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| 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.
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| 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.
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| 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.
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| 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.
Cheers
Q&A:
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 Fan⁴ (go directly to Figure 2B). They
used datasets from Vassena et al.⁵, 2011, Xie et al.⁶, 2010 and Yan et al.⁷, 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:
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
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Genomic analysis using single cell RNA (2013)
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Naive cells in hESC culture using a HERVH promoter & gene analysis of ICM & early embryo cells
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1st Naive Paper MIT (w/ Hanna now in Israel, Weizmann)
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A.Smith Cambridge downstream transcription factor Tfcp2l1 in Naive conversion
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Singapore use of 3iL creates a closer native epiblast state of pluripotency "Naive" (rewiring of regulatory circuitry)
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Hanna Weizmann Institute use of 2iL & in-vitro derivation of mouse like naive cells capable of forming inter-species mouse–human chimeric embryos
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Seattle Washington alternative derivation method to Naive state
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Whitehead MIT Talen mediated reporter system for naive derivation medium 5iL (R. Jaenisch)
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A. Smith Cambridge team uses simple transient expression of two transcription factors to rewire back to Naive
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Valencia early embryo gene analysis
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Developmental biology focus on human tissue
Existing legislation in Western countries has provided a basis for clarification on the scope of scientific and translational activities, as reviewed here. Europe expressly prohibits at present germ line editing for reproduction. The UK has always had a pro-knowledge framework for discovery using early stage fertilized pre-embryos up to 14 days. The US presently restricts Federal Funding on embryo creation for research and when destructive practices are employed in the lab. 
Both the ISSCR and CIRM have previously stated that they support research on early stage pre-embryos for scientific purposes and have reiterated that position also this week while calling for renewed study of the societal implication of gene therapy and germ line editing.
Commercially the recent change in the position of the European Patent Office on the acceptable use of non-viable pre-embryos methods via germ-line modification brings the alignment closer together and bodes well for the application of various stalled avenues of translational science for the benefit of patients in need. 
