On 2017 Sep 28, Jacob H. Hanna commented:

Follow-up papers from Hanna group further critique this study: http://www.biorxiv.org/content/early/2017/09/07/184135

Jacob (Yaqub) Hanna M.D. Ph.D.

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2016 Jan 23, Jacob H. Hanna commented:

A critique of this paper is available on bioRxiv: http://dx.doi.org/10.1101/013961

We copy below the most important section indicating that the entire findings by Dos Santos et al. claiming that Mbd3 facilitates epigenetic reprogramming can be trivially explained by inducing cell proliferation block as a result of 100% ablation of Mbd3 early in the process:

"Complete depletion of Mbd3 blocks somatic cell proliferation and viability"

In Rais et al. Nature 2013 clearly indicated that near 100% complete inhibition of Mbd3 in somatic cells or EPiSCs results leads to abrupt decrease in cell proliferation and viability. As such we greatly focused on using hypomorphic Mbd3flox/- cell lines and compared them to Mbd3+/+ WT cells. In cases where we achieved high reprogramming efficiency of Mbd3-/- cells (Rais et al, Figure1-2, Extended Data Figure 3B,C), we always started with Mbd3flox/- cells (and NOT Mbd3-/- cells as used in Dos Santos et al.) in order to ensure low and residual Mbd3 protein levels already during the first 48 hours of OSKM induction. Afterwards, 2i was supplemented with tamoxifen to achieve complete ablation of Mbd3. This is a critical point that was highlighted in our paper - Rais et al Nature 2013:

“Mbd3-/- MEFs (but not pluripotent cells) experience accelerated senescence and proliferation capacity loss, and thus Mbd3-/- somatic cells were reprogrammed by applying tamoxifen on Mbd3flox/- cells only after 48 h of OSKM induction”. (A direct quote from Rais et al. Nature 20131 Methods section)

The results presented by Dos Santos et al. systematically used different conditions where they did either one of the following:

1) Dos Santos et al.2 used Mbd3-/- cells that were maintained for multiple passages as null cells and only afterwards OSKM were introduced. As we had previously indicated1, these cells dramatically lose their proliferation capacity, and indeed all growth proliferations curves shown in Dos Santos et al. validate this result (even though they were carried for 4 days only). Therefore the entire findings by Dos Santos et al. claiming that Mbd3 facilitates epigenetic reprogramming can be TRIVIALLY explained by inducing cell proliferation block, and in our opinion, has nothing to do per se with epigenetic reprogramming. Further, the latter decrease in cell proliferation occurs even without OSKM induction, further supporting the notion that the inhibition of reprogramming results from simply hampering cell proliferation in donor cells, rather than epigenetic reprogramming per se.

2) For Tamoxifen induced deletion experiments, Dos Santos et al.2 used Mbd3flox/flox cells (and not Mbd3flox/- cells), and applied tamoxifen at different time points. However, these experiments lead to complete deletion of Mbd3, and not during the critical window of early reprogramming, which we highlighted as critical1.

3) Finally, Dos Santos et al. did not compare WT vs. Mbd3flox/- cells as starting somatic donors for reprogramming, which is the most relevant comparison and the most robust comparative one presented in Rais et al. Nature 2013.

In summary, our findings in fact are consistent with Dos Santos et al, as they use conditions that ensure either complete inhibition of Mbd3 and block cell proliferation, or avoid optimal depletion of Mbd3/NuRD activity during a critical early reprogramming window, which we previously highlighted1 as critical determinants for capturing the beneficial effect of Mbd3/NuRD depletion on iPSC reprogramming.

For full version of our critiques you are invited to read bioRxiv preprint: http://dx.doi.org/10.1101/013961

Jacob (Yaqub) Hanna M.D. Ph.D.

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2015 Mar 07, Jacob H. Hanna commented:

A critique of this paper is available on bioRxiv: http://dx.doi.org/10.1101/013961

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2015 Mar 07, Jacob H. Hanna commented:

A critique of this paper is available on bioRxiv: http://dx.doi.org/10.1101/013961

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2016 Jan 23, Jacob H. Hanna commented:

A critique of this paper is available on bioRxiv: http://dx.doi.org/10.1101/013961

We copy below the most important section indicating that the entire findings by Dos Santos et al. claiming that Mbd3 facilitates epigenetic reprogramming can be trivially explained by inducing cell proliferation block as a result of 100% ablation of Mbd3 early in the process:

"Complete depletion of Mbd3 blocks somatic cell proliferation and viability"

In Rais et al. Nature 2013 clearly indicated that near 100% complete inhibition of Mbd3 in somatic cells or EPiSCs results leads to abrupt decrease in cell proliferation and viability. As such we greatly focused on using hypomorphic Mbd3flox/- cell lines and compared them to Mbd3+/+ WT cells. In cases where we achieved high reprogramming efficiency of Mbd3-/- cells (Rais et al, Figure1-2, Extended Data Figure 3B,C), we always started with Mbd3flox/- cells (and NOT Mbd3-/- cells as used in Dos Santos et al.) in order to ensure low and residual Mbd3 protein levels already during the first 48 hours of OSKM induction. Afterwards, 2i was supplemented with tamoxifen to achieve complete ablation of Mbd3. This is a critical point that was highlighted in our paper - Rais et al Nature 2013:

“Mbd3-/- MEFs (but not pluripotent cells) experience accelerated senescence and proliferation capacity loss, and thus Mbd3-/- somatic cells were reprogrammed by applying tamoxifen on Mbd3flox/- cells only after 48 h of OSKM induction”. (A direct quote from Rais et al. Nature 20131 Methods section)

The results presented by Dos Santos et al. systematically used different conditions where they did either one of the following:

1) Dos Santos et al.2 used Mbd3-/- cells that were maintained for multiple passages as null cells and only afterwards OSKM were introduced. As we had previously indicated1, these cells dramatically lose their proliferation capacity, and indeed all growth proliferations curves shown in Dos Santos et al. validate this result (even though they were carried for 4 days only). Therefore the entire findings by Dos Santos et al. claiming that Mbd3 facilitates epigenetic reprogramming can be TRIVIALLY explained by inducing cell proliferation block, and in our opinion, has nothing to do per se with epigenetic reprogramming. Further, the latter decrease in cell proliferation occurs even without OSKM induction, further supporting the notion that the inhibition of reprogramming results from simply hampering cell proliferation in donor cells, rather than epigenetic reprogramming per se.

2) For Tamoxifen induced deletion experiments, Dos Santos et al.2 used Mbd3flox/flox cells (and not Mbd3flox/- cells), and applied tamoxifen at different time points. However, these experiments lead to complete deletion of Mbd3, and not during the critical window of early reprogramming, which we highlighted as critical1.

3) Finally, Dos Santos et al. did not compare WT vs. Mbd3flox/- cells as starting somatic donors for reprogramming, which is the most relevant comparison and the most robust comparative one presented in Rais et al. Nature 2013.

In summary, our findings in fact are consistent with Dos Santos et al, as they use conditions that ensure either complete inhibition of Mbd3 and block cell proliferation, or avoid optimal depletion of Mbd3/NuRD activity during a critical early reprogramming window, which we previously highlighted1 as critical determinants for capturing the beneficial effect of Mbd3/NuRD depletion on iPSC reprogramming.

For full version of our critiques you are invited to read bioRxiv preprint: http://dx.doi.org/10.1101/013961

Jacob (Yaqub) Hanna M.D. Ph.D.

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2017 Sep 28, Jacob H. Hanna commented:

Follow-up papers from Hanna group further critique this study: http://www.biorxiv.org/content/early/2017/09/07/184135

Jacob (Yaqub) Hanna M.D. Ph.D.

Department of Molecular Genetics

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna@weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna