L3- Induced pluripotent stem cells Flashcards
What are iPSC’s?
Induced pluripotent stem cells which are artificially made pluripotent stem cells.
How are iPSC’s generated? N
Mouse fibroblasts are cultured in vitro with iPS reprogramming factors (Yamanaka factors)- Oct3/4, Sox2, Myc, Klf4.
These are expressed normally during embryonic stem cell growth.
These convert fibroblast to iPSC colonies which have self renewal capabilities and can differentiate into different cell types (all 3 germ layers).
What are the different reprogramming approaches?
- Integrating methods using retrovirus, lentivirus
- Non-integrating methods using plasmids, proteins, mRNA
- Reprogramming time scales vary but are around 30 days on average
What are the experiments done to validate an iPSC line?
- Testing for pluripotency factors in vitro using staining
- Testing for the formation of 3 germ layers in vitro
- Formation of teratoma’s when injected into mice
- Karyotyping
What are the differences between pluripotent and multipotent stem cells?
- Different sources of origin
- High rate of proliferation in pluripotent
- Low rate of proliferation in multipotent
- No spontaneous differentiation in multipotent
- Lower capacity to produce diverse cell types in multipotent
What are the applications of iPSC’s?
• Disease modelling to discover the molecular mechanism of a disease
• Drug screening and discovery
• Cardiac, neural and liver toxicity tests
(in drug development)
What is an example of cardiac modelling using iPSC’s?
Skin fibroblasts from a patient affected by type 1 long QT syndrome (LQT1), carrying a mutation in the KCNQ1
potassium channel gene, were reprogrammed into iPS.
iPS cells were then differentiated into cardiomyocytes. Phenotype seen in the heart of patients with LQT1 was mimicked by isoprenaline, which induced arrhythmic events in these cells.
Treatment with the β-blocker propranolol suppressed arrhythmia.
What are examples of cell therapy using iPSC’s?
• Endogenous mouse haemoglobin genes replaced by human alpha and beta globin. Causes beta-A to be replaced by beta-S gene. Beta-S/Beta-S homozygotes have sickle cell anaemia.
What is autologous stem cell therapy?
Using the patients own somatic cells and reprogramming them into iPSC’s to cure the disease.
Not good for emergencies- impractical, expensive, time consuming
What is allogenic therapy?
Transplanting from human leukocyte antigen (HLA) matching donor.
Matching HLA can be difficult.
How are HLA screening expenses reduced?
Strict criteria for pre-screening of potential donors:
• Homozygocity of atleast 3 HLA loci (A, B, DR)
• Blood type O
• Clean medical and family disease history
What is the bias in HLA matching?
Some ethnic groups are less likely to find a donor with matching HLA.
Whites are more likely to find donors as compared to ethnic minorities.
Socioeconomic status may also play a role.
How is it ensured that clinical grade iPS cells do not contain mutations?
Exome sequencing
Karyotyping
DNA profiling
What is an example of autologous stem cell therapy?
iPSC-derived (derived from skin biopsy) retinal pigment epithelial (RPE) cells for treatement of age related macular degeneration of retina (2010)
Study suspended due to safety issues (genetic changes found in the cells).
What are examples of allogenic stem cell therapies?
iPSC-derived cells to treat graft-versus-host disease (2016)
First person to receive RPE cells derived from iPSC donated by another person (2018)
iPSC-based treatment was approved to treat ischemic cardiomyopathy (2018)
iPSC-based cancer immunotherapies using CAR-T cells
