Paper reveiw: Converting human pluripotent stem cells into B-cells: recent advances and future challenges. Christopher N. Mayhew and James M. Wells

The main focus of this paper was to investigate the current research, challenges, and the future research needed to allow replacement of B-cells from human pluripotent stem cells (hPSC). This is a important topic because diabetes is a worldwide health problem affecting over 200 million individuals and is associated with several long term health problems. Type 1 diabetes mellitus results from the autoimmune destruction of the B-cells of the islets of Langerhans that produce insulin in the pancreas. Type 1 diabetes can be treated with insulin injections to control blood glucose levels but these injections can not fully compensate for B-cell loss. Thus replacement of B-cells from hPSC may provide a better solution for the treatment of diabetes.

Current Research:

Human embryonic stem cells (hESC) can be differentiated into glucose responsive B-like cells by incorporating signalling molecules required for B-cell development in vivo to the differentiation process.

4 stages for the development of B-cells from hESC
1. Endoderm Formation: The nodal signalling pathway controls endoderm formation in most vertebrate species. The nodal related protein activin can be applied to hESC to promote their differentiation into definitive endoderm.
2. Pancreas Specification: Definitive endoderm can produce a vast array of cell types . hESC derived endoderm can be differentiated into pancreatic progenitors by applying signalling molecules such as fibroblast growth factor (FGF), bone morphogenic protein (BMP), retonic acid, and hedgehog signalling molecules.
3. Endocrine Specification: Pancreatic progenitors can be promoted to adopt a endocrine cell fate by inhibiting notch signalling and preventing exocrine cell formation. Epidermal growth factor (EGF) can then be used to expand the endocrine progenitors.
derived
4. B-cell Maturation: Some pathways thought to be involved in B-cell maturation include the incretin signalling pathway mediated by glucagon-like peptide-1 (GLP1) and nerve growth factor (NGF). However B-cell maturation is not well understood so the promotion of hESCpancreatic endocrine cells into B-like cells has had limited success. Thus a better understanding of B-cell maturation is needed to more successfully guide hPSC into B-cells in vitro.

Challenges and Future Research:

The recombinant proteins needed to direct the differentiation of PSC are expensive to manufacture and thus so far scientists have been unable to produce a sufficient number of B-cells for human transplantation. Thus there is a need to develop a large amount of cost-effective small molecules to direct the PSC differentiation into B-cells. Recent studies screening chemical liberies have found compound such as IDE1 and IDE2 that promote hESC differentiation into definitive endoderm. In addition indolactum was found to promote the differentiation of definitive endoderm to pancreatic progenitors.
Teratomas have been found to be associated with PSC derived cell transplants. These teratomas are thought to be associated with undifferentiated hESC thus better cell-sorting techniques need to be applied to hESC cultures to reduce the chance of teratoma formation. Cell sorting techniques using immunocytochemistry and or immunohistochemstry have shown promise for future research. But all PSC will need to be tested for cancer forming cells in future research.
The transplantation site for PSC derived B-cells should ideally promote the long term survival of the grafted tissue while maximizing the patients safety. In current animal trials PSC derived B-cells are transplanted into the liver from the portal vein due to the fact that the majority of insulin is utilized in the liver. However research has shown that B-cell transplantation to this site has resulted in death of approximately half of the B-cells, thus there is a need to investigate other transplantation sites for B-cells.
Patients that receive hESC derived B-cells transplants will require life long immunosepressive drugs to prevent graft rejection. Thus research is needed to minimize or eliminate the use of immunospressive drugs in these patients. Research areas to combat the immune response to B-cell transplantation include the use CD3 specific antibodies; microencapsulation of grafted tissue; transplantation of cells to immuno privileged sites; and creating large banks of hESC and matching human leukocyte antigen (HLA) to recipient patients. In addition current research has shown that human somatic cells can be reprogrammed into a ESC like state. Induced PSC (iPSC) are ideal because they could be derived from patients thus generating patient specific B-cells for autologus transplant and combating the patients immune response.

Critique:

In a personal critique of this publication, Christopher N. Mayhew and James M. Wells provide a easy to read and understandable review of the use of human pluripotent stem cells to replace B-cells in diabetic patients. Although a more detailed description of the signalling pathways involved would have helped my understanding of the differentiation process of human pluripotent stem cells into B-cells the diagram provided in the paper worked well to illustrate the molecules involved in the process. The authors also did a great job of illustrating the problems associated with the transplantation of hESC derived B-like cells into diabetic patients and highlighting the research that is necessary to combat these problems and make this procedure possible in the future. I particularly find this paper of extreme interest since research involving the differentiation of hPSC can potentially be used to treat a vast array of human health problems, in addition to diabetes.

This paper can be located at http://www.ncbi.nlm.nih.gov/pubmed/19855279

The Pancreas

Development of the Pancreas

• The pancreas is derived from endoderm in the primitive gut. Two primordia (protrusions) from the primitive gut are precursors for the pancreas.
• The ventral primordia moves over to the dorsal side were the primordia fuse and form the pancreas.
  
• The liver, gall bladder, and bile duct also develop from the ventral primordia.
• The dorsal and ventral primordia grow and differentiate into different cell types.

• Interactions from gene products in the developing pancreas produce the first of the primative tubules, islet cells, acinar cells, and mature ducts. The blood vessels, lymphatics, and connective tissue develop from mesodermly derived mesenchyme within the developing pancreas.
• The ductal cells produce the definitive ductular and ductual epithelial cells that form the ducts. The acinar cells remain within the confines of the primitive ductuals. While the endocrine system cells from the primitive epithelial cells migrate into the mesenchyme and aggregate into groups, producing the islets of Langerhans.
  
• Mesodermly derived cells in the mesenchyme differentiate into blood vessel forming cells. These vessels form a network of arteries, capillaries, and veins which connects with and is supplied by the aorta. The network connects with the venous system and drains through the hepatic portal vein.
• The extensive nervous system from the nerve fiber cells (neural crest cells) from different parts of the general nervous system. These nerves form connections with the central nervous system as well as the enteric nervous system (Beger ET AL 2008).
  

Histology/cytology of the pancreas

• The normal pancreas is pink-tan to yellow color and measures 15-20cm in length and weighs 80-120g. It is a unpaired organ located at the left superior retroperitonium. The pancreas is basically a epithelial organ that includes both exocrine (acini and ducts) and endocrine elements (islets of Langerhans) (Mills ET AL 2007).
  

• The pancreas has a well developed lobular arrangement (microscopically the pancreas is arranged in 1-10mm lobules) of highly glandular cellular tissue.
  
• The pancreas is covered by a thin layer of peritoneum and is invested by a thin layer of fibroconnective tissue containing vessels and nerves that separate the lobules (Mill ET AL 2007).
  

Acinus

• 85% of the pancreas is composed of acinar cells which are the excretory portion of the gland
• The acinus has a single layer of polygonal cells surrounding a small central lumen. There are numerous different pathways by which the secretions of the acinus may reach the ductal system.

• Acinar cells contain

1. A large central nucleus
2. Lots of ER arranged in parallel stakes
3. apical granular eosinophilic cytoplasm with
4. lots of zygmogen granules
5. lots of secretory granules

• The secretory granules of the acinar cells fuse with the apical plasma membrane and expel their contents into the lumen.
• Adjacent acinar cells are joined by apical junction complexes zonula occludens and adherans and desmosomes (macula adherans) join the acinar cells around their lateral surfaces.
• Each acinus is surrounded by a continuous basement membrane (Mills ET AL 2007).
  

Ducts

• The ducts transport the acinar cell secretions to the duodenum. The ductal epithelium cells secrete water, chloride, and bicarbonate to buffer the acidity of the pancreatic juices and stabilize the pro enzymes until activation in the duodenum.

• The Ductal system has five portion

1. Centroacinar cells
2. interacleted ducts
3. intralobular ducts
4. interlobular ducts
5. main ducts

• The duct system starts with the centraciner cells, which are flat to cuboidal cells located in the middle of the acini. The lumen surrounding the acini and the centroacinar cells drains into the intercleted ducts (smallest) just outside the acini. These ducts fuse to form the intralobular ducts neither of these ducts have a significant collagenous matrix around them. Once the ducts leave the lobules they become surrounded by a thick rim of collagen they are now termed the intralobular ducts. The cells of the intralobuler and the interacleted ducts resemble centoacinar cells but are slightly different in their granule constituents, the transition of cells types can not be seen. The main pancreatic duct receives tributaries from the interlobular ducts, the lining epithelium of these ducts is flat except in the distal portion were papillae are found. The Cells of these larger ducts are low columnar with basal round nuclei, special stains are needed to see mucin in these ducts.
• Cells starting from the small interlobular ducts may have cilia that function to mix the pancreatic juices (Mills ET AL 2007).
  

Islets of Langerhans

• Endocrine component of the pancreas, comprises 1-2% of the glands in adults.
• There are over one million islets of Langerhans in the pancreas, they are found all over the pancreas but are more numerous in the tail.
• A thin layer of connective tissue separates the islets from the surrounding connective tissue, but they are not truly encapsulated.
• Two types of islets

1. Compact (90%)
2. Diffuse (restricted to the head of the gland)

• Each endocrine cell produces produces one specific kind of peptide

1. Beta cells: insulin
2. Alpha cells: glucagon
3. Delta cells: somatostatin
4. PP cells: pancreatic polypeptide

• There is also a small number of individual endocrine cells scattered among the acini (Mills et al 2007).
  

Connective Tissue

• In the normal adult pancreas there little connective tissue between the lobules and almost none around them. The acini are surrounded by a basement membrane but little other collagen exists.

Nerves

• The acini and ducts are innervated by bundles of unmeylinated nerves that travel through the intralobular connective tissue. The islets are innervated by both the sympathetic and the parasympathetic nerves.

Adipocytes

• There is 3-20% adipocyte cells in the pancreas depending on the age and the nutritional condition of the individual.

Goblet Cells

• May be found in the ductal epithelium especially in the main ducts (Mills ET AL 2007).
  

Functions of the pancreas

• The pancreas is a duel endocrine and exocrine gland with important functions in both the endocrine and digestive systems (Campbell ET AL 2005).
  

Digestive (exocrine) functions

• The pancreas produces several hydrolytic enzymes to aid in digestion and a alkaline solution that is rich in bicarbonate. The bicarbonate acts as a buffer offsetting the acidity of the stomach. The pancreatic enzymes include protease's (protein-digesting enzymes) (Campbell ET AL 2005) endopeptidase pro enzymes include trypsin, chymotrypsin, elastase, collagenase, exopeptidase, and carboxypeptidase A and B (Hill ET AL 2008). These protease's are secreted into the duodenum in the inactive form and activated once they are safely located in the extracellular space within the duodenum (Campbell ET AL 2005). In addition the pancreas also secretes amylase (starch or glycogen digestion) and lipase's (lipid digestion) into the midgut (Hill ET AL 2008).
  

Endocrine functions

• Pancreatic endocrine cells are clustered in the islets of Langerhans, that are embedded in the pancreatic exocrine tissue that secrete digestive enzymes.

• Endocrine cells include:

1. Beta cells that produce insulin a peptide that promotes the uptake and storage of nutrients by most cells.
2. Alpha cells that produce glucagon a peptide that maintains blood levels of nutrients after a meal and during stress.
3. Delta cells that produce somatostatin a peptide that inhibits digestion and absorption of nutrients by the gastrointestinal tract (Hill ET AL 2008).
   

• These hormones like all endocrine signals are secreted into the extracellular fluid and enter into the circulatory system.

Homeostatic regulation of cellular fuel:

• Insulin and glucagon are antagonistic hormones that regulate the concentration of glucose in the blood. The metabolic balance of cellular fuel in humans depends on maintaining a blood glucose level of approximately 90mg/100ml. When blood glucose levels exceed this level the pancreas secretes insulin into the blood. Insulin enhances the transport of glucose into the body cells and stimulates the muscle and the liver to store glucose as glycogen, thus lowering blood glucose levels. When blood glucose levels drop below set levels the pancreas secretes the hormone glucgon, which opposes the effect of insulin. Glucagon promoted the breakdown of glycogen in the liver and the release of glucose into the blood thus increasing blood glucose levels (Campbell, 2005).
  

Pathology of the pancreas

There are many pathological problems associated with the pancreas, I have just attempted to touch on some of the most known in this blog.

Developmental anomalies

• Annular or ring pancreas: very rare partial or complete circling of the second part of the duodenum by pancreatic tissue, commonly causes a duodenal obstruction.
• Pancreas divisium: failure of duct fusion between the pancreatic duct and the bile duct. this condition occurs in 5-10% of individuals and patients have a increased incidence of pancreatitis, abdominal pain, jaundice, and gallbladder cancer.
• Pancreatic heterotrophia: pancreatic tissue located outside of the normal anatomical position of the gland, most commonly found in the duodenum. This is easy to mistake as a carcinoma, though in some cases adenocarcinomas have arisen in pancreatic heterotrophia patients (Mills ET AL 2007).
  

Diabetis Mellitus

• Endocrine disorder that is caused by a deficiency in insulin or a decreased response to insulin in target tissues. Diabetes is marked by high glucose levels in the blood and the urine.
• Type 1 Diabetes is a autoimmune disorder in which the immune system destroys the beta cells of the pancreas. Diabetes usually appears during childhood and destroys a persons ability to produce insulin.
• Treatment consists of insulin injections usually several times a day, human insulin can be obtained by genetically engineering bacteria (Campbell, 2005).
  

Chronic/Acute pancretitis

• There are several types of chronic pancretitis and they tend to mimic pancreatic carcinomas.
• Causes fibrosis and chronic inflammatory cells are usually present and a marked distortion of the lobule is usually present.
• Eventually chronic pancreatitis will lead to a complete loss of aciner elements and ducts as the gland is replaced by fibrous tissue.
• Acute pancreatitis involves a rapid inflammation of the pancreas which is usually caused by gallstones or alcoholism.
• Symptoms of Pancreatitis may include abdominal pain, nausea, considerable weight loss, and the development of diabetes.
• Elastic ducts often due to ductal obstruction are often found in patients with pancreatitis. (Mills ET AL 2007).
  

Pancratic neoplasm

• Metaplasia when cuboidal non-mucinous epithelial change to tall columnar cells with abundant mucin. This condition can form the beginning of a ductal neoplasm. Mucinous metaplasia is now regarded as the early stages of pancreatic intraepithial neoplasm.
• Oncoytic changes are also common in centroacinar cells, oncoytic metaplasia may effect intercalated and intralobular ducts. This condition may be associated with chronic inflammatory process.
• Islet Hyperplasia as a increase in the size or number of the islets relative to the normal islet volume at a given age. Conditions associated with islet hyperplasia include Beckwith-Weidmann syndrome, maternal diabetes, Eryroblastosis fetalis, and hyperinsulinemic hypogycemia (Mills ET AL 2007).

References:

1. Beger H.G., Matsuna S., Cameron J.L. ED. (2008) Diseases of the pancreas current surgical therapies. Springer.
2. Campbell and Reece. (2005) Biology Seventh edition. Pearson Education Inc, San Francisco.
3. Hill., Wyse., and Anderson. (2008) Animal Physiology Second Edition. Sinauer Associates Inc, USA.
4. Mills. and Stacy. (2007) Histology for Pathologists. Lippincott Williams and Wilkins.