Stem Cells for Clinical Use Flashcards
How can stem cells be used in biopharma?
- Toxicity testing → differentiate hESCs into cardiomyocytes and hepatocytes. Test drugs to see what effects they have on these cells.
- Screening for thereapeutic compounds → create disease specific iPSCs using somatic cell nuclear transfer. These will carry the genotypic characteristic of the genetic disease. Can use this to aid in the development of compounds that target the abnormal cells.
- Identification of molecules that promote differentiation → use healthy pluripotent stem cells and look at compounds that promote lineage specific differentiation. Could be useful in treating developmental disease.
Overview 3 ways stem cells can have clinical use
- Replacement of diseased or injured cells eg)
− Injury → non-union bone fractures (non-healing) , 2nd or 3rd degree burns, spinal cord injury
− Autoimmune disease → eg) osteo/rheumatoid arthritis
− Genetic disease → leukemia, heart disease, muscular dystrophy
− Neurodegenerative disease → parkinsons, alzheimers - Immunomodulation (MSCs) → inflammatory bowel disease and other inflammatory disorders
- Vehicles for gene therapy (MSCs, PSPs, HSCs)
How can human ES cells/iPS cells be used as a cell therapy?
• With ES cells, have the potential to generate committed precursors for transplantation therapy
• However:
− We are only just now starting to control differentiation precisely
− Problem of rejection → solve by tissue typed stem cell banks
− Need to be sure cells get to, and remain in, the correct location
− Will they survive? If so, for how long?
− Will they retain the phenotype?
− Are they homogenous?
− Will they cause tumours?
• As rejection is a problem, possibility to generate self ES cells which could be induced to differentiate into specific committed precursors for therapy
− Biopsy patient somatic cells
− Take the nucleus and put it into an enucleated oocyte
− Reprogramme and develop to blastocyst state (only occasionally possible)
− induce pluripotent stem cells (achieved in 2013 but hESCs carry genetic abnormalties – resultant cells may need gene correction)
Where do the oocytes come from?
• Legal using animal eggs under licence from the HFEA → may not be acceptable for therapy as will have mitochondrial and cytoplasmic components from an animal, and the mitochondrial genome contributes to the transcriptome
• Noggler et al, 2011 added a somatic nucleus to an intact oocyte and there were able to get blastocyst formed.
• Targeted differentiation is often inefficient and cannot be properly controlled
• Will iPSCs prove the solution? Will they be identical to normal ES cells and give normal differentiated cells?
− Would hope they would not be rejected as this is an autologous transplant
− Dogma → but in 2011 a group reported this was not the case
− Tested in mice:
− ES cells:
− B6 ESC → B6 host = teratoma formation
− 129 ESC → B6 host = rejection
− 129 or B6 ESC → SCID host = teratoma formation
− iPS cells:
− B6 iPSC → SCID host = teratoma formation
− B6 iPSC → B6 mouse = rejection
Why did this happen?
• iPS cells express embryonic antigens not normally expressed during differentiation
• 2 of these minor antigens stimulated T cell infiltration and rejection
• So human iPSCs may not be suitable for therapy
However
• Yamanaka suggested this may not be a big issue for neural injection
− Plasmid based iPSCs from autologous cynomologous monkeys differentiated to neurons and when injected back into donor brains, did not cause a significant immune response
− Even allogenic, deliberately non-matched neurons from iPSCs did not give a huge immune response
− Plasmids lack viral inserts which may have led to the retention of transgenes → virally programmed iPS cells did cause an immune response
− Group are now going on to look at allogenic tissue matched cells – a better model for allogenic transplant in humans
• Suggests that for humans, iPS therapy may not require immunosuppression
How can human iPS cells be used as a disease model?
- Reprogramme diseased cells from a patient to give diseased iPS cells, and can differentiate them → need stem cells as disease models as they will propagate indefinitely!!
- Also have normal control cells, which can be reprogrammed to iPS cells and differentiated
- Then compare the two to see if there are differences
- Found that the diseased cells don’t make incorrect amounts of a particular protein. Can then look at various drugs to see if you can correct expression.
Problem
• Control and diseased cell have different genotype
• Wont just vary by one mutation – there will be a lot of different genetic changes → even true when you use siblings
How can we be certain the disease phenotype is actually due to that mutation?
• Use CRISPR genome editing
• Correct the mutated gene in the disease phenotype → so now have a cell that, apart from the corrected mutation, has the same genotype as the diseased cell
− Compare this to the disaesed cell and see if has the same phenotype
− Can also use CRISP to insert the mutation in the control cells and then compare this with the diseased phenotype
• CRISP can now be used as a therapy as well as a disease model
• Generated chondrodysplasia disease iPSCs from a patient with a mutation in collagen oligomeic protein
What are the 3 requirements for cell therapy?
• GMP standards assure the quality of the therapeutic product for the safety and protection of the patient
• Meticulous records and a clear adult trail during all stages of production and clinical use is essential
− documents everything that has happened to those cells whilst in the lab
• Requires rigorous testing of intermediates and the therapeutic product
What is GMP?
• GMP standards assure the quality of the therapeutic product for the safety and protection of the patient
• Meticulous records and a clear adult trail during all stages of production and clinical use is essential
− documents everything that has happened to those cells whilst in the lab
• Requires rigorous testing of intermediates and the therapeutic product
Cells need to be produced under ‘clean room’ conditions:
• Purpose built to provide GMP conditions
• Air standards → very low levels of particulate matter with no contamination
• Environmental monitoring
• Regulated flow (people, materials, equipment)
• Train people in gowning procedures
• Competency assessment for eg) aseptic tecniques
What are the culture conditions needed to produce stem cells for therapy?
- Xenobiotic free – materials produced without any animal derived substances → animal products can be recognized as foreign, or animal pathogens may be carried over
- Feeder free → use recombinant human proteins, clinical grade small molecules and human substrate proteins, or synthetic substrates.
How are stem cells for therapy characterised?
• Self-renewal
• Phenotype and developmental profile
• Stability → karyotype and phenotype. Don’t want to administer cells that have developed chromosomal aberrations – and this is common in culture.
• Safety:
− Transformation (cancer)
− Transdifferentiation → no chance of them differentiating in the body to something different
− Contamination with pathogens
− Immunological properties
− Migration → will they stay where transplanted or go somewhere else?
Test these
- in vitro
- in animal models
- in clinical trials
How do we make differentiated cell types with a view to therapy?
• Cant use spontaneous differentiation → uncontrolled, heterogenous • Cant use embryoid body → uncontrolled • Have to use directed differentiation − Stepwise induction of TFs − Exposure to GFs and small molecules − Modulation by ECM − Co-culture with appropriate cell types − Use of 3D scaffolds
How have iPS cells been used to treat parkinsons?
Parkinsons
• Degeneration of the substantia nigra – lack of dopaminergic neurons leading to the loss of movement coordination
• Effects in 1 in 500, around 10,000 per year in the UK
• No cure – but drug replacement of L-dopa manages symptoms
• Fetal dopaminergic neurons have been triad, with some success, but side effects include graft induced dyskineses. Source is also ethically and practically problematic.
Proof of principle in rodents and primates
• Kriks et al
− Use of small molecule to activate the Wnt pathway
− Generated floorplate neurons from fibroblast iPS cells → then turned these to functional dopamine neurons.
− By precise differentiation to precise neurons required, the researchers overcame past failures in engrafting ES derived neurons
− Gave functional repair of movement disorder
− Neurons intregrated and corrected the defects in both primates and rats
Describe the use of stem cells in treating Alzheimers
• Neurons in the brain die, leading to progressive loss of cognitive ability (dementia)
• Generally >65, but there is a juvenile onset disease – ataxia telangiectasia
• 4 million people in the UK have alzheimers
• No cure – symptoms controlled by cholinesterase inhibitors to increase ACh
• Stem cells have been in trial to try and regenerate the neurons lost, but have to be mindful of safety:
− 2009: patient developed a brain tumour some years after treatment for ataxia telangiectasia
− Patient died from the tumour
− When analysed, tumour was found to be not of patient origin, but from multiple donors
Describe the use of stem cells in treating spinal cord injury
Proof of principle
• Long distance growth and connectivity of neural stem cells after severe spinal cord injury
− Neural stem cells repair spinal cord injury in rats
− Electrophysiological relays across the site of injury
− 200 fold increase in axons
− Formation of synapses
− Emergence of new endogenous NSCs
− Improvement of hindlimb locomotion over control by week 5
How have ESCs been used in spinal cord injury and macular degeneration?
• Oct 2010:
− American company Geron announced phase I trial for use of ES cell derived oligodendrocytes of the nervous system
− Safety trial to see if the cells are safe and can enhance nerve repair in spinal cord injury
− Trialed in patients who had exhausted all possible therapies
− Nov 2011: Geron pulled out of stem cell therapies to concentrate on cancer
• The London Vision Project
− Cell therapy for macular degeneration
− Retinal pigment cells from ES cells successful
− Therapy is at the stage of safety trials
− First in man studies and phase I/II clinical trial data published:
− 50-150 x103 cells hESC-RPE cells transplanted into patient eye
− No adverse proliferation, rejection or other issues
− 78% of 18 patients had increased sub retinal pigmentation
− Visual acuity improved in 10 eyes, same in 7 and worse in 1
− Vision related quality of life increased
How can stem cells be used to treat diabetes?
- Effects >5% UK population
- Costs NHS £10million a day
- Recent major increase in Type 2
- Current therapy for Type 1 is life-long insulin injection
- Also the pioneering pancreatic islet transplantation – but you need about 4 pancreases for this and may only need to a reduction in insulin requirement for a limited time.
• hESCs and hiPSCs produce functional glucose responsive beta cells in vitro and in vivo in mice (loss of differentiation lecture)
− Cells only produce insulin, no other pancreatic hormone
− Cells respond to different doses of glucose
− Rescue insulin deficiency when implanted under the kidney capsule
How can stem cells be used to treat liver failure?
iPSCs generated 3D vascularized liver bunds in vitro:
• hIPSC → HNF4a + hepatic endoderm
• H+ endoderm cultured with HUVECs and hMSCs
• Self organization to form liver buds with integration of vasculature
• Showed vascular perfusion + albumen + a1-AT secretion
• Improved survival of immundeficient mice after gangcyclovir induced liver failure
