How do you think their results would have changed, if at all, if they had used a different cell type (here they used a fibroblast)? Would it be logical that cellular reprogramming to pluripotency would be the same for all cell types?
Why wasn't Snail (a "key transcription factor" for EMT as the authors proclaim) detectable in their analysis? Does the discrepancy lie in the way they developed their analysis or is it for some other reason?
This study identified that many proteins involved in changing MEF cells to iPSCs have opposing results in protein expression between the first 3 days of reprogramming and the final 3 days. Why do most of the proteins studied stay fairly constant during reprogramming in the intermediate stages from day 3 to day 12?
This study states that in the intermediate stages of iPSC generation, protein folding in the ER, processing in the Golgi, and protein transport are inhibited. Does the down-regulation of these processes during this time of large-scale cellular change seem contradictory? Do other findings in this study support these inhibition of protein processing results?
Many proteins related to cell proliferation are upregulated in the first few days of reprogramming. What role does cell proliferation play in reprogramming? Do cells divide at certain time points as they are being reprogrammed? Or is there a fairly constant rate of cell proliferation throughout the 15 day reprogramming process?
I'm a bit confused by the results in the primary article concerning the metabolic switch to glycolysis. The researchers note that there is an increased dependence on glycolysis rather than oxidative phosphorylation in iPSCs and I'm just wondering why this is the case given that glycolysis only produces 2 ATP/glucose and oxidative phosphorylation gives much more than that. Additionally, what do you think is the purpose of the shift in the electron transport chain's complex expression and its correlation to the efficiency of oxidative phosphorylation as a whole?
In the primary article by Hansson et al. why did the authors choose to focus on Nup210? Was it because it had recently been shown to function in differentiation? I would assume that many proteins are essential for reprogramming so was this a proof of principle that could be applied to other proteins in their data set?
In the primary article, the authors mention that it is useful to study proteomic changes in order to identify ways that the process of making iPSCs could be more efficient. It seems like they unearthed a lot of information that might be useful, although I'm wondering how they might begin to pare this information down to the steps most important for influencing pluripotency? In other words, how can they use the data they collected to improve the process of making iPSCs in the future?
The authors state that they looked at the proteome at intermediate stages of pluripotency progression to see if they could elucidate reprogramming mechanisms (page 1587). However, earlier in the paper they stated that the biggest changes in the proteome were observed in the first and last 3 days (pg 1580). Can we really understand anything about the reprogramming mechanisms from intermediates if not much appears to be happening in the middle of reprogramming? Or am I misunderstanding what they meant by "intermediates"?
The article states that extreme (max or min) protein levels are during the intermediate phases of the cell cycle to create iPSC. Do you believe that these proteins are more important for pluripotency than the early and late stage proteins, since they are more consistent and last longer within the cells?
Would you please briefly explain the FACS method that was used? Also, do you think any of the methods used could have caused the protein expression of the cells to be different from what it would be under normal circumstances?
In the paper, there were big differences between the first 3 days of reprograming and the final step after day 12. Was it because major reorganization of the proteome occurs during the first 3 days and final step after day 12 of reprograming while subtle changes occur in the intermediate phase Could you clarify how there were differences?
The article stated that the major reorganizations that occur take place during the first 3 days of reprogramming and after day 12 as well, while the other days show more subtle changes occur in the intermediate phase. Is there a key reason that this occurs during these times or is it simply because more happens at the beginning and the end. I feel like there has to be more of a reason otherwise evolutionarily the process would occur more quickly.
When they categorized their proteins into eight different clusters, they noted that proteins of cluster two showed and overrepresentation of mitochondria. It makes sense to me why mitochondria would be important for reprogramming, but why isn't this present in the other seven clusters.
It was stated that among the genes that are upregulated upon
Nup210 knockdown during cellular differentiation,
most of the corresponding proteins in their data showed an
upregulation during reprogramming. Why does this finding suggests
that Nup210 may have opposing effects on the regulation of
target genes depending on cellular context? What opposing effects, and depending on what cellular context?
In the introduction, the authors suggest that this data might become useful for streamlining the process for creating iPSCs. The conclusion did not return to this point. Are there any points in the process of inducing pluripotency that could be improved upon after the generation of the data?
While reading the review, I wondered how sensitive mass spectrometry is and how accurate readings are if it isn't. If such small deviations are indicative of reprogramming, it seems as though many mistakes could be made during an analysis.
I found it very interesting that research in the primary Hansson et al. paper was conducted on fibroblasts, with no qualifications within the paper as to why fibroblasts were the appropriate substrate for the study. Why did the researchers use this particular cell type and what differences could potentially arise if the same studies were to be performed using different cell types?