ES and iPS cells

Question 1

Where do ES cells come from?

Answer 1

originate from the inner cell mass of the early embryo (the cluster of cells that give rise to the body of the embryo proper, as opposed to extraembryonic structures), and given appropriate culture conditions they will divide indefinitely

Question 2

Why are ES cells pluripotent and not totipotent?

Answer 2

Their only limitation is that they do not give rise to extraembryonic tissues such as those of the placenta. Thus they are classified as pluripotent, rather than totipotent.

Question 3

When were ES cells first described?

Answer 3

ES cells first described in 1981

Question 4

What happens when you take blastocyst cells from one mouse and inject it into another one?

Answer 4

we knew that during the development of a mouse we would reach the stage of a blastocyst and from the bastocyst; we also knew thast if we took the cells from the blastocyst and injected it in situ to other mice we would get a teratocarsinoma

Question 5

What needs to be done to prove that you have pluripotent cells?

Answer 5

• they set a standard for the field of what has to be done to prove that you have pluripotent cells
  
  • you need to prove that you can do in vitro differencation that gives you all three layers of the embryo
  • teratoma formation
  • chimeric mice

Question 6

Do we really have ES cells in an embryo?

Answer 6

• ES cells don’t really exist in the embryo
• the pluripotency stage is very transient in the emrbyo

Question 7

How were ES cells cultured in the past?

Answer 7

• initially the cells were cultured in embryonic bodies (you ut cells in a drop on a petri dish, you turn them upside down and they basically hang in there); however this is very laborious and difficult to do
• it was important to find a way to culture the cells in 2D on plastic so that required the presence of feeder cells (for example embryonic fibroblasts) which we put on petri dish for them to generate a mass of factors and then that would keep the ES cells in the pluripotent cells

Question 8

Why are feeder cells annoying to work with?

Answer 8

feeder cells are a little bit annoying cuz they have to be the same kind as the ES cells and that pushed the field to find out what the factors that they secreted were

Question 9

What do mouse ES cells require to stay ES cells?

Answer 9

• mouse ES cells require LIF (leukimia inhibitory factor) or 3i medium (three inhibitors). they inhibit signalling of :
  
  • GSK beta - involved in many developmental events in the brainsuch as neuronal migration and differentiation
  • ERK/MEK- survival, proliferation and differentiation
  • FGFR-TK - proliferation, differentiation and survival

Question 10

What is nuclear transfer?

Answer 10

nuclear transfer - you take the nucleus of the egg out of the egg and you take the nucelus of some other differentiated cell and put it in the denucleated egg which then gives you a morula and then a blastocyst

Question 11

How were the factors characteristic to embryonic stem cells identified?

Answer 11

• he used Fbx15 as a marker of pluripotence in the ES cells (the marker is not essential to the cells existence so it is truly just a marker)
• he got 24 facotrs that could be important for the ES cells and he got cells that were very similar to the ES cells
• he then proceeded to examine which ones of the 24 facotrs were truly important for the ES cells
  he was making libraries of the factors - one of them so when the pluriotent cells were still generated the missing factor was not that important

Question 12

What are the four factors responsible for pluripotency of cells?

Answer 12

• c-Myc
• Sox2
• Oct4
• KLF4

Question 13

What is the difference between ES and iPS cells?

Answer 13

• when he looked at the histone marks of the ES and IPS and MEF cells he realised that they were quite similar but when he looked at the methylation of Oct3/4 and Nanog the results were not that clear. MEF cells were quite heavily methylated while ES cells were almost completely unmethylated. IPS cells were somewhere in between → IPS cells are not exctly the same as ES cells
• then the science improved, the culture conditions were better and now IPS cells are almost identical to ES cells
• then he did some transcriptomics and showed that iPS cells were more similar to ES cells than to MEFs

Question 14

How can ES cells avoid senescence so effectively?

Answer 14

ES cells must avoid senescence - fibroblasts and many other types of somatic cells are limited in the number of times they will divide, in part at least because they lack telomerase activity, with the result that their telomeres become progressively eroded in each division cycle, leading eventually to cell-cycle arrest. ES cells, by contrast, express high levels of active telomerase, allowing them to escape senescence and continue dividing indefinitely

Question 15

How effective is induction of iPS cells?

Answer 15

• Only a few of the cells that receive the OSKM factors will actually become iPS cells
• Conversion to an iPS character by the OSKM factors is not only inefficient but also slow: fibroblasts take ten days or more from introduction of the conversion factors before they begin to express markers of the iPS state
• changes are being extensively studied, and they affect both the expression of individual genes and the state of the chromatin.

Question 16

Describe the process of induction of iPS cells

Answer 16

The process begins with a Myc-induced cell proliferation and loosening of chromatin structure that promotes the binding of the other three master regulators to many hundreds of different sites in the genome. At a large proportion of these sites, Oct4, Sox2, and Klf4 all bind in concert. The binding sites include the endogenous Oct4, Sox2, and Klf4 genes themselves, which eventually creates the types of positive feedback loops just described that makes expression of
these genes self-sustaining
- The three core factors activate some target genes and repress others, producing a cascade of effects that reorganize the gene control system globally and at every level, changing the patterns of histone modification, DNA methylation, and chromatin compaction, as well as the expression of innumerable proteins and noncoding RNAs.

Question 17

What happens at the end of iPS induction

Answer 17

By the end of this complex process, the resulting iPS cell is no longer dependent on the artificially generated factors that triggered the change: it has settled into a stable, self-sustaining state of coordinated gene expression, making its own Oct4, Sox2, Klf4, and Myc (and all the other essential ingredients of a pluripotent stem cell) from its own endogenous copies of the genes.

Question 18

What can we deduce from the fact that iPS induction is not the most effective process

Answer 18

• The low efficiency and slow rate of conversion suggest that there is some barrier blocking the switch from the differentiated state to the iPS state in these experiments, and that overcoming this barrier is a difficult process that involves a large element of chance.
• From our discussion of nuclear transplantation, one might expect that any reprogramming of a differentiated cell would require a radical and widespread change in the chromatin structure of selected genes. Not only are such changes observed, but a large number of different experiments reveal that the efficiency of the reprogramming process can be substantially increased by altering the activity of proteins that affect chromatin structure

Question 19

What happens if you implant iPS/ES cells into an older embryo or an adult?

Answer 19

if you implant iPS cells or ES cells into an embryo or adult at a later stage then they will not get appropriate signals to differenciate into something We can think of embryonic development in terms of a series of choices pre- sented to cells as they follow a road that leads from the fertilized egg to terminal differentiation. After their long sojourn in culture, the ES cells or iPS cells and their progeny can still read the signs at each branch in the highway and respond as normal embryonic cells would.

Question 20

Hw can we manipulate iPS cells to differentiate to what we want

Answer 20

• In culture, by exposing the ES or iPS cell to an appropriate sequence of signal proteins and growth factors, delivered with the right timing, it is possible to guide the cell along a pathway that approximates a normal developmental pathway, so as to convert it into one of the standard specialized adult cell types

Question 21

Can we not convert cell A to cell B instead of going through the iPS?

Answer 21

Could we not convert cell type A into cell type B directly, without backtracking to the embryonic-like
iPS state? For many years, it has been known that such transdifferentiation can be achieved in a few special cases, such as the conversion of fibroblasts into skeletal muscle cells by forced expression of MyoD (see p. 396). But now, with the insights that have come from the study of ES and iPS cells, ways are being found to bring about such interconversions in a much wider range of cases.

Question 22

Give an example transdifferentiation

Answer 22

• An elegant example comes from studies of the heart. By forcing expression of an appropriate combination of factors—not Oct4, Sox2, Klf4, and Myc, but Gata4, Mef2c, and Tbx5—it is possible to convert heart fibroblasts directly into heart muscle cells. This has been done in the living mouse, using retroviral vectors, and the transformation occurs with high efficiency when the vectors carrying the transgenes are injected directly into the heart muscle tissue itself.
• Although they occupy only a small fraction of the tissue volume, the fibroblasts in the heart out- number the heart muscle cells, and they survive in large numbers even where the heart muscle cells have died. Thus, in a typical nonfatal heart attack, where heart muscle cells have died for lack of oxygen, the fibroblasts proliferate and make collagenous matrix so as to replace the lost muscle with a fibrous scar. This is a poor sort of repair. By forcing expression of the appropriate factors in the heart, as described above, it has proved possible, in the mouse at least, to do better than nature and regenerate lost heart muscle by transdifferentiation of heart fibro- blasts.

Question 23

Are NSCs easier to reprogram?

Answer 23

NSC have more Sox2 and cMyc than most other somatic cells and can be reprogrammed by adding Oct4 and Klf4 only

Question 24

How can we use iPS cells in studies of genetic diseases, give an example

Answer 24

• Where a disease has a genetic cause, we can derive iPS cells from sufferers and use these cells to produce the specific cell types that malfunction, to investigate how the malfunction occurs, and to screen for drugs that might help to put it right.
• Timothy syndrome provides an example. In this rare genetic condition, there is a severe, life-threatening disorder in the rhythm of the heart beat (as well as several
  other abnormalities), as a result of a mutation in a specific type of Ca2+ channel. To study the underlying pathology, researchers took skin fibroblasts from patients with the disorder, generated iPS cells from the fibroblasts, and drove the iPS cells to differentiate into heart muscle cells. These cells, when compared with heart muscle cells prepared similarly from normal control individuals, showed irregu-
  lar contractions and abnormal patterns of Ca2+ influx and electrical activity that could be characterized in detail. From this finding, it is a small step to development of an in vitro assay for drugs that might correct the misbehavior of the heart muscle cells.

Question 24

How does reprorgamming really happen?

Answer 24

A Lot of chromatin needs to be reorganised and it doesn’t always work out fine
because you can start from almost any cell there is a lot of chaos invilved at the begining of reprograming but once the cells reach a certain state the process becomes much more organised

Question 25

Compare ES cells and adult stem cells

Answer 25

• ES cells
  
  • pluripotent (contribute to all germ layers)
  • easily cultured
  • in vtro they tend to differentiate into all kinds of tissues
  • in vivo they exist only transiently
• adult stem cells
  
  • multi/unipotent (contribute to the tissue they reside in)
  • many are impossible or hard to culture
  • in vitro, they tend to differentiate into one or few lineages
  • in vivo they persist throughout the life of the organism (and they age with it)

Question 26

What are the advantages of using ES cells?

Answer 26

• embryonic stem cells have the potential to replicate indefinitely under defined conditions without differentiation, making an “off the shelf” preparation possible.
• cells are able to form many committed cell types for regenerative tissue repair when differentiated prior to implantation
• minimal genetic manipulation of ES cells and there is a consequently decreased risk of aberrant tumor formation compared to iPS cell lines.

Question 27

What are the disadvantages of using ES cells?

Answer 27

• disadvantages
  
  • Routinely utilized methods for inner cell mass isolation necessitate destruction of an embryo, leading to ethical concerns across species, but especially in human ES cell research.
  • potential for allogenic immunogenicity
  • need for specific, complicated culture conditions to propagate and maintain ES cell lines in an undifferentiated state and the requirement for frequent monitoring of cultured cells for changes in genomic state to assure phenotypic stability.
  • there is a risk for tumor formation if ES cells are not fully directed into a differentiated cell type prior to surgical implantation

Question 28

What are the advantages of using iPS cells?

Answer 28

• less prone to immunorejection since they can be patient-derived or MHC class I-matched for compatibility.
• Production of iPS cell lines also avoids the ethical controversy of embryo destruction associated with ES cell generation

Question 29

What are some disadvantages of using iPS cells?

Answer 29

• the use of viral vectors (especially retro or lenti viruses that randomly incorporate into the host genome) for iPS colony generation increases the risk of tumor formation and leads to concern for transplantation of reprogrammed cells in clinical trials
• iPS cells can also show dramatic variability in the completeness of reprogramming and require extensive screening to select the most ES-like cells