Cellular Reprogramming and prenatal Therapy group Flashcards Preview

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Flashcards in Cellular Reprogramming and prenatal Therapy group Deck (64):
1

what are iPSC stem cells?

induced pluipotent stem cells:Differen?ated cells can be reprogrammed to pluripotency and other cell fates by treatment with defined factors

2

what are the three main discoveries which contributed tot e work on iPSCs?

1. he discovery of somatic ce nuclear transfer which allows the researchers to find that differentiated cells retain the same eugenic information as the early embryonic stem cells
2. the development of techniques allowing to derive culture and study pluripotent cell lines.
3. the observation that transcription factors are key determinants to cell fate. enforced expression of TF can switch mature cell type into another

3

what is the principal behind nuclear transfer?

when you take a nucleus from an adult differentiated cell and place it into an oocyte- a clone can be made

4

what can pluripotent at stem cells give rise to?

- only cells within their lineage- HS and NSC etc

5

what did guardian find?

Showed that differen?ated cells retain the genetic informa?on necessary for cloning and that development imposed reversible, and not irreversible, epigenetic changes on the genome during cellular differentiation

6

what phenotype did clones show?

clones animals show subtle to severe phenotypic and gene expression abnormalities, which result in faulty epigenetic reprogramming.

7

how was dolly the sheep cloned?

- take a black faced sheep and take the nucleus out and put it in a whit faced seep egg. Then implant egg into white face sheep and you get a black faced daughter!

8

what is pluripotency?

the ability to differentiate into all of the three germ layers

9

what 5 stem cellsharbor an epigene?c conforma?on that is permissive for a spontaneous reversion to pluripotent state and expression of OCT4

Embryonal carcinoma cells
• Embryonic stem cells (pre-implantation embryo)
• Epiblast stem cells (post-implanta?on embryo)
• Primordial germ cells (mid-gestation embryo)
• Multipotent germ line stem cells

10

what is the most important gene in reactivating pluripotency?

oct4

11

what does epigenetic changes mean?

heterochromatinisation and DNA folding that prevent access of the TF to the DNA

12

what 4 genes are involved in pluripotency?

Oct4, Sox2, Nanog, c-Myc (klf4?)

13

what is the general premise of iPSC production?

- you want to reactivate the TFs involved in pluripotency

14

what is the main method of activating the pluripotency genes use first?

- insert genes via virus into the genome which encode the Tfs involved in PSC formation. the idea that these would give rise to the proteins and these could stimulate the green pathways

15

what has the method of producing iPSC cells by introducing exogenous genes been successful in?

mice and humans

16

is the production of iPSCs efficient by using fibroblasts that are differeniated?

no

17

if making iPSCs from differentiated fibroblasts is inefficient, what is an alternative?

stem cells

18

what are the 4 difference ways of inducing the expression of the pTFs?

- plasmids- not insertional and not viral. This is less efficient because the ectopic genes are expressed for a shorter period of time
- viruses- will insert genes into the genome. But this also involves viral genes being inserted and can disrupt the genes in the genome because they are there forever and can cause oncogenic mutation.s. insertional methods are not good
- proteins- put directly or RNA direct into the parental cells
- small molecules - epigenetic modifiers to help ectopic expression of factors

19

do cells vary in their efficiency in being reprogrammed?

yes

20

how can yo evaluate if the cell is fully pluripotent? (2 main methods)

Molecular:
ü Morphology
ü Alkaline phsophatase assay ü Pluripotency markers
ü Retroviral silencing- not depend on ectopic genes you have put in
ü DNA methylation
ü Factor independence
Functional:
ü In vitro differentiation- try to differentiate into each germ layer
ü Teratoma formation- bal of cels that has spontaneously differentiated- look to see genes expressed in all three germ layers
ü Chimera development- inject into immnocompromised mouse and the cells will spontaneously differentiate into any lineages
ü Germline transmission
ü Tetraploid complementation

21

what needs to be considered when thinking of techniques of delivery of factors to cells? (2)

efficiency of reprogramming and quality of the resul?ng iPSCs (has no ectopic DNA in its genome- no risk of oncogenes or viral genes being reactivated or mutations)

22

how are retrovirus/ lentivirus vectors used?

table integraGon in the genome and activation of DNA & histones (process oeen incomplete, failure to activate endogenous genes, dependence on exogenous factors expression, residual ac?vity of viral transgenesàtumor formation)

23

how are integration free iPSCs formed?

IntegraGon-free iPSC: avoid (1) leaky transgene expression, (2) insertional mutagenesis.
ü Non-integra?ng vectors
ü Integrating vectors that can be removed
ü Not using acid-based vectors

24

what are three examples of integration free vectors?

adenovirus, sendai virus, polycistronic mini circle vectors: transient expression of OKSM is sufficient to produce iPSC.

25

what is the efficiency of non-integrating vectors? why is this?

0.001% compared to 0.1%-1% with integrating vectors. This is due to factor expression not being maintained for a sufficient length of time to allow complete epigenetic reprogrammin.

26

why is integration sometimes preferable to non integration vectors?

- because the timing of expression is longer- yamanaka says that time is very important

27

how can a vector that has the timing of expression of integrated vectors, but the lack of integration be produced? what are the pitfalls?

- integrating vectors that can be removed.
- contain sites that can be excised from the host genome
- this increases the efficiency
- pitfall:short vector sequences can remain in the host cell, which can affect cell function.

28

can you use a combination of chemicals and exogenous/ endogenous vectors?

- yes

29

what are chemicals the way to go?

when you use in combination the efficiency in increased- activate p53 pathway or those involved in metabolism, or the acetylation for unwinding etc.

30

in what cells have iPS cells been produced by using chemicals?

in the mice

31

what are the uses on the iPSC?

study mammalian development
study epigenetic reprogramming
therapeutic potential

32

how can iPSC cells be used in cell therapy?

- take cells from a diseased person which as a problem in a certain area but you can't take for example neurons from their brains- but you can make an iPSC and produce a neuron from it and then look to see whether the drugs can be used to treat these neurons.

33

how can iPSC be used for cell therapy?

you can ta take cells from a diseases person and make them iPSCs then repair the disease causing mutation and then differentiation them back and then transplant them back in

34

what are the limits or organ transplanation and how can iPSCs circumvent this?

- availability of matches tided and requirement of life long treatment with immunosuppressive drugs
- iPSCs can be differentiated into desired cell type
- iPSCs can be manipulated to repair disease- causing mutations by homologue recombination

35

describe an example in which iPSCs were used to treat a disease.

iPSCs to treat sickle cell anemia (results from a point mutation in the hemoglobin gene which renders the red blood cells nonfunctional)
Step 1: disease-causing mutation fixed in iPSCs from autologous skin cells
Step 2: coax the cells into blood-forming progenitors
Step 3: transplantation into anemic mice
Conclusion: the mice produced normal red blood cells and were cured of the disease

36

what is the study and treatment of many degenerative diseases limited by?

– the accessibility of the affected ?ssues.
– Inability to grow relevant cell types in culture for extended periods of ?me.

37

what is disease modelling?

iPSCs are derived from a pa?ent’s skin and differen?ated in vitro into the affected cell types, thereby recapitula?ng the disease in a Petri dish.
Useful to iden?fy novel drugs to treat the disease.
Example: drugs that prevent the death of motor neurons, or abnormal loss of insulin-producing β cells in diabe?c pa?ents.
Useful to recapitulate the early stages of the disease.

38

for what disease has iPCs been used for disease modelling?

iPSCs have been derived from pa?ents suffering from Parkinson’s disease, juvenile diabetes, Fanconi anemia, Down syndrome

39

what are the 4 challenges facing the use of iPSCs for cell therapy?

iPSCs form teratomas: risk of presence of residual undifferentiated cells (see positive and negative selection)
• Improve transgene-free approaches
• Develop more efficient targeting strategies to
repair mutant alleles
• Epigenetic memory of the donor cells: which one to use?

40

wha are the four challenges facing using iPSCs for drug development?

- it remains unclear whether multigenic diseases such as diabetes or alzheimers disease can be recapitulated in vitro within a few weeks- many diseases develop in a non cell autonomous way and involve interaction of different cell types
-Even though it should be possible, in principle, to derive all of the relevant cell types involved in disease from iPSCs, current differentiation strategies into functional cell types are inefficient and limited to a few tissues.

41

what should be kept in mind when seeking to understand development using in vitro differentiation of ES cells?

although they are derived from the ICM or from primordial germ cells, their growth and molecular characteristics are the product of tissue culture section for rapid in vitro proliferation, and this invariably will result in cells areepigenetically and biologically different from their corresponding cells of origin.

42

what is generally thought about the types of cells relative to rhe ease of reprogramming

the differentiation state of the donor cell affects the efficiency of producing cloned animals, with less differentiated cells being more amenable to epigenetic reprogramming. For example, the generation of cloned ES cells from neurons was less efficient than that from neural stem cells

43

what has been suggested about the type of egg that is able to reprogramme nuclei and what has bee argued against this idea?

- it has been suggested that only unfertilised oocytes can reprogramme nuclei and this poses a problem for human clinical application, as human unfertilised oocytes are hard to get. But in mice it was shown that an enunciated zygote could be used to reprogram

44

what are the 3 main strategies for reprogramming somatic cells? what re the problems with these

- nuclear transfer
- cell fusion: fusion of ES cells with somatic cells- very inefficient. tetraploidy of the reprogrammed cells presents a major shortcoming for using this approach for cus- tomized cell therapy.
- reprogramming by defined Tfs

45

how did yamanaka and takashi create iPSCs?

they sed retroviral infection to activate the 4 main TFs and then selected for those expressing the oct4 target gene- fb14

46

what evidence is there that c-myc may not be needed for reprogramming ?

, as both mouse and human iPS cells have been obtained in the absence of c-myc transduction, although with low efficiency

47

how can you look at DNA to determined reporogramming?

Global gene ex- pression and the chromatin configuration of Oct4 or Nanog- selected iPS cells were indistinguishable from those of ES cells.

48

how long is it suggested that the 4 factors need to be expressed in order to get iPSCs?

12 days

49

what is the problem with using c-myc in iPSC production?

can result in tumours and is oncogenic

50

what alternative combination of factors has been shown to produce IPSCs?

sox2 oct4 and lin 28

51

what 3 things have been uncovered about the way that oct4, sox2 ad nano work in the cell?

(1) Oct4, Sox2, and Nanog bind together at their own promoters to form an interconnected autoregulatory loop, This autoregulatory circuitry suggests that the three factors function collaboratively to maintain their own expression.
(2) the three factors often co-occupy their target genes, and (3) Oct4, Sox2, and Nanog collectively target two sets of genes, one that is actively expressed and another that is silent in ES cells but remains poised for subsequent ex- pression during cellular differentiation

52

how are pluripotency factors genrally thought to work ?

pluripotency factors gener- ally do not control their target genes independently, but rather act coordinately to maintain the transcriptional program required for pluripotency. They activate genes involved n puripotency and inhibit factors involved n differentiation

53

what Tfs did yamanaka use?

oct4, sox2, c-myc and klf4

54

where is iPSC research particulalry good?

iPS-cell technology is especially attractive for researchers in countries
in which the use of embryonic cells is restricted.

55

how have iPSCs been shown to be functional equivalent to ESCs?

Mouse iPS cells have been shown to be functionally equivalent to mouse ES cells3,19,20, as they express mouse ES-cell markers, have similar gene- expression profiles, form teratomas when injected into nude hosts and contribute to the cell types of chimeric animals, includ- ing the germ line.

56

what are the four main technical problems that need to be addressed?

he use of retroviral vectors to introduce reprogramming fac- tors into cells; the need to use a selection marker (either inserted into the starting cell by homologous recombination or included as part of the vector) to identify reprogrammed cells; the use of the onco- gene MYC to achieve reprogramming; and the integration of retroviral vectors into the genome.

57

how has the problem with selection criteria been addressed so far?

Two groups have developed morphological criteria for selecting mouse iPS cells without the use of a selection marker

58

why are iPSCs maybe better than ESCs in terms of differentiation lineages?

Acknowledgement of these lineage-specific differences is a reason for the establishment of stem-cell banks22. By contrast, iPS cells might offer the tantalizing possibility that their differentiation can be completely directed to any developmental lineage.

59

give an example of a disease that could not be treated by the iPSC therapy approach?

Cell therapy might not be effective in the context of an autoimmune disease such as type 1 diabetes (which would involve the use of replacement pancreatic β-cells), because
the transplanted cells may be destroyed
by the host immune response.

60

describe an example where disease modelling could be done using iPSCs

A good example would be Huntington’s disease32,
a neurodegenerative disease that is
caused by CAG repeats in the gene that encodes huntingtin protein. The process that leads to cell death by expression of polyglutamine-containing molecules remains unclear, despite the presence of various cellular and animal models. Part of the difficulty is the absence of an appropri- ate human experimental system. If iPS cells from patients could be induced to develop into the relevant types of neuron, it would be the optimal study material for investi- gating the process of neural degeneration induced by these extra-long CAG repeats. The only other way to produce such cells would be by somatic cell nuclear transfer,
a procedure that has not been successful in human cells. Neural cells from patients with Huntington’s disease would be important tools for searching for drugs that reduce the toxicity of polyglutamine

61

describe an interesting way that iPSCs could be used fro drug effect investigation

iPS cells can be used to examine the effects of known genotypes on the cell- ular phenotype. An important possible application comes from the fact that iPS cells can be generated from any human who is taking a medicine. Thus, any effect or lack of effect of a particular drug that is detected during clinical treatment can be re-analysed using iPS cells from patients. This would greatly facilitate studies such as those to monitor drug safety once drugs are on the market.

62

how can iPSCs be used to stuy development and how can this be clinically applied?

In type 1 diabetes, where it is necessary
to replace β-cells that are destroyed by disease, knowledge of the steps that are necessary to produce progenitor, precur- sor and functional β-cells would be crucial for developing replacement therapy.

63

how can iPSC be used to look at cancer?

Another area that might be ripe for iPS-cell technology is cancer research. By illuminating the genetic and epigenetic changes that occur during normal development, human ES cells
have provided opportunities to study the oncogenic process in a prospective manner. iPS-cell technology further expands what can be done: iPS cells can be derived from cancerous somatic cells to investigate the significance of the genetic background of the patient in the development of cancer. These data would contribute to the existing large datasets of genetic profiles of various cancers34, and could also be used in comparative analyses.

64

how can cancer cell studies be used to look at the development of iPSCs?

Cancer cells themselves would also be
a useful starting material from which to derive iPS cells. Previous attempts to repro- gramme cancer cells by nuclear transfer into unfertilized eggs showed that the gen- eration of ES cells from a nuclear transfer embryo is more difficult using cancer cell nuclei35 than normal somatic cell nuclei. Because of technical problems with nuclear transfer, the molecular basis underlying this difficulty remains uninvestigated. we now know that cancer cells inhibit iPS-cell induction, whereas normal cells do not. Here again the underlying mechanism is unknown. Given that cancer development involves the accumulation of mutations,
we could evaluate which genetic alterations in cancer cells inhibit iPS-cell induction.