This document includes the summary (info slides + lesson notes) of lesson 9 of the course 'genome technology and applications'. Lesson 4 was simply an introduction by NGS to the task. Here, you must give a group presentation about a certain NGS technique.
Induced pluripotent stem cells (iPSCs)
1. What
Stem cells
- Can divide and create an iden-cal copy of themselves, a process called self-renewal
- Can differen-ate/mature into cells that make up every type or several types of
-ssue(s) and organ(s) in the body, a process called
pluripotency/to-potency/mul-potency
o Pluripotent: differen-ate to many other celtypes
o To-potent: differen-ate to all celtypes
o Mul-potent: selec-on of celtypes
Potency
- Different types:
o Early embryonic cells – to-potency
§ First cells created -> generated placenta and embryo-c cells
o Embryonic stem cells – pluripotency
§ AJer one week you have cells surrounding the the embryo
o Adult stem cells: e.g., hematopoie-c stem cell – mul-potency
§ Differen-ate to certain celtypes
o Induced pluripotent stem cells – pluripotency
Induced
- You start from a soma-c cell -> bring them back to a stem cell
- You add Yamanaka cells
IPSC discovery
- Different TF will bring your soma-c cells back to stem cells
- Human IPSC
o Start from fibroblast
o Couple of TF that are crucial
o It needs to be able to form 3 germ layers
2. How?
IPSC crea5on
Soma,c cells
- Start from fibroblast
- Puncture skin
o Advantages: easily to get
o Disadvantage: for some people it is s-ll to invasive and UV exposure
§ Muta-on from sun exposure is and you don’t want this
- Alterna-ves
Yamanaka factors
- 4 factors
- Different hypothesis how it is possible to go to stem cells with only 4 factors
o S-ll need to be inves-gated
, - How do you get them in
o Integrated in the genome
§ You can do it with viral methods
• + transduc-on efficiency
• + You can do it for dividing and non-dividing cells
• - Random integra-on in te genome -> disadvantage
o cMyc is a oncological driver -> can be a problem, oncological
proper-es
• - Reac-va-on of transgene
§ Non viral methods
• - Nucleofec-on/transfec-on efficiency
• - They can also re-integrate in the genome
o Non-integra-ve system
§ Viral
• It can stay quite long in the cell -> it taked quite of passages to get it out
or you can do a heat shock
• Good transduc-on efficiency
§ Non-viral
• Effect is gone quite rapidly
• Transient
• Lower reprogramming efficiency
-
Valida,on
iPSC-specific
- Morphology: visual inspec-on of colonies
o Group of cells that are iden-cal = colony
o Well defined boarder without spikes
o Cells -ghtly packed
o Ra-o of the nucleus in comparison with the cytoplasm is very high
o Nucleoli are very prominent
o Bright center in the middle
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