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Evaluation of Stuart et al.: Distinct Molecular Trajectories Converge to Induce Naive Pluripotency
Cell Stem Cell
Peer Review Evaluation of Stuart et al.: Distinct Molecular Trajectories Converge to Induce Naive Pluripotency Matthias Stadtfeld1,* 1Department of Medicine, Weill Cornell Medicine, New York, NY 10021, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2019.08.009
An example of the peer review process for ‘‘Distinct molecular trajectories converge to induce naive pluripotency’’ (Stuart et al., 2019) is presented here. Editor’s note: These are the first-round reviewer comments provided to the authors of ‘‘Distinct molecular trajectories converge to induce naive pluripotency’’ from Silva and colleagues. They were written for Cell Stem Cell during the first round of the peer review process and have been lightly edited for clarity. We chose to highlight this review since it focuses on ensuring that the main points of the paper are appropriately supported by the underlying data and that different aspects of the analyses are mechanistically reconciled, together helping readers understand how different molecular trajectories from the same starting point can result in a common pluripotent end state. The authors submitted a revised version of the manuscript that addressed the reviewers’ comments, and that was re-reviewed, accepted for publication, and published in the September 2019 issue of Cell Stem Cell. Overview and Summary In their manuscript, Stuart et al. compare conversion trajectories of primed mouse pluripotent stem cells into a naive state when driven by different individual transcription factors (TFs). This is a well-established and suitable experimental model, and the authors provide largely convincing data that different TFs trigger unique trajectories. Further investigation reveals specific molecular requirements of these trajectories and provides intriguing evidence for an important role of OCT4 levels in sustaining the primedto-naive conversion. While several of the authors’ observations are novel and likely will be of broad interest for the stem cell community, some key conclusions are not fully supported by their experimental
evidence and a stronger mechanistic basis for their findings should be established. Strengthening Characterization of the Cellular Starting, Intermediate, and End States to Understand Distinct Trajectories The initial observation that iEsrrb, iKlf2, and iPStat3 EpiSCs achieve naive pluripotency via distinct molecular trajectories is interesting and overall convincing. However, due to the central importance of this conclusion to the authors’ claims, their inducible transgene approach should be characterized more thoroughly. Is a similar level of induction (in terms of % expressing cells and levels of expression) achieved with all inducible transgenes, including those that are inefficient at inducing reprogramming? What is the time course of exogenous protein induction (this is relevant for interpretation of the data discussed on the bottom of page 10 and shown in Figure 4F)? Do ‘‘iPSCs’’ derived with iEsrrb, iKlf2, and iPStat3 differ in their molecular signatures (Figures 1H and S1A suggest similarity but do not rule out reproducible and significant differences) and/or chimera forming potential? A time course of successful reprogramming with different constructs would be a useful addition to the 96-h snapshot in Figure 1C. The two major conclusions based on scRNA-seq data—that iPStat3 intermediates resemble the early embryo and that iKlf2 intermediates resemble mesoderm—do not seem entirely justified at this point. For iKlf2 intermediates, only a distinct and minor subpopulation expresses markers such as T at elevated
levels (Figure 2G), suggesting that these cells might be on an entirely different trajectory. Additional direct evidence, based on surface marker sorting or lineage tracing, would be required to better support this point. Focus on the Mechanistic Underpinnings of the Core Findings Rather than Expanding the Paper’s Scope to Other Contexts Several of the authors’ observations are interesting and very worthwhile reporting, including the specific requirements of primed-to-naive reprogramming mediated by different factors and, in particular, the elegant observation that maintaining OCT4 levels abolishes the need for any inducing TF in the presence of 2i/LIF. However, the expansion of these observations into other contexts are significantly less convincing and currently weaken the manuscript. For example, in the context of the proposed role of BMP signaling for the human primed-to-naive conversion, crucial controls such as the quantification of colony formation upon exposing ‘‘steady-state’’ primed and naive human PSCs to DMH2 are missing (Figure 5G). Also, the observation that hOct4 is reduced in presence of DMH2 suggests that the effects of the inhibitor are not specific to transitioning cells, as this locus cannot be considered a specific component of the naive network (in contrast to what is implied by the authors on page 12). The experiments aimed at addressing the role of ‘‘fine-tuned’’ Oct4 expression during somatic cell reprogramming are also not convincing, as the starting cells used for these experiments are poorly defined, derived from early embryos,
Cell Stem Cell 25, September 5, 2019 ª 2019 Elsevier Inc. 297
Cell Stem Cell
Peer Review and, most importantly, should not be considered normal somatic cells (or somatic at all) as they were exposed to constant transgenic OCT4 expression. Instead of expanding into other experimental contexts, it would be more informative if the authors had opted to conduct a deeper molecular investigation of their core findings. What is the trajectory in FixedOct4 reprogramming? What is the molecular underpinning of the different requirements of iKlf2 and iEssrb? Deepening the Characterization and Focus on Rex1+ Intermediate States The Rex1 reporter should be better characterized in the context of the authors’ transgenic approach. What is the percentage of Rex1+ cells at different times of the conversion with the different cassettes? What is the efficiency of Rex1 intermediates (Figure 2B) to form Rex1+ naive colonies? The decision to focus solely on Rex1+ intermediates for the scRNA-seq analyses is a little unfortunate. This focus forced the authors to ignore early-stage
298 Cell Stem Cell 25, September 5, 2019
post-transgene induction (which would have substantially strengthened their point of distinct trajectories, in particular as the kinetics of induction with the different factors differs greatly) and also prevented them from detecting alternative cell fates induced by the factors (which, based on the abundance of Rex1 cells shown in Figure 2B, might be abundant).
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How do the authors explain the substantial reduction in iEsrrbmediated reprogramming in the presence of DMH2 (Figure 5C) in the apparent absence of activation of BMP signaling in iEsrrb intermediates (Figure 5A)? The authors’ claim that maintenance of endogenous (=ESC-like) Oct4 levels is a hallmark of all reprogramming routes, but Figure S6C shows substantial upregulation in iPStat3 intermediates and Figure S6H protein reduction in iEsrrb cells.
d
Quantification of the data shown in Figure S6F would be nice. Statistics are missing in several figures (for example, S5E, 5J, etc.). Can ESCs be derived in 2i/LIF from blastocysts temporarily exposed to BMP inhibition? Are non-pluripotent tissues (TE and PE) unaffected in these embryos? Abbreviations iGOI and bsd not explained in legend to Figure 1A. The authors should strongly consider using a different term than iPSCs for the cells generated in their approach (which primarily does not entail the reprogramming of somatic cells). Many of the subheadings appear incomplete and do not convey sufficient information (‘‘Routes are functionally distinct’’).
REFERENCES Stuart, H.T., Stirparo, G.G., Lohoff, T., Bates, L.E., Kinoshita, M., Lim, C.Y., Sousa, E.J., Maskelenka, K., Radzisheuskaya, A., Malcolm, A.A., et al. (2019). Distinct molecular trajectories converge to induce naı¨ve pluripotency. Cell Stem Cell 25, this issue, 388–406.
Peer Review Evaluation of Stuart et al.: Distinct Molecular Trajectories Converge to Induce Naive Pluripotency Matthias Stadtfeld1,* 1Department of Medicine, Weill Cornell Medicine, New York, NY 10021, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2019.08.009
An example of the peer review process for ‘‘Distinct molecular trajectories converge to induce naive pluripotency’’ (Stuart et al., 2019) is presented here. Editor’s note: These are the first-round reviewer comments provided to the authors of ‘‘Distinct molecular trajectories converge to induce naive pluripotency’’ from Silva and colleagues. They were written for Cell Stem Cell during the first round of the peer review process and have been lightly edited for clarity. We chose to highlight this review since it focuses on ensuring that the main points of the paper are appropriately supported by the underlying data and that different aspects of the analyses are mechanistically reconciled, together helping readers understand how different molecular trajectories from the same starting point can result in a common pluripotent end state. The authors submitted a revised version of the manuscript that addressed the reviewers’ comments, and that was re-reviewed, accepted for publication, and published in the September 2019 issue of Cell Stem Cell. Overview and Summary In their manuscript, Stuart et al. compare conversion trajectories of primed mouse pluripotent stem cells into a naive state when driven by different individual transcription factors (TFs). This is a well-established and suitable experimental model, and the authors provide largely convincing data that different TFs trigger unique trajectories. Further investigation reveals specific molecular requirements of these trajectories and provides intriguing evidence for an important role of OCT4 levels in sustaining the primedto-naive conversion. While several of the authors’ observations are novel and likely will be of broad interest for the stem cell community, some key conclusions are not fully supported by their experimental
evidence and a stronger mechanistic basis for their findings should be established. Strengthening Characterization of the Cellular Starting, Intermediate, and End States to Understand Distinct Trajectories The initial observation that iEsrrb, iKlf2, and iPStat3 EpiSCs achieve naive pluripotency via distinct molecular trajectories is interesting and overall convincing. However, due to the central importance of this conclusion to the authors’ claims, their inducible transgene approach should be characterized more thoroughly. Is a similar level of induction (in terms of % expressing cells and levels of expression) achieved with all inducible transgenes, including those that are inefficient at inducing reprogramming? What is the time course of exogenous protein induction (this is relevant for interpretation of the data discussed on the bottom of page 10 and shown in Figure 4F)? Do ‘‘iPSCs’’ derived with iEsrrb, iKlf2, and iPStat3 differ in their molecular signatures (Figures 1H and S1A suggest similarity but do not rule out reproducible and significant differences) and/or chimera forming potential? A time course of successful reprogramming with different constructs would be a useful addition to the 96-h snapshot in Figure 1C. The two major conclusions based on scRNA-seq data—that iPStat3 intermediates resemble the early embryo and that iKlf2 intermediates resemble mesoderm—do not seem entirely justified at this point. For iKlf2 intermediates, only a distinct and minor subpopulation expresses markers such as T at elevated
levels (Figure 2G), suggesting that these cells might be on an entirely different trajectory. Additional direct evidence, based on surface marker sorting or lineage tracing, would be required to better support this point. Focus on the Mechanistic Underpinnings of the Core Findings Rather than Expanding the Paper’s Scope to Other Contexts Several of the authors’ observations are interesting and very worthwhile reporting, including the specific requirements of primed-to-naive reprogramming mediated by different factors and, in particular, the elegant observation that maintaining OCT4 levels abolishes the need for any inducing TF in the presence of 2i/LIF. However, the expansion of these observations into other contexts are significantly less convincing and currently weaken the manuscript. For example, in the context of the proposed role of BMP signaling for the human primed-to-naive conversion, crucial controls such as the quantification of colony formation upon exposing ‘‘steady-state’’ primed and naive human PSCs to DMH2 are missing (Figure 5G). Also, the observation that hOct4 is reduced in presence of DMH2 suggests that the effects of the inhibitor are not specific to transitioning cells, as this locus cannot be considered a specific component of the naive network (in contrast to what is implied by the authors on page 12). The experiments aimed at addressing the role of ‘‘fine-tuned’’ Oct4 expression during somatic cell reprogramming are also not convincing, as the starting cells used for these experiments are poorly defined, derived from early embryos,
Cell Stem Cell 25, September 5, 2019 ª 2019 Elsevier Inc. 297
Cell Stem Cell
Peer Review and, most importantly, should not be considered normal somatic cells (or somatic at all) as they were exposed to constant transgenic OCT4 expression. Instead of expanding into other experimental contexts, it would be more informative if the authors had opted to conduct a deeper molecular investigation of their core findings. What is the trajectory in FixedOct4 reprogramming? What is the molecular underpinning of the different requirements of iKlf2 and iEssrb? Deepening the Characterization and Focus on Rex1+ Intermediate States The Rex1 reporter should be better characterized in the context of the authors’ transgenic approach. What is the percentage of Rex1+ cells at different times of the conversion with the different cassettes? What is the efficiency of Rex1 intermediates (Figure 2B) to form Rex1+ naive colonies? The decision to focus solely on Rex1+ intermediates for the scRNA-seq analyses is a little unfortunate. This focus forced the authors to ignore early-stage
298 Cell Stem Cell 25, September 5, 2019
post-transgene induction (which would have substantially strengthened their point of distinct trajectories, in particular as the kinetics of induction with the different factors differs greatly) and also prevented them from detecting alternative cell fates induced by the factors (which, based on the abundance of Rex1 cells shown in Figure 2B, might be abundant).
d d d
d d
Other Points d
d
How do the authors explain the substantial reduction in iEsrrbmediated reprogramming in the presence of DMH2 (Figure 5C) in the apparent absence of activation of BMP signaling in iEsrrb intermediates (Figure 5A)? The authors’ claim that maintenance of endogenous (=ESC-like) Oct4 levels is a hallmark of all reprogramming routes, but Figure S6C shows substantial upregulation in iPStat3 intermediates and Figure S6H protein reduction in iEsrrb cells.
d
Quantification of the data shown in Figure S6F would be nice. Statistics are missing in several figures (for example, S5E, 5J, etc.). Can ESCs be derived in 2i/LIF from blastocysts temporarily exposed to BMP inhibition? Are non-pluripotent tissues (TE and PE) unaffected in these embryos? Abbreviations iGOI and bsd not explained in legend to Figure 1A. The authors should strongly consider using a different term than iPSCs for the cells generated in their approach (which primarily does not entail the reprogramming of somatic cells). Many of the subheadings appear incomplete and do not convey sufficient information (‘‘Routes are functionally distinct’’).
REFERENCES Stuart, H.T., Stirparo, G.G., Lohoff, T., Bates, L.E., Kinoshita, M., Lim, C.Y., Sousa, E.J., Maskelenka, K., Radzisheuskaya, A., Malcolm, A.A., et al. (2019). Distinct molecular trajectories converge to induce naı¨ve pluripotency. Cell Stem Cell 25, this issue, 388–406.