lecture 21: stem cells and diabetes

Question 1

What are the main characteristics of stem cells?

Answer 1

• stem cells can renew themselves
• can differentiate to make mature cell types
• needed for organs to repair after injury

Question 2

What are the two main types of stem cells?

Answer 2

• pluripotent stem cells (PSCs)
  
  • can be cultured for extended periods in the laboratory and retain stem cell characteristics
  • actually very difficult to grow a cell in culture for a long time, but relatively well done in PSCs
  • can turn into any cell type
    
    • embryonic stem cells made from early embryos
    • induced pluripotent stem cells made by reprogramming adult cells
• adult stem cells (ASCs)
  
  • difficult to grow in the laboratory and remain as stem cells
  • purified from adult tissues and tend to make a restricted range of cell types
    
    • haematopoietic stem cells make blood cells
    • mesenchymal stem cells make bone, fat, and cartilage
  • currently used clinically (bone marrow transplants)

Question 3

What is diabetes?

Answer 3

• diabetes is a chronic condition in which the body deals with glucose ineffectively
• rather than being taken up and used by the tissues, glucose remains in the blood
• high blood glucose is called hyperglycemia
  
  • symptoms of hyperglycemia
    
    • feeling excessively thirsty
    • frequently passing large volumes of urine
    • feeling tired
    • blurred vision
    • infections (e.g. thrush, cystitis, wound infections)
    • weight loss

Question 4

What are the two main types of diabetes?

Answer 4

• type 1: specific immune mediated destruction of beta cells
  
  • type one unlucky person
  • unable to produce insulin and therefore respond to blood glucose levels
• type 2: chronic energy overload associated with diet and lifestyle
  
  • type too much food

Question 5

What is type 2 diabetes?

Answer 5

• usually caused by prolonged excess energy intake
• characterised by:
  
  • sustained elevated blood glucose
  • insulin resistance in peripheral tissues
  • diminished function of beta cells
  • treatment involves dietary modification and sometimes insulin injection
  • epidemic transmitted by “Do you want diabetes with that”?

Question 6

What is type 1 diabetes?

Answer 6

• cause unknown
• characterised by autoimmune attack on insulin producing beta cells in the pancreas (current best hypothesis)
• usually occurs during childhood/adolescence
• traditional treatment involves regular insulin injections (many times a day)/monitoring blood glucose – this is particularly hard for young children and their parents
• thought to be an autoimmune disorder resulting in destruction of insulin-secreting beta-cells
• ~122,000 people in australia (~0.6%)
• complications from type 1 diabetes
  
  • retinopathy
  • neuropathy
  • depression or anxiety
  • end-stage kidney disease

Question 7

What are some fun facts about diabetes?

Answer 7

• 4% of australians have diabetes
• 10% of all diabetes is type 1
• vast majority have type 2
• gestational diabetes → prone to type 2 later in life
• type 2 has other factors associated - CVD and so on

Question 8

Compare type 1 and 2 diabetes

Answer 8

type 1 vs type 2

• onset
  
  • sudden vs gradual
• cause
  
  • unknown vs lifestyle
• age at onset
  
  • mostly in children vs mostly in adults
• appearance
  
  • thin or normal vs often obese
• ketoacidosis
  
  • common vs rare
• autoantibodies
  
  • usually present vs absent
• endogenous insulin
  
  • low or absent vs normal, decreased
• prevalence
  
  • ~10% vs ~90%

Question 9

What does the pancreas do?

Answer 9

• controls food digestion and distribution
• located under the stomach adjacent to the duodenum

Question 10

What are the islets of the pancreas?

Answer 10

• only small part of the pancreas
• acinar/exocrine cells secrete digestive enzymes into ducts leading to duodenum/intestines
• islets/endocrine cells secrete hormones into the blood stream
  
  • beta cells
  • endocrine cells sit in close proximity to blood vessels

Question 11

What are the hormones produces by the islets?

Answer 11

• hormones produced in the islets are secreted into the blood
• there are five different types of cells
  
  • alpha cells producing glucagon (~15%)
  • beta cells producing insulin (~75%)
  • delta cells producing somatostatin (~5%)
  • PP cells producing pancreatic polypeptide (~3%)
  • epsilon cells producing ghrelin (less than 1%)

Question 12

What are current treatments for type 1 diabetes?

Answer 12

• insulin injection (many times a day), or infused through catheter (a small needle) attached to an insulin pump
• frequent monitoring of bloodsugar levels by finger prick glucose testing
• can be long term complications even if tight blood sugar control is maintained
• many live in constant fear of ‘hypos’ - severe hypoglycaemia (low blood sugar) which can result in loss of consciousness and sometimes death
• there is a need for new forms of treatments

Question 13

What are problems with current treatment options?

Answer 13

• injection of insulin
  
  • onerous
  • imperfect control of blood glucose
  • does not prevent complications
• islet transplantation
  
  • high rate of graft failure
  • requires immunosuppression
  • lack of donor material
  • very tight window to collect after someone has died

Question 14

What are potential stem cell alternatives to insulin injections?

Answer 14

• would a new blood system help?
  
  • if type 1 diabetes is caused by errant lymphocytes then why can’t we just replace these by getting rid of the old blood system and putting in a new one?
    
    • bone marrow transplantation?
    • has too many adverse affects to be a viable option
• would bew beta cells help?
  
  • if we can tame the immune system then we might be able to replace the lost insulin producing cells with new ones
    
    • islet transplantation?

Question 15

What are transplant options?

Answer 15

• pancreas transplant
  
  • most often performed when a patient also needs a kidney transplant
  • the success rate (long-term insulin independence) is increasing
  • after one year about 85% of pancreas transplant recipients are insulin independent
  • a few thousand pancreas transplants a year worldwide, mostly in the U.S.
• islet transplantation and other experimental treatment options
  
  • does not require a major operation and the procedure has a small complication rate
  • results generally not as good as pancreas transplantation
• individuals receiving a pancreas or islet transplant must take immuno suppressive medications to prevent graft rejection

Question 16

What is islet transplantation?

Answer 16

• increasingly successful but limited by lack of donors
• donor (usually dead)
• pancreas removed
• islets isolated and injected into portal vein of liver
• lodge in there and find a place to live and start working

Question 17

What are isolated islets?

Answer 17

• isolated islets stained with a dye that reacts with zinc
• insulin is stored in granules which are high in zinc

Question 18

How could we replace beta cells with cells made from pluripotent stem cells?

Answer 18

• given we know islet transplantation works a little bit then the aim is alleciate the shortage of donor material by generating the appropriate cells in teh laboratory using stem cells
• the pancreas has its own stem cells but these are difficult to grow in the laboratory and, at the moment, coaxing these cells to become large numbers of endocrine cells is very difficult
• pluripotent stem cells represent an attractive starting point because they can grow in large quantities – large numbers of cells will be needed to treat a person

Question 19

What are human embryonic stem cells?

Answer 19

• normal diploid cells grown from a 6 day embryo called a blastocyst
• can differentiate into any cell found in adults

Question 20

What are induce pluripotent stem cells?

Answer 20

• adult cells
• add reprogramming genes or chemicals
• colony of iPS cells
• similar to ES cells called iPSCs can be made in the lab by adding extra genes or chemicals to adult cells in a process called reprogramming
• iPS cells behave like ES cells and are a genetic match to the person whose cells were reprogrammed → however in diabetes immune system has already rejected own cell, so benefits of using iPS cells over hESCs are not immediately apparent

Question 21

What are important considerations of using stem cells?

Answer 21

• need to be able to make enough cells to be effective
• cells need to be safe – left over undifferentiated hESCs could give rise to teratomas (a form of tumour)
• cells will still be subject to immune surveillance
• cells actually have to work
• so how do we go about making beta cells from hESCs?

Question 22

How do beta-cells arise?

Answer 22

• an orderly developmental process during embryogenesis
• embryonic stem cell differentiation recapitulates aspects of this process
• morala
  
  • ICM
    
    • epiblast
      
      • definitive endoderm
        
        • foregut
          
          • pancreas
            
            • endocrine
• if each step is only 10% efficient, by the time you get to beta cells you have almost nothing left
• even if each step is 90% efficient, far fewer at the end than the begining
• people working on these kinds of protocols

Question 23

What is PSC differentiation to pancreatic endoderm?

Answer 23

• incorporates growth factors implicated in development
• people have looked at which growth factors are present at particular times along the development of embryo nd tried to add these in at correct times during the growth of PSCs
• ES cell → activin, Wnt3a → definitive endoderm → RA, Cyc, FGF → posterior foregut → DAPT → pancreatic endoderm → Ex4, IGF1 → endocrine cell

Question 24

What are growth factors controlling the differentiation of hESCs toward pancreatic endoderm?

Answer 24

• developed a very influential protocol
• developmental biologists
• each cell type along the path of differentiation needs a specific set of genes expressed

Question 25

What is the production of pancreatic progenitors?

Answer 25

* viacite were the ones who developed that influential protocol * using scaled banks of CyT49 → scaled thaw and expansion in cell flasks → cell factories → suspension aggregation (aggregation spinner flask) → scale that up → suspension differentiation → takes about 12+ days * critical/cunning thing these guys have done is not try to differentiate their cells all the way to beta cells * human embryonic stem cells can differentiate into pancreatic progenitors in vitro * look for markers of differentiation/undifferentiation * OCT4 - undifferentiated cells → need to have few/none of these for FDA approval * FOXA2 → endoderm * NKX6.1/PDX1 → pancreatic endoderm * CHGA → endocrine * NKX2.2 → endocrine * grafted pancreatic progenitors develop into functional insulin producing cells in mouse models of diabetes * most of the work done since then is about ensuring safety not efficacy

Question 26

How can we evade the immune system?

Answer 26

* although immunosuppression is workable it is still undesirable * encapsulation devices are designed to hide the transplanted cells from the immune system * T cells cannot get in * physical barrier * prevents immune cells from contacting Beta cells * prevents unwanted cells from escaping into the bloodstream (e.g. undifferentiated teratoma forming cells) * keeps the graft in one place so it can be monitored * easy to remove the graft if problems occur

Question 27

What are problems with encapsulation?

Answer 27

* a number of problems have been observed with current generation encapsulation devices and these may ultimately limit the effectiveness of this delivery system * the device may still admit anti-islet antibodies that could interfere with beta cell function → holes need to be big enough for insulin to get out * some devices elicit a fibrotic response * fibrotic response to transplanted material → wraps it up in fibroblasts and connective tissue etc, effectively shutting it down

Question 28

What about iPSCs as a genetically matched source of beta-cells?

Answer 28

* in theory, genetically matched beta cells may not help in the case of type 1 diabetes because it is an autoimmune disease, but: * gene editing technology could be used to remove or alter antigens targeted by the immune system * cells could be genetically modified to make them resistant to immune attack * cells could be fitted with suicide cassettes that could be activated if the cells underwent an oncogenic transformation

Question 29

What are the pros and cons of pancreatic progenitors?

Answer 29

* advantages * because progenitors retain some proliferative capacity fewer cells are needed * progenitors are more resilient than beta cells – they can survive the shock of the transplantation process * disadvantages * require instructive signals in the serum of patients to permit the final stages of differentiation → don't know if this is present in humans * other unwanted cell types could differentiate from the starting population * only a clinical trial will answer these questions (aiming for 2014)

Question 30

What about fully differentiated beta cells?

Answer 30

* fully differentiated cells might work better because that is what is in islet – and we know islets work sometimes * at the moment we can't do that * cells would be further along the differentiation pathway and less likely to contain unwanted cell types (undifferentiated cells) * functionality of cells could be tested before transplantation

Question 31

What are general requirements for any stem cell based therapies?

Answer 31

* stem cells must efficiently make the required mature cell type * cells must be available in sufficient quantities and purity for therapeutic use * problems of immune rejection must be overcome * the transplanted cells must be safe - free of infectious agents and free of remaining stem cells that might cause tumours

Question 32

So what is the potential of stem cells in treating diabetes?

Answer 32

* although controllable with insulin injections, Type 1 diabetes is a prime target for stem cell based therapies of the future * human ES and iPS cells can be differentiated into pancreatic progenitors that can generate functional beta cells following transplantation

Question 33

things to remember

Answer 33

* difference between type 1 and type 2 diabetes * three major hormone producing cells of the islets * why is a cell based treatment for type 1 diabetes desirable * two major issues associated with type 1 diabetes in terms of alternative treatments (immune attack and lack of beta cell mass) * why haematopoietic stem cell transplantation has been trialled as a treatment and why it is not a viable option * what are the underlying principles of PSC differentiation * what is the major difference between iPSCs and ESCs * what concerns are there about cell based treatments * what are the facotrs that may affect the success of PSC derived treatments * what issues could encapsulation address * what could go wrong with encapsulation approaches * could anything be done to prevent immune destruction of un-encapsulated cells