On 2016 Jun 19, Jacob H. Hanna commented:

We noted that Dr. Hendrich states : "Furthermore, we previously showed that Mbd3 null ES cells generated by genetic manipulation in vitro are viable. ".

I find using the above-mentioned result as a predictor and indication that it must be possible to derive Mbd3 null ESCs from ICMs, as wrong and misleading. We all know that in the case of Nanog, it can be deleted in previously established ESCs, however it cannot be derived from Nanog null ICMs since pluripotency is not formed in the absence of Nanog in vivo. This is not the case for Mbd3. In my opinion, you should retract this paper that purports Mbd3 as an essential factor for eastblishing pluripotency and ES cells.

On 2016 Jan 26, K Kaji commented:

Hi Jacob, FYI, our conclusion in this paper is that Mbd3 is required for development of pluripotent cells in peri-implantation embryos, from ICM in blastocyst to post-implantation epiblast.

On 2016 Jan 25, Jacob H. Hanna commented:

Thank you for the response. As a scientist I have been trying to understand the biology of Mbd3/NuRD in pluripotency during early mouse development in vivo and in vitro, and it is not about whether a certain sentence is actually explicitly stated or not (this is typically the job of patent lawyers:) ). We have absolute confidence in the data presented in this paper, however we believe that the interpretation is misleading and wrong.

There are two aspects of pluripotency formation that are being addressed:

1) In vivo formation of pluripotency at E3.5: your paper concludes that Mbd3 is required for formation of pluripotent cells in vivo. However, by viewing immunostaining for Oct4/Nanog in E3.5 epiblasts, the formation of pluripotent cells is not compromised at all based on the data presented in Figure 2B from Kaji et al. Development 2007. We have highlighted this in the following summary slide ( http://imgur.com/lsM6kbV ), and contrasted this with data on Nanog null ICMs, where pluripotent cell stability is compromised in vivo (and subsequently Nanog knockout ESCs could not be derived from Nanog null embryos). You recent gene-expression data on Chd4 null ICMs further validates the emergence of the pluripotent epiblast at E3.5-E4.5 O'Shaughnessy-Kirwan A, 2015. Therefore in our opinion, the adequate conclusion based on your data is that pluripotency formation is not compromised in vivo by ablation of Mbd3/Chd4/NuRD complex.

2) In vitro formation and derivation of ESCs: There have been many instances in science where new technologies emerge and allow us to revisit old findings (e.g. new microscopy, new growth conditions etc.) and prove that old conclusions were in fact erroneous due to the inability of the tools available at that time to conduct the best experiment. As we are interested only in unraveling the true biology (which is timeless), the fact that Oct4+/Nanog+ cells emerge in vivo in Mbd3 and Chd4 null embryos (see comment 1 above - http://imgur.com/lsM6kbV ), and that ESCs can accordingly be derived under optimized conditions (Rais et al. Nature 2013), in our opinion this indicates that Mbd3/NuRD is not required for in vitro derivation of pluripotent ESCs and does not even compromise this process.

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 25, Brian Hendrich commented:

Our paper describes the function of Mbd3/NuRD in pluripotent cells in the early mouse embryo. Whether one can derive ES cells from Mbd3 null blastocysts is not relevant to the message of this paper.

Figure 4 shows that Mbd3-/- ICMs taken from C57Bl/6 mouse embryos did not proliferate when cultured in Serum/LIF conditions. This paper predates the advent of 2i culture media, reported by Ying et al. 2008, and therefore the possibility that Mbd3-/- ES cells can be derived in 2i/LIF conditions is not inconsistent with what is reported in our paper. Furthermore, we previously showed that null ES cells generated by genetic manipulation in vitro are viable. Nowhere in this paper do we state “it is absolutely impossible to derive Mbd3-/- ESCs from Mbd3 null E3.5 embryos”.

On 2016 Jan 23, Jacob H. Hanna commented:

After posting our comment and critiques below (http://1.usa.gov/1HeRjpX), we have noted a similar doubt was raised by Dr. Silva and Sr. Smith in 2007 in their Essay titled: "Capturing Pluripotency" Cell 2007 - http://www.sciencedirect.com/science/article/pii/S0092867408002079 Silva J, 2008

"A critical test is whether repair of a gene defect is sufficient to restore differentiation capability. For example, ES cells in which the NuRD repressor complex is inactivated by deletion of the Mbd3 subunit are compromised in prosecuting differentiation, but this defect is eliminated upon re-expression of Mbd3, meaning that the pluripotent state has been preserved throughout ( Kaji et al., 2006). A specific requirement in maintaining the pluripotent state should only be claimed when a change in developmental potential is irreversible, as for example when Oct4 or Sox2 is deleted (Niwa, 2007). Interestingly, Mbd3 appears necessary for derivation of ES cells, but this is because in its absence the epiblast does not form properly (Kaji et al., 2007) and not because Mbd3 is required by ES cells."

Regarding the last sentence, we now know that Mbd3 is NOT necessary for deriving mouse Mbd3 null ESCs from KO mouse ICMs that retain Nanog and Oct4 expression in vivo (Rais et al. Nature 2013).

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 08, Jacob H. Hanna commented:

This Kaji et al. 2007 paper claims that it is absolutely impossible to derive Mbd3-/- ESCs from Mbd3 null E3.5 embryos. This result is surprising considering that the authors show in the same paper presence of Oct4+/Nanog+ cells in Mbd3-/- ICMs. Further, Mbd3-/- ESCs are "hyper-naive" and resist differentiation even in the absence of LIF (Kaji et al. Nature Cell Biology 2006, Reynolds et al. Cell Stem Cell 2012). Our group has revisited this result in Rais et al. Nature 2013, and was able to efficiently derive Mbd3-/- ESCs in serum free enriched 2i/LIF conditions. This suggests that the main conclusion of Kaji et al. 2007 Development paper titled "Mbd3 is required for development of pluripotent cells", is invalid and constitutes an artifact likely due to using a low quality fetal bovine serum (FBS) batch.

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 08, Jacob H. Hanna commented:

This Kaji et al. 2007 paper claims that it is absolutely impossible to derive Mbd3-/- ESCs from Mbd3 null E3.5 embryos. This result is surprising considering that the authors show in the same paper presence of Oct4+/Nanog+ cells in Mbd3-/- ICMs. Further, Mbd3-/- ESCs are "hyper-naive" and resist differentiation even in the absence of LIF (Kaji et al. Nature Cell Biology 2006, Reynolds et al. Cell Stem Cell 2012). Our group has revisited this result in Rais et al. Nature 2013, and was able to efficiently derive Mbd3-/- ESCs in serum free enriched 2i/LIF conditions. This suggests that the main conclusion of Kaji et al. 2007 Development paper titled "Mbd3 is required for development of pluripotent cells", is invalid and constitutes an artifact likely due to using a low quality fetal bovine serum (FBS) batch.

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:

After posting our comment and critiques below (http://1.usa.gov/1HeRjpX), we have noted a similar doubt was raised by Dr. Silva and Sr. Smith in 2007 in their Essay titled: "Capturing Pluripotency" Cell 2007 - http://www.sciencedirect.com/science/article/pii/S0092867408002079 Silva J, 2008

"A critical test is whether repair of a gene defect is sufficient to restore differentiation capability. For example, ES cells in which the NuRD repressor complex is inactivated by deletion of the Mbd3 subunit are compromised in prosecuting differentiation, but this defect is eliminated upon re-expression of Mbd3, meaning that the pluripotent state has been preserved throughout ( Kaji et al., 2006). A specific requirement in maintaining the pluripotent state should only be claimed when a change in developmental potential is irreversible, as for example when Oct4 or Sox2 is deleted (Niwa, 2007). Interestingly, Mbd3 appears necessary for derivation of ES cells, but this is because in its absence the epiblast does not form properly (Kaji et al., 2007) and not because Mbd3 is required by ES cells."

Regarding the last sentence, we now know that Mbd3 is NOT necessary for deriving mouse Mbd3 null ESCs from KO mouse ICMs that retain Nanog and Oct4 expression in vivo (Rais et al. Nature 2013).

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 25, Brian Hendrich commented:

Our paper describes the function of Mbd3/NuRD in pluripotent cells in the early mouse embryo. Whether one can derive ES cells from Mbd3 null blastocysts is not relevant to the message of this paper.

Figure 4 shows that Mbd3-/- ICMs taken from C57Bl/6 mouse embryos did not proliferate when cultured in Serum/LIF conditions. This paper predates the advent of 2i culture media, reported by Ying et al. 2008, and therefore the possibility that Mbd3-/- ES cells can be derived in 2i/LIF conditions is not inconsistent with what is reported in our paper. Furthermore, we previously showed that null ES cells generated by genetic manipulation in vitro are viable. Nowhere in this paper do we state “it is absolutely impossible to derive Mbd3-/- ESCs from Mbd3 null E3.5 embryos”.