B. ISLET AND STEM CELL THERAPY Flashcards
1
Q
problems with T1DM insulin therapy
A
doesn’t eliminate long complications:
- retinal damage
- diabetic foot ulcers
- costly
2
Q
what are the 3 strategies to regenerate beta-cell function
A
- organ transplants
- islet transplants
- stem cell therapies
3
Q
organ transplants
A
- rarely performed as shortage of organs/donors
- need to be tissue compatible so no risk of rejection and in perfectly good use
- morbidity associated with surgery as its very long
- risks associated with long-term immune suppression (infection, malignancy, bone disease, CVD)
- reserved for end-stage renal failure patients requiring a kidney and pancreas transplantation
4
Q
Islet of Langerhans transplantation
A
- isolated islet cells from donor tissue engrafted into liver (done on NHS)
- or insulin producing beta-cells
5
Q
what is the protocol for islet transplantation
A
The Edmonton Protocol
- collagenase in pancreas releases intact islet cells
- the islets are purified and injected through a percutaneous catheter into portal vein of liver
- can produce insulin
6
Q
what is required for the islet transplantation
A
- islet cells purified from donor organ
- perfused into liver under local anaesthetic
- immunosuppression required: 5 doses of Daclizumab at 1mg/kg IV over 8 weeks followed by maintenance on Tacrolimus
7
Q
what was the outcome of the Edmonton protocol
A
- initially poor success rates
- clinical trial primary end-point defined as insulin independence at 1 year
- 44% of patients met primary endpoint
- 76% required insulin therapy within 2
years
8
Q
when and for who are islet transplants performed
A
- available on NHS since 2008 but only 152 procedures from 2008-2015
- sometimes offered to T1DM patients who suffer regular hypos
- they often have poor hypoglycaemic awareness (will have seizures and collapse)
- more likely in patients on long-term insulin treatment
9
Q
benefits of islet transplants
A
- reduced frequency of hypos
- improved awareness of hypo symptoms
- normally functional for 6-10 years
- patients continue to need low dose insulin therapy
10
Q
who is islet transplants not suitable for
A
- weight>85kg
- poor kidney function
- requires >50 units insulin daily
11
Q
risks of islet transplants
A
- infection from procedure
- small increased cancer risk
- rejection of transplant
12
Q
what are the three main types of stem cells
A
- embryonic stem cells (pluripotent)
- adult stem cells (multipotent)
- induced pluripotent stem cells
13
Q
what are pluripotent stem cells
A
- cells that divide (self-renew) indefinitely and can potentially differentiate into the 3 primary germ layers (ie: endoderm, mesoderm, ectoderm) and hence all other cell types in body
- no lifespan
14
Q
what are pluripotent stem cells currently used for
A
- cornea regeneration
- skin repair
- bone repair etc
- bone marrow blood cells
15
Q
embryonic stem cells
A
- pluripotent: can form all cells of body
- self-renewal or differentiation
- expression of specific gene networks (response to environmental signals) determines self-renewal (stem-cell) or differentiation
- there are certain TFs in stem cells which can activate stem cell genes to keep it as a stem cell
- when it differentiates, those TFs are suppressed and others come in and activate differentiated cell gene expression programmes
16
Q
what is the source of ESCs
A
- patients undergoing IVF treatment
- Surplus IVF embryos (blastocytes) donated with consent to hospital
- inner mass cells removed before destruction
- cultured ESCs for research, clinical applications
17
Q
adult stem cells
A
- multipotent: can generate certain types of cells
- bone marrow, intestine, brain, fat tissues contain adult stem cells
- they regenerate the cells of that tissue (committed progenitors)
- can undergo self-renewal, commitment and differentiation
18
Q
what is reversing differentiation
A
- induction of PSC from ie: mouse embryonic and adult fibroblast cultures
- need defined factors
- ie: skin cell to stem cell
19
Q
induced pluripotent stem cells
A
- cultured fibroblast can be reprogrammed to become iPSC then differentiation occurs (particular tissue patient needs?)
- come from patient
- pluripotent associated factors: Oct4, Sox2, cMyc, Klf4, Nanog (stem cell specific TFs) - switch off all differentiated gene programs and switch on stem cell programs
- culture conditions adjusted to generate different cell types
- challenges: low efficiency, controlling differentiation
20
Q
disadvantages of human ESCs
A
- not human matched
- immunosuppression may be needed
- ethical concerns? (objection to using embryo tissue)
21
Q
advantages of iPSCs
A
- resemble ESCs
- genetically identical to patient
- amenable to genome editing - correcting genetic defects (then put back in patient)
22
Q
disadvantages of iPSCs
A
- may have incomplete reprogramming (experimental technique)
23
Q
are iPSCs a ideal cell therapy for islet regeneration
A
- IPSCs from patient are grown in lab
- differentiation into beta-cells (or other cells of islet)
- engraft into patient’s liver via Edmonton Protocol via perfusion technique
- stem cells stored for future rounds of islet regeneration (-80 degrees Celsius or under liquid N)
- autoantibodies still attack stem cells (beta-cells)
- give another transfusion in 5 years
24
Q
future prospects and challenges with stem therapy
A
- preclinical trials are very promising for hES and IPS cells as insulin producing beta cells produced in vivo
- unclear which strategy will be most effective in the clinic
- underlying autoimmune disease will still be present so likely to require repeated administration (1 dose per year)
- beta-cells encapsulated within devices can be implanted in the body but can induce fibrotic immune reaction
- novel (ie - clotting) less immunogenic materials may improve these devices
- genome editing (CRISPR CAS9 technology) might be used to protect the encapsulated cells from immune system
25
Q
what is CRISPR CAS9
A
- genome editing tool
- molecular scissors (enzyme) which is guided into DNA and knows which specific gene sequence to cut by guide RNA
- guide RNA = copy of sequence want to cut (20 bp)
- CRISPR CAS9 is suitable for adenovirus vector delivery
26
Q
applications of CRISPR CAS9
A
- gene (base) editing: allele specific
- corrections of mutations
- enhance or suppress specific gene expression
27
Q
macro-encapsulated beta-cells
A
- vascularised pouch with beta-cells in it
- wear inside or outside body
- develop blood capillary system
- can sense blood glucose and produce insulin
- overcomes need for injections
28
Q
micro-encapsulated beta-cells
A
- porous barrier to immune cells
- beta-cells in capsules so antibodies can’t attack them
29
Q
‘super beta-cells’
A
- CRISPR gene edited “Super beta-cells” (could it cause cancer)
- not recognised by immune cells (resistant to autoantibodies)
- inbuilt suicide mechanism to eradicate if needed (cells will die if become tumorous etc)
