Understanding and curing diabetes

Question 1

What are the ongoing attempts to find a cure for type1 diabetes ?

Answer 1

• Islet cell regeneration
• Induction of tolerance in the immune system
• Islet or pancreas transplantation
• Xenotransplantation
• Stem cells, adult, pluripotent

Question 2

What is type 1 diabetes characterized by?

Answer 2

The body’s immune system attacks the insulin producing beta cells in the pancreas. Thus, little to no insulin is produced by the pancreas.

Question 3

What is type 2 diabetes characterized by?

Answer 3

The body’s cells become resistant to the effects of insulin. Over time, the pancreas doesn’t produce enough insulin to overcome the resistance.

Question 4

What are the challenges of cell transplantation ?

Answer 4

It’s a valid practice (bone marrow, organ transplantation…), however challenges include :
- Immune rejection
- Availability and quality control of donor cells/organs

Question 5

Which immunosuppresive drugs are used in treating T1D ? And their mechanisms.

Answer 5

Rapamycin :
- Inhibits mTOR = stops T and B cells cycles = reduces T and B cell proliferation

Tacrolimus :
- Blocks IL-2 transcription = reduced T cell activation = reduced immune response

Question 6

What are the roles of insulin regarding blood glucose levels ?

Answer 6

Insulin regulates blood glucose levels by providing muscle and fat cells with glucose, by triggering for example GLUT4.
Insulin inhibits gluconeogenesis and glycogenolysis, preventing the liver from releasing too much glucose into the bloodstream.

Question 7

Due to lack of islet/organ shortage, what other solution is there for treating type 1 diabetes ?

Answer 7

Derive pluripotent stem cells into insulin-secreting cells (islets of langerhans). Promise as paper in 2008 transplanted pancreatic endoderm (precursor to islets) in mice and observed decrease in blood glucose levels.

Question 8

What are the vertex diabetes trials ?

Answer 8

Vertex pharmaceutical is undergoing clinical trials for treating T1D by using islet cells derived from stem cells with the aim of restoring endogenous insulin production.
Participants chosen had a clinical history (over 5 years) of T1D and and documented episodes of severe hypoglycemia.

Question 9

What are the complications of immune suppression ?

Answer 9

• Mouth ulcers
• Diarrhea
• Acne
• Edema
• Ovarian cysts
• Increased cancer rate (40% after 20 years of immunosuppression) vs 6%

Question 10

How can the immune compatiblity of iPSCs be enhanced ?

Answer 10

Human leukocyte antigens (HLA) present antigens to T-cells to mount an immune response. If transplanted cells present HLA molecules that are different from the recipient, they are recognized as foreign. Through the targeted disruption of HLA genes via crispr-cas9 in pluripotent stem cells, T cell responses, NK and macrophages killing can be diminshed.

Question 11

What is MODY ?

Answer 11

Monogenic diabetes of the young is a form of inherited diabetes because of autosomal dominant inheritance (single copy of the mutation is enough).
Many forms of MODY exist :
- MODY 2 : mutation in the glucokinase gene, responsible for glucose sensing in beta cells = disregulated insulin secretion. 22%
- MODY 3 : mutation in the HNF1A gene, responsible for the regulated expression of several genes (INS) = reduced insulin secretion.
Transcription factors mutations represent 66% of MODY

Question 12

Why is C-peptide used as measurement in diabetes patients ?

Answer 12

C-peptide is produced in equimolar amounts with insulin in pancreatic beta cells. C-peptide has a longer half-life than insulin in the bloodstream, it is thus an indirect assessment of endogenous insulin secretion, reflecting the functional status of pancreatic beta cells.

Question 13

How are c-peptide levels in diabetes patients ?

Answer 13

T1D : low or undetectable C-peptide
T2D : normal or elevated due to insulin resistance

Question 14

How can c-peptide measures help in evaluating insulin therapy ?

Answer 14

C-peptide levels can help distinguish endogenous and exogenous insulin. Also helps in clinical trials to assess efficacy of therapies aimed at restoring beta-cell function.

Question 15

What is permanent neonatal diabetes ?

Answer 15

Rare form of diabetes manifesting within 6 first months of life due to hyperglycemia because of impaired insulin secretion.

Question 16

How can PND be treated ?

Answer 16

You find the specific insulin mutation, for e.g. subtitution of Cys for Tyr and grow stem cells with the corrected mutation into beta-like cells.

Question 17

What’s an interesting observation about insulin mutations ?

Answer 17

Insulin mutations do not seem to affect beta cell differentiation, but rather insulin dysfunction.

Question 18

What is proinsulin ?

Answer 18

Proinsulin is the precursor to insulin, synthesized in pancreatic beta cells. Specific proteases cleave proinsulin at 2 sites, removing the C-peptide and forming of mature insulin (A and B chains)

Question 19

What is cystic fibrosis related diabetes (CFRD) ?

Answer 19

Cystic fibrosis is a multisystem genetic disorder causing thick and sticky mucus blocking the airways but also the pancreatic duct.

Question 20

What is the cystic fibrosis transmembrane regulator (CFTR) ?

Answer 20

It’s a chloride channel protein which is mutated in CFRD patients

Question 21

Does a CFTR deficiency affect development of beta like cells ?

Answer 21

No, culture experiments of CFRD mutated pluripotent stem cells do not seem to establish or proliferate less.

Question 22

Is CFTR required for beta cell function ?

Answer 22

No, in fact there is little to no expression of CFTR protein in normal human beta cells

Question 23

Conclude about the CFTR protein gene.

Answer 23

• Mutations in CFTR do not affect differentiation to beta cell
• CFRD patient ipsc derived beta cells are glucose responsive, thus beta cell function is not affected by CFTR deficiency
• CFRD is not caused by autoimmunity, grafting autologous stem cells may not require immune protection

Question 24

What is likely causing Beta cell dysfunction in cystic fibrosis ?

Answer 24

Chronic inflammation due to pancreatic duct blockage because of the mucus. Released cytokines can interfere with insulin secretion (IL-1B and TNF-alpha)

Question 25

What is HbA1c and why is it measured ?

Answer 25

It’s a form of hemoglobin that attaches to glucose. These red blood cells have a lifespan of about 120 days, giving a snapshot of glucose levels over a long period of time. The ideal percentage of HbA1c is below 7%.

Question 26

Describe the steps of insulin secretion induced by glucose.

Answer 26

1. Glucose enters beta cells via GLUT2
2. Glycolysis : glucose converted to pyruvate = production of ATP
3. Increase in ATP closes K-ATP (potassium) channel = membrane depolarization
4. Depolarization triggers influx of calcium ions (Ca2+)
5. Insulin is released into the bloodstream

Question 27

Explain the mechanisms of beta-cell mass maintenance

Answer 27

• Self-replication of already existing beta cells
• Trans-differentiation from delta and alpha cells (after extreme beta cell loss)
• Expression of stem cell markers (PDX1 and SOX9) points to stem cell contribution

Question 28

What fact goes against the idea that stem cells contribute to beta cell maintenance ?

Answer 28

Terminally differentiated beta cells retain proliferative capacity in vivo.

Question 29

Why are islets transplanted in the portal vein ?

Answer 29

• The portal vein brings blood from the gastrointestinal tract, rich in nutrients, enhancing islet insulin secretion and function
• Less invasive than transplanting into the pancreas
• Liver has factors promoting cell survival

Question 30

What does daclizumab do ?

Answer 30

It’s an antibody that targets the CD25 subunit of IL-2 on T cells. It prevents subsequent T cell activation and proliferation.

Question 31

Why co-culture islets with MSCs?

Answer 31

Human MSCs enchance insulin secretion when in contact with human islets (N-cadherin interaction)

Question 32

Why use pig islets for xenotransplantation ?

Answer 32

• Similar organ size
• Cost-efficient
• Physiological similarities with humans
• Insulin differs by 1 amino acid

Question 33

What’s the major problem of pig islets ?

Answer 33

Humans have a severe immuno reaction against “alpha gal” (we lack the corresponding enzyme and produce anti-gal antibodies.
The alpha gal gene was successfully knocked out of pigs for transplant.
The 10 gene edited pig now exists, with deleted and added genes.

Question 34

What strategy exists for overcoming graft rejection without the need for immunosuppresion ?

Answer 34

Islet encapsulation in hydrogels. Islets are safely encapsulated in a permeable membrane allowing the exchange of nutrients and oxygen, and the diffusion of insulin while protecting the iselts from immune cells and antibodies.
The drawback is that it can cause fibrosis around the capsule.

Question 35

Why would using juvenile pigs be more interesting ?

Answer 35

• More efficient digestion of the pancreatic tissue
• Higher yield of cell clusters, but islets would be immature
• Less expensive cause young pigs

Question 36

How can juvenile pigs be used (MSCs) ?

Answer 36

Co-culturing porcine islets and porcine/human MSC from pancreas supports insulin secretion. Encapsulate everything into microbeads = improves diabetes

Question 37

What is Car-Treg ?

Answer 37

Chimeric antigen receptors in regularatory T-cells. CAR-Tregs can bind to antigens on T cells, secrete cytokines (IL-10, TGF-Beta) and supress T cell activation.