On 2014 Nov 25, Hongkui Deng commented:

We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?

[reply] When we started to screen for Oct4 replacers, we didn’t know at that time which small molecules would ultimately be used in chemical reprogramming.

Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)

[reply] In our experiments, we used pOct4-GFP (GOF18) transgenic mice, but not endogenous knockin reporter mice. It is possible that the transgenic reporter may not accurately indicate the endogenous expression of Oct4, which was also reported in other studies (Cell. 2010 143:617-27; Cell Stem Cell. 2008; 3:603).

We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?

[reply] In the initial screen, we used a viral transduction system to introduce the DOX-inducible expression of Oct4 (Maherali et al., Cell Stem Cell, 2008) in OG-MEFs. To make the Oct4 expression level stable from batch to batch, we then used the transgenic mice with inducible Oct4 expression (Hochedlingeret al., Cell, 2005). These two Oct4-inducible systems were used to screen different small molecule libraries.

We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?

[reply] Before we found DZNep, we didn’t know whether a single additional small molecule was sufficient to induce CiPSCs in together with these small molecules. At that time, we only intended to screen for small-molecule candidates that facilitate late stage reprogramming.

What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?

[reply] We presented detailed data about concentrations titration in Figure S7A and listed the concentrations that we used in Table S1B. The optimized duration was also shown in figure S7B-C. In the main text, limited by the space, we did not state the optimized concentrations and durations of the small molecules. But the results were clearly presented in these figures.

Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?

[reply] We found FSK was required in stimulating Sall4, as shown in figure S24B. There is no evidence that FSK acts directly at the Oct4 promoter, as FSK could not induce pOct4-GFP positive cells directly.

How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?

[reply] The efficiency was calculated according to Yamanaka’s method, by which the number of cell colonies was counted and divided by the number of cells plated (Takasashi et. al., 2007). In addition, I recommend a review paper which carefully discussed how to calculate the reprogramming efficiency: “Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells”, Nimet Maherali and Konrad Hochedlinger, Cell Stem Cell, 2008. We followed these principles: (1) calculation of plating efficiency, which can be done via cell counts or single-cell plating; (2) eliminating the count of sister clones through single-cell plating; and (3) use of a reliable and stringent method to identify and quantify iPSC colonies. In our study, we only calculated the numbers of CiPSC colonies, characterized by 2i-competent, ESC-like in morphology, and GFP-positive.

Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?

[reply] We have tested these small molecules on human fibroblasts, and found that they were not sufficient to induce pluripotency. Maybe some different small molecule combinations are needed for human cells.

Did you evaluate any off-target effects of these compounds?

[reply] For the two novel small molecules FSK and DZNep, we have revealed their potential targets in CiPSC induction (Fig. S17 and S18). We did not further evaluated their off-target effects.

We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

[reply] The original sentences in our paper are: “To better understand the pluripotency-inducing properties of these small molecules, we profiled the global gene expression during chemical reprogramming and observed the sequential activation of certain key pluripotency genes, which was validated by real-time PCR and immunofluorescence (fig. S21). The expression levels of two pluripotency-related genes, Sall4 and Sox2, were most significantly induced in the early phase in response to VC6TF, as was the expression of several extra-embryonic endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and figs.S22 to S24).”

In the sentence referenced in figure 21, we only intended to show the global gene expression profiling data during chemical reprogramming and the initial observation of the sequential activation of certain key pluripotency genes. Actually, “which was validated by real-time PCR and immunofluorescence” was an attributive clause that modifies the initial observations, and is redundant with the next sentence, where the detailed validation results by real-time PCR and immunofluorescence was stated, as referenced to Fig. 4, D to F, and figs.S22 to S24. As the two sentences were written together, we thought the figure citations are not necessary to be duplicated in these two sentences.

On 2013 Oct 23, Gholson J Lyon commented:

We reviewed this paper for our lab journal club and we were left with a few questions. We decided to post these questions here in case others have the same questions and/or in case the authors feel like responding to any of them. We posted the same thing to PubPeer.

1. We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?
2. Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)
3. We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?
4. We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?
5. What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?
6. Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?
7. How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?
8. Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?
9. Did you evaluate any off-target effects of these compounds?
10. We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

Anyway, we thought we would post our questions and comments here, in case others in the world read this paper and have similar questions. We would of course welcome answers to any of our questions from the authors.

On 2013 Oct 23, Gholson J Lyon commented:

We reviewed this paper for our lab journal club and we were left with a few questions. We decided to post these questions here in case others have the same questions and/or in case the authors feel like responding to any of them. We posted the same thing to PubPeer.

1. We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?
2. Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)
3. We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?
4. We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?
5. What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?
6. Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?
7. How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?
8. Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?
9. Did you evaluate any off-target effects of these compounds?
10. We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

Anyway, we thought we would post our questions and comments here, in case others in the world read this paper and have similar questions. We would of course welcome answers to any of our questions from the authors.

On 2014 Nov 25, Hongkui Deng commented:

We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?

[reply] When we started to screen for Oct4 replacers, we didn’t know at that time which small molecules would ultimately be used in chemical reprogramming.

Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)

[reply] In our experiments, we used pOct4-GFP (GOF18) transgenic mice, but not endogenous knockin reporter mice. It is possible that the transgenic reporter may not accurately indicate the endogenous expression of Oct4, which was also reported in other studies (Cell. 2010 143:617-27; Cell Stem Cell. 2008; 3:603).

We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?

[reply] In the initial screen, we used a viral transduction system to introduce the DOX-inducible expression of Oct4 (Maherali et al., Cell Stem Cell, 2008) in OG-MEFs. To make the Oct4 expression level stable from batch to batch, we then used the transgenic mice with inducible Oct4 expression (Hochedlingeret al., Cell, 2005). These two Oct4-inducible systems were used to screen different small molecule libraries.

We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?

[reply] Before we found DZNep, we didn’t know whether a single additional small molecule was sufficient to induce CiPSCs in together with these small molecules. At that time, we only intended to screen for small-molecule candidates that facilitate late stage reprogramming.

What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?

[reply] We presented detailed data about concentrations titration in Figure S7A and listed the concentrations that we used in Table S1B. The optimized duration was also shown in figure S7B-C. In the main text, limited by the space, we did not state the optimized concentrations and durations of the small molecules. But the results were clearly presented in these figures.

Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?

[reply] We found FSK was required in stimulating Sall4, as shown in figure S24B. There is no evidence that FSK acts directly at the Oct4 promoter, as FSK could not induce pOct4-GFP positive cells directly.

How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?

[reply] The efficiency was calculated according to Yamanaka’s method, by which the number of cell colonies was counted and divided by the number of cells plated (Takasashi et. al., 2007). In addition, I recommend a review paper which carefully discussed how to calculate the reprogramming efficiency: “Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells”, Nimet Maherali and Konrad Hochedlinger, Cell Stem Cell, 2008. We followed these principles: (1) calculation of plating efficiency, which can be done via cell counts or single-cell plating; (2) eliminating the count of sister clones through single-cell plating; and (3) use of a reliable and stringent method to identify and quantify iPSC colonies. In our study, we only calculated the numbers of CiPSC colonies, characterized by 2i-competent, ESC-like in morphology, and GFP-positive.

Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?

[reply] We have tested these small molecules on human fibroblasts, and found that they were not sufficient to induce pluripotency. Maybe some different small molecule combinations are needed for human cells.

Did you evaluate any off-target effects of these compounds?

[reply] For the two novel small molecules FSK and DZNep, we have revealed their potential targets in CiPSC induction (Fig. S17 and S18). We did not further evaluated their off-target effects.

We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

[reply] The original sentences in our paper are: “To better understand the pluripotency-inducing properties of these small molecules, we profiled the global gene expression during chemical reprogramming and observed the sequential activation of certain key pluripotency genes, which was validated by real-time PCR and immunofluorescence (fig. S21). The expression levels of two pluripotency-related genes, Sall4 and Sox2, were most significantly induced in the early phase in response to VC6TF, as was the expression of several extra-embryonic endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and figs.S22 to S24).”

In the sentence referenced in figure 21, we only intended to show the global gene expression profiling data during chemical reprogramming and the initial observation of the sequential activation of certain key pluripotency genes. Actually, “which was validated by real-time PCR and immunofluorescence” was an attributive clause that modifies the initial observations, and is redundant with the next sentence, where the detailed validation results by real-time PCR and immunofluorescence was stated, as referenced to Fig. 4, D to F, and figs.S22 to S24. As the two sentences were written together, we thought the figure citations are not necessary to be duplicated in these two sentences.