OK, that is enough, I will concede the point about dedifferentiation to pluripotent extents in Amphibians, the interesting question though would be how they'd go about verifying if there was limited dedifferentiation involved or whether adult stem cells are involved, maybe engineer fluorescent proteins to be expressed in adult cells and see whether the same is true of blastema cells? (this would support the dedifferentiation hypothesis, if engineered adult cells gave rise to dedifferentiated blastema cells which in turn would express said fluorescent protein)

Thoughts?

I understand that the main concern of neurologists planning trials of Parkinson's Disease treatment with ESC is indeed the development of brain tumors.
For this reason, it is indeed good news that a summary can be found in today's issue of Medical News of an article published in Genes and Development. which reports that a gene has been identified that prevents stem cells from turning cancerous.

"Research associate Maria Garcia-Fernandez, Hermann Steller, head of the Strang Laboratory of Apoptosis and Cancer Biology, and their colleagues explored the activity of a gene called Sept4, which encodes a protein, ARTS, that increases programmed cell death, or apoptosis, by antagonizing other proteins that prevent cell death. ARTS was originally discovered by Sarit Larisch, a visiting professor at Rockefeller, and is found to be lacking in human leukemia and other cancers, suggesting it suppresses tumors. To study the role of ARTS, the experimenters bred a line of mice genetically engineered to lack the Sept4 gene....
The work supports the idea that the stem cell is the seed of the tumor and that the transition from a normal stem cell to a cancer stem cell involves increased resistance to apoptosis.
ARTS interferes with molecules called inhibitor of apoptosis proteins (IAPs), which prevent cells from killing themselves. By inhibiting these inhibitors, under the right circumstances ARTS helps to take the brakes off the process of apoptosis."

GenesForLife wrote:Thoughts?

My thoughts are that this thread has got way more technical than I can begin to understand.

GenesForLife wrote:

Ragsnoei wrote:

Actually, it's not even that unlikely and considering the fact that we are currently limited to only a few species from which stem cells have been generated (human, mouse, rat, rhesus monkeys, and one or two other simian (or 'higher primate') species), we still have a long way to go in terms of understanding the underlying processes.

We have, however, had success with inducing embryonic stem cells to differentiate into defined tissue types, the concerns I had were about the problems of guiding that differentiation in-vivo as opposed to predifferentiated (in-vitro) stem cell lines, the paper I am looking for (unsuccessfully so far) involved the failure of ESC transplantation for Parkinson's in China that ended up in the patient's death because a variety of tissue types had grown in his brain,

Yeah, just placing undifferentiated cells in an environment that supports the desired tissue type does not cause those cells to form said tissue type. In fact, recent findings suggest that embryonic stem (ES) cell cultures consist of a mix of populations of cells that have already obtained some of the characteristics of specific germ layers (see Toyooka Y, Shimosato D, Murakami K, Takahashi K, Niwa H (2008) Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135: 909–918.). So are all cells in a culture of ES cells really capable of forming all tissue types? It would seem that this is not the case, or at least that subsets of cells have a very strong inclination to form certain tissue types.

GenesForLife wrote:
There is evidence to suggest that regeneration as opposed to scar tissue may be down to the signalling processes involved in wound healing, this case might be of interest, although I haven't been able to track down the corresponding peer reviewed paper, but I'll let you hunt for it

I'll have a look if I can find something, but I seriously doubt that it will be as simple as turning a few genetic switches on or off to cause an amputee to regrow anything resembling the original structure. I haven't watched the video yet, but I will do as soon as I get a chance, I'm sure it'll aid my search.

GenesForLife wrote:

The genesis of cancer generally involves mutations in genes that regulate cell division and cause specialized cells, that are not very well equipped to divide so frequently, to start dividing much more. Because they're so ill-equipped, these cells then tend to accumulate more and more mutations because of mistakes made during the division process, which involves copying the entire genome. That's why most cancers are so difficult to treat, they consist of a 'mosaic' of all kinds of different cells with lots of different mutations, which allows them to adapt to medication quite quickly.

Correct, tumorigenesis requires escape from cell cycle controls, firstly, and immortalization, secondly, this is the reason it often needs to progress from dysplasia through to hyperplasia through to malignancy before it is truly dangerous, and in such cases, the process of metastasis which makes it so deadly again involves the positive somatic selection of cells which can grow quicker and traverse the body through various vascular and lymphatic channels, this requires a class of cell changes called Epithelial Mesenchymal Transitions in cases of all carcinomas.

Yes, but that's a very technical way of putting it, or should I say 'off-putting'? My motivation for using layman's language was to try and convey the message to our fellow forum users in a reasonably understandable way. Granted, I have no way of knowing that my section above is actually understandable to the general forum user, but I think you will be able to at least detect the attempt

GenesForLife wrote:
Regarding the difficulty with drugs and treatment, think of it as being driven by evolution in the same way as antibiotic resistance is driven, for instance, the extremely high mutation rate and the adaptive benefit conferred by the ability to either render drugs superfluous by evolving other routes to the same phenotypes (signalling cascades are a bit wonky, there is more than one way to a particular phenotype, for instance p53 knockout can be achieved either by mutations in p53, promoter methylation of p53, or mdm2 amplification) , there also is the problem of cells developing the ability to pump drugs out, MDR class of efflux pumps comes to mind. This would make for interesting reading at this juncture...

I must admit my knowledge on cancer, much like the signalling cascades involved, is a bit wonky... I'd say that the comparison to antibiotic resistance is not the best you could make though, as this usually involves exchange of plasmids between individual bacteria that can have quite a different genetic makeup, whereas cancer shows large variation, but that variation is mostly restricted to the genetic material it has to 'work' with and no large chunks of DNA can come to a cancer cell's aid upon exposure to a specific toxic substance (or medicine, if you will). Anyway, I can agree with the rest of your summary of the way this works, I just think we should try to avoid jargon, particularly since the OP seems to have gotten lost in translation:

rJD wrote:

GenesForLife wrote:Thoughts?

My thoughts are that this thread has got way more technical than I can begin to understand.

GenesForLife wrote:
And on a closing note, two things...

1) Check out cancer stem cells.
2) I found this paper free for download from Google Scholar, I will put up the abstract and the citation.

1) I know about them and occasionally gain some information on them from reviews on stem cells in general. The time required to really dive into the subject is lacking though and I must also admit that clinical application is of secondary importance to me personally, as I have gone into research for the fundamental biology. In other words, I just want to know what makes life 'tick', but if that tells us something about diseases in the process, I'm perfectly happy with that. Having said that, cancer stem cells do represent an interesting case in point as the derailing of a process can tell us a lot about the process itself. Anyway, thanks for the suggestion

2) Addressed in my last post, but since I hate it when people quote a list of points and then neglect to answer one of them I thought I'd mention it.

GenesForLife wrote:OK, that is enough, I will concede the point about dedifferentiation to pluripotent extents in Amphibians, the interesting question though would be how they'd go about verifying if there was limited dedifferentiation involved or whether adult stem cells are involved, maybe engineer fluorescent proteins to be expressed in adult cells and see whether the same is true of blastema cells? (this would support the dedifferentiation hypothesis, if engineered adult cells gave rise to dedifferentiated blastema cells which in turn would express said fluorescent protein)

Thoughts?

I'm quite sure the verification is in the works as we speak, the groups that did the initial research certainly seem competent and motivated enough to pull it off. Although your idea could be a good way of trying to find out, I suspect it will be very difficult, as engineering adult stem cells and reintroducing them into the right location gives no guarantee of accurately representing the situation as it is in the animal when it loses a limb. Also, adult stem cells are much more difficult to isolate and manipulate than ES cells... Now that we've found ways of inducing dedifferentiation though, one would think that a way to block dedifferentiation might not be so far off and if we can do that, we can easily distinguish the contribution(s?) of blastema cells that require dedifferentiation and/or adult stem cells that expand (create a lot more adult stem cells by rapidly dividing) and then differentiate into the required tissue types.

Ooh, your solution is elegant, maybe just block homologues of the four transcription factors in question? I imagine it shouldn't be too hard designing morpholinos/LNA/siRNA to achieve results to that end

Regarding my proposal, why not link the fluorescent protein to cell specific promoters and introduce them by a viral vector or so in order to avoid the extraction - reintroduction problem?

I also wonder if looking at expression profiles in blastema cells using Comparative Genome Hybridization may reveal anything of value.

Ragsnoei wrote: My motivation for using layman's language was to try and convey the message to our fellow forum users in a reasonably understandable way.

Much appreciated!

I know it is often much more economical to use the jargon - it's short, precise and effective, and moreover it's a habit that is difficult to shake off. I know from my own experience how difficult it can be to not use it. So I'm very grateful when people make the effort to translate their highly technical language into layspeak. Even if something is lost in the process (the precision), surely much more is gained (wider understanding of the topic).

Granted, I have no way of knowing that my section above is actually understandable to the general forum user, but I think you will be able to at least detect the attempt

As a layperson - very much so - I can say that it at least seemed to be understandable. Granted, I have no way of knowing that I actually understood everything correctly.

GenesForLife wrote:Ooh, your solution is elegant, maybe just block homologues of the four transcription factors in question? I imagine it shouldn't be too hard designing morpholinos/LNA/siRNA to achieve results to that end

Again, the idea is basically good, but you will find that in this case it won't work. The reason is that the four transcription factors are also involved in the partial dedifferentiation that occurs in blastema cells (Christen et al., BMC Biol, 2010). It seems that it's not so much the presence or absence of these factors that matters, but more the level at which they are expressed, which is why they are often referred to as molecular rheostats (rheostat = a variable resistance electric device that can be adjusted to control the strength of a current) that change to controlling different sets of target genes based on the level at which they are expressed. In this way, these factors can control a wide range of processes in many different cells, but at the same time it is very hard to manipulate what they do other than turning them on (for example by introducing viral vectors like Takahashi did in the paper you mentioned) or off (using for example the techniques mentioned in the above quoted post) in cells that would normally express them at intermediate levels. For reference, see for example Niwa et al., Nat Genet, 2000.

By the way, that Takahashi paper you cited is very famous for being the first to show how normal cells can be converted to pluripotent cells, but since then, a lot of progress has been made. For example, we are now able to reprogram cells by introducing mRNA in stead of viral vectors (which are potentially dangerous if you intend to place cells back into a patient), see Warren et al., Cell Stem Cell, 2010, and this is being done at a much higher efficiency.

GenesForLife wrote:Regarding my proposal, why not link the fluorescent protein to cell specific promoters and introduce them by a viral vector or so in order to avoid the extraction - reintroduction problem?

I also wonder if looking at expression profiles in blastema cells using Comparative Genome Hybridization may reveal anything of value.

Unfortunately, cell-specific promotors tend to be very inefficient in such protocols, as cells possess mechanisms to turn any introduced copies of a gene off (I don't have a reference for this, as it's a bit hard to find, but I will again refer you to the Takahashi paper; check out the efficiency of the procedure and then imagine applying that to cells that cannot be grown for prolonged periods because they do not have the self-renewing ability that ES cells have. Also, even if such cells could be generated, one would have to place these cells into the recipient organism at exactly the right time to make it work. I think there are more efficient ways of distinguishing between partial dedifferentiation of blastema cells or contribution of adult stem cells.

Comparative Genome Hybridization is a technique specifically used to detect changes in copy number of DNA and since this would not apply to these cells, I doubt it would provide significant contribution. If you could find antibodies that will specifically recognize blastema cells in the process of dedifferentiation or adult stem cells though, you could compare them using similar high-throughput techniques, like ChIP-on-chip or ChIP-seq.

katja z wrote:

Ragsnoei wrote: My motivation for using layman's language was to try and convey the message to our fellow forum users in a reasonably understandable way.

Much appreciated!

I know it is often much more economical to use the jargon - it's short, precise and effective, and moreover it's a habit that is difficult to shake off. I know from my own experience how difficult it can be to not use it. So I'm very grateful when people make the effort to translate their highly technical language into layspeak. Even if something is lost in the process (the precision), surely much more is gained (wider understanding of the topic).

Granted, I have no way of knowing that my section above is actually understandable to the general forum user, but I think you will be able to at least detect the attempt

As a layperson - very much so - I can say that it at least seemed to be understandable. Granted, I have no way of knowing that I actually understood everything correctly.

That's good to know, thanks.

My bad, I got my methods mixed up, :blush: it would have to be normal microarrays then.

Regarding the exogenous DNA silencing issue, Ragnoei, I think someone did come up with a solution involving some specific RNA sequences or something, I will look through my archive to see if I can find that paper.

Cannot locate that paper as of now, but I found this...

Preventing gene silencing with human replicators

Haiqing Fu1,3, Lixin Wang1,3, Chii-Mei Lin1, Sumegha Singhania1, Eric E Bouhassira2 & Mirit I Aladjem1
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Transcriptional silencing, one of the major impediments to gene therapy in humans, is often accompanied by replication during late S-phase. We report that transcriptional silencing and late replication were prevented by DNA sequences that can initiate DNA replication (replicators). When replicators were included in silencing-prone transgenes, they did not undergo transcriptional silencing, replicated early and maintained histone acetylation patterns characteristic of euchromatin. A mutant replicator, which could not initiate replication, could not prevent gene silencing and replicated late when included in identical transgenes and inserted at identical locations. These observations suggest that replicators introduce epigenetic chromatin changes that facilitate initiation of DNA replication and affect gene silencing. Inclusion of functional replicators in gene therapy vectors may provide a tool for stabilizing gene expression patterns.

http://www.nature.com/nbt/journal/v24/n ... t1202.html

I also found this

Introduction of genetic material into an organism or vector construct and proper expression of that material is critical to cellular reprogramming approaches. The lack of stable expression of these transgenes in target cell lines remains a serious problem for researchers. Once integrated into chromosomes, expression may be regulated by various position effects associated with the surrounding chromatin that are capable of inhibiting gene expression and neutralizing the intended effect of the inserted transgene.

Experimental results suggest that gene position effects can be partially overcome by flanking the transgene with regulatory elements called chromatin insulator. These insulators can overcome position effects by shielding the promoters from the influence of neighboring regulatory elements, or by preventing the spread of heterochromatin which can lead to subsequent gene silencing.

This invention discloses the use of gamma satellite DNA as highly efficient chromatin insulators that have a remarkable ability to overcome position effects and prevent the silencing of transgenes. Stable transgene expression was recorded for well over eight months when human chromosome 8 gamma satellite sequences were used as flanking DNA in mouse cells. Until recently, no chromatin insulator sequences were known to completely prevent gene silencing on a long term basis in transfected cells. The human gamma-satellite sequences demonstrate a higher efficiency than any known chromatin insulator identified so far in intergenic regions, and may have invaluable applications in the fields of gene therapy, protein expression, and cellular reprogramming where adequate expression of the transgene is essential for long term therapeutic or developmental success.

http://ttc.nci.nih.gov/opportunities/op ... opp_id=744