Naїve Human Pluripotency & The Broad Shoulders of Science

Stevenage, UK BioScience Campus
Scientific debate in the pursuit of knowledge by way of accumulated evidential data is fundamental, just as socio-economic competition is needed to spur innovation, product development & growth in commercial business. Distinct and largely operating on their own, these two worlds have now collided and become integrated in a synthetic process that is driving 21st century evolution.

As a pillar of progress and community success, medical science is a central focus of tomorrow’s design. One in which the health and well-being of society can be calculated and factored into the spreadsheets of sustainability. The footnotes in such macros are bolded as requirements to achieve, yet are a challenge to deliver.  

Salk iPS_Ruiz-StemCell
Given this backdrop the nature of a pluripotent cell, with its ability to generate all tissue types, has been a hot topic of debate and investigation for many years. Its potential is often cited but the field remains a few steps away with many questions still to be answered. 

Source, Stability and Scale – the trinity of our destiny caught in a matrix of possibilities where clarity of method is needed. 

Science knows no bounds when it comes to unresolved issues of definition and process, so the discussion continues. However, with advents in genomic analysis the cell systems of our inner being are becoming clearer and these new insights are helping to provide the answers. 

The human "naїve" cell state in the earliest stages of human embryogenesis is one such focus. The identification and establishment of cell lines along the pluripotent continuum has been a foundational endeavor of the community. Ever since the mouse modelling proved the existence of these powerful engines of growth have the leading labs sought to isolate and engineer the human equivalents. This ongoing work has inspired the field to challenge each other to discover and answer the unresolved questions that will unlock the full potential of pluripotency. 

Whitehead Institute MIT
In its so called "naїve" state the pre-programming of the early cell development machinery hasn't kicked off yet and committed to its natural tissue generating pathways, hence the terminology. The use of cells at this early moment in the cycle could alleviate some of the drawbacks of the standard later stage "primed" version, allowing for more efficient homologous recombination in a therapeutic setting using reprogramming technologies. The naїve state also has a greater proliferation capability and can differentiate more effectively into all desired tissue types. In addition, these cells are able to form inter species chimeras for research and tissue engineering, a highly valuable addition to the toolbox.    

Over the years I have looked for data on early stage embryonic states, specifically any variations in the genetic profiles of pre-compaction blastomeres and ICM hESCs. The Galan, Á. et al (2010) Valencia paper was one such document I found. Of note here in the more recent research done on early human development was that the variation in profiling was correlated to naїve at a specific stage of human embryogenesis at around the 8 cell stage (referred to in Q&A). This moment evidently coincides to the withdrawal of maternal influence yet prior to the blastocyst wave of fate expression.  

Benjamin Dodsworth
I touched on the pluripotent topic during my interviews in Sweden during this year’s annual ISSCR 2015 conference and followed up by reading a then just published paper entitled “The Current State of Naїve HumanPluripotency¹.Benjamin Dodsworth of Oxford co-authored the work with his colleagues Rowan Flynn and Sally Cowley (team leader and head of the James Martin Stem Cell Facility, affiliated to the Oxford Stem Cell Institute, at the Sir William Dunn School of Pathology, University of Oxford).

Sally Cowley Ph.D
The passage in the paper's abstract about the naïve state not being an “artifact” caught my attention and intrigued me given the differing opinions on the subject, the extent to which mouse modelling is representative of human developmental biology and the evolving genetic data analysis of early stage embryonic cell states.

I connected with Ben in a Twitter exchange and he was open to doing a Q&A on the topic, which we started prior to some subsequent developments in the area (iPS “2C” totipotent reprogramming² and the Karolinska paper³ on early human development). Comments on the 2C paper are included in the interview below in [brackets]. 

Thank you Ben for your feedback & good luck with your research.



M - With regard to the human Naive state generally and attempts made to create hNaïve cell lines, are we really mainly discussing iPS reprogramming techniques to revert to an earlier point of embryogenesis or would you envision a new methodology for ICM hESC cell lines with them being converted backwards post extraction also? If so do you envision any technical issues associated with than or in their maintenance?

B - Very good point. There are clear parallels to iPS reprogramming techniques. We are currently looking at a method to convert already established human pluripotent stem cell (hPSC) lines to the naïve state. However, if the naïve state is indeed as useful as we anticipate and becomes our new standard, I would expect the emergence of protocols to generate naïve induced pluripotent stem cells directly from primary cells (such as fibroblasts) which skip the primed state. If this holds true, I do expect technical issues. Many protocols for handling hPSCs have been optimised for cells in the primed state. These will not be ideal for naïve cells. Maintenance of naïve human cells might also be challenging and current standard operating procedures will have to be adapted.

M - You mention hESC differentiation pathways that are unreachable - which are those?

B - Endodermal and germline lineages are difficult to access with our current primed hPSCs. This means that although possible, it is inefficient. The Hanna lab have actually used naïve cells to generate primordial germ cells (PGCs) very efficiently. In comparison, primed cells do not efficiently differentiate into PGCs.

Just as important as accessing these differentiation pathways is the maturity of the cells we then produce. Maturity is the extent to which their functions resemble the in vivo cell type. Naïve hPSCs might increase the level of achievable maturity (for example of hepatocytes).

But what I find a lot more interesting is that we have excellent protocols for the differentiation into cells (for example dopaminergic neurons) which work robustly with some hPSC lines but not with others. This heterogeneity could be removed with a protocol which uses cells that are developmentally at the same starting point and without epigenetic bias. The naïve state could deliver on both of these aspects.

M - Has there been any focus on comparative analysis done using hESCs derived from various cell stages of the early human embryonic Blastomere cell stages 2, 4, 8, 16?

B - To my knowledge this has not been performed using hESCs derived from different developmental time points. However, a very useful direct comparison of current naïve and primed hES lines to early human embryonic blastomere cell stages has been performed using single cell transcriptomics by Huang, Maruyama, and Fan (go directly to Figure 2B). They used datasets from Vassena et al., 2011, Xie et al., 2010 and Yan et al., 2013 and compared gene expression to various naïve cells.

M - Why is the Naive state also referred to as Ground State? Is there any technical reason? 

B - Ground state and naïve state both describe the earliest accessible and unbiased cellular state. These terms are interchangeable.

M - Do you believe the reprogramming concept being studied will ultimately be pursued to the point where reversion produces a Totipotent state in order to fully map the process?

B - Possibly, but there are many technical hurdles to overcome and ethical issues to consider.

[M - Do you have a as follow-up comment on this point with regard to the recent Inserm Totipotent development?

B - The 2C paper is indeed a very interesting piece of work which I have been following closely. However, I would like to see more evidence for totipotency, in particular higher efficiency differentiation down difficult lineages such as PGCs. There is not enough evidence to show that these cells are indeed totipotent. For our lab, totipotent cells are unnecessary and we won’t be using these.]

M - Do existing techniques adequately result in Pluripotent cells able to be scaled and applied effectively to therapeutic programs?

B - Current techniques allow the production of induced pluripotent cells to be scaled up. However, before iPS cells can be used therapeutically, the field needs to overcome some fundamental issues. Two main challenges revolve around the host eliciting an immune response to hES or iPS cells even when sourced from the same individual and on the other hand, pluripotent cells have been changed to allow proliferation. This raises concern that these cells could be more susceptible to becoming cancerous. There is a lot of preclinical work to be done.

M - In your conclusion you point to the protocols yielding different results which has yet to be interpreted conclusively, as well as the transient nature of the actual biological moment in-vivo which it may occur. In addition you point to the possibility of a scale of different states along the defined continuum. In that respect would you say any in-vitro activity to reproduce these embryo-genesis states are by defacto man made events and the best we can expect ultimately is a "like" status?

B - Absolutely. Any cell grown into the lab is unlikely to be exactly the same as the in vivo counterpart. As long as we keep this in mind and factor it into our data interpretation, this is not a problem.

M - The data you cite regarding the primate transcript HERVH indicates that mouse systems are distinct to that of primates in this specific area (at least that monkey species). This would indicate that aspects of the human embryo-genesis system are biologically different to that of mouse, in certain ways. Does that perhaps also apply to cell prodigy behavior in your opinion?

B - The paper discussing HERVH is an excellent piece of work which shows compellingly that pluripotency networks are indeed different between human and mouse. And you are right, we can also see these differences in the cellular behaviour. Mouse and human ES cells cannot be cultured in vitro in the same way. The networks which allow capture of naive pluripotency in mouse are not identical to the human system.

M - The utility advantages you mention of Naive versus Primed indicate manufacturing bias towards use of Naive in the future. Can you outline the utility issues specifically for naive cell use and do you view this for specific clinical purposes or for certain discovery processes.

B - Although some labs are currently working on clinical applications, we are focusing on using hPSCs for modelling only. The human naive state promises a lot of benefits – if it is indeed similar to the naive state in mouse. Extrapolating from the mouse, homogeneity would be expected to be improved in naive cell populations. This means that cells are held not in a spectrum of states but all at exactly the same developmental time point. Differentiation protocols could be a lot more effective when applied to a uniform starting point. Other benefits include higher cell yields due to faster doubling times and easier handling.

M - The statement that "TGFβ might not be essential in the human system" caught my attention. Can you elaborate on that in light of published data.

B - In the past, TGFβ signalling was required to maintain hPSCs in culture. However, the requirement of TGFβ signalling is a trait associated with the primed state. In addition, the inhibition of TGFβ signalling increases efficiency of mouse iPSC reprogramming. This is why it would be interesting if we can culture hPSCs without TGFβ.

[Follow-up Q relating to the Karolinska analysis paper³ on early human development was left unanswered prior to publishing]

Q&A Refs:

1. Dodsworth, B. et al. (2015). The Current State of Naïve Human Pluripotency. Stem Cells. doi: 10.1002/stem.2085

2. Ishiuchi, T. et al (2015). Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Nature Structural & Molecular Biology 22, 662–671 (2015) doi:10.1038/nsmb.3066

3. Töhönen, V. et al. Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development. Nat. Commun. 6:8207 doi: 10.1038/ncomms9207 (2015).

4. Huang, K. et al. (2014). The Naïve State of Human Pluripotent Stem Cells: A Synthesis of Stem Cell and Preimplantation Embryo Transcriptome Analyses. Cell Stem Cell 15(4): 410-415.

5. Vassena, R. et al. (2011). Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development Development 138, 3699–3709

6. Xie, D. et al. (2010). Rewirable gene regulatory networks in the preimplantation embryonic development of three mammalian species" Genome Res. 20, 804–815.

7. Yan, L. et al. (2013). Single-cell RNA-Seq profiling of human pre-implantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131–1139.

Selected Other Refs (in no particular order):

Takahashi/Yamanaka review of the iPS reprogramming pluripotency

Takahashi,K., et al. A developmental framework for induced pluripotency. Development 2015 142: 3274-3285; doi: 10.1242/dev.114249 
Salk paper on region specific PSCs (2015):

Wu, J. et al. An alternative pluripotent state confers interspecies chimaeric competency. Nature 521, 316–321 (21 May 2015) doi:10.1038/nature14413
Genomic analysis using single cell RNA (2013)

Xue, Z. et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 500, 593–597 (29 August 2013) doi:10.1038/nature12364
Naive cells in hESC culture using a HERVH promoter & gene analysis of ICM & early embryo cells

Wang, J. et al. (2014). Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. Nature 516, 405–409, doi:10.1038/nature13804
1st Naive Paper MIT (w/ Hanna now in Israel, Weizmann)

Hanna, J. et al. (2010). Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci U S A. 2010 May 18; 107(20): 9222–9227.
A.Smith Cambridge downstream transcription factor Tfcp2l1 in Naive conversion

Martello, G. et al (2013). Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor. EMBO J. 2013 Oct 2; 32(19): 2561–2574. doi: 10.1038/emboj.2013.177
Singapore use of 3iL creates a closer native epiblast state of pluripotency "Naive" (rewiring of regulatory circuitry)

Chan, Y-S. et al. (2013). Induction of a Human Pluripotent State with Distinct Regulatory Circuitry that Resembles Preimplantation Epiblast. Cell Stem Cell. 2013 Dec 5; Vol 13, Issue 6. doi:10.1016/j.stem.2013.11.015
Hanna Weizmann Institute use of 2iL & in-vitro derivation of mouse like naive cells capable of forming inter-species mouse–human chimeric embryos

Gafni, O. et al (2013). Derivation of novel human ground state naive pluripotent stem cells. Nature. 2013 Dec 12;504(7479):282-6. doi: 10.1038/nature12745.
Seattle Washington alternative derivation method to Naive state

Ware, C. et al. (2014). Derivation of naïve human embryonic stem cells. Proc Natl Acad Sci U S A. 2014 Mar 25; 111(12): 4484–4489. doi:10.1073/pnas.1319738111
Whitehead MIT Talen mediated reporter system for naive derivation medium 5iL (R. Jaenisch)

Theunissen, T. et al. (2014). Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency. Cell Stem Cell doi: 10.1016/j.stem.2014.07.002
A. Smith Cambridge team uses simple transient expression of two transcription factors to rewire back to Naive

Takashima, Y. et al (2014). Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human. Cell, Vol.158, Issue 6, 2014 Sept 11. DOI: 10.1016/j.cell.2014.08.029
Valencia early embryo gene analysis

Galan, Á. et al (2010). Functional Genomics of 5- to 8-Cell Stage Human Embryos by Blastomere Single-Cell cDNA Analysis. PLOS | One 2010, Oct 26. DOI: 10.1371/journal.pone.0013615
Developmental biology focus on human tissue

Gerrelli, D. et al. (2015). Enabling research with human embryonic and fetal tissue resources. Development 2015, Sept 15. doi: 10.1242/dev.122820
Harvard led w/ Daley/Jaenisch/Rossant - Comments by Hanna

De Los Angeles, A. et al (2015). Hallmarks of pluripotency. Nature 525, 469–478 (24 September 2015) doi:10.1038/nature15515