Development of MAPC derived induced endodermal progenitors Sambathkumar, Rangarajan
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Sambathkumar, R. (2017). Development of MAPC derived induced endodermal progenitors: Generation of pancreatic beta cells and hepatocytes. University of Groningen.
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Chapter 1
General Introduction and Background
General Introduction and background 1.1. Pancreas structure and function
The pancreas is a glandular digestive and endocrine organ. It is about 15 cm long and located across the back of the upper left abdomen, behind the stomach. The head of the pancreas, on the right side of the abdomen, is connected to the duodenum via the pancreatic duct. The tail of the pancreas extends to the left side of the body abutting the spleen. The pancreas has both exocrine and endocrine functions. The exocrine pancreas comprises of acinar cells that produce multiple digestive enzymes such as proteases, lipases, amylases, and nucleases. These secretary cells surround the intercalated ductal cells. Acinar cells contain small granules of zymogens (inactive proenzymes). Once released in the duodenum, the zymogens are activated by enteropeptidases, which cleave the zymogens creating active enzymes, such as trypsin and chymotrypsin. A highly branched ductal epithelial network transports zymogens and bicarbonate ions into the intestine for digestion of food. The endocrine pancreas consists of five different cell types namely alpha (α), beta (β), delta (δ), pancreatic polypeptide (PPY) and epsilon (ε) cells, which produce glucagon (GCG), insulin (INS), somatostatin (SST), PPY, and ghrelin (GHRL), respectively. Endocrine cells exist in clusters of cells called “Islets of Langerhans”. β-‐cells form the core of the islet and are surrounded by the other endocrine cells. Within human islets, 60-‐80% of the cells are INS producing β-‐cells, 15-‐20% GCG-‐producing α-‐ cells, and 5-‐10% SST-‐producing δ-‐cells. Approximately 1 million islets of Langerhans are present in the pancreas and each islet contains 100-‐3000 cells [1](Fig.1). Endocrine islet clusters are scattered throughout the exocrine and ductal compartment of the pancreas. Endocrine cells are in close contact with endothelial cells [2]. β-‐cells produce INS when blood glucose levels increase after a meal. INS allows transport of blood glucose into the liver, muscle, and fat tissue. INS inhibits glycogenolysis and beta-‐oxidation of fatty acids in the liver, but increases glycogen storage, increases fatty acids and cholesterol biosynthesis. When blood glucose levels are low, e.g. during fasting, α-‐cells produce GCG, which promotes liver glycogenolysis and beta-‐ oxidation of fatty acids into glucose [3]. The other islet hormones, SST, PPY and
GHRL, regulate the endocrine alpha and β-‐cell function. SST is a neuroendocrine hormone that regulates secretion of growth hormone and thyroid stimulating hormone. It may acts as a neurotransmitter in the nervous system. In the pancreas, SST inhibits the secretion of GCG from α-‐cells and INS from β-‐cells during excessive hormone production. In the gastrointestinal tract, it inhibits the secretion of gastrointestinal hormones including gastrin and secretin. Excessive SST levels can be found in patients with an endocrine tumor, a “somatostatinoma” [4]. PPY, a 36 amino acids polypeptide, is induced upon exercise, fasting and acute hypoglycemia [5]. The vagal nerve is a major stimulator of PPY secretion. PPY secretion is absent in obese children with Prader-‐Willi syndrome [6]. Finally, GHRL is produced by ε-‐cells. GHRL is an appetite-‐promoting peptide. This hormone inhibits INS secretion and stimulates GCG production cells during fasting and hypoglycemia [7].
Figure 1: The Pancreas structure contains exocrine and endocrine cells a) the adult mature pancreas located next to duodenum and most anterior part of small intestine. b) The function of exocrine cells – (acinar cells) is to supply digestive enzymes, which are transported to the intestine via pancreatic ductal cells. c) The endocrine pancreas consists of ‘’Islets of Langerhans’’. Each islet cluster consists of five hormone-‐producing cells present: α-‐, β-‐, δ-‐ and pancreatic polypeptide (PP) cells. α-‐cells (red) secrete glucagon and make up 15–20% of the endocrine pancreas. β-‐cells (green) secrete insulin and make up 60–80% of the endocrine pancreas. δ-‐cells (yellow) secrete somatostatin and make up 5– 10% of the endocrine pancreas, whereas PP cells (blue) secrete PP and make up less than 2% and epsilon (ε) cells occupy less than 1% of the endocrine pancreas (adapted from Helena Edlund et al., Nature Review Genetics, 2002)[3]. (Figure and Legend reproduced with permission of the copyright holder, Nature Publishing Group).
1.1.1. Diabetes mellitus: types, signs, symptoms and complications
Diabetes mellitus (DM) is a chronic disease characterized by elevated levels of blood glucose. The International Diabetes Federation (IDF) estimated that 387 million people were affected with DM worldwide, in 2014, and 55 million people in Europe.
In 2035, the number of people with DM is predicted to reach > 592 million patients [8]. Generally, DM is divided into two major types, Type 1 and Type 2 diabetes. Type 1 diabetes (T1D) is caused by an autoimmune attack against β-‐cells. Once the islet mass decreases to 20% of normal, patients become insulin dependent and need to have insulin injections to control blood glucose levels. T1D is more prevalent in children or young adults [9]. Pathologically, the islets T1D display insulitis, i.e. inflammatory cell infiltration resulting in β-‐cell destruction. CD8-‐T cells are found predominantly in islets of T1D patients, with as well as macrophages, CD4-‐T cells, B-‐ lymphocytes, plasma cells, forkhead box P3 (FOXP3) regulatory T cells, and natural killer cells. The result is necrosis and apoptosis of β-‐cells and reduced insulin secretion [10].
Type-‐2 diabetes (T2D) is also termed non-‐insulin dependent DM or adult onset DM. T2D is characterized by insulin resistance, due to decreased sensitivity of liver, muscle and fat cells to insulin, leading to decreased glucose uptake. This results in hyperglycemia due to relative insulin deficiency. At later stages the persistent hyperglycemia can cause loss of the β-‐cell mass. People with T2D initially manage with exercise and diet. However, in later stages, once the β-‐cell mass decreases, they may become insulin dependent.
Other forms of diabetes occur during pregnancy, termed gestational diabetes (GDM), which is found in 1/25 pregnancies and can be associated with complications for both the mother and baby. High blood glucose concentrations can damage the fetal organs, and as babies are commonly overweight, this can cause problems with child delivery. Gestational diabetes usually disappears after pregnancy [11]. Other, rare forms of diabetes are listed in table-‐1.
Signs and symptoms of DM include frequent urination, excessive thirst, increased hunger, blurred vision, and weight loss. These signs and symptoms are more sudden and dramatic in T1D but in the T2D they are often very mild or absent [11]. People with DM have an increased risk of developing a number of serious health complications, including infections, cardiovascular diseases (cardiac ischemia, stroke,
peripheral vascular disease), diabetic retinopathy, kidney failure, and peripheral nerve disease (PND). Maintaining blood glucose levels, blood pressure, and cholesterol as close to normal as possible can prevent diabetes complications. Normal fasting blood glucose concentrations are 70-‐99mg/dL and after a meal they range between 120-‐140mg/dL. The diagnosis of DM is made when fasting blood glucose are above 100mg/dL to 125mg/dL and post meal higher than 140mg/dL to 200mg/dL. The glycated haemoglobin-‐A1C (HA1C) test is a standard biomarker used for diagnosis of chronic hyperglycemia, as it reflects average blood glucose levels over a 2-‐3 month period of time and correlates with microvascular and to lesser extent macrovascular complications. HA1C value ≥ 6.5% is the threshold for diagnosis for DM. This test is an important factor in the management of patients with diabetes [11].
Table-‐1 Etiologic classification of DM [11]. S.no Types of diabetes Cause and Subtypes
1 Type 1 diabetes β-‐cell destruction, leads to insulin deficiency
a) immune-‐mediated
b) Idiopathic
2 Type 2 diabetes Insulin resistance with relative insulin deficiency to secretory defect
3 Gestational diabetes 4 Genetic defects β-‐cell
function a)Maturity Onset Diabetes of the Young (MODY)-‐3:(chromosome-‐12, hepatocyte nuclear factor-‐1 alpha (HNF-‐ 1α))
b) MODY-‐1:(chromosome 20, hepatocyte nuclear factor-‐4 alpha (HNF-‐4α))
c) MODY-‐2: (chromosome 7, glucokinase-‐(GCK))
d) Other rare forms of MODY-‐4 (chromosome 13, insulin promoter factor-‐1 (IPF1)), MODY-‐6 (Chromosome 2, Neuronal differentiation 1 (NEUROD1), MODY-‐7 (chromosome 9, carboxyl ester lipase-‐(CEL))
e)Transient neonatal diabetes (TND)-‐ zinc finger protein associated with apoptosis and cell cycle arrest (ZAC)/ imprinted in hydatidiform mole (HYAMI) imprinting defect
f) Permanent neonatal diabetes-‐ the ATP-‐sensitive K+ channel KCNJ1 gene encoding kIR6.2 of β-‐cell KATP channel
g) Mitochondrial deoxyribonucleic acid (DNA),
h) others-‐Mutation on insulin gene.
5 Genetic defects in
insulin action Type A insulin resistance, Leprechaunism, Rabson-‐Mendenhall syndrome, Lipoatropic diabetes , others
6 Endocrinopathies Several growth hormones like cortisol, glucagon, epinephrine antagonize insulin action. Acromegaly, Cushing’s syndrome, Glucagonoma, Pheochromocytoma, hyperthyroidism, somatostainoma, Aldosteronoma, Others
7 Drug or chemical
induced Vacor, Pentamidine, Nicotinic acid, Glucocorticoids, thyroid hormone, Diazoxide, β-‐Adrenergic agonists, Thiazides, Dilantin, gamma-‐interferon , others
8 Infections Cogenital rubella, Cytomegalovirus, Others 9 Uncommon forms of
immune mediated diabetes
“Stiff-‐man”” syndrome b) Anti-‐insulin receptor antibodies c) Others
10 Other genetic syndromes associated with diabetes
Down syndrome, Klinefelter syndrome, Turner syndrome, Wolfram syndrome, Friedreich ataxia, Huntington chorea, Laurence-‐Moon-‐Biedl syndrome, Myotonic dystrophy, Porphyria, Prader-‐Willi sundrome, Others
11 Diseases of the
exocrine pancreas Pancreatitis, Trauma/Pancreatectomy, Neoplasia, Cystic fibrosis, Hemochromatosis, Fibracalculous pancreatopathy, Others
1.1.2. Current treatment for diabetes mellitus
The discovery of insulin in 1921-‐22 was a major breakthrough in the treatment of T1D. However, multiple daily insulin injections do not provide perfect metabolic regulation. This has been significantly improved over the last years, thanks to the development of insulin pumps combined with continuous glucose monitoring, with computer algorithms or integrated closed loop systems that can more finely regulate insulin administration in response to blood glucose levels [12, 13]. Nevertheless, despite these advances, complications of hypo-‐ and hyperglycemia eventually ensue. T1D management may also include the use of insulin analogues (Incretins, Glucagon-‐ like peptide-‐1 (GLP1), and Leptin) and pramlintide or amylin (a 37-‐residue peptide hormone that delays gastric emptying, inhibits glucagon secretion; averting postprandial increases in blood glucose levels).
For T2D diabetes, before exhaustion of the β-‐cell pool, patients can be treated with Metformin, which augments insulin release; 2-‐Sulphonylureas, which increases insulin sensitivity; Bromocriptine, which antagonizes dopamine D2 and serotonin receptors; GLP1 analogues, Alpha-‐glucosidae inhibitors, Dipeptidyl peptidase 4 (DPP4) inhibitors; or Sodium dependent glucose cotransporter 2 (SGLT2) inhibitors [10, 14].
The most physiological method of achieving normoglycemia without the risk of hypoglycemia in T1D patients is to restore the β-‐cell pool. This can be achieved by whole-‐pancreas transplantation. This improves the metabolic control of blood glucose without the need of exogenous insulin and can lead to long-‐term insulin independence [15]. The major limitations associated with this therapy are the shortage of organs, the invasiveness, toxicity of immunosuppression regimens, risk of immunorejection, and nephrotoxic side effects of the immunosuppressive drugs [16]. Also the procedure is associated with major surgery and high morbidity. For that reason transplantation of only the islets is preferred. This can occur via the Edmonton protocol developed by the Shapiro group [17, 18]. This approach is less invasive as islets retrieved from donor pancreata can be infused into portal system, where they survive long-‐term in the liver sinusoids. Long-‐term insulin independence was achieved following islet transplantation in selected T1D recipients. However to reach sustained metabolic control for one year, at least 2 million β-‐cells/kg of body weight are needed, which requires 2-‐3 donor pancreata. After 5 years, only 20% of transplanted patients maintained a functioning graft. One drawback remains the loss of high amounts of islets (up to 80%) in the immediate period after transplantation due to the instant blood mediated reaction but also graft rejection and recurrent autoimmunity are an issue [17-‐19]. To overcome the problem of graft rejection and recurrent autoimmunity, methods have been developed for immunoisolation of β-‐ cells, by encapsulation in a biomaterial that can protect the graft from immune attacks [20]. However, this approach does not protect the graft from cytokine induced β-‐cell stress [21-‐27]. Despite this periods of graft survival up to years have been reported. This technology of encapsulation also holds the promise of the use of alternative sources for islets such as the use of xenogeneic sources or stem cells to alleviate the shortage of human islets [28].
1.1.3. Rationale for creation of β-‐cells from stem cells
The limited number of available donor organs as well as the immunological issues restricts current treatments such as whole pancreas or islet transplantation for T1D. An alternative source for human cadaveric islets would be to generate INS-‐producing β-‐cells from stem cells. Over the last decade generation of functional pancreatic β-‐
cells from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) has become possible, using methods mimicking in vivo pancreatic development [29-‐32]. However, differentiation from adult human bone marrow (BM) mesenchymal stem cells and human MAPCs is not possible. However, the low risk for tumorigenesis associated with adult stem cells (ASCs) make them a favorable choice. To extend the use of ASCs, many groups are evaluating whether it is possible to extend their tissue restricted differentiation ability to also generate β-‐cells.
1.2 Liver structure and function
The liver is the second largest organ in the human body, located in the upper right portion of the abdominal cavity, beneath the diaphragm. The liver has multiple functions including metabolization of glucose, lipids, proteins and amino acids, and detoxification of xenobiotics such as drugs, alcohol and toxins as well as urea production. The liver also produces and secretes bile, and plasma proteins, such as clotting factors and albumin and stores vitamins A, D, E, K and B12 [33]. The liver is composed of parenchymal and non-‐parenchymal cells. Parenchymal cells are represented by the hepatocytes and the non-‐parenchymal cells by biliary epithelial cells (BEC) or cholangiocytes, kupffer cells (KCs), liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSCs), and pit cells (intrahepatic lymphocytes or natural killer cells). The Internal structure of the liver is made up of around 100,000 small hexagonal functional units known as lobules. Each lobule consists of a central vein, which is surrounded by 6 hepatic portal veins, combined with a hepatic artery and a hepatic bile duct at each of the six corners of the lobule, known as the portal triad (Fig.2). The portal vein and hepatic artery supply blood to each lobule. The portal vein receives partially deoxygenated nutrient rich blood from the stomach, duodenum, gall bladder, pancreas, spleen and small intestine. The hepatic artery receives oxygenated blood from the dorsal aorta. These blood vessels are connected by a series of capillary like tubes, called sinusoids, which extend from the portal veins and arteries to meet in the central vein from which nutrients are taken up by the hepatocytes and xenobiotics are detoxified. The blood leaving the liver tissue collects into the hepatic veins that lead to the vena cava and returns to the heart. The liver cell plate consists of 15-‐25 hepatocytes organized alongside a sinusoidal
capillary from the portal triad to the central vein [34, 35]. Bile secreted by the hepatocytes is collected in the gall bladder, and secreted in the duodenum for digestion of fats. Putative hepatic progenitors cells (HPCs) reside in the Canals of Herring present between the peripheral branches of bile duct and hepatocytes. They are considered a source of hepatic progenitors that, upon severe (>80%) damage of the liver, can differentiate into hepatoblasts and subsequently into hepatocyte or cholongiocytes, [34, 36-‐38]. The hepatic progenitors cell niche consists of LSECs,
KCs, HSCs, pit cells, and other inflammatory cells that produce hormones and growth factors that induce HPC proliferation and differentiation.
Figure 2: Schematic overview of liver lobule structure a) Structure of a portion of liver lobule[35] b) the portal triad includes the portal vein, hepatic artery, and bile ducts. Blood from the portal vein and the hepatic artery flows toward the central vein between the hepatocytes through the sinusoids surrounded by fenestrated liver sinusoidal endothelial cells (LSECs). Bile produced by hepatocytes is collected into bile ducts via the bile canaliculi. Kupffer cells, resident macrophages of the liver, are located at the luminal side of sinusoids, while hepatic stellate cells (HSCs) are positioned in close proximity to LSECs at the ‘‘space of Disse,’’ a location between hepatocytes and a sinusoid. The canal of Herring is the junctional region between hepatocytes and bile ducts (Adapted from Karim Si-‐Tayeb et al. Developmental Cell, 2010 [3] and Atsushi Miyajima et al. Cell Stem Cell, 2014)[34] (Figure and legend reproduced with permission of the copyright holder, Elsevier).
1.2.1 Parenchymal liver cells: Hepatocytes
Hepatocytes are polarized parenchymal epithelial cells with cuboidal or polygonal shape, which form the liver cell plate. They represent 60% of all liver cells and 80% of the liver volume. They regulate most of the biochemical and metabolic functions of the liver. Most hepatocytes have a single nucleus, but binucleated cells are common. Hepatocytes contain large numbers of mitochondria, lysosomes and peroxisomes for metabolic and detoxification functions. Approximately 15% of the hepatocyte volume consists of smooth and rough endoplasmic reticulum (SER/RER). K.
Jungermann et al., demonstrated that the function of hepatocytes depends on their position along the periportal to central vein axis, a concept named liver zonation [39]. Zone 1 (periportal (PP) region) comprises 6 to 8 hepatocytes where oxygenated blood enters. Consequently, periportal hepatocytes display oxidative metabolism (carbohydrates, lipids and aminoacids, fatty acids), gluconeogenesis and glycogen synthesis from lactate or dietary products, urea synthesis, cholesterol and lipid biosynthesis, and albumin and blood clotting factors production. Zone 3 (centrilobular or perivenous (PV) region) comprises of 2-‐3 hepatocytes located around the central veins, where oxygenation is poor. Consequently, perivenous hepatocytes display different functions than periportal hepatocytes, including glycolysis, lipogenesis and ketogenesis, glutamine synthesis, bile acid synthesis, and xenobiotic metabolism by cytochrome p450 enzymes as well as monooxygenases (Fig. 3). Zone 2 (midlobular region) comprises of 6-‐10 hepatocytes located between zone 1 and zone 3 that have mixed functions [40, 41]. Maturation of hepatocytes begins from the periportal zone and moves across the liver plate towards the perivenous zone. Regardless of their position, all hepatocytes can self-‐renew for liver homeostasis and to repopulate the pool of cells upon minor hepatocyte injury or loss [42].
Figure 3: The structure and function of a zonated liver lobule. a) The liver cell plate blood circulation indicated in red moves from the PP region to the PV region, and bile flow indicated in green moves from the PV region to the PP, in opposite direction to the blood. Oxygen and hormone concentrations decrease from the PP to the PV area (Adapted from book chapter-‐2, Liver Zonation, Sabine colnet & Christine Perret, 2011)[41]. b) Liver functions are zonated: PP hepatocytes display β-‐oxidation of fatty acids, and gluconeogenesis while glycolysis, lipogenesis, ketogenesis, triglyceride synthesis are typical for PV hepatocytes (Adapted from Birchmeier et al, Nature cell biology, 2016)[43]. (Figure and legend reproduced with permission of the copyright holder, Springer publishing group and Nature publishing group).
However, a recent study by Wang et al., using lineage tracing for Wnt and Axin-‐2 in mice suggested that proliferating and self-‐renewing hepatocytes can be found in the PV region of the liver lobule. Perivenous hepatocytes are diploid and express the hepatic stem cell marker Tbx3, whereas mature hepatocytes are polyploid and are Tbx3 negative. They further demonstrated that central vein endothelial cells secrete Wnt proteins that provide the niche to maintain the PV cells proliferative, whose descendants can replenish 40% liver cell mass in normal conditions (Fig.4)[44].
Figure 4: Perivenous hepatocytes can contribute to everyday liver regeneration. The endothelial cell lining of the central vein produces Wnt signals that activate the expression of Wnt responsive genes in adjacent PV hepatocytes. Wang et al. [44]. reported that these signals stimulate the proliferation of PV hepatocytes. The cells give rise to descendants that reside beyond the reach of Wnt signals, and that replicate more slowly than their parents (some of the descendants have more than one nucleus).
In this way, perivenous hepatocytes contribute to the maintenance of liver mass. (Adapted from Zaret
et al. Nature, 2015 [45]). (Figure and Legend reproduced with permission of the copyright holder, Nature Publishing Group).
1.2.2 Non-‐parenchymal liver cells
The non-‐parenchymal fraction represents 40% of the total number of liver cells and occupies 6.5% of the liver volume, while the remaining 13.5% of the liver volume consists of vascular and ductular networks. The non-‐parenchymal cells include bile duct epithelial cells or cholangiocytes, LSECs, HSCs, KCs and Pit cells. These cells play important roles in the regulation of hepatocyte proliferation and function, including growth factor production, transport and metabolism, scavenging of foreign material, storage of extracellular matrix, vitamins and fats, and inflammation responses [46].
1.2.3 Biliary Epithelial ductal tree or cholangiocytes
Tight junctions formed between hepatocytes create microscopic bile canaliculi. Several bile canaliculi combine to form larger bile ducts forming the left and right intrahepatic bile duct tree found throughout the liver. This bile duct trees collect bile from the different liver lobes and join together to form the extra hepatic bile ducts consisting of the common hepatic ducts, gall bladder, cystic duct, bile duct, and the common hepato-‐pancreatic duct, which reaches the duodenum. Most of the bile produced by the liver is stored in the gall bladder, until it is needed for digestion. The bile duct trees are formed by cholangiocytes [47]. Intrahepatic bile ducts consist of small and large cholangiocytes. Small cholongiocytes are considered committed biliary progenitors. They have a cuboidal morphology, a relatively high nucleus to cytoplasm ratio, and express more cell proliferation genes. By contrast, large cholongiocytes have a columnar morphology, a low nucleus to cytoplasm ratio, abundant Golgi and RER, and express functional mature genes. A large number of perbilliary (PB) glands are found within the duct wall of intra-‐ and extra-‐hepatic, cystic, and the common hepato-‐pancreatic ducts (Fig.5). They contain multipotent stem and progenitor cells in humans of all ages [48], which self-‐replicate and can differentiate into hepatocytes and cholangiocytes or pancreatic islets depending on the niche microenvironment [47].
Figure 5: Schematic overview of stem cell niches in the intrahepatic and extrahepatic biliary tree. Stem cell niches within the liver are located in the Canals of Herring (red circles). Hepatic stem cells are precursors to hepatoblasts, which are presumed to be the transit amplifying cells that first give rise to committed progenitors, and then to hepatocytes and cholongiocytes. Perbilliary glands contain stem cell niches within the biliary tree (blue circles). These glands are along the biliary tree from the hepatopancreatic common duct near the duodenum up to the septal ducts. High numbers of peribiliary glands occur in the cystic duct, hilum and periampular regions. The progenitor and/or stem cells within peribiliary glands probably act as sources for cell turnover of the entire biliary tree distal to the interlobular bile ducts. (Adapted from Vincenzo Cardinale et al., Nature Reviews Gastroenterology and Hepatology, 2012)[47] (Figure and Legend reproduced with permission of the copyright holder, Nature Publishing Group).
1.2.4. Liver diseases
Liver diseases can be inherited or caused by a variety of factors, such as viruses, drugs or alcohol. They are extremely costly in terms of human suffering, premature loss of productivity and affect millions of people worldwide. In 2013, liver cirrhosis accounted for 1.8% of all deaths (170,000 deaths per year) and liver cancer accounted for around 47, 000 deaths in Europe, according to the World Health Organization (WHO). Approximately, 29 million people in the European union suffer from chronic liver disease [49].
1.2.4.a. Metabolic liver diseases: Metabolic diseases are characterized by a deficiency in a hepatic enzyme or protein leading to hepatic and/or extrahepatic diseases such as: Crigler Najjar syndrome type I (CN1) (lack of functional uridine diphosphate glucuronosyltransferase (UDPGFT) enzyme); urea cycle disorders (deficiency in one of the six enzymes of the urea cycle); familial hypercholesterolaemia (absence of the low-‐ density lipoprotein receptor (LDLR)); α1-‐antitrypsin (A1AT) deficiency (caused by autosomal recessive disorder caused by
mutations in the A1AT encoding SERPINA1 gene; retention of A1AT misfolded polymers within the hepatic ER cause hepatic dysfunction); familial transthyretin amyloidosis (FTA), caused by mutations in the transthyretin-‐encoding gene (TTR) leading to secretion of monomeric misfolded TTR proteins by the liver and formation of extracellular fibrils as amyloid in target organs in the brain and heart); Wilson’s Disease (WD) (caused by mutations in the of ATP7B gene, an ATPase expressed in hepatocytes that aids in excretion of copper into the bile and blood stream); glycogen storage disease type I (GSD1a) (deficiency of the hepatic enzymes glucose-‐ 6-‐phosphatase or the glucose-‐6-‐phosphate transporter due to rare autosomal disorder caused by mutations in G6PC encoded gene); infantile Refsum disease (reduced peroxisome function); coagulation factor deficiencies, like hemophilia A (lack of factor VIII) and hemophilia B (lack of factor IX); progressive intrahepatic cholestasis; phenylketonuria (deficiency of enzyme phenylalanine hydroxylase (PAH); tyrosinaemia (deficiency of the enzyme fumarylacetoacetate hydrolase (FAH)); acute intermittent porphyria (deficiency of the hepatic haemenzyme porphobilinogen (PBG) deaminase; maple syrup urine disease (accumulation of branched chain amino acids (BCAAs) because of a deficiency of the enzyme branched chain keto acid dehydrogenases (BCKDH))[50-‐52]. All these diseases could be treated by transplantation of hepatocytes. In fact, after several studies in mouse models, transplantation of adult hepatocytes has been used relatively successfully in these settings [50, 52, 53].
1.2.4.b. Acute Liver disease (ALD): is characterized by rapid decline in hepatic synthetic function (loss of function of 80-‐90% liver cells), with significant risk of mortality. Nowadays, treatments are largely supportive. An alternative is the use of bioartificial liver devices (BAL), in which the patients blood or plasma is perfused through an extracorporeal bioreactor filled with hepatocytes [54], or isolated human hepatocyte transplantation[55]. Drug induced liver injury (DILI) is the most common reason for pharmaceutical drug withdrawal from the market. DILI accounts for 50% of acute liver failure [49].
1.2.4.c. Chronic Liver Disease (CLD): CLD is caused by viral hepatitis, alcoholic liver disease or non-‐alcoholic fatty liver disease (NAFLD). These cause hepatic injury, which when sustained for long time, leads to progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC). In the chronic stage, there is infiltration of polymorphonuclear leukocytes in the liver, with focal or zonal necrosis, destruction of hepatocytes and architectural disarray. The liver is susceptible to multiple viral infections, including Yellow fever virus, Dengue virus, and hepatitis virus A -‐ E, of which B (HBV) and C (HCV) are major causes for CLDs and HCC. Alcoholic liver disease occurs after long periods of alcohol abuse, whereas and Non-‐Alcoholic liver diseases (NALD) is a term that includes several phenotypes ranging from simple steatosis (deposition of fat in the hepatocytes) to non-‐alcoholic steatohepatitis, progressive fibrosis, cirrhosis and HCC. HCC accounts for 70-‐90% of primary liver cancers. HCC is rapidly fatal without any treatment with 5-‐year survival rates of around 5% [49, 56, 57].
1.2.4.d. Liver regeneration and transplantation
It is well known that the liver has a high regenerative capacity. The first response, if damage is limited, is re-‐entry of quiescent hepatocytes into the cell cycle, and replacement of the damaged and lost hepatocytes. When hepatocyte loss is more profound or when hepatocyte proliferation is impaired due to infection, HPCs present in the “canals of Herring”, biliary tree stem cells in the perbiliary glands of the intra-‐ and extra-‐hepatic biliary ducts, or cholongiocytes (SOX9+, keratin-‐19 (KRT19), epithelial cell adhesion molecule (EPCAM), CD133-‐prominin positive cells) are activated and differentiate into hepatocytes and cholongiocytes [34, 47, 58]. When both repair systems are exhausted, acute and/or chronic liver failure ensues. The only cure is orthotropic liver transplantation of which >5,500 are performed in Europe per year, with a survival rate of 83% after one year. Major imitations are shortage of organs, adverse effects due to long-‐term immunosuppression, and graft rejection.
1.2.5. Use of hepatocytes in the pharmaceutical industry
As discussed above, DILI accounts for 50% of the acute liver failure. One reason for DILI to occur is that preclinical drug testing uses animal models that do not fully mimic the physiology and function of the human liver, due to species difference in, for instance, drug metabolization gene expression. Therefore, there is a need for mature human hepatocytes for predicting drug toxicity and bioavailability. Currently, freshly isolated primary hepatocytes (PHH) or cryopreserved PHHs are used for testing drug metabolism and toxicity, because they express the complete set of phase 1 and phase 2 metabolization enzymes (e.g cytochrome P450 (CYPs)) and drug transporters (e.g. organic anionin polypepetide 1B1 (OATPB1), Na+-‐ taurocholate co-‐ transporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance protein -‐2 (MRP-‐2) involved in hepatic drug clearance [59, 60].
However, PHH de-‐differentiate very fast in culture with loss of metabolic enzymes and transporters. Moreover, there is a scarcity of healthy donor organs for use in the pharmaceutical industry and there is considerable variability between donors [61-‐ 64]. Alternatives for PHH are liver tumor-‐derived or immortalized cells, such as the HepG2 and HuH7.5 cell lines, which do, however, have minimal to no drug metabolization and detoxification ability. Alternatively, the Fa2N-‐4 (derived from PHH immortalized by transfection with SV-‐40 larger T antigen) and HepaRG cells possess substantially higher drug metabolization and transporter functions, including CYP1A2, CYP2B6, CYP2C9, CYP2E1 and CYP3A4, constitutive androstane receptor
(CAR), pregnane X receptor (PXR) and aryl hydrocarbon receptor (AHR) at levels similar to PHH. However, these cell lines are derived from only a single donor and are transformed [36, 65].
1.2.6. Rationale for creation of hepatocytes from stem cells
Due to the shortage of human healthy livers and the fast dedifferentiation of PHHs in culture, the number of patients that can be treated with hepatocyte or whole liver transplantation is limited, and reliable drug-‐screening models are not readily available for the pharmaceutical industry. To overcome this problem, generation
hepatocytes from stem cells is seen as an alternative method to generate hepatocytes for drug screening and cell based therapy applications. Many groups have developed protocols to differentiate human pluripotent stem cells (PSC), including embryonic stem cells (ESCs) to into hepatocyte like cells (HLC)[66-‐72]. Human PSCs-‐derived HLCs express hepatocyte marker genes and display some mature hepatocyte functions such as ALB secretion, urea synthesis, and glycogen storage but however, they still mimic a fetal phenotype compared to the PHH [73, 74]. Adult stem cells such as mesenchymal stem cells (MSCs) from bone marrow [75-‐ 81], wharton jelly [82, 83], umbilical cord [84, 85], amniotic fluid [86], adipose tissue[87-‐90], and multipotent adult progenitors (MAPCs)[91] do not robustly differentiate into HLCs. This demonstrates a continued need for the creation of fully functional hepatocytes suitable for transplantation and drug metabolization and toxicity studies.
1.3. Pancreas and Liver development
To use stem cells for the generation of specific cells, it is important to identify extrinsic factors that stimulate differentiation. Therefore, knowledge of the processes that operate during normal embryogenesis that regulate cell proliferation, differentiation and specialization is required as many signaling pathways play important roles during development are highly conserved. In this introduction I will discuss some important findings regarding endoderm development and hepatocyte and pancreas organogenesis.
1.3.1 Endoderm development
During the third week of human development or at E6.5 in mouse development, pluripotent epiblast cells undergo a series of gastrulation events, including epithelial to mesenchymal transition (EMT) and migration to form the primitive streak (PS) at the posterior region of the epiblast. PS formation is an essential step for gastrulation to occur correctly. When embryos fail to form a PS, gastrulation fails [92]. The PS is marked by expression of Mix paired-‐like Homeobox 1 (Mixl1), Eomesdermin (Eomes), LIM homeobox 1 (Lhx1), Brachury-‐T, Goosecoid (Gsc) and the Forkhead Box A2 (Foxa2) genes. In the process of differentiation, mesendoderm (ME) precursors migrate through the PS to create mesoderm in the middle and endoderm in the
outer layer of the embryo. Based on Nodal signaling in the embryo, the PS is patterned into anterior and posterior regions. A mouse epiblast fate map, created using intracellular tracer studies, showed that at E7.0-‐E7.5, anterior definitive endoderm (DE) arises from the most anterior primitive streak, expressing (Foxa2, Hematopoietically expressed homeobox (Hhex), Gsc, and Eomes), where nodal signaling is high [93]. SRY (sex determining region Y) related HMG (high mobility group)-‐Box 17 (Sox17) is required in the development of the posterior DE [94-‐96]. Their timing of expression correlates with their activities; Foxa2 is expressed first, while Sox17 is expressed slightly later, when posterior definitive endoderm emerges from the anteriormost primitive streak. The posterior primitive streak generates mesoderm expressing Even-‐Skipped Homeobox 1(Evx1) and mesoderm posterior
basic helix-‐loop-‐helix transcription factor 1 (Mesp1) [97, 98], as a result of lower
levels of nodal signalling. Initial cells that exit the PS give rise to anterior DE and axial mesoderm. The cells that exit later from the PS form the posterior DE. The primitive endoderm (PrE) gives rise to the extraembryonic endoderm, which later contributes to the yolk sac. The parietal endoderm cells grow with minimal cell-‐cell contact and are scattered on the inner surface of the trophoblast. They secrete copious amounts of basement membrane proteins to form the Reichert’s membrane in conjugation with the trophoblast cell layer. The PrE, in contact with extra embryonic ectoderm and epiblast differentiates into an epithelial cell layer called visceral endoderm (VE). VE cells covering the trophoblast have a columnar and cuboidal morphology and express the TF SRY-‐Box 7 (SOX7). VE cells covering the epiblast have a more epithelial like morphology and express alpha-‐fetoprotein (AFP)[99]. At the end of gastrulation, DE cells invade and replace the extraembryonic VE cells. However, there is evidence that some VE cells can be found in the DE layers and in the primitive gut in mouse [100]. At the end of gastrulation events, the DE sheet of cells surrounds the outer surface of mouse embryo [101, 102].
1.3.1.1 Molecular mechanisms underlying endoderm morphogenesis
During gastrulation, cell migration, cell adhesion and cytoskeletal dynamics are linked with endoderm formation and patterning. Nodal, fibroblast growth factor
(FGF), and Wingless-‐type MMTV integration site family member (Wnt) signaling is essential for the coordinated series of cell movements that drive ME morphogenesis. The dorsal ME elongates via polarized cell intercalations in a process known as convergent-‐extension, which is controlled by FGF and non-‐canonical Wnt/planar cell polarity (PCP) signaling. On the other hand, the anterior endoderm cells exhibit directional migration [103] controlled by Nodal signaling and are mediated by dynamic cell-‐cell adhesion and integrin-‐ extra cellular matrix (ECM) interactions [104, 105]. Further, ME migration is controlled by Mixl1, Eomes, Lim1, Foxa2 and GATA Binding-‐protein 4-‐6 (Gata4-‐6) [106-‐111]. Nodal also activates the C-‐X-‐C chemokine receptor type 4 (Cxcr4) [112] and the ligand stromal derived factor (Sdf1) [113, 114], which serves as chemoattractant for CXCR4 expressing endoderm cells. Another target of Nodal is Fibronectin-‐leucine rich transmembrane (Flrt3), which regulates cadherin-‐dependent cell adhesion and ME migration via the small guanosine triphosphate (GTPase) RAS-‐related Nuclear protein (Ran 1) [115]. Flrt3-‐/-‐ knock-‐out
(KO) mouse embryos have defects in DE migration [116, 117]. In the mouse gastrula, FGF, mitogen-‐activated protein kinase (MAP) kinase signaling, and Eomes are essential to downregulate E-‐cadherin in epiblast cells and allow them to undergo EMT and ingress through the primitive streak [108, 118, 119]. However, what regulates the subsequent migration of definitive endoderm and their incorporation into the visceral endoderm is less known [102] (Genes involved in endoderm formation are listed in table-‐2).
1.3.1.2 Endoderm patterning
At the end of gastrulation, the developing embryo consists of an inner germ layer of definitive or naïve endoderm cells. After 48 hours, the endoderm layer forms the primitive gut tube from which endodermal organ buds emerge. Endoderm patterning occurs by a series of growth factors signals from the adjacent mesoderm along the anterior-‐posterior (A-‐P) axis, into foregut, mid-‐ and hindgut domains and then, subsequently, into committed organ primordia.
The anterior foregut gives rise to the lungs, trachea, thyroid, esophagus, and thymus. The posterior foregut gives rise to the liver, biliary system, pancreas, stomach and
duodenum, while the midgut (MG)/hindgut (HG) give rise to the small and large intestine [102, 120](Fig.6).
Figure 6: Schematic overview and timeline of endoderm organ formation: a) The major events in endoderm organ formation are listed in order of development b) Images of mouse embryo at E7.5 (top), E8.5, and E9.5 of development, (endoderm regions shaded in yellow), A schematic illustration of a cross section of a E9.5 embryo illustrates the characteristic arrangement of the germ layers with the endoderm lining of gut tube (yellow), surrounded by MD (red), and ectoderm (blue). C) Schematic illustrations of endoderm cell lineage of the gastrointestinal tract, Fg; foregut, mg; midgut; and Hg; hindgut (Adapted from Aaron Zorn and James wells et al, Annual Reviews, 2009)[102]. (Figure and Legend reproduced with permission of the copyright holder, Annual Reviews).
The different gut tube domains can be identified by the expression of Hhex, SRY (Sex determining region Y) related HMG (high mobility group)-‐Box 2-‐(Sox2), and Foxa2 in the anterior half of the embryo, and Caudal type Homeobox 1(Cdx1), 2 (Cdx2) and 4 (Cdx4) found in the posterior half of the embryo [94]. During gastrulation, dynamic tissue movements result in the juxtaposition of the endoderm with different mesodermal tissues that secrete patterning factors [106, 121, 122](Fig.7). Mesodermal fibroblast growth factor 4 (FGF4), (Wnt)/β-‐Catenin, bone morphogenetic protein (BMP4) and Retinoic acid (RA) signaling promotes the expression of hindgut endoderm and represses the anterior foregut genes Hhex and Foxa2 [123-‐127].
