On 2015 Sep 25, Jacob H. Hanna commented:

The Hanna group congratulates our friends, colleagues and collaborators on their elaborate, timely and elegant review. We wish to alert the readers to four minor constructive comments and points, which may be useful for future discussions:

1) On page 473 the authors state: “Direct derivation of ground state ES cells from human embryos would be a landmark”

We fully agree with the authors that for any newly established stem cell growth conditions, it is important to show technical feasibility for such conditions to obtain newly derive human ESCs from pre-implantation blastocysts. However, it should be kept in mind that the pluripotent state identity is going to be dictated by the derivation growth conditions and not by whether their source was from the human inner cell mass (ICM). The fact that human ICM cells explanted in conventional/primed FGF2/TGFB1 containing conditions, undergo a dramatic in vitro adaptation process and have yielded only primed human ESCs for the last 20 years (Thomson et al. Science 1998), certainly does not prove that conventional human ESCs are in an ICM-like/naïve state. Similarly, mouse ICM derived cells yield naïve stem cells when derived in mouse stem cell naïve conditions in vitro, or give rise to primed Epiblast Stem Cell (EpiSCs) when explanted and derived under mouse primed pluripotency growth conditions (Hanna et al. Cell Stem Cell 2009, Najm et al. Cell Stem Cell 2011). http://www.cell.com/cell-stem-cell/abstract/S1934-5909(11)00049-X http://www.cell.com/cell-stem-cell/abstract/S1934-5909(09)00169-6

The latter examples constitute a clear cautionary note against using ability for ESC derivation from human ICMs in itself as a validation criterion for endowing naïve-like pluripotent state characteristics in any in vitro expanded stem cell type or condition. In fact, we modestly believe that applying such a criterion to annotate the state of an in vitro captured pluripotent cell would be wrong. ESC derivations from human ICM are only technical validations for new growth condition capabilities and applications, and can yield naive or primed pluripotent cells depending on the conditions applied.

2) Less importantly, on page 473 the authors indicate: “Nonetheless, a recent study described altered primate PS cells that can incorporate into host embryos and develop into chimaeric fetuses with low-grade contribution to all three germ layers and early germ cell progenitors57. As in mice, high-grade contribution and germline transmission remain as more stringent tests to demonstrate naive pluripotency in primate ES cells“

We find it unfortunate that this review fails to clearly indicate that the “altered” primate PS cells used in Reference 57 and capable of generating monkey chimeric fetuses for the first time ever, were expanded in negligibly “altered” NHSM conditions previously described by our group in Gafni et al. Nature 2013 (Reference 52). For clarification, the latter fact is clearly indicated by the author in Chen et al. Cell Stem Cell 2015 (Reference 57). http://www.cell.com/cell-stem-cell/abstract/S1934-5909(15)00264-7

3) On Page 473 the authors indicate: “More compelling evidence for cross-species blastocyst chimaerism has been reported following injection of primate naive iPS cells into mouse blastocysts, leading to clonal contribution to solid tissues56.”

We find it unfortunate that this review fails again to indicate that the naïve monkey iPSCs used in Reference 56, and capable of generating cross-species chimerism after micro-injection in mouse blastocysts, were also expanded in presumably “altered” NHSM conditions previously described by our group in Gafni et al. Nature 2013 (Reference 52). Instead of providing exogenous low-dose TGFB1 as devised in NHSM conditions, Fang et al. Cell Stem Cell 2014 (Reference 52) simply used mouse embryonic feeder cells that are known to secrete other TGF family members ligands (including Activin A), and similarly substitute for TGFβ1 in supporting pluripotent stem cell expansion. http://www.cell.com/cell-stem-cell/abstract/S1934-5909(14)00395-6

4) At the end of Page 471 the authors indicate: “The observation that naive cells tolerate depletion of epigenetic regulators supports the concept of naive pluripotency as a configuration with a reduced requirement for epigenetic repression compared to primed PS cells and somatic cells.”

The authors did not provide a citation for the the first and only study thus far conducting side by side comparison on mouse naive and primed cells and showing for the first time opposing tolerance of epigenetic repressor depletion in naive and primed cells from the same species, which was conduced by our group (Geula et al. Science. 2015 Feb 27;347(6225):1002-6. doi: 10.1126/science.1261417).

We had indicated in the discussion by Geula et al Science 2015: “The fact that murine naïve cells, rather than primed cells, are tolerant to depletion of epigenetic and transcriptional repressors supports the concept of naïve pluripotency as a configuration with a relatively minimal requirement for epigenetic repression (in comparison to primed pluripotent and somatic cells)”.

Relevant data and discussion backing these original conclusions are presented in Figure 1 http://www.sciencemag.org/content/347/6225/1002.long, Supplementary Page 17 and Figure S1 http://www.sciencemag.org/content/suppl/2014/12/30/science.1261417.DC1/1261417.Guela.SM.revision1.pdf of Geula et al Science 2015.

With great appreciation,

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

Department of Molecular Genetics

Weizmann Institute of Science

Email: jacob.hanna@weizmann.ac.il

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

Twitter: @Jacob_Hanna

On 2015 Sep 25, Jacob H. Hanna commented:

The Hanna group congratulates our friends, colleagues and collaborators on their elaborate, timely and elegant review. We wish to alert the readers to four minor constructive comments and points, which may be useful for future discussions:

1) On page 473 the authors state: “Direct derivation of ground state ES cells from human embryos would be a landmark”

We fully agree with the authors that for any newly established stem cell growth conditions, it is important to show technical feasibility for such conditions to obtain newly derive human ESCs from pre-implantation blastocysts. However, it should be kept in mind that the pluripotent state identity is going to be dictated by the derivation growth conditions and not by whether their source was from the human inner cell mass (ICM). The fact that human ICM cells explanted in conventional/primed FGF2/TGFB1 containing conditions, undergo a dramatic in vitro adaptation process and have yielded only primed human ESCs for the last 20 years (Thomson et al. Science 1998), certainly does not prove that conventional human ESCs are in an ICM-like/naïve state. Similarly, mouse ICM derived cells yield naïve stem cells when derived in mouse stem cell naïve conditions in vitro, or give rise to primed Epiblast Stem Cell (EpiSCs) when explanted and derived under mouse primed pluripotency growth conditions (Hanna et al. Cell Stem Cell 2009, Najm et al. Cell Stem Cell 2011). http://www.cell.com/cell-stem-cell/abstract/S1934-5909(11)00049-X http://www.cell.com/cell-stem-cell/abstract/S1934-5909(09)00169-6

The latter examples constitute a clear cautionary note against using ability for ESC derivation from human ICMs in itself as a validation criterion for endowing naïve-like pluripotent state characteristics in any in vitro expanded stem cell type or condition. In fact, we modestly believe that applying such a criterion to annotate the state of an in vitro captured pluripotent cell would be wrong. ESC derivations from human ICM are only technical validations for new growth condition capabilities and applications, and can yield naive or primed pluripotent cells depending on the conditions applied.

2) Less importantly, on page 473 the authors indicate: “Nonetheless, a recent study described altered primate PS cells that can incorporate into host embryos and develop into chimaeric fetuses with low-grade contribution to all three germ layers and early germ cell progenitors57. As in mice, high-grade contribution and germline transmission remain as more stringent tests to demonstrate naive pluripotency in primate ES cells“

We find it unfortunate that this review fails to clearly indicate that the “altered” primate PS cells used in Reference 57 and capable of generating monkey chimeric fetuses for the first time ever, were expanded in negligibly “altered” NHSM conditions previously described by our group in Gafni et al. Nature 2013 (Reference 52). For clarification, the latter fact is clearly indicated by the author in Chen et al. Cell Stem Cell 2015 (Reference 57). http://www.cell.com/cell-stem-cell/abstract/S1934-5909(15)00264-7

3) On Page 473 the authors indicate: “More compelling evidence for cross-species blastocyst chimaerism has been reported following injection of primate naive iPS cells into mouse blastocysts, leading to clonal contribution to solid tissues56.”

We find it unfortunate that this review fails again to indicate that the naïve monkey iPSCs used in Reference 56, and capable of generating cross-species chimerism after micro-injection in mouse blastocysts, were also expanded in presumably “altered” NHSM conditions previously described by our group in Gafni et al. Nature 2013 (Reference 52). Instead of providing exogenous low-dose TGFB1 as devised in NHSM conditions, Fang et al. Cell Stem Cell 2014 (Reference 52) simply used mouse embryonic feeder cells that are known to secrete other TGF family members ligands (including Activin A), and similarly substitute for TGFβ1 in supporting pluripotent stem cell expansion. http://www.cell.com/cell-stem-cell/abstract/S1934-5909(14)00395-6

4) At the end of Page 471 the authors indicate: “The observation that naive cells tolerate depletion of epigenetic regulators supports the concept of naive pluripotency as a configuration with a reduced requirement for epigenetic repression compared to primed PS cells and somatic cells.”

The authors did not provide a citation for the the first and only study thus far conducting side by side comparison on mouse naive and primed cells and showing for the first time opposing tolerance of epigenetic repressor depletion in naive and primed cells from the same species, which was conduced by our group (Geula et al. Science. 2015 Feb 27;347(6225):1002-6. doi: 10.1126/science.1261417).

We had indicated in the discussion by Geula et al Science 2015: “The fact that murine naïve cells, rather than primed cells, are tolerant to depletion of epigenetic and transcriptional repressors supports the concept of naïve pluripotency as a configuration with a relatively minimal requirement for epigenetic repression (in comparison to primed pluripotent and somatic cells)”.

Relevant data and discussion backing these original conclusions are presented in Figure 1 http://www.sciencemag.org/content/347/6225/1002.long, Supplementary Page 17 and Figure S1 http://www.sciencemag.org/content/suppl/2014/12/30/science.1261417.DC1/1261417.Guela.SM.revision1.pdf of Geula et al Science 2015.

With great appreciation,

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

Department of Molecular Genetics

Weizmann Institute of Science

Email: jacob.hanna@weizmann.ac.il

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

Twitter: @Jacob_Hanna