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Embryogenesis and neogenesis of the endocrine pancreasGangaram-Panday, Shanti Tireshma

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Embryogene sis and neogenesis of the endocrine pancreas

The role of c-met, c-kit and nestin positive cells

Shanti Tireshma Gangaram-Panday

The author gratefully acknowledges the nancial support for the printing of this thesis by Astellas Pharma Europe BV.

Cover design: Vidya MD Gangaram PandayLay-out: Legatron Electronic Publishing, Rotterdam, The NetherlandsPrinted by: Ipskamp Drukkers BV, Enschede, The Netherlands

ISBN: 978-90-367-4401-0 (printed version)ISBN: 978-90-367-4400-3 (digital version)

© 2010, Shanti Tireshma Gangaram-Panday, The Netherlands.All rights reserved. No part of this thesis may be reproduced or transmitted in anyform or by any means without the prior written permission of the author, or, whereappropriate, of the publisher of the articles

RIJKSUNIVERSITEIT GRONINGEN

Embryogenesis and neogenesis of the endocrine pancreas

The role of c-met, c-kit and nestin positive cells

Proefschrift

ter verkrijging van het doctoraat in de

Medische Wetenschappen

aan de Rijksuniversiteit Groningen

op gezag van de

Rector Magni cus, dr. F. Zwarts,

in het openbaar te verdedigen op

maandag 31 mei 2010

om 11.00 uur

door

Shanti Tireshma Gangaram-Panday

geboren op 14 juni 1978, te Amsterdam

Promotor: Prof. dr. L.F.M.H. de Leij

Copromotores: Dr. P. de Vos Dr. M.M. Faas

Beoordelingscommissie: Prof. dr. T.P. Links Prof. dr. J.H. Strubbe Prof. dr. G. Molema

“Nothing is impossible, the word itself says ‘I’m possible’!” Audrey Hepburn

Voor mijn ouders

Contents

Chapter 1 Introduction: design and rationale of this study 9

Chapter 2 Towards stem cell therapy in the endocrine pancreas 17

Chapter 3 Regenerative effect of c-met and c-kit carrying rat neonatal 43 cells on the endocrine pancreas (preliminary data)

Chapter 4 The number of nestin, c-met, and c-kit positive cells is 59 associated with growth and differentiation of the embryonic, neonatal, and adult rat pancreas

Chapter 5 Nestin, c-met, and c-kit expression in differentiating neonatal 83 beta-cells

Chapter 6 Ex vivo differentiation of pancreas derived c-met and c-kit 109 carrying cells to insulin producing cells

Chapter 7 Summary, general discussion and future perspective 127

Nederlandse samenvatting 135

Dankwoord 139

Curriculum Vitea 143

chapter 1

Introduction: design and rationale of

this study

Chapter 1

10

Diabetes mellitus is a chronic disorder that results from a de ciency of insulin. Insulin is produced by the beta-cells of the pancreas and regulates uptake of glucose from the blood into cells. Diabetes mellitus is manifested in two distinct forms: an absolute de ciency of insulin (Type 1) and a relative de ciency of insulin (Type 2) diabetes. Type 1 diabetes mellitus, also known as Juvenile or insulin-dependent diabetes, results from an autoimmune destruction of the beta-cells. Approximately 10% of all diabetics are suffering from Type 1 diabetes. The more common manifestation of diabetes is Type 2 diabetes or non-insulin-dependent diabetes. Type 2 results from insulin resistance, pancreatic beta-cell dysfunction or both. Type 2 diabetic patients are nowadays recommended to change their lifestyle to decrease their symptoms and to avoid too many uctuations in their glucose levels. There are special lifestyle therapies for these patients. These lifestyle therapies are usually not only successful in decreasing the frequency of episodes of hyperglycaemia but also in decreasing the chance on the associated high blood pressures or high lipid pro le. In case the goals are not met, pharmacologic therapy (usually insulin sensitisers) is combined with the lifestyle therapy. In case lifestyle and pharmacologic therapy are not successful insulin therapy is mandatory. A very comprehensive overview of diabetes, its complications and treatment options is given in a supplement of diabetes care (diabetes care. 33, Suppl 1) issued in January 2010. In the following a short review is presented to introduce the experiments given in this dissertation.

Since Type 1 diabetic patients are unable to produce insulin it is necessary to treat these diabetic patients with exogenous insulin. Nowadays most patients are treated with insulin therapy. However, this treatment is not able to regulate the glucose levels on minute-to-minute basis and therefore still exposes the patient to hyper- and hypoglycaemia episodes. Long term episodes of hyperglycaemia are associated with various complications such as, cardiovascular diseases (hypertension and/ or dyslipidemia), retinopathy or glaucoma, renal diseases, neuropathy, and diabetic ketoacidosis. The chance on the development of these complications can be decreased by tight glycaemic control by intensive insulin therapy. Unfortunately, however, also intensive insulin therapy is not without risks since it is associated with hypoglycaemic unawareness which is disabling a growing number of patients.

Introduction: design and rationale of this study

11

A principally different approach to achieve euglycaemia is to provide the diabetic patient with an endogenous source of insulin by transplantation of endocrine pancreatic tissue. There are two options, i.e. transplantation of the whole pancreas and transplantation of only the islets of Langerhans. The rst option, pancreatic organ transplantation, is an established mode of treatment with a world-wide experience of more than 20.000 cases. At present, patient and graft survival rates almost equal those of more conventional grafts such as kidney transplantation. A successful pancreas transplant provides almost normal glucose homeostasis, but it requires major surgery and life-long immunosuppressive medication. The side-effects of life-long immunosuppression in combination with the mandatory major surgery make it doubtful whether this approach will ever be an acceptable alternative for life-long insulin therapy. At present, the majority of research centres restrict themselves to combined pancreas and kidney transplantation in diabetic patients with end-stage renal failure.

Islet transplantation, in contrast to pancreas transplantation, requires no major surgery and there are conceivable approaches that allow for transplantation of islets in the absence of immunosuppression. Approaches currently under investigation are immuno-isolation by encapsulation, immunotolerance, and immunomodulation. The advantage of islet-cell transplantation is that it requires a minimally invasive procedure which can be repeated without major consequences for the recipient. The downside of the method is that it requires 2-4 human donors per recipient. Islets disappear during isolation from the pancreas, but also during and after transplantation. This is not only due to immunological problems, but also because of several viability and metabolic problems due to ischemic periods in the immediate period after transplantation. Recent improvement in this transplantation technology is the administration of non-glucocorticoid immunosuppression (sirolimus, tacrolimus, daclizumab) by the Edmonton-group, which is associated with graft survival in the majority of the transplanted diabetic patients for up to 24 months. This improvement has led to a tremendous growth in the number of research groups involved in human islet transplantation. Nevertheless, due to the limitation of donor shortage islet-transplantation cannot be performed on a world-wide basis.

Chapter 1

12

Therefore, at the moment many research groups are exploring new ways to produce insulin producing cells that can be used for transplantation. One of the technologies that is being investigated is the ability differentiate stem cells into mature beta-cells. Despite the progresses that have been made in the insights and technologies to differentiate stem cells into mature beta-cells a number of challenging questions remain to be answered:

• What are the requirements to manipulate a stem cell to become a fully functional insulin producing beta-cell?

• Which biological factors are involved in the end-stage differentiation of a precursor towards a fully glucose responsive beta-cell?

• How can senescence and genetic disorders like unlimited proliferation in stem cells be avoided?

In order to answer these questions knowledge about the fundamentals of precursor cells is required.

Precursor cells are involved in pancreas development, i.e. during embryogenesis, and during regeneration or growth of the pancreas, i.e. neogenesis. During embryogenesis various phases can be described, i.e. the formation of the primitive ducts, the movement of endocrine and exocrine precursor cells to form islet cell clumps and pre-acini. The nal organisation of cells for further differentiation into last stage endocrine and exocrine cells is completed. Precursor cells are not only assumed to be actively involved during embryogenesis, but they are also suggested to be involved during regeneration of the beta-cell mass of the pancreas. During regeneration, new beta-cells can be formed by proliferation of existing beta-cells or by a process called neogenesis, formation of new beta-cells from precursor cells. Moreover, it has been shown that the endocrine pancreas can enlarge the beta-cell mass during increased metabolic demand. This growth is due to proliferation of existing beta-cells and neogenesis. Processes and cells involved in growth of the beta-cell mass can therefore be studied in the pancreas of pregnant animals. The advantage of using pregnancy as a model for pancreatic growth above other models of pancreatic growth, i.e. obesity or beta-cells damage is that the growth of the beta-cell mass during pregnancy is a quick process, which takes place in a well de ned time frame. Therefore, in our studies we have used the models of fetal

Introduction: design and rationale of this study

13

and neonatal embryogenesis and pregnancy to study the role of precursor cells in development and growth of the pancreas.

DESIGN AND RATIONALE OF THIS STUDY

Pancreatic precursor cells have been suggested to have a therapeutic value for the treatment, or even cure, of diabetes mellitus or other pancreas related diseases. Pancreatic precursor cells are responsible for the natural process of pancreas development, but also for regeneration after damage or for natural growth of the pancreas as is taking place for example during pregnancy. Three pancreatic precursor cell populations have been found in the pancreas and several studies showed that these cells play an important role in development of endocrine cells, in particular beta-cells. Although the presence and differentiation potential of these cells have been reported, a detailed description of their localization, phenotypic and genotypic appearance, differentiation capacity, and the potential to contribute to regeneration is still lacking. In the development of the pancreas and of other organ structures the speci c proteins nestin, c-met, and c-kit are expressed and some of them had even been proposed to be present on precursor cells.

Therefore, the aim of this thesis was to study the expression of nestin, c-met and c-kit in the embryogenesis and the development and growth of the adult pancreas and the capacity of nestin, c-met and c-kit carrying cell to differentiate toward functional beta-cells.

Chapter 2 reviews what is known about the different precursor stem cells and the description of the features to ful ll the criteria of being a potential source for the generation of insulin producing cells. At present embryonic stem cells are thought to be the most suitable source for generation of insulin producing cells. Whether pancreatic, thus organ bound precursor cells can play a role in the formation of insulin producing cells via differentiation, remain to be elucidated.

In chapter 3 we investigated whether cells carrying the c-met and c-kit proteins indeed have some ef cacy in stimulating regenerative processes in the adult rat

Chapter 1

14

pancreas. This was done by infusion of pure, isolated populations of cells in adult rat pancreata previously treated with the beta-cell toxin streptozotocin.

In chapter 4 we performed a histological analysis of nestin, c-met, and c-kit positive cells to investigate the localization of these cells in the endocrine, i.e. the islets, and exocrine, i.e. ductules, exocrine tissue, and blood vessels, compartments in the embryonic, neonatal and physiological growing rat pancreas.

In chapter 5 we investigated whether nestin, c-met, and c-kit positive cells in the neonatal pancreas express molecules that are associated with speci c time points in the differentiation process and can therefore be applied as a measure for the differentiation state of the cells. This was done by investigating the expression pro le of a number of genes expressed during the early, mid and late phase of differentiation towards endocrine and exocrine cells, and by immuno uorescence detection of their respective proteins.

In chapter 6 we investigated whether isolated c-met and c-kit positive cells from neonatal rats have the pro le of pancreatic precursor cells and whether these cells have the potential to differentiate into insulin producing cells after ex vivo manipulation. This was done by positive ow cytometric selection of these cells from neonatal pancreata and subsequent culturing of these cells under beta-cell speci c growth factors.

In chapter 7 the results described in this thesis are summarised and discussed, both from a technical point of view as well as from a cell biological point of view.

Shanti T. Gangaram-Panday, MSc, Marijke M. Faas, PhD and Paul de Vos, PhD

University Medical Center Groningen, University of Groningen, Department of Pathology

and Medical Biology, Division of Medical Biology, Immunoendocrinology, Hanzeplein 1,

9700 RB Groningen, The Netherlands.

Published in a modi ed form in Trends Molecular Medicine 2007: 13(4):164-73

chapter 2

Towards stem cell therapy in the endocrine pancreas

Chapter 2

18

ABSTRACT

Many approaches of stem cell therapy for the treatment of diabetes have been described. The rst is the application of stem cells for replacement of nonfunctional islet-cells in the native endogenous pancreas and the second is to serve as an inexhaustible source for islet-cell transplantation. During recent years three types of stem cells have been under investigation; embryonic-stem cells, bone-marrow derived stem cells, and organ-bound stem cells. We discuss the advantages and obstacles of these different cell types. The principle applicability for the treatment of de ciencies in the pancreas has been shown for all three cell types, but more detailed understanding of the sequence of events during differentiation is required to produce fully functional insulin producing cells.

DIABETES AND THE CALL FOR STEM CELL THERAPY

A major challenge in the treatment of diabetes is to provide patients with an insulin source that regulates glucose levels on a mandatory minute-to-minute basis. Conceivable approaches to achieve this are restoring an endogenous source and/or implanting an autologous- or non-autologous-derived source. At present there are different strategies under investigation such as stimulation of regeneration of the residual islet-cells in the diabetic pancreas, increasing the regenerative capacity by infusion of precursor-cells [1-3], and islet-cell transplantation [4]. The principle application of these approaches has been shown in both experimental animals and humans.

The studies addressing the implantation of an autologous- or non-autologous-derived source for treatment of diabetes have always been of limited clinical relevance due to shortage of suf cient supply of islet-cells. This has been the main rationale for researchers to design means to generate inexhaustible islet-sources. During recent years many have focussed on application of stem cells as an inexhaustible source. Stem cells are self-renewing progenitor cells that can differentiate into one or more specialised cell types. Stem cells have a number of characteristics that qualify them as a potential inexhaustible source for islet-cells. Theoretically they have the capacity for unlimited replication


19

and with adequate differentiation they can become fully mature and functional islet-cells.

Several strategies and different stem cell sources for islet-cell substitution have been proposed. Not all have shown the same degree of success. In the present manuscript we will discuss the successes and failures of the different approaches in view of future clinical application. Also, we will discuss the present insights in developmental biology of the endocrine pancreas since this knowledge is mandatory for understanding and designing strategies to create fully functional islet-cells from stem cells of both non-pancreatic and pancreatic origin.

LESSONS FROM EMBRYOGENESIS AND ORGANOGENESIS OF THE ENDOCRINE PANCREAS

The characteristic organisation of the pancreas, i.e. the highly branched ductal structure, (Figure 1) is formed after the endodermal gut tube is created from the endoderm. The dorsal and the ventral pancreatic buds are pouched out from the endodermal epithelium. Subsequently, the ventral bud rotates towards the dorsal bud and fuses as the epithelium invaginates. Next, the pancreatic precursor-cells differentiate into the endocrine and exocrine structures [5] after which the various cell types are formed (Table 1).

The subsequent development of the endocrine pancreas is under tight control of a number of pancreas speci c transcription factors. Among these factors are Pdx-1, ngn-3, Pax4 and Pax6, Isl-1, Beta2/NeuroD, Maf family, Nkx2.2, and Nkx6.1. The most well-de ned transcription factors and their expression are presented in Table 2 and some are described in the text below. These transcription factors regulate migration, differentiation, and proliferation during development. Understanding the sequence, the signals for, and quantitative pattern of expression is essential for designing ex vivo approaches for creating functional islet-cells.

Chapter 2

20

Figure 1: Pancreas development. The human pancreas rst develops as 2 distinct buds, which subsequently fuse to one organ. The dorsal pancreatic bud is the rst to appear around day 26 of human development (E9.5 in rodents), as it buds from the endoderm close to the foregut (2). The ventral pancreatic bud arises less than a day later from the endoderm (in rodents at E10.5). One important component in the regulation of this program is the exclusion of the hedgehog gene family of signaling molecules. These molecules, Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are involved in the promotion of intestinal differentiation. When the Shh and Ihh are downregulated the dorsal and ventral pancreatic buds start to grow. During the fth week of development, the ventral pancreatic bud migrates around the foregut until the ventral bud comes into contact with the dorsal bud. By the beginning of the sixth week both buds have fused (in rodent E17) (2). The dorsal and ventral buds grow into their surrounding mesoderm to form the main ducts. As the main ducts elongate, secondary ducts form and branch off, they in turn elongate and a further generation of ducts branch off (3). By the beginning of the ninth week, the tubules terminate in distinct clumps of cells, these are the presumptive acini (4). By week 12, the interlobular ducts are established, forming the pattern of the future lobular structure of the pancreas. Differentiation of the primitive acini starts around week 12 (in rodents the ductal structure and acini are clearly distinguishable at E14.5). By week 16, the rst mature acini are formed and this continues until birth. At further elongation and dilatation of the ducts, cell clumps, distinct from those of the terminal structures, bud off from the walls of the smaller branches, these are the presumptive islets. The presumptive islet-cell clumps migrate away from the tubules into the stroma of the developing gland,


21

while new clumps continue to form and bud off. The islets expand through proliferation of the islet-cell precursors and by merging of cell clumps in close proximity. During week 10 type alpha-cells differentiate; during week 11 type delta-cells appear and two weeks later, beta-cells start to appear (in rodents early endocrine cells appear from E18-19).

Table 1: Pancreatic cells; their phenotypes and function. Overview of the cells, and their function, which reside in the pancreas.

Pancreas Cells FunctionEndocrine beta-cells Production of insulin for sustaining euglyceamia

alpha-cells Secrete glucagons which counteract insulin’s hypoglycemic effectsdelta-cells Secrete somatostatin which inhibit insulin secretionPP-cells Pancreatic polypeptide function remains unclearghrelin-cells Production of ghrelin can inhibit glucose induced insulin release

and can stimulate glucagon secretion [70]Exocrine Acinar cells

Duct cells

Produce at least 22 digestive enzymes such as, proteases, amylases, lipases and nucleasesNon-enzymic components of the pancreatic juice, including bicarbonate

One of the most intensively studied gene is Pdx-1 (Table 2). During organogenesis Pdx-1 is widely expressed in cells which eventually differentiate into endo- and exocrine pancreatic-cells, whereas in the adult pancreas the protein expression of Pdx-1 is restricted to beta- and delta-cells [6]. It was shown that Pdx-1 is involved in islet-cell speci c expression of various essential genes for glucose metabolism such as insulin and somatostatin [7,8]. The disruption of Pdx-1 in mice and humans resulted in absence of development of the pancreas [9].

Also, ngn-3 is crucial for pancreas development [10]. Ngn-3 is found in both the embryonic and postnatal pancreas where it contributes to islet-cell renewal under physiological conditions [10]. Ngn-3 protein expression leads to protein-expression of extracellular Notch ligands in precursor-cells. This subsequently leads to activation of Notch-receptors which is another critical factor in pancreas development [10].

Chapter 2

22

Table 2: Summary of well-known transcription factors involved in pancreas development. Transcription factors, their function, and their timing of expression during embryogenesis. The table summarizes which transcription factors can be found in which precursor cells. Notably, some of these transcription factors remain expressed in mature endocrine cells.

Pancreatic cells

Tran-scription factors

Function Species Expression in rodents

Ref

Endocrine progenitors

Ngn-3 Directs differentiation of pancreatic precursor cells towards endocrine lineages

mouse, rat, human

Starting E9.5, diminishes after birth, not present in adult

[10]

Pax4 Formation of beta- and delta-cells; represses glucagon transcription

mouse, rat, human

Starting E10, decreasing from late neonatal

[13]

HNF6 Required for normal endocrine development. Appears to activate ngn3 expression

mouse, rat, human

Starting E9.5, neonatal, adult unknown

[71]

Isl-1 Early endocrine cell differentiation

mouse, rat, human

Starting E9, decreasing from neonatal, in adult restricted to islets

[15]

Beta2/NeuroD

Islet growth and differentiation

mouse, rat, human

Starting E9.5, from E17.5 and in adult restricted to islets

[11]

Pax6 Formation of alpha-cells Activates glugacon transcription

mouse, rat, human

Starting E9, present during neonatal, assumed in adult

[13]

Nkx2.2 Necessary for beta-cell precursors to express Nkx6.1 and Ins

rat, human Early embryonic, decreasing in neonatal, in adult restricted to islets

[72]

Nkx6.1 Final differentiation of beta-cells

rat, human Starting E10.5 in whole pancreas, from E15.5 and in adults restricted to beta-cells

[21]

MafA Controls Ins gene expression

mouse, rat, human

Starting E14, decreasing in neonatal, in adult restricted to beta-cells

[73]

MafB Formation of alpha- and beta-cells

mouse, rat, human

Starting E15, decreasing in neonatal, adult restricted to alpha-cells

[16]

Pdx-1 Formation of beta- and delta-cells

mouse, rat, human

Starting E8.5, neonatal, adult restricted to islets

[6]

Exocrine progenitors

Hes1 Directs differentiation of pancreatic precursor cells towards exocrine lineages

mouse, human

Starting E9.5, decreasing in neonatal, adult not present

[74]

Pdx-1 Formation of exocrine tissue

mouse Starting E8.5, neonatal, adult restricted to islets

[6]


23

Pancreatic cells

Tran-scription factors

Function Species Expression in rodents

Ref

Mature beta-cells

Lmx1.1 Ins activation in mature beta-cells

human Not valid [75]

HNF1 Activation of Ins and Pdx-1

mouse, rat, human

Not valid [76]

HNF4 Activation of HNF1 , Ins and GLUT-2

mouse, rat, human

Not valid [77,78]

Pdx-1 Important activator of Ins mouse, rat, human

Not valid [7, 79]


rat, human Not valid [72]

Nkx6.1 Final differentiation of beta-cells


Beta2/NeuroD

Ins activation mouse, rat, human

Not valid [11]

MafA Controls Ins gene expression

mouse, rat, human

Not valid [73]

Mature alpha-cells



MafB Controls glucagon expression

mouse, rat, human

Not valid [16]

Mature PP-cells



Abbreviations: Ngn-3, neurogenin 3; Pax, paired homeobox; Hes1, hairy of enhancer of split 1; Lmx1.1, LIM homeobox factor 1.1; HNF hepatocyte nuclear factor; Pdx-1, pancreatic en duodenal homeobox 1; Nkx, NK homeobox; Isl-1, islet-1; Beta2/NeuroD, beta-cell e-box transactivator 2/ neurogenic differentiation.

The bHLH Beta2/NeuroD gene is associated with activation of the insulin gene [11]. During mouse embryogenesis Beta2/NeuroD protein is expressed in a subset of pancreatic epithelial cells on E9.5, while later its protein-expression is restricted to islets [11,12]. Beta2/NeuroD de cient mice died 3-5 days post-partum due to severe hyperglycaemia and islet anomalies [11].

Pax4 and Pax6 proteins are speci cally expressed in the developing endocrine pancreas and regulate differentiation and proliferation of islets [13]. During embryogenesis Pax6 protein is found from E9 on a small subset of the pancreatic endoderm [13]. Pax4 protein-expression can be found from E10 in the pancreatic bud [13]. At the end of the development Pax4 decreases, whereas Pax6 remains

Chapter 2

24

elevated [13]. Knock-out studies have con rmed that Pax4 -/- mice do not generate beta- and delta-cells, while Pax6 -/- mice are unable to form any alpha-cells [13].

The LIM-domain (Lin-11, Isl-1, and Mec3 genes) protein Isl-1 is expressed in endocrine cells and a subset of neurons in the adult rat [14]. During development Isl-1 is detected from E9 in the dorsal pancreatic epithelium of mice [15]. At E11, Isl-1 is detected in the ventral pancreatic epithelium which is required for the differentiation of islet-cells. In Isl-1 mutant mice-embryos, islet development was completely absent [15].

Also, Maf-proteins are essential for development of the endogenous pancreas. In adults and during embryogenesis MafA is speci c for insulin expression [16-18]. Although MafB is speci c for glucagon expression in adults [16], recently MafB-protein was also found at E12.5 where it was co-expressed with insulin [16]. This MafB-insulin co-expression was persistent throughout subsequent development. In the postnatal pancreas, MafB is downregulated in beta-cells, but not in alpha-cells. In a later developmental stage (E18.5) a few Pdx-1 and MafB-positive cells were found, which was interpreted as differentiation into insulin-producing cells [16]. A follow-up study showed that in embryos MafB is expressed before MafA suggesting that differentiation of beta-cells proceeds through a MafB+MafA-Ins+ intermediate cell to MafB-MafA+Ins+ cells [19]. These results suggest that MAfB has a dual role in embryonic differentiation of both beta- and alpha-cells.

The NK homeodomain family is also involved in the development of islets. Nkx2.2 protein-expression can be detected during early embryogenesis in all endocrine cell types [20] while in adult islets it becomes restricted to beta-cells, alpha-cells, and PP-cells [21]. Knockout-studies have shown that Nkx2.2-null mutants mice develop severe hyperglycaemia [21] due to the fact that beta-cell precursors survived, but failed to mature [21]. Another member of the NK-homeodomain family is the transcription factor Nkx6.1. In the developing mouse pancreas Nkx6.1-protein expression can already be detected at E10.5 [21]. From E15.5 the expression of Nkx6.1-protein becomes restricted to insulin- producing cells, scattered ductal cells, and periductal cells [21]. At later stages and in the adult pancreas Nkx6.1 protein becomes restricted to


25

insulin producing cells [21]. Disruption of Nkx6.1 leads to blockage of beta-cell neogenesis [21].

Adequate knowledge of the transcription factor expression is not only essential for stem cell research, but also for understanding the pathophysiology of diabetes. This is illustrated by the discovery of 6 human genes called the MODY genes which are responsible for the onset of a subtype of diabetes Type 2 [22]. Among these genes are, MODY4 mutations in the insulin promoter factor 1 (IPF-1/Pdx-1), and MODY6 mutations which is a neurogenic differentiation factor (Beta2/NeuroD).

STEM CELLS FOR ISLET-CELL SUBSTITUTION

The embryonic stem cells Different sources of stem cells have been proposed for the production of beta-cells. The application of ESC in regenerative medicine is currently under investigation in many elds [23]. ESC can be derived from blastocyst and can in vivo differentiate into cells of endoderm, mesoderm, and ectoderm origin and thereafter into cells speci c for the various tissues in the body (Figure 2a). Murine and human ESC can in vitro generate embryoid bodies containing cells with a beta-cell like phenotype [24-27]. Differentiated ESC have also been shown to be able to lower blood glucose levels in rodents [28,29]. Various groups have shown that the number of insulin producing cells during in vitro differentiation can be enhanced by over expressing Pax4, Pdx-1 or Nkx6.1 in ESC [29-31]. An increase in the number of differentiated beta-cells can also be accomplished by culturing ESC with differentiation factors. Murine ESC-derived embryoid bodies cultured in the presence of 15% FCS, followed by serum-free conditions, give rise to a population of cells that express insulin and Pdx-1 [32]. Also Segev et al. [27] reported enhanced expression of beta-cell speci c genes in ESC after manipulation of culture medium.

The potential application of ESC for producing beta-cells has brought enormous optimism into the eld. However, there is a backside that also requires some considerations. Despite enormous progress in knowledge of the developmental biology of beta-cells, there are still large gaps in our insight in the processes

Chapter 2

26

involved in differentiation of ESC to beta-cells. It has been suggested that beta-cells derived from ESC do not represent bona de embryonic or foetal phenotypes, but rather are aberrant products from misregulated or scrambled differentiation programs [33]. The lack of knowledge about normal differentiation is probably also responsible for the observation that in vitro generated products of stem cells show many features of senescence such as multiple fragments of chromosomes [34]. Moreover, evidence that the ‘generated’ beta-cells produce insulin in a normal biphasic way is completely lacking up to now. Not only functional problems may arise, also the change of malignancies should be considered as a threat. ESC closely resemble embryonal carcinoma cells which are known to have the capacity to form teratocarcinomas. It has been shown that animals develop tumours when they are transplanted with ESC-derived insulin-producing cells [35]. From the above follows that it is advisable to perform detailed ef cacy studies on ESC before proposing ESC as a suitable source for insulin producing cells in the treatment of diabetes.

Not only ESC, but also umbilical cord blood cells have been shown to have the capacity to differentiate into islet-cells [2,36-38]. Cells of the umbilical cord express nestin and ngn-3 on protein levels, while on a mRNA level nestin, cytokeratin 8, 18, and 19 as well as the beta-cell speci c transcription factors Isl-1, Pax4, ngn-3 are expressed [2]. After culturing for 8 days the umbilical cord blood cells also started to express Pax4 mRNA. The principle applicability of the umbilical cord blood cells for treatment of diabetes was shown in various animal studies in which infusion of these cells lowered the blood glucose levels in diabetic mice [36-38]. Moreover, it was shown that the infused cells had differentiated into insulin producing cells [37,38]. Therefore, umbilical cord blood cells deserve more attention as a source for generation of cells of the endocrine pancreas.

Bone marrow derived stem cells The BMDS cells have been subject of haematopoietic studies for decades and are therefore the best characterized stem cell source. BMDS cells are multipotent and capable of self-renewing and well known as stem cells for blood cells (Figure 2b).

However, they can also differentiate into other cells, since BMDS cells can migrate towards a site of damage and can differentiate under the in uence of factors from the microenvironment (cell-cell, cell-extracellular matrix interactions and growth


27

factors) [39,40]. This process, in which precursor cells differentiate into another cell type, is called transdifferentiation [40,41]. Besides transdifferentiation, BMDS cells may fuse with specialized cells in the vicinity in order to substitute damaged cells [42]. This, however, is suggested to be a rare event in the pancreas [43].

Infusion of bone marrow cells can restore chemically-induced diabetes in mice [1,3]. Notably, however, these studies do not indisputably show that BMDS cells differentiate into beta-cells. A more recent study showed engraftment of endogenous bone marrow in damaged islets [44]. However, the engraftment did not give rise to cells expressing insulin or beta-cell speci c transcription factors like Pdx-1 or Nkx6.1 [44]. This may suggest a supportive role of BMDS cells in regeneration rather than direct substitution of damaged cells. Other studies do not corroborate the above mentioned ndings, since they did not nd a single endogenous BMDS cells in injured pancreata [45]. In spite of con icting ndings the role of BMDS cells in regenerative processes in vivo remain subject of study mainly because the in vitro ndings are very supportive [3,46]. In vitro BMDS cells are able to produce de novo insulin and are able to express insulin and GLUT-2, when challenged in high glucose [3,46].

In summary, current research may point towards an absence of a role for BMDS cells in in vivo differentiation into beta-cells. Simultaneously, however, it has been shown that BMDS cells can in vivo support pancreatic growth and can be manipulated in vitro to differentiate into beta-cells.

Organ-bound or endogenous stem cells It is generally accepted that the pancreas in the adult organism has a small population of cells that are capable of continuous self-renewal and can differentiate into cells of the pancreas. These organ-bound stem cells or pancreatic stem cells have received a lot of attention of the scienti c community during the past 5 years since they hold a number of advantages over the other sources. They combine the capacity for prolonged proliferation with an already partial differentiation towards an endocrine phenotype. This may facilitate a more readily and less complicated differentiation strategy toward islet-cells than with the other sources. Also, the cells can be target for speci c clinical therapies to stimulate the endogenous stem cells

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28

Figure 2: The embryonic, bone-marrow derived, and organ-bound stem cells. Embryonic stem cells (a) can derive from the embryoblast. The embryoblast is the inner cell layer of the blastocyst. The outer cell layer of the blastocyst is the trophoblast. The embryoblast forms the three germ layers; ectoderm, mesoderm, and endoderm which develop into the various tissues as shown in the gure. The trophoblast develops into the placenta. Bone marrow derived stem cells (BMDS) (b) are multipotential stem cells, which can differentiate into multiple cell lineages. A well known line of stem cell, which differentiates from the multipotential stem cell is the hematopoetic stem cell line which can differentiate


29

into lymphoid and myeloid progenitor cells. These cells can further differentiate into cells of the lymphoid lineage (immune cells such as, T-cells, B-cells, NK-cells involved in adaptive immune responses) and myeloid lineage (blood cells and phagocytes involved in innate immune responses). Other lines of stem cells are mesenchymal stem cells and endothelial progenitor cells. BMDS can also differentiate into other cells or tissues such as probably beta-cells. Organ bound stem cells (c) are unspecialised cells or cells with some but not all features of specialised cells. These cells can renew themselves and can differentiate into fully specialised cells mostly of the organ they reside in. It is assumed that pancreatic stem cells reside in the ducts and in the islets of Langerhans. Cells from ducts can differentiate into exocrine cells and endocrine cells. Stem cells in the islets can differentiate into the endocrine cells and possibly also into supporting cells such as endothelial cells. The function of these cells is plausibly to replace damaged cells in case of organ damage.

to induce compensatory growths [47]. At least two populations of pancreatic stem cells have been described to be present in the pancreas (Figure 2c). The rst group of stem cells can be found in the ductal cells and acinar cells. It has been suggested that these cells are pancreatic ductal epithelium cells, expressing the ductal marker CK-19 and Pdx-1 [48]. These cells can expand and differentiate into endocrine cells [49]. The second group of stem cells are the islet-derived stem cells. The evidence for the existence of an islet-derived population of stem cells comes from studies showing a population of insulin containing cells reappearing in the islets after total destruction of the beta-cell mass with streptozotocin [47]. A few studies have addressed the identi cation of residential islet-bound stem cells [50,51]. These studies have identi ed several distinct populations as will be discussed in the following sections.

For many years nestin positive cells were proposed to represent a population of islet-derived stem cells [51]. It has been shown that NIP cells express the ATP binding cassette (a stem cell marker) [50] and have an extended capacity to proliferate in vivo. Also, NIP cells derived from the islets can in vitro differentiate into cells with liver, pancreatic exocrine/ductal, and endocrine phenotypes [51]. After differentiation the cells were found to produce insulin, glucagon, GLP-1, and the transcription factor Pdx-1 [51]. Nestin, however, is not speci c for the

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endocrine pancreas, but also found in ductules [52]. These ductal nestin positive stem cells also have characteristics of beta-cells precursors such as high expression levels of Pdx-1 [52,53]. Unfortunately, many have abandoned studies on nestin positive cells since some studies show that nestin in the pancreas is only expressed in cells that are not destined to become beta-cells [54-56]. However, recent evidence suggest that there are different populations of nestin positive cells in the pancreas [57]. In summary, according to the present insight nestin seems to be a functional protein that under speci c circumstances is expressed in beta-cell precursors while it may also be found in other cell types [57]. Our opinion therefore is that nestin is not a suitable marker for endocrine stem cells, but may be an important functional protein which can be found in a wide variety of stem cell sources. It is therefore mandatory to further investigate the role of nestin in beta-cell precursors since its expression pattern suggests an essential function in the maturation of precursor cells.

The pancreas also contains a population of cells carrying the HGF receptor, i.e. the c-met receptor [58]. In the pancreas HGF has been suggested to be a potent regulator of development of beta-cell function, differentiation, and proliferation [59]. For long it has been assumed that the c-met positive cells in the pancreas are the oval hepatic cells [58]. However, recent studies suggest that c-met is not speci c for the liver and can also be found in other tissues [58]. In the neonatal mice pancreas c-met positive cells are found to reside in acinar tissues, but not in the islets [60]. Also some cells were found in ducts, but these cells did not have an endocrine nor acinar phenotype. In the adult pancreas c-met is mainly expressed in and around the ducts, vascular endothelial cells, and cells around acinar tissues (Gangaram-Panday et al., unpublished). This should not be interpreted as a suggestion that c-met positive cells are not involved in regeneration of adult islets. In a recent study we have shown that c-met positive cells are present in the islets during the growth of the adult pancreas during pregnancy (Gangaram-Panday et al., unpublished). We also found that pancreatic c-met positive cells express endocrine speci c transcription factors ngn-3, Beta2/NeuroD, Pax6, and endocrine hormones like insulin, and glucagon and can thus be considered to be pancreatic stem cells (Gangaram-Panday et al., unpublished). Others have shown that c-met positive cells can differentiate into endocrine cells in vivo [58]. Although this study may suggest that c-met positive cells can differentiate into islet-cells it does not proof


31

that the cell is residential in islets. It cannot be excluded that the c-met positive cells during increased metabolic demand migrate from the extra-islet compartment or from the bone marrow to the islets in order to increase the mass of the endocrine cell-compartment.

A third population of cells in the pancreas that quali es for residential islet-bound stem cell is the c-kit positive cells. C-kit is known to be a universal marker for stem cells. Binding of stem cell factor to the c-kit receptor leads to cell proliferation, cell survival, and chemotaxis [61]. The c-kit receptor is expressed on a variety of cells and was recently shown to be expressed in the prenatal and postnatal rat pancreas [62]. Co-localisation studies in the 7-day-old postnatal rat pancreas demonstrated that c-kit is expressed in the periphery of islets and co-localised with glucagon positive cells [62]. We found that c-kit positive cells are also found in adults near the ductules and blood vessels, while during increased metabolic demand (i.e. during pregnancy) we found an increase of c-kit positive cells in islets, ductules, and near blood vessels (Gangaram-Panday et al., unpublished). Studies on epithelial monolayers derived from postnatal rat pancreatic islets demonstrate the presence and increase of c-kit expressing cells [63]. These epithelial monolayers expressed c-kit and developmental transcription factors such as Pdx-1 and ngn-3 during a culture period of 8 weeks. After further differentiation, insulin and glucagon were weakly expressed, whereas c-kit mRNA disappeared [63]. Although, the role of c-kit in the development of beta-cells remains to be further elucidated, these studies illustrate that c-kit positive stem cells certainly have characteristics of precursors of endocrine cells and can be found in islets. As for c-met it has not been demonstrated that c-kit positive cells are residential cells or whether they migrated from other sources.

SOURCES OF NON-PANCREATIC ORGAN BOUND STEM CELLS

Not only pancreas-derived cells, but also extra pancreatic organ-bound stem cells can differentiate into islet-cells. Recently, liver cells [41,64-66], human adipose tissue-derived mesenchymal stem cells (MSC) [67], gut stem cells [68], and even splenocytes [69] have been shown to bare this capacity. The rationally to

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study extra pancreatic sources is that organ-bound stem cells from these organs can be harvested from patients in a less invasive fashion and may be available in higher amounts. Strategies to induce this transdifferentiation from extra pancreatic sources are similar to those with ESC. Cultured hepatic stem cells from rodent or human origin will differentiate towards a beta-cell phenotype in a high-glucose environment. Also, over expression of the transcription factors Pdx-1 [66] and the combination of over expression of Beta2/NeuroD and addition of betacellulin to the medium [41,65] is a successful approach to promote a differentiation towards a beta-cell phenotype. Unfortunately, there are no functional studies yet showing biphasic insulin release upon glucose challenge available with these cells.

FUTURE DIRECTIONS

Researchers have shown the principle applicability of embryonic-stem cells, bone-marrow derived stem cells, and organ-bound stem cells for treatment of the diseased endocrine pancreas. At the same time they show a number of challenges. The most important challenge is to produce a fully functional insulin-producing cell with a normal biphasic insulin release and the capacity to regulate blood glucose in a minute-to-minute dynamic fashion. To achieve this goal it is to our opinion mandatory to gain insight in the basic biology of differentiation towards insulin-producing cell. That this is a successful approach is illustrated by the studies in which novel transcription factors have been over expressed in precursor cells after which the cells differentiated towards an endocrine phenotype. Without doubt there are functional genes to be identi ed that are responsible for the induction of the glucose-induced insulin response.

In the discussion between the potentials and pitfalls of the different stem cells sources we have illustrated the importance of giving the endogenous stem cell source more attention for clinical application. Although it is a relative new stem cell source, it provides a number of advantages over the other systems. They can possibly be applied autologous since they can be of either pancreatic or extra pancreatic origin. Also, since these cells are already partially differentiated they can readily differentiate into fully functional beta-cells. These endogenous stem cells can be found in the ductal system of the pancreas, plausibly in the endocrine


33

pancreas, and in other organ-structures. Conceivable applications of endogenous stem cells are enhancement of the regenerative capacity of the endocrine pancreas in non-autoimmune diabetes by stimulation of differentiation using tropic factors or by infusion of these endogenous stem cells into diabetic patients. Also, these cells can plausibly be applied in islet-transplantation to increase the regenerative capacity of the graft in the ischemic period between transplantation and full vascularisation of the graft. Also, ex vivo islet-cell generation may be an option, but up to now there are no studies available showing an unlimited proliferation of endogenous stem cells.

A potential pitfall that requires more attention is that many cultured stem cells show not only features of senescence, but also genetic disorders such as unlimited proliferation leading to tumour formation. Technologies should be designed to delete these cells. Another challenge is to overcome autoimmune destruction when stem cells are proposed not only for type 2 but also for type 1 diabetes. The exogenously differentiated beta-cells are phenotypically identical to beta-cells and therefore prone to autoimmunity.

CONCLUDING REMARKS

Many have described the potentials and bene ts of stem cells for the treatment of diabetes. Several approaches have been described and reviewed. Options for the use of stem cells are increasing the regenerative capacity of the diabeticpancreas [1,3], and as source for islet-cells to solve donor shortage in islet transplantation [26]. All these applications have been extensively reviewed during recent years. Fundamental biological processes however have gained not enough attention, since we still do not have insight in which mechanisms are involved in differentiation into fully functional insulin-producing beta-cells. A strict sequence and pattern of expression of transcription factors is required for producing islet-cells with normal biphasic insulin responses. At present it is far from simple to stimulate this process exogenously. Therefore, the progress has not yet met the clinical expectations. This however does not mean that progress is lacking. During recent years many new insights in the development biology of the pancreas and improved culture technologies has brought clinical application to a realistic option.

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Stepwise analysis on expression patterns of transcription factors has brought some insight into the sequence of events resulting in beta-cell formation and also allows quanti cation of differentiation toward beta-cells. These new insights were followed by studies on exogenous manipulation of the expression of transcription factors in stem cells to promote differentiation of stem cells towards islet-cell phenotypes. However, there is still a gap in knowledge on development biology of beta-cells, resulting in our disability to produce fully functional beta-cells from stem cells in vitro. Therefore much more knowledge and step-by-step scienti c research is mandatory to extend our knowledge. Nevertheless, a promising and bright future lies in front of us now that doors have been opened by stem cells as alternative sources for grafting of novel beta-cells.

ACKNOWLEDGEMENTS

This work is supported by the Graduate School for Drug Exploration (GUIDE) institute of University of Groningen and University Medical Center Groningen.


35

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GLOSSARY BOX

Beta2/NeuroD: beta-cell e-box transactivator 2/ neurogenic differentiation

BMDS cells: bone marrow derived stem cells

CK-19: cytokeratin-19 a ductal cell marker

E: embryonic day

Ectoderm: the outer germ layer of the embryo from which the epidermis, the nervous

tissue and sense organs develop

Endoderm: the inner germ layer of the embryo from which the gastrointestinal and

digestive tract, and the lungs develop

Embryogenesis: the development and growth of an embryo principally from the second

week through the eight weeks following conception

ESC: embryonic stem cells

Euglycaemia: normal concentration of glucose in the blood. Also called normoglycaemia

GLP-1: glucagon like peptide-1

HGF: hepatic growth factor the ligand for the c-met receptor

Hyperglycaemia: the presence of an abnormally high concentration of glucose in the blood

Hypoglycaemia: an abnormally low concentration of glucose in the blood

LIM domain: Lin-11, Isl-1, and Mec3 genes

Maf: avian musculoaponeurotic brosarcoma oncogene homolog

Mesoderm: the middle germ layer of the embryo from which the connective tissue,

muscle, bone, the urinary and circulatory systems develops

MODY: the Maturity-Onset Diabetes of the Young genes

Ngn-3: neurogenin-3

NIP cells: nestin positive islet-derived progenitor cells

Nkx proteins: NK homeobox family of proteins

Organ-bound or

endogenous stem cells: a small population of cells in each organ in the adult organism that are

capable of continuous self-renewal and can differentiate into cells of the

tissue they reside in

Organogenesis: the formation and development of organs in living organisms

Pax genes: paired box genes

Pdx-1: pancreas duodedum homeobox-1

Stem cells: self-renewing progenitor cells that can differentiate into one or more

specialised cell types


41

Transcription factors: proteins regulating the activation of transcription of DNA in the eukaryotic

nucleus

Transdifferentiation: a process in which precursor cells differentiate into another cell type

Shanti T. Gangaram-Panday, MSc, Bart J. de Haan, Arjen Petersen, Marijke M. Faas, PhD and

Paul de Vos, PhD

University Medical Center Groningen, University of Groningen, Dep. Pathology and Medical

Biology, Div. of Medical Biology, Immunoendocrinology, Hanzeplein 1, 9700 RB Groningen,

The Netherlands.

chapter 3

Regenerative e ect of c-met and c-kit carrying rat neonatal

cells on the endocrine pancreas (preliminary data)

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ABSTRACT

Objective: In the present pilot study we investigated whether c-met and c-kit positive cells may stimulate regeneration of the damaged adult pancreas. Research Design and Methods: Low dose streptozotocin injected animals were infused with c-met or c-kit positive cells obtained from neonatal pancreata. Results: We found large amounts of the infused c-met and c-kit positive cells in the pancreas of infused rats. C-kit and to a much lesser extent also c-met positive cells induced a decrease in the blood glucose levels at 4 days after injection. However, we did not observe any difference in intensity in insulin staining. Conclusions: Our study suggests that pure population of c-met and c-kit carrying neonatal rat cells can contribute to the regeneration of the adult pancreas.

INTRODUCTION

During embryogenesis the pancreas evolves according to a number of strictly controlled and prede ned steps. It starts with the epithelial evagination of the foregut endoderm into the surrounding splanchnic mesoderm. This epithelial-mesenchymal interaction is followed by acinar and islet cell differentiation [1]. During this early stage, a multipotential stem cell may differentiate into cells that possess either an endocrine or exocrine phenotype [1]. The endocrine precursor cells develop further by budding from embryonic duct-like cells. This process leads to the formation of primitive islets in the mesenchyme adjacent to the ducts. Much new insight has been gained during recent years in the processes responsible for development of the pancreas in embryogenesis and a number of cell types have been identi ed that that are being hold responsible for development of the primitive pancreas.

A series of recent reports [2-5] have proposed that such cells that are involved in the development of the endocrine pancreas carry the receptor for hepatocyte growth factor (c-met) and the stem-cell factor (c-kit). These markers have been shown to be associated with stem cells [2-5]. C-met and c-kit positive cells have also been shown in the adult pancreas, especially in situations of pancreatic regeneration. Some authors even suggested that infusion of clonally expanded populations of

Regenerative effect of c-met and c-kit carrying cells on the endocrine pancreas

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these cells may stimulate regeneration of the damaged adult pancreas. To test whether these cells carrying the c-met and c-kit proteins indeed have some ef cacy in stimulating regenerative processes in the adult pancreas we infused pure, isolated populations of these cells in adult rat pancreata previously treated with the beta-cell toxin streptozotocin. Since c-met and c-kit positive cells can be found in high quantities in the neonatal pancreas [2,5, this thesis] we isolated these cells from the neonatal pancreas.

MATERIALS AND METHODS

Design of the studySince it is suggested that c-met or c-kit positive cells can stimulate regeneration of the damaged pancreas, we choose to use rats in which the pancreas was slightly damaged by a low dose of streptozotocin (STZ), rather than using a high dose of STZ to induce a diabetic state. Rats were treated with 40 mg/kg STZ two days prior to the infusion with c-met or c-kit positive cells. At the day of the infusion the rats were infused with 400.000 isolated c-met or c-kit positive cells from neonatal pancreata which were labelled with the PKH26 Red Fluorescent Cell linker Kit. This labelling was performed to be able to trace back the cells in the pancreata after sacri ce of the rats. The blood glucose levels of the rats were measured at the day of the infusion, and 4 days after infusion (day of termination). We also weighed the rats at these various time points.

At day 4 after infusion the rats were sacri ced and the pancreata were harvested. Using immuno uorescence, we investigated whether the infused c-met or c-kit positive cells could be visualized in the pancreas. We also studied the expression of insulin in the pancreata of c-met (n=5) or c-kit (n=5) injected and non-injected STZ-treated rats using immunohistology. For comparison of insulin in c-met or c-kit injected and non-injected STZ-treated rats, we also stained the pancreata of non-injected, non-STZ-treated rats for insulin.

AnimalsTo obtain neonatal rat pancreata, pregnant rats were purchased from Harlan. These rats were allowed to deliver naturally. Neonatal rats were used 1-2 days after

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delivery. All experiments were conducted in accordance with NIH-guidelines for the care and use of laboratory animals. All rats were kept in a temperature- and

light-controlled room (lights on from 6 AM to 6 PM).

Female Wistar rats (Harlan; age 3-4 months and weighing ~200 g) were used for the injection of c-met (n=5) or c-kit (n=5) positive pancreatic precursor cells. As controls, we included non-injected STZ-treated rats (control rats) in which we performed sham operations. For c-met injections, we included n=5 and for the c-kit injections we included n=4 control rats.

SurgeryThe neonatal rat pancreata were obtained from 1-2 days old rat pups. The pups were decapitated and the pancreas was surgically removed. This was done by laparotomy, replacing the stomach aside, and taking the pancreas out by cutting it loose from the spleen, duodenum, and stomach wall. The neonatal pancreata were processed for cell-isolation. For cell-isolation, the pancreata were stored in 5 ml of 0.20 m ltered Krebs-Ringer-Hepes buffer (KRH: 133 mM NaCl, 4.69 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4.7H2O, 10 mM HEPES and 2.52 mM

CaCl2.2H2O (pH 7.4)) containing 5% bovine serum albumin (BSA) on ice.

Cell-isolationNeonatal pancreata were digested with collagenase to transform them into single cells. The pancreata were rst cut in small fragments of approximately 1 mm. The fragments were then washed once with KRH containing 5% BSA for 5 minutes at 4°C. The pancreas fragments were digested with 5.5 mg/ml Collagenase P (Boehringer Mannheim, Germany) in KRH containing 1% BSA for 12 minutes at 37°C under continuous agitation in a water bath. For the separation of the single cells from the debris, the mixture was centrifuged at 500 rpm for 30 seconds at 20°C. The supernatant (containing the single cells) was aspired. The single cell suspension was washed 3 times with KRH containing 1% BSA for 5 minutes at 4°C. After washing, the cells were resuspended in KRH containing 1% BSA. Cell viability was determined with Trypan blue dye exclusion, viability was always 90%. After this procedure the single cells were stained for c-met or c-kit in order to be able to sort the cells.


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Staining for c-kit and c-met According to standard methods the following procedure was performed for c-met or c-kit cell-sorting. First the single cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the pellet was pre-incubated with undiluted swine serum for 30 minutes. After incubation the cells were washed with PBS containing 0,5% BSA and 0,1% sodium azide. Subsequently, the mixture was centrifuged at 1500 rpm for 5 minutes at 4°C. Next the cells were incubated with the primary antibody anti-c-met (1:100)(H-190, Santa Cruz Biotechnology) or anti-c-kit (1:100) (C-19, Santa Cruz Biotechnology) for 60 minutes. Then the cells were incubated with secondary antibody swine-anti-rabbit FITC (1:50) (DakoCytomation, Denmark) for 30 minutes in the dark. The whole procedure was performed on ice. Positive selection for c-met or c-kit was performed using a Fluorescence Activated Cell Sorter (FACS, MoFlo ow cytometer) (Cytomation, USA). The sorted cells were collected in a tube with sterile RPMI 1640 containing 10% FCS, and10 mg/ml Gentamycine (GIBCO).

PKH26 labeling for c-met and c-kit sorted cellsFreshly isolated c-met or c-kit positive cells were stained with the PKH26 Red Fluorescent Cell linker Kit (Sigma) according to the manufacturer’s protocol. Brie y, the cells were collected and centrifuged at 1500 rpm for 5 minutes. After centrifuging the cells, the supernatant was aspired leaving no more than 25 l of supernatant on the pellet. The cells were resuspended in 1 ml of Diluent C. Next 1 ml of diluted PKH26 dye in Diluent C (1:1000) was added to the cells. The sample was mixed by gently pippeting and the sample was incubated for 5 minutes. The staining reaction was stopped by adding 1% BSA to the sample and incubating the sample for 1 minute. To dilute the sample an equal volume of medium, sterile RPMI 1640 containing 10% FCS, and 10 mg/ml Gentamycine (GIBCO), was added and the sample was centrifuged at 1500 rpm for 5 minutes. The supernatant was aspired and the pellet with cells was washed 2 times with medium, sterile RPMI 1640 containing 10% FCS, and 10 mg/ml Gentamycine (GIBCO), and 1 time with medium, sterile RPMI 1640 and 10 mg/ml Gentamycine (GIBCO). After the last wash the cells were resuspended in 150 l of sterile RPMI 1640 and 10 mg/ml Gentamycine (GIBCO). The whole procedure was performed at room temperature.

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Induction of mild diabetes ratsThe pancreata of the rats were slightly damaged by given them a low dose of 40 mg/kg STZ (Zanosar) 2 days prior to the infusion. The rats were anaesthetised with a combination of iso urane, oxygen, and NO2. After the rats were anaesthetised, they were injected with 40 mg/kg STZ via the tail vene.

Injection of c-met or c-kit positive cells via the pancreatic arteryAt the day of infusion the rats were injected with 400.000 isolated c-met (n=5) or c-kit (n=5) positive cells from neonatal pancreata which were labelled with the PKH26 Red Fluorescent Cell linker Kit. The rats were anaesthetised with a combination of iso urane, oxygen, and NO2 with a small incision in the abdomen, the abdominal cavity was opened and the pancreas was lifted. Next the pancreatic artery was located near the pancreatic duct and the artery was punctured injected for the infusion of the cells. Sham surgery was performed on control rats which were treated in a similar fashion, except that only medium was injected into the pancreatic artery.

Suppression of immune systemSince we used outbred wistar rats, we had to suppress the immune system of the infused and control rats. Therefore, the rats were injected with Ciclosporin A (5 mg/kg) subcutaneously. During the experiment the rats obtained 2 injections of Ciclosporin A, i.e., on the day of injection, and 2 days after the injection.

Glucose measurementThe blood glucose levels of the rats were measured by tailsnapping at 2 days before the injection, the day of the injection and 4 days after the injection (day of termination). The measurement was done by using the ACCU-CHEK Advantage blood glucose meter and the accessory glucose strips.

Isolation of pancreata from injected and control animals Pancreata were obtained from c-met and c-kit injected-STZ treated or non-injected/STZ-treated rats. Therefore, these rats were anaesthetised with a combination of iso urane, oxygen, and NO2 and the pancreas was removed by laparotomy and subsequently snap-frozen in liquid nitrogen and stored at -80°C until sectioning.


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Visualization of c-met and c-kit infused cellsPancreata of c-met and c-kit injected STZ-treated or non-injected/STZ-treated rats were sectioned at 4 m and stored at -80ºC until use. Tissue cryosections were air dried and then xed in acetone for 10 minutes, followed by air drying for 30 minutes. Then the sections were incubated with 4’, 6-diamidino-2-phenylindole (DAPI) (1:2500) (Roche) for 10 minutes and mounted with Citi uor (Agar Scienti c). The whole procedure was performed at room temperature. Analysis was performed using the Leica DMRXA uorescent microscope and Leica Qwin Pro software.

Immunohistological staining for insulinPancreata of c-met or c-kit injected STZ-treated and non-injected STZ-treated rats and non-injected non-STZ-treated rats were sectioned at 4 m and stored at -80ºC. Tissue cryosections were air dried and then xed in Bouin for 10 minutes and followed by washing with PBS. Next, the sections were incubated with primary antibody anti-insulin (1:750) (Sigma-Aldrich, The Netherlands) for 60 minutes. After three times washing with PBS, The endogenous peroxidase activity was blocked by 30 minutes incubation in PBS with 30 % H2O2. Thereafter, the sections were incubated with normal goat serum for 30 minutes. Then the sections were incubated with secondary antibody goat-anti-mouse-IgG1-HRP (1:50) (DakoCytomation, Denmark) for 30 minutes. Peroxidase activity was visualised by applying 3-amino-9-ethyl-carbazole (Sigma, Steinheim, Germany). Background staining was performed with heamotoxilin-eosin staining for 3 minutes. The whole procedure was performed at room temperature. The sections were analysed using Leica DMLB light microscope (including Leica DC 300 camera).

RESULTS

As shown in gure 1A, both the infusion of c-kit and c-met positive cells induced a decrease in the blood glucose levels at 4 days after infusion of 400.000 cells. In this graph, we show delta values, due to the large variation in the glycaemic levels after STZ treatment. Figures 1B and 1C show the individual glycaemic levels of the animals. We found as expected a more profound effect on the glucose levels in rats with a high glucose level at the day of injection of the cells. Also in the animals with a less pronounced increase in glycaemia we found a decrease in

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glycaemic values after infusion of c-met and c-kit cells, but these effects were less pronounced. This should not be interpreted as a suggestion that the infusion did not contribute to regeneration as we found effects in immunocytochemistry (vide infra). When compared with the rats that received a sham-surgery the difference in basal glycemic levels did not show such clear improvement. In addition, we observed that neither the control groups nor the infused rats show a change in body weight (data not shown).

C-met and c-kit infused cells in the pancreasThe uorescent PKH26 labelling allowed us to study the accumulation of the infused cells in the diabetic pancreas. We found large amounts of the infused c-met and c-kit positive cells in the pancreas, even at 4 days after injection of the cells via the pancreatic artery (Figure 2A). In c-met infused pancreata, PKH26 uorescent cells were found scattered around in the pancreas (Figure 2B), while also c-met injected cells were found in the islets of Langerhans (Figure 2C). This was quite similar in the STZ-treated c-kit-infused pancreas. Also here we found PKH26 uorescent cells scattered through the pancreas (Figure 2D), while also c-kit positive cells were found in the islets of Langerhans (Figure 2E).

Insulin staining Since the above mentioned effects may result in increased amounts of insulin in the pancreas we stained slices of the pancreas for insulin. Figure 3 shows a comparison between control non-injected-non-STZ treated animals, sham-treated STZ rats, c-kit, and c-met treated animals. We did not observe any difference in intensity of staining.


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Figure 1: Glucose concentrations of c-kit and c-met infused rats before injection of streptozotocin (day -2), 2 days after injection of streptozotocin (i.e. just before injection of c-kit and c-met positive cells; day 0) and 4 days after the injection of c-met or c-kit positive cells (day 4) (A). The delta glucose values. (B and C) Absolute individual glucose values. The delta gure shows that both the infusion of c-kit and c-met positive cells induced a decrease in the blood glucose levels at 4 days after injection. The individual glucose values showed that 3 out of 5 c-kit- and 2 out of 5 c-met infused rats showed a decrease in the blood glucose levels at 4 days after injection of 400.000 c-kit or c-met positive cells (B and C). On the day of injection animals that received an injection of c-kit positive cells had a mean glucose level of 10,56 ± 0,88 mM. After 4 days the mean glucose level was decreased to 7,94 ± 1,83 mM. Animals receiving an injection with c-met positive cells had a mean glucose level of 11,38 ± 1,53 mM. After 4 days the mean glucose level was decreased to 10,66 ± 1,23 mM. Some animals that received a sham-surgery showed some differences in basal glucose levels.


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Figure 2: Photomicrographs of pancreata retrieved from c-met and c-kit infused cells in the pancreas. The red staining is the uorescent staining of PKH, with which the c-met and c-kit positive cells were stained. The staining represents the c-met or c-kit infused cells. Large amounts of the infused c-met and c-kit positive cells in the pancreas were observed at 4 days after injection. Figure A shows a representative of the pancreas of sham operated animals (A). In c-met infused pancreata, the cells were found scattered around in the pancreas (B) and they were also were found in the islets of Langerhans (C). Like the c-met infused pancreas, c-kit positive cells were also scattered through the pancreas (D) and were found in the islets of Langerhans (E). Original magni cation 200X

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Figure 3: Representative photomicrographs of the non-injected-non-STZ treated rat pancreata and STZ treated rat pancreata after immunostaining for insulin. No difference in intensity of insulin staining was observed. Original magni cation 200X

DISCUSSION

Our data suggests that infused, c-met and c-kit carrying cells isolated from the neonatal rat pancreas may contribute to regeneration of the damaged endocrine pancreas. This provides an interesting perspective for the possible future use of these cells in the therapeutical induction of regeneration of the endocrine pancreas. Since the present study is only a pilot experiment, we realise that for a proper interpretation of the data a number of questions still needs to be answered. Our study demonstrates a number of interesting observations that indicate the involvement of cells carrying c-met or c-kit in the regeneration and ontology of the endocrine pancreas. First, we show that after intravenous infusion a large group of cells home back to the damaged pancreas. Obviously the damaged pancreas has a chemo-attractive potential for at least a portion of the c-kit and c-met carrying cells.


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Secondly, a striking observation was that we sometimes found accumulation of the cells in structures that had many characteristics of so-called buds. These buds are found in the developing and regenerating pancreas1. Small groups of ductal epithelial cells that have recently been identi ed as cells hav

university of groningen embryogenesis and neogenesis of the endocrine pancreas ... · 2016. 3....

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