig. 7B and C).

As previously described for islet-derived MSCs (28) the exocrine derived MSCs

formed cluster-like structures in serum free media (SFM, Fig. 5A), and this was accompanied

by the up-regulation of several pancreatic markers, including the main endocrine hormones

glucagon, insulin and somatostatin (Suppl Fig. 5A), albeit at very low levels. This suggested

that the action of the exogenous TFs might be further enhanced if the cells were cultured in

the absence of serum. In SFM, the 4TFs were able to induce glucagon and somatostatin

expression but insulin levels were not significantly altered (Fig. 5B). However, in SFM, but

not serum containing medium (SCM), glucagon, insulin and somatostatin expression was

increased by treatment with the 4TFs followed by addition of betacellulin, exendin-4 and

nicotinamide (BEN, Fig. 5B). These compounds have previously been shown to induce

formation of insulin producing cells from murine exocrine cells or adult islet cultures (22,

30). Individually these compounds had little effect (Suppl Fig. 5B). Of importance was the

observation that the combination of the 4TFs and BEN in SFM increased the endogenous

expression levels of a number of endocrine transcription factors, such as Pdx1, Pax4, Nkx6.1,

and Pax6 (Fig. 5B), suggesting that the cells had undergone transdifferentiation.

Chromatin-modifying agents enhance reprogramming. To determine the effect of

modulating the chromatin structure within the MSC population prior to addition of the

adenoviruses, the cells were incubated for 72 h with the DNA methyltransferase inhibitor 5-

Aza-2’deoxycytidine (A) and the histone deacetylase (HDAC) inhibitor sodium butyrate

(Bu). Under these conditions the expression of all of the three hormones was markedly

enhanced when compared to the addition of 4TFs and BEN alone (Fig.5C). It was possible to

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detect immunoreactive glucagon, but not insulin or C-peptide in the media of the resultant

cells. Interestingly, the secretion of glucagon was inhibited by high glucose, suggesting that

the reprogrammed cells contained some of the properties of functional α-cells (Fig. 5D).

Immunocytochemistry demonstrated, however, that the majority of the cells were multi-

hormonal, expressing both glucagon and C-peptide (Fig. 5E). No endocrine hormones were

found in non- treated exocrine cultured cells (data not shown). Blood glucose levels were

constantly higher in diabetic or non-diabetic NOD/SCID mice, in which the multi-hormonal

cells had been grafted under the kidney capsule. This is consistent with the elevated serum

glucagon levels detected in fasted diabetic and non-diabetic animals (Suppl. Fig. 6).

Collectively, these results indicate that the treatment of pancreatic exocrine derived

MSCs with chromatin modifying agents, followed by TFs and BEN in SFM resulted in the

formation of α-like cells that expressed glucagon along with insulin. The cells exhibited some

of the functions of α-cells in an in vitro glucose release study and increased blood glucose

levels when grafted into NOD/SCID mice. Reprogramming potential was specific to

pancreatic exocrine derived MSCs, since neither human skin fibroblasts nor human bone

marrow MSCs responded in a similar manner to the reprogramming protocol (Suppl Fig. 8).

Thus the cultured pancreatic exocrine derived MSCs must retain some memory of their

pancreatic origins.

Reversing or preventing EMT enhances reprogramming towards functional β-like cells.

The above experiments demonstrated that the pancreatic exocrine derived MSCs could be

induced to transdifferentiate and that optimal effects were achieved under serum free

conditions when the cells formed epithelial-like clusters. This suggested that reprogramming

might be further optimised by promoting a mesenchymal to epithelial transition or inhibiting

the dedifferentiation of fresh exocrine-enriched epithelial tissue.

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The strategy towards suppressing this process of dedifferentiation involved culturing

the cells in the presence of small molecules that have been previously shown to inhibit

mesenchymal cell differentiation (39, 40). Thus inhibition of activin receptor-like kinases by

the selective inhibitor SB431542 (S) and the Rho-associated protein kinase inhibitor Y27632

(Y) resulted in maintenance, for a 10 day period, of the acinar and ductal cell markers

amylase and CK19 at levels similar to those observed in fresh tissue (Fig. 6). In keeping with

our conjecture, treatment of freshly plated exocrine enriched tissue in SFM with S, Y, A, Bu,

4TF and BEN resulted in high levels of insulin, glucagon and somatostatin along with Pdx1,

Pax4, MafA and Nkx6.1 when the cells were analysed after 10 days (Fig. 7A). These

reprogrammed cells were glucose responsive, releasing C-peptide in a glucose dependent

manner and their insulin content was ∼ 4 fold higher than that of untreated cells (Fig. 7B and

C). Clusters of cells that were highly positive for insulin and C-peptide were readily found

in reprogrammed cultures (Fig. 7D, a-f) and these cells were double positive for Pdx1 and

insulin (Fig. 7D, g-i). Interestingly, some glucagon positive cells were found in the C-peptide

positive cell clusters, in a structure that closely resembles that of a mature islet (Fig. 7D).

Both types of the reprogrammed endocrine cells generated by this protocol were

monohormonal and the majority of the cells in the clusters were insulin positive. This

protocol generated an average of 18.3% (±4.9, n= 452 cells from 3 donor preparations) of

insulin producing cells in the reprogrammed cultures. No significant levels of glucagon,

insulin or other pancreatic markers were found in untreated or in exocrine cells transduced

with a control Ad-GFP vector at day 10 (.Suppl. Fig. 7) In summary, maintenance of the

exocrine phenotype of cultured exocrine cells at the start of the treatment greatly improved

the efficiency of reprogramming of exocrine pancreatic cells towards monohormonal insulin

positive cells, leading to the generation of glucose responsive β-like cells.

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The in vivo function of the reprogrammed insulin-producing cells was further

determined by transplanting these cells under the kidney capsule of NOD/SCID mice that had

been rendered diabetic with STZ one day prior to surgery (Fig. 8A). Animals that were

transplanted with reprogrammed cells retained normal blood glucose levels and maintained

body weight throughout the course of the experiment. Animals that were transplanted with

non- reprogrammed exocrine cells, or those that were not transplanted with cells under the

kidney capsule, exhibited markedly elevated blood glucose levels associated with weight loss

(Fig. 8A). Removal of the transplanted kidney after 30 days resulted in an increase in the

blood glucose levels of the animals transplanted with the reprogrammed cells (Fig. 8A).

Human C-peptide was present only in the serum of fed mice that were transplanted with the

reprogrammed insulin producing cells (Fig. 8B) but absent from the blood when fasted,

suggesting that the reprogrammed cells released insulin in a glucose responsive manner in

vivo. Immunostaining of the grafted kidneys showed that the transplanted cells formed a

cluster like structure under the kidney capsule, where the centre of the structure was mainly

composed of strongly positive insulin positive cells, with the majority of the glucagon

positive cells localized in the periphery of the cluster (Fig. 8C). The majority of the cells in

this structure were also positive for the pancreatic transcription factor Pdx1 (Fig. 8D).

Collectively these data support the conclusion that the exocrine pancreatic cells of the adult

human pancreas can be reprogrammed towards functional insulin producing cells. The

reprogrammed cells are able to ameliorate diabetes in a diabetic mouse model and generate a

cluster-like structures reminiscent of islets of Langerhans.

Finally, to determine whether the reprogramming cocktail might be mediating an effect on

the proliferation of pre-existing β-cells, reprogramming was performed on fresh exocrine

tissue in which residual β-cells were genetically tagged with dsRed (Suppl. Fig. 9). Staining

of the traced cells revealed that insulin producing cells were not derived from pre-existing β-

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cells, as none of the tagged dsRed+

cells were insulin positive after the reprogramming

protocol. Even in the absence of reprogramming, insulin expression was lost in pre-existing

β-cells after 10 days in culture (Suppl. Fig. 9). In addition, these lineage tracing experiments

further support the role of the soluble factors added in preventing EMT, since dsRed+ cell

proliferation as a mesenchymal population was observed in untreated cultures, but not in

cultures treated with the soluble factors alone (Suppl. Fig. 9). To investigate if residual

acinar amylase, which could have a deleterious effect upon transplantation, was present in the

reprogrammed cells, amylase expression was investigated at the end of the reprogramming

protocol (Suppl. Fig. 9C). As observed for residual β-cells, amylase expression disappeared

in both reprogrammed and untreated cells after the 10 day protocol (Suppl. Fig. 9C),

indicating that no traces of exocrine markers were present in the reprogrammed cultures. No

evidence was found, by tracing of amylase positive cells during the reprogramming protocol,

to support the view that the newly generated β-like cells were derived from pre-existing

acinar cells (Suppl. Fig. 10). These data suggest that ductal cells are the most likely source of

the new insulin producing cells, however lineage tracing tools were not available to confirm

this hypothesis.

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DISCUSSION

In this study we cultured exocrine cells from the fractions that are a by-product of the

islet isolation procedure. Of particular importance is the fact that all of these samples were

obtained from a single centre prepared under standardised operation procedures. When placed

in culture, the exocrine pancreatic cells rapidly began to co-express the mesenchymal marker

vimentin with a quick decline in epithelial markers as the culture became uniformly single

positive for vimentin. Similar results were observed when a human islet enriched fraction was

used as starting material. Our immunocytochemistry and genetic lineage tracing results

showing that cells transiently express both epithelial and mesenchymal markers clearly

support the view that the human pancreatic MSC population arises from a combination of

endogenous stromal cells and through EMT of all pancreatic epithelial cell types. This

differs from the case in rodents where genetic lineage tracing studies have found no evidence

for pancreatic MSCs arising via EMT from β-cells (41, 42).

It has previously been shown that pancreatic MSCs derived from human isolated islets

can be induced to redifferentiate when plated on extracellular matrix in response to a cocktail

of growth factors under serum free conditions (28, 30, 43, 44), although it has been argued

that this occurs only to a limited extent (45). We hypothesised that the capacity of pancreatic

exocrine derived MSCs to transdifferentiate could by enhanced by using ectopic expression

of pancreatic transcription factors (TFs) that are known to define and maintain the identity of

islet cell types (46). Our approach was influenced by in vivo studies on the

transdifferentiation of liver (15-17) and pancreatic exocrine cells (24) in mouse. Our first

major finding was that pre-treatment of the cells with chromatin modifying reagents followed

by adenoviruses expressing Pdx1, Ngn3, Pax4 and MafA, along with two growth factors,

betacellulin and exendin 4, and the water soluble vitamin nicotinamide could convert the

pancreatic MSCs into glucagon positive cells α-like cells. These multihormonal cells secreted

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glucagon in a physiological manner, where hormone release was inhibited by high glucose

concentrations in the medium. When grafted into NOD/SCID mice, these α-like cells

continued to secrete glucagon in response to changes in serum glucose levels, even when the

animals were rendered diabetic with streptozotocin. It was surprising that the resultant cell

population was mainly composed of α-like cells, as the inclusion in our reprogramming

cocktail of Pax4, which favours the production of β- cells (47), was designed to tip the

balance towards the β-cell lineage. During embryogenesis α- and β- cells arise from a

common progenitor that expresses Ngn3. There is a narrow competence window for Ngn3

expression, the length of which can markedly affect the generation of different endocrine cell

types (48). In the case of the reprogrammed exocrine cells perhaps the temporal expression of

Ngn3 may dominate and this could be addressed in further experiments by modulating

expression of the exogenous Ngn3.

Our second major finding, however, was that the production of functional β-cells

could be achieved by preventing dedifferentiation of the exocrine cells towards MSCs.

Treatment of the exocrine cultured cells with factors that inhibit TGF-β1 and Rho-kinase

pathways (39, 40), which are known to be key players in mesenchymal differentiation,

prevented a decrease in exocrine marker expression in the cultures of exocrine derived cells

and the substantial expansion of pancreatic MSCs. When applied to these cells that more

closely resemble the exocrine pancreas, the reprogramming protocol that had previously

resulted in the formation of functional glucagon producing cells gave rise to functional

insulin producing cells that were able to secrete insulin in response to glucose and normalise

glucose levels in a streptozotocin diabetic NOD/SCID mouse model. Importantly, the levels

of human C-peptide that were secreted in vitro in the presence of 20 mM glucose were 0.4

ng/ml, which compares well with the amount of insulin released by equivalent numbers of

cultured human islets (50).

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In conclusion, we have developed a protocol that led to the successful reprogramming of

human exocrine pancreatic cells towards functional β-like cells capable of preventing the

onset of diabetes in a mouse diabetic model. The important finding was that fresh tissue that

was predominantly epithelial could be efficiently reprogrammed towards functional β-cells,

whereas expansion as a mesenchymal monolayer was less efficient, generating predominantly

α-like cells. The clinical importance of these findings is that it may be possible to

reprogramme the exocrine pancreatic tissue that is normally discarded after the islet isolation

procedure, and in a matter of weeks provide a second batch of islets for the recipient derived

from the same donor. This would obviate the need for a second donor preparation, thus increasing

overall supply and also preventing additional potential donor specific immune problems. Overall,

cryopreserved exocrine tissue holds promise as a donor-recipient specific top-up supply of islets for

further use as the graft function deteriorates.

Acknowledgements

This work was supported by the Wellcome Trust (085664) through the Scottish

Translational Medicine and Therapeutics Initiative (KM) and grants from the Medical

Research Council J015377/1), Grampian National Health Service Trust and Tenovus

Scotland. Author contributions: MJL, KRM and , HMD performed experiments on the

exocrine tissue, RD and JAR undertook the FACS analysis and differentiation studies on

MSCs, NWAM, SF and JC were involved in the isolation and characterisationof human

islet- and exocrine- enriched fractions, while YH, IH, HH and PR provided lentiviruses and

other reagents for the reprogramming and lineage tracing experiments. MJL, KRM and KD

wrote the manuscript, MJL, KRM, SJF, JC, and KD were involved in the planning and

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interpretation (IH, YH and HH) of experiments. No potential conflicts of interest relevant to

this article were reported.

KD is the guarantor of this work and, as such, had full access to all the data in the study and

takes responsibility for the integrity of the data and the accuracy of the data analysis.

We thank Jan De Jonge, Vrije Universiteit Brussel, for technical assistance.

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Figure Legends

Figure 1 – Pancreatic exocrine fractions dedifferentiate towards mesenchymal cells in

culture.

(A) Phase contrast images of exocrine fractions when cultured in tissue culture dishes over a

14 day period. Scale bar = 50 µm. (B) QRT/PCR analysis of pancreatic, epithelial and

mesenchymal markers in cultured exocrine fractions from day 2 up to passage 7 after plating

in tissue culture dishes. Data are presented as mean ± standard error of the mean (SEM).

Expression levels are relative to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), n=

3.

Figure 2 – Dedifferentiation of pancreatic exocrine fractions is accompanied by

epithelial to mesenchymal transition.

Immunocytochemistry of plated pancreatic exocrine fractions was performed from day 2 to

day 18 in culture. The pancreatic exocrine markers Amylase (AML) and Cytokeratin 19

(CK19), the epithelial marker E-cadherin (ECAD) and the mesenchymal marker Vimentin

(VIM) expression were analysed at days 2, 4, 10 and 18 of culture. Nuclei were

counterstained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar = 20µm.

Figure 3 – Genetic lineage tracing of acinar amylase+ cells in pancreatic exocrine

fractions.

(A) Schematic representation of the two viral vectors used for tracing amylase+ cells. (B)

dsRed+ cells were monitored in culture for a 10 day period. dsRed fluorescence (top row) and

brightfield images (bottom row) were analysed at days 4, 6, 8 and 10. Scale bar = 50 µm. (C)

Immunocytochemistry was performed at day 3, 7 and 10 on traced amylase+ cells. The

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exocrine pancreatic markers Amylase (AML) and Cytokeratin 19 (CK19) were analysed

along with the mesenchymal marker Vimentin (VIM) and the proliferation marker ki67.

Nuclei were counterstained with DAPI. Scale bar = 20 µm.

Figure 4 – Ectopic expression of pancreatic transcription factors reprogrammes

exocrine pancreatic MSCs into glucagon positive cells.

(A) Passaged human exocrine pancreatic fractions were plated in tissue culture dishes and

subsequently transduced with different combinations of adenoviruses expressing the

pancreatic transcription factors Pdx1 (P), MafA (M), Pax4 (Px) and Ngn3 (N), each with an

MOI of 100. After 7 days, QRT/PCR was performed and the data expressed relative to

GAPDH and presented as mean ± SEM, where n=3. (*) p-value < 0.05; (**) p-value < 0.01;

(***) p-value < 0.001 non treated samples (N/A).. (B) Exocrine cells treated with different

combinations of adenoviruses expressing Pdx1 (P), MafA (M) and Ngn3 (N), in the presence

or absence of Pax4 (Px4) were stained for the expression of glucagon and cell nuclei were

counterstained with DAPI. Scale bar = 20 µm.

Figure 5 – Serum free medium and chromatin modifying reagents enhance

reprogramming of exocrine pancreatic MSCs towards glucagon producing cells.

(A) Representative phase contrast images of passaged exocrine pancreatic fractions cultured

in serum containing medium (SCM) or in serum free medium (SFM). Scale bar = 50 µm. (B)

Passaged exocrine pancreatic cells were cultured in SCM or SFM and transduced with

adenoviruses expressing Pdx1, MafA, Ngn3 and Pax4 (4TFs). After 24h 1 nM betacellulin,

10 nM exendin-4 and 10 mM nicotinamide (BEN) were added to both SCM and SFM

cultures. After 7 days the cells were harvested and QRT/PCR was performed. The data are

expressed relative to GAPDH and presented as mean ± SEM, with n=3 where (*) p-value <

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0.05; (**) p-value < 0.01; (***) p-value < 0.001. (C) Passaged exocrine pancreatic cells were

cultured in SFM supplemented with 1 µM 5-Aza-2’-deoxycytidine (A) and/or 1 mM sodium

butyrate (Bu), transduced with adenoviruses (4TF) and treated with BEN as indicated. QRT-

PCR was performed and the data expressed relative to GAPDH and presented as mean ±

SEM, with n=3, (***) p-value < 0.001 relative to non-treated samples (N/A). (D) Glucagon

secretion in culture medium of non-treated (N/A) or reprogrammed (A + Bu + 4TFs + BEN)

exocrine cells, in the presence of basal (2.5 mM) or after stimulation for 1h with high (20

mM) glucose. Glucagon levels were measured by ELISA and data represents the mean ±

SEM, with n = 3 , p-value (*) < 0.05. (E) Glucagon and C-peptide immunofluorescent

staining in exocrine cells treated with 4TFs+BEN, in the presence of 5-aza-2’-deoxycytide

and sodium butyrate. Scale bar = 20 µm.

Figure 6 – Rho-kinase and TGF-β1 pathway inhibitors suppress dedifferentiation of

cultured pancreatic exocrine cells.

(A) Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. After 48hr to

allow attachment, cells were treated without (N/A) or with 2 µM Rho-kinase inhibitor

Y27632 (Y) and 10 µM TGF-β1 inhibitor SB431542 (S) individually or in combination and

the cells were incubated for a further 5 days. Treated and untreated (N/A) cells as well as

baseline samples after 48 h in culture (Day 0) were then harvested and RNA extracted for

QRT/PCR analysis for expression of pancreatic, epithelial and mesenchymal markers. Data

are expressed relative to GAPDH and presented as mean ± SEM, with n=3. A one-way

ANOVA was performed with Dunnet post hoc test comparing treatment groups with N/A. A

t-test was used to compare Day 2 with YSB. For all analyses (*) p-value < 0.05; (**) p-value

< 0.01; (***) p-value < 0.001. (B) Immunocytochemistry for the pancreatic markers amylase

(AML) and Pdx1, the epithelial marker E-cadherin (ECAD) and the mesenchymal marker

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vimentin (VIM) in cultured exocrine pancreatic cells after 10 days in the presence of Y27632

and SB431542 (Y+S). Untreated (N/A) cells were also analysed for the same markers. Nuclei

were counterstained with DAPI. Scale bar = 20µm.

Figure 7 – Rho-kinase and TGF-β1 pathway inhibitors enhance reprogramming

towards insulin producing cells.

(A) Unpassaged exocrine pancreatic cells were plated in tissue culture dishes. The cells were

then cultured for 72 h in SFM containing Y27632 (Y), SB431542 (S), 5-aza-2’-deoxycytidine

(A) and sodium butyrate (Bu). They were then transduced with adenoviruses expressing

Pdx1, MafA, Ngn3 and Pax4 (4TFs) and cultured for 7 days in SFM containing 1 nM

betacellulin, 10 nM exendin-4 and 10 mM nicotinamide (BEN). Treated and untreated (N/A)

cells were then harvested and RNA extracted for QRT/PCR analysis. Data are represented as

mean ± SEM and expressed relative to GAPDH, where p<0.05 (*) or p<0.01 (**)relative to

N/A samples.. (B) Release of C-peptide to the medium in transdifferentiated

(S+Y+A+Bu+4TFs+BEN) and non-treated cells (N/A), after incubation with 2.5 or 20 mM of

D-glucose for 1 h. The dashed line indicates the assay detection limit. Data are representative

of triplicate experiments , where (***) p-value < 0.001. (C) Insulin content of

transdifferentiated (S+Y+A+Bu+4TFs+BEN) and non-treated cells (N/A) normalized to the

DNA content of each sample. Data are representative of triplicate experiments, where (*) p-

value < 0.05. (D) Immunostaining for insulin (INS), C-peptide (C-PEP), Pdx1 and glucagon

(GLC) of transdifferentiated cells in culture. Nuclei were counterstained with DAPI. Data are

representative of triplicate experiments. Inlets show a 2x higher magnification image of

stained clusters. Scale bar = 50 µm.

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Figure 8 – Reprogrammed insulin producing cells prevent STZ-induced diabetes in

vivo.

(A) Body weight and blood glucose levels were measured in NOD/SCID mice grafted with

transdifferentiated cells (Transdif Cells), exocrine pancreatic cells (Exoc Cells) or in non

grafted mice (Ctrl) over a 38 day period after surgery. A single dose (150 mg/kg)

streptozotocin was administered one day prior to surgery. n=5 Transdiff cells; n=3 Exoc

cells; n=2 Ctrl. (B) Serum C-peptide levels were measured in NOD/SCID mice grafted with

transdifferentiated cells (Transdif Cells) or exocrine pancreatic cells (Exoc Cells) and in non

grafted mice (Ctrl) after a 4h starvation period (fast) or under ad libitum feeding (fed)

conditions. n=5 Transdiff cells; n=3 Exoc cells; n=2 Ctrl. (C) Immunostaining for insulin and

glucagon of grafted kidneys following kidney removal. Yellow dash lines indicate the border

between the kidney (k) and the graft. The red circle in panel (A) indicates the difference in

glucagon staining observed within the cluster. A 5x higher magnification of the cells inside

this circle is shown in panel c. 10x higher magnifications of the cells inside (e) and outside (f)

the circle is shown. Panel d shows a 5x higher magnification of insulin staining within the

area marked by the red square in panel b. A 10x higher magnification of insulin positive cells

present in the centre of the cluster is shown in panel g. Scale bar for a and b = 100 µm. Scale

bar for c-g = 20 µm. (D) Immunofluorescent staining for Pdx1 in kidneys grafted with

transdifferentiated cells. Scale bar = 50 µm. A 5x higher magnification inlet is shown.

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Fig.1

Day1 Day2 Day4

Day8 Day10 Day14

a b c

d e f

A

B AML

D2

D4

D6

P2 P3 P4 P5 P6 P7

0

1

2

3

4

CK19

D2

D4

D6 P

2P3

P4

P5

P6

P7

0

5

10

15

INS

D2

D4

D6 P

2P3

P4

P5

P6

P7

0

5

10

15

20

25

PDX1

D2

D4

D6 P

2P3

P4

P5

P6

P7

0.0

0.5

1.0

1.5

ECAD

D2

D4

D6

P2 P3 P4 P5 P6 P7

0

5

10

15

EPCAM

D2

D4

D6

P2

P3

P4

P5

P6

P7

0

100

200

300

400

VIM

D2

D4

D6 P2 P3 P4 P5 P6 P7

0

100

200

300

SNAI2

D2

D4

D6

P2 P3 P4 P5 P6 P7

0

10

20

30

a-SMA

?D4

D6

P2

P3

P4

P5

P6

P7

0.0

0.5

1.0

1.5

Rel

ativ

e Ex

pre

ssio

n

Day/Passage in culture

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VIM

/EC

AD

/DA

PI

AM

L/C

K1

9/D

AP

I V

IM/C

K1

9/D

AP

I A

ML/

VIM

/DA

PI

Day 2 Day 4 Day 10 Day 18

C

Fig. 2

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Fig.3

dsRed

A

B Day 4 Day 6 Day 8 Day 10

Day 3 Day 7 Day 10

DA

PI/

AM

L/d

sRED

C

K1

9/V

IM/d

sRED

V

IM/k

i67

/dsR

ED

CK

19

/ki6

7/d

sRED

C

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Fig. 4

A

N/A P+M+N P+M+N+Px

DA

PI

GLC

M

ERG

ED

B

Rel

ativ

e Ex

pre

ssio

n

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SCM SFM A

B

C

E C-PEP MERGED GLC

Rel

ativ

e Ex

pre

ssio

n

D

Rel

ativ

e Ex

pre

ssio

n

Fig. 5

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Fig. 6

A a b c

d e f

a b c

d e f

AML

DAY 0

N/A Y S

Y+S

0

1

2

3

4

****

*

CK19

DAY 0

N/A Y S

Y+S

0

5

10

15

20ns

PDX1

DAY 0

N/A Y S

Y+S

0.0

0.1

0.2

0.3**

**

*

INS

DAY 0

N/A Y S

Y+S

0

5

10

15

20

**

ns

GLC

DAY 0

N/A Y S

Y+S

0

1

2

3

**

ns

SST

DAY 0

N/A Y S

Y+S

0.0

0.5

1.0

1.5

**

ns

VIM

DAY 0

N/A Y S

Y+S

0

50

100

150

200

250 ns

EPCAM

DAY 0

N/A Y S

Y+S

0

100

200

300

400

500

*

nsRel

ativ

e ex

pre

ssio

n

Y +

S

N/A

AML/ECAD/DAPI PDX1/VIM ECAD B

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Fig. 7

D

B C

C-P

epti

de

(ng

/ml)

Insu

lin (

ng

/µg

DN

A)

PD

X1

/IN

S G

LC/C

-PEP

D

AP

I/IN

S

d

a b c

f

g h i

e

A R

elat

ive

exp

ress

ion

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Fig. 8

PD

X1

/DA

PI

D a b c

A B

C

c

k

k

b a

d c

e f g

1x

5x

10

x

GLC

GLC

GLC GLC INS

INS

INS

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1

ONLINE SUPPLEMENTAL MATERIALS

Table 1 – List of primary antibodies used for FACS analysis.

Antibody IsoType Supplier Dilution

CD13 Mouse IgG1 ABDSerotec 1/100

CD29 Mouse IgG1 ABDSerotec 1/100

CD31 Mouse IgG1 ABDSerotec 1/50

CD34 Mouse IgG1 ABDSerotec 1/100

CD44 Mouse IgG2a ABDSerotec 1/10

CD45 Mouse IgG2a ABDSerotec 1/10

CD49b Mouse IgG1 ABDSerotec 1/50

CD54 Mouse IgG1 ABDSerotec 1/50

CD73 Mouse IgG1 Hycult Biotech 1/50

CD90 Mouse IgG1 ABDSerotec 1/10

CD105 Mouse IgG1 ABDSerotec 1/50

CD117 Mouse IgG1 ABDSerotec 1/5

IgG1 Neg Mouse IgG1 ABDSerotec 1/100

IgG2 Neg Mouse IgG2a ABDSerotec 1/100

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Table 2 – List of TaqMan® gene expression primers used for QRT-PCR.

Gene Assay Number

PDX1 Hs00236830_m1

MAFA Hs01651425_s1

PAX4 Hs00173014_m1

NGN3 Hs00360700_g1

NKX6.1 Hs00232355_m1

PAX6 Hs00240871_m1

INSULIN Hs00355773_m1

AMYLASE Hs00949916_m1

GLUCAGON Hs00174967_m1

SOMATOSTATIN Hs00174949_m1

CK19 Hs00761767_s1

VIMENTIN Hs00185584_m1

E-CADHERIN Hs01023894_m1

EPCAM Hs00901885_m1

GAPDH Hs99999905_m1

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Table 3 – List of primary antibodies used for immunohistochemistry and

immunocytochemistry.

Antigen Host Species Dilution Supplier

Pdx1 Rabbit 1/800 Abcam, Cambridge, UK

MafA Rabbit 1/200

Santa Cruz Biotechnology, Heidelberg,

Germany

Pax4 Goat 1/200 Santa Cruz Biotechnology

Ngn3 Rabbit 1/200 Abcam

Insulin Mouse 1/1000 Sigma Aldrich

C-Peptide Rabbit 1/100 Cell Signalling, Hitchin, UK

Amylase Rabbit 1/200 Abcam

Glucagon Mouse 1/1000 Sigma Aldrich

CK19 Mouse 1/200 Dako, Denmark

Vimentin

Vimentin

Mouse

Rabbit

1/200

1/100

Dako

Proteintech

E-Cadherin Rabbit 1/100 Becton Dickinson, Oxford, UK

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Supplementary Figure Legends

Supplementary Figure 1 – Characterization of pancreatic exocrine fractions.

(A) Dithizone staining of two unplated pancreatic exocrine fractions on arrival at the

laboratory. Scale bar = 50 µm. (B) Immunocytochemistry for insulin (INS) and amylase

(AML) of a pancreatic exocrine fraction cultured for 2 days. Scale bar = 20 µm. (C) Flow

cytometry analysis of passaged exocrine fractions (n=2, both at passage 5) for cell surface

markers that are typical of mesenchymal stem cells, endothelial and hematopoietic cells. (D)

Differentiation of passaged pancreatic exocrine cells towards the adipocytic (a), osteoblastic

(b) and chondrogenic (c) lineages. Scale bar = 20 µm.

Supplementary Figure 2 – Genetic lineage tracing of AR42J-B13 and MIN6 cells.

(A) Schematic representation of the two viral vectors used for amylase+ cells tracing in the

AR42J-B13 cell line. dsRed+ cells were monitored in culture for a 10 day period and analysed

at days 4, 6, 8 and 10. Scale bar = 100 µm. (B) Schematic representation of the two viral

vectors used for insulin+ cells tracing in the MIN6 cell line. dsRed

+ cells were monitored in

culture for a 10 day period and analysed at days 4, 6, 8 and 10. Scale bar = 50 µm.

Supplementary Figure 3 – Genetic lineage tracing of Insulin+ cells in pancreatic islet

fractions.

(A) Schematic representation of the two viral vectors used for insulin+ cells tracing. (B)

dsRed+ cells were monitored in culture for a 10 day period. dsRed fluorescence (top row) and

brightfield images (bottom row) were analysed at days 4, 6, 8 and 10. Scale bar = 50 µm. (C)

Immunocytochemistry was performed at day 3, 7 and 10 on traced insulin+ cells. The β-cell

marker insulin (INS), the mesenchymal marker vimentin (VIM) and the proliferation marker

ki67 were analysed. Nuclei were counterstained with DAPI. Scale bar = 20 µm.

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Supplementary Figure 4 – Adenoviral uptake by cultured pancreatic exocrine cells.

(A) Exocrine fractions were plated in tissue culture dishes and subsequently transduced with

adenoviruses expressing the transcription factors Pdx1, MafA, Pax4 or Ngn3, each with an

MOI of 100. Immunocytochemistry was performed after 72h for each transcription factor.

Non transduced (N/A) cells were also stained for the four transcription factors. (B)

Quantification of the percentage of cells that were expressing the exogenous pancreatic

transcription factors. Data is presented as mean ± SEM, where n = 400 cells for each staining.

Supplementary Figure 5 – Serum free medium increases endocrine hormone expression

levels in cultured human pancreatic exocrine cells.

(A) Human exocrine pancreatic cells were cultured in serum complete medium (SCM) or

serum free medium (SFM) for 10 days. Cells were harvested and RNA extracted for QRT-

PCR analysis of pancreatic markers. Data are expressed relative to GAPDH and presented as

mean ± SEM, where n=3, (*) p-value < 0.05; (**) p-value < 0.01; (***) p-value < 0.001. (B)

Human exocrine pancreatic cells were cultured in SCM or SFM and transduced with

adenoviruses expressing Pdx1, MafA, Ngn3 and Pax4 (4TFs). After 24h betacellulin,

exendin-4 or nicotinamide (BEN) was added to both SCM and SFM cultures. After 7 days the

cells were harvested and QRT-PCR was performed. The data is expressed relative to GAPDH

and presented as mean ± SEM, with n=3.

Supplementary Figure 6 – Transdifferentiated pancreatic MSCs release glucagon in a

regulated manner in vivo.

(A) Blood glucose levels were measured in NOD/SCID mice grafted (A+B+4TFs+BEN) and

in non grafted mice (Ctrl) over an 18 week period. n=5 animals in each group. (B) Glucagon

was measured from the serum of grafted animals after a 4h starvation period (fast) or under

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ad libitum feeding (fed) conditions. n=5 animals in each group. (C) NOD/SCID mice were

rendered diabetic with one dose (150 mg/kg) of streptozotocin, one day prior to surgery.

Blood glucose levels were measured in grafted (A+B+4TFs+BEN) and in non grafted mice

(Ctrl) over a 5 week period. n=5 animals in each group. (D) Glucagon was measured from the

serum of grafted animals after a 4h starvation period (fast) or under ad libitum feeding (fed)

conditions. n=5 animals in each group.

Supplementary Figure 7 – Ad-GFP transduction in exocrine pancreatic cells does not

upregulate expression of pancreatic markers.

(A) Human exocrine fractions were plate in tissue culture dishes in SFM and transduced with

Ad-GFP at an MOI of 400. Transduced (Ad-GFP) and untreated (N/A) cells were harvested 7

days after infection and analysed by QRT/PCR. Data is expressed as mean ± SEM and

expression is relative to GAPDH, with n = 3. (B) Immunostaining for insulin (Ins) and C-

peptide (C-Pep) of exocrine cells transduced with Ad-GFP. Nuclei were counterstained with

DAPI. Data are representative of triplicate experiments. Scale bar = 50 µm.

Supplementary Figure 8 – Bone marrow derived mesenchymal stem cells and human

skin fibroblasts cells are less amenable to transdifferentiation towards endocrine

pancreatic lineages.

(A) Human neonatal skin fibroblasts were cultured in SFM containing 5-aza-2’-

deoxycytidine and sodium butyrate for 72 hours, transduced with adenoviruses (4TFS) and

cultured for 7 days in SFM containing betacellulin, exendin-4 and nicotinamide (BEN).

Treated and untreated (N/A) cells were then harvested and RNA extracted for QRT/PCR

analysis. Data are represented as mean ± SEM and expressed relative to GAPDH, where

p<0.05 (*), p<0.01 (**) or p<0.001 (***). (B) Human bone marrow mesenchymal stem cells

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(hBM-MSCs) were cultured in SCM containing 5-aza-2’-deoxycytidine and sodium butyrate

for 72 hours, transduced with adenoviruses (4TFS) and cultured for 7 days in SFM

containing betacellulin, exendin-4 and nicotinamide (BEN). Treated and untreated (N/A)

cells were then harvested and RNA extracted for QRT/PCR analysis. Data are represented as

mean ± SEM and expressed relative to GAPDH, where p<0.05 (*), p<0.01 (**) or p<0.001

(***).

Supplementary Figure 9 – Genetic lineage tracing of insulin+ cells in reprogrammed

pancreatic exocrine cultures.

(A) Schematic representation of the viral vectors used for lineage tracing. Unpassaged

exocrine pancreatic cells were plated in tissue culture dishes and transduced after 24h with

both lineage tracing vectors. After another 24h, the cells were cultured for 72h in SFM

containing Y27632 (Y), SB431542 (S), 5-aza-2’-deoxycytidine (A) and sodium butyrate

(Bu). They were then transduced with adenoviruses expressing Pdx1, MafA, Ngn3 and Pax4

(4TFs) and cultured for 7 days in SFM containing betacellulin, exendin-4 and nicotinamide

(BEN). Insulin+

traced cells were also cultured in the absence of the 4TFs but in the presence

of all soluble factors (SFs), as well as in the absence of any factors (NA). (B)

Immunostaining for insulin (INS) and C-peptide (C-PEP) was performed at the end of the

protocol in insulin+ traced cells under the indicated conditions. Scale bar = 20 µm. (C)

Immunostaining for amylase (AML) was performed at the end of the protocol in insulin+

traced cells under the indicated conditions. Scale bar = 20 µm.

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Supplementary Figure 10 – Genetic lineage tracing of amylase+ cells in reprogrammed

pancreatic exocrine cultures.

(A) Schematic representation of the viral vectors used for lineage tracing. Unpassaged

exocrine pancreatic cells were plated in tissue culture dishes and transduced after 24 h with

both lineage tracing vectors. After another 24 h, the cells were cultured for 72 h in SFM

containing Y27632 (Y), SB431542 (S), 5-aza-2’-deoxycytidine (A) and sodium butyrate

(Bu). They were then transduced with adenoviruses expressing Pdx1, MafA, Ngn3 and Pax4

(4TFs) and cultured for 7 days in SFM containing betacellulin, exendin-4 and nicotinamide

(BEN). Amylase+

traced cells were also cultured in the absence of the 4TFs but in the

presence of all soluble factors (SFs), as well as in the absence of any factors (NA). (B)

Immunostaining for insulin (INS) and C-peptide (C-PEP) was performed at the end of the

protocol in amylase+ traced cells under the indicated conditions. Scale bar = 20 µm.

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Re

lati

ve

exp

ress

ion

A BSKIN FIBROBLASTS BONE MARROW MSCs

INS

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs +

BEN

0.00

0.02

0.04

0.06

0.08

0.10

***

GLC

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs +

BEN

0

5

10

15

20

***

SS

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs +

BEN

0

5

10

15

***

PDX1

N/A

A + B

u

4TFs +

BEN

A+Bu +

4TFs

+ B

EN

0.00

0.01

0.02

0.03

0.04

0.05

INS

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs +

BEN

0.00

0.02

0.04

0.06

0.08

0.10

GLC

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs + B

EN

0

5

10

15

20

*** ***

SS

N/A

A + B

u

4TFs +

BEN

A+Bu + 4

TFs +

BEN

0

5

10

15

PDX1

NA

A + B

u

4TFs +

BEN

A+B +

4TFs

+ B

EN

0.00

0.01

0.02

0.03

0.04

0.05

Suppl Fig. 8

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