Balancing Paradigms with Mesenchymal Stromal Cells

Steve Gschmeissner/Science Photo Library

Innovation isn't uniquely devoid of commonality of adoption by discipline. Rather the likelihood of acceptance generally tracks evenly to historical norms in parallel with society's openness to progress and the search for solutions. However, the impact of technological change is variable and dependent on societal factors related to income and health. One could argue the greatest benefit comes when change drives both economic prosperity and improved health standards.   

While the average pace of technological innovation slowed some decades ago the recent rapid rise of medical science has taken on the mantle of sustainability for growth. The dramatic impact potential of fundamentally transformative practices in healthcare is being fueled by access to new knowledge and a greater sharing of insight. 

Today, due to the convergence of various technology led disciplines, there are many important catalysts for paradigm shifting change. A key criteria common to all are the Drivers - fundamental products or processes that opens up the gates to new realms of understanding and acceptance. At each juncture a bridge must span the divide and a stake ground into new terrain. 

Are MSCs a Driver that can forge a paradigm shift in stem cell healthcare & how did we get here?
         

The investigation of bone marrow (“BM”) stem cells led to the establishment and widespread clinical practice using cells of the mesodermal blood lineage via bone marrow transplantation – known as hematopoietic cells (“HSCs”).

The first use of these BM  stem cells as therapy was pioneered over 50 years ago when transplants were first introduced experimentally to treat leukemia. However, as with most donor tissue the understanding of immune rejection of foreign non-self cells was and still is of major concern for the successful treatment of disease using allogeneic (donor) tissue. This is even the case when immuno-histocompatibility is done via matching of the cells to the host. This complication has stymied the field of cellular therapeutics due to the severe adverse events that can result from the administration of donor derived cellular treatments. In the case of BM transplantation they routinely cause Graft versus Host Disease (“GvHD”) as a result of the treatment, with approximately 50% of all such patients reporting complications. The percentage of mortality as a result of this last resort treatment intervention even today is staggering with up to 17% of all severe liver/gut GvHD cases resulting in death(1).

NIH.gov

As a field the discovery and isolation of Mesenchymal Stromal Cells (“MSCs”), a small subset of BM niches representing less than 0.01% of all HSCs, was a watershed moment. It was a true breakthrough as these cells were found to be able to replicate as multipotent precursors and can be differentiated into fat, bone and cartilage. The isolation and clonal nature of these MSCs opened up a whole new avenue for cellular investigation. Further sources of MSCs were discovered in a range of bodily tissues, including fat, perinatal tissue and dental pulp. The technology for human application of these adult cells gave rise to the stem cell industry we know today. Upwards of 500+ clinical trials using MSCs are registered currently in the US central database clincaltrial.gov for a variety of unmet disease indications (2).

In addition, there is a large growing trend of undocumented cases using MSC products in private medical offices as marketed treatments via autologous (self-to-self) therapies (3). These unlicensed medical practitioners using MSCs products are the subject of considerable debate as to where the line should be drawn between required regulatory oversight and freedom of medical use in private clinics for autologous treatments. The US FDA is currently reviewing draft guidelines (4,5,6,7) for treatment products using MSCs. They are preparing to define what constitutes more than minimal manipulation and cell use parameters. This is with a view to determining clinical trial requirements for MSC biologics, in keeping with current drug development procedures already in place.

Safe and Effective?   

The prospect of MSC utility for therapeutics has been due in large part to the evident immunological privileged nature of MSCs and their potential for universal application without immunosuppressive drugs – unlike HSCs themselves. Although MSCs have an antigen profile they lack major class antigens which makes them relatively immune-privileged to the host system thereby allowing for donor derived cell treatments without treatment rejection in low dose regimes.      

The Scientist - Keith Kasnot

The properties of MSC have been appropriately described as “ambulatory” and “paramedic” – i.e. they’re built to respond to injury in the body and assist in its repair. How they detect, migrate and signal, in addition to what biological manner they act, and what way in different circumstances, is a source of considerable study. It seems clear though now that their “method of action” (“MOA”) is modulatory in nature via complex regulatory mechanisms (8). One such mechanistic attribute is via the excretion of bioactive factors (vesicles, exosomes et al) and work to facilitate cell to cell communication networks (9).

Much has been written about the potential of tissue derived MSCs as a treatment option for a host of acute, immune and degenerative conditions. However, the field is still developing and protocols are being tested and adjusted to maximize possible outcomes. I’ve added an overview video below on the challenges and issues faced by MSCs product developers’ to-date by a leading expert in the field Dr. Jacques Galipeau of Emory University. The presentation highlights a number of findings on research and data in this sector and is well worth watching

Dr. Jacques Galipeau of Emory University

As mentioned, and referred to in the video, numerous clinical studies are underway on the use of MSCs and case reports have been published on both the potential benefits and in certain cases a lack of statistical benefit in patients receiving these cells from a variety of tissue sources.

With regard to the clinical trial results there is clear validation of MSCs safety profile, which is fundamental to their successful translation. Potential treatment efficacy of MSCs is suggestive to-date of positive activity on various outcome measures in a number of reported studies. These positive results are counter-balanced with questions on method of action (“MOA”) and some failed studies. This somewhat mixed picture generally points to issues relating to the development of medicinal products and cellular biologics should be viewed as no different.

A few of the better known company examples of MSC sector developments in the sector are briefly summarized below with links to the company for further details on the data.

• TiGenix (adipose/fat) – has moved on from the 1st EU approved and marketed autologous (“auto”) MSC cell therapy called ChrondroCelect for cartilage repair to an allogeneic (“allo”) product strategy with solid Phase III results in hand for Cx601 in Crohn’s Disease. This will mark their first allo indication nearing approval with other adipose stem cell products in the pipeline. 

• Mesoblast (BM) – bought the first approved western auto cell therapy Prochymal for GvHD from Osiris which had mixed results and was never released. They are developing a full in-house line-up of allo product candidates with good support data and are partnered with a Teva Pharma. Notable pipeline news include marketing approval of TemCell in Japan for GvHD with local partner JCR Pharma (repackaged Osiris product) and solid data in late stage trials (MSC-100-IV for GvHD also, MPC-150-IM for heart and MPC-06-ID for back pain, amongst others).

• Athersys (BM) – lost Pfizer as a program partner for MultiStem after releasing mediocre data in ulcerative colitis. A second Phase II read-out, this time in stroke, also failed to meet endpoints. However, newly released interim data in its ongoing stroke study is now suggestive of positive results from the homing-in strategy on potential earlier treatment window benefit. Also of note are the additional clinical programs in development for cardiovascular and inflammatory/immune indications. In addition there’s a solid validation deal with Healios of Japan for MultiStem in that market and use of the product for Healios’ ongoing development programs.

• Pluristem (placenta) – “PLX” product line for vascular, muscular and immune indications in early stage clinical trials (PI & PII) with solid data in muscle and critical limb ischemia. Promising preclinical results for bone marrow repair with government sponsorship for rapid route to market in acute radiation syndrome.

• 
  Vericel (BM for heart program) – previously known as Aastrom with a long history of development of auto MSCs for heart and CLI indications with poor accumulated data continues to develop the heart product in clinical studies with recent positive data after previous endpoint failure, indicative of statistical benefit. In 2014 they secured additional auto cell therapy products from Sanofi (Carticel & MACI – cartilage and Epicel – skin) which had previously received certain market authorizations and are generating revenue with patient benefit.

Indicative data sets for comparative analysis and ratio breakout are yet to be tabulated with regard to which conditions and methodologies the cells work well for and in which cases they don’t help all that much or at all. However, one must be cautious when assessing the efficacy value of cellular products as they are biologics and there are many issues relating to their degree of effectiveness, such as: their source; derivation method; inherent donor variability; passage potency; culture conditions & scale-up manufacturing; cold chain methodology; target indication; patient population; disease states and application methods, amongst others. As a result not all cellular products will perform well in human studies. These issues play a significant role in whether they achieve benefit in tests on patients, and to what extent in relation to standard of care. Although the jury is still out there is a general agreement based on empirical data that these cells are on the whole safe, when developed and used appropriately. Where they have been shown to have positive outcome and biological activity there is acknowledged room for improvement with regard to enhancing efficiency, potency and cell mechanistic action, which is encouraging.

One aspect of the development of industrial scale cellular therapies speaks to the need for increased replicative capacity, lower passaged products and standardization via use of optimization technologies and shifting to pluripotent cell sources instead of donor derived batch processing of multipotent cells.

Octane Bioreactors

As a result of this progressive development culture method adjustments gleaned from the early pioneering work of MSC development are giving rise to efficiencies of process and improved manufacturing protocols for next generation methods in both multipotent and pluripotent products. The above mentioned early leaders in MSC product offerings are beginning to line up their treatments for entry to the market, while the sector looks to prepare and trial the more advanced cell factories of the future.

UC Davis MSC Investigators

This momentum is also being driven by the rise of synthetic constructs using MSCs - the personalized tailoring of targeted medicines for improved performance. MSCs possess inherent homing and immunomodulatory properties and therefore are ideal for use in combination with gene and nano technologies. In addition, the extraction of the inherent cell properties of MSCs for standalone biologic products adds to the overall picture and excitement in the field.

MSC products are representative of the wider cell therapeutic field and are the standard bearers in the effort to bridge the shifting paradigms of new treatment modalities for patients in need.

Cheers

Ref: Sector Update on Asian Market for MSCs > "Cell Therapy in Asia Erupts with Partnerships and Joint Ventures"

Progress and the Circle of Scientific Medical Innovation

Designing an adaptable system without the known variables is often an exercise in caution, unless of course you have been down a similar road before and can rely on established templates for creating repurposed guidelines. In some cases however old parameters simply don't fit any longer and the opportunity to engage in the development of more flexible rules with the benefit of hindsight becomes a net positive in the never ending cycle of innovation driven progress.

A series of high profile developments along this path have taken place of late on the topic of genetics and the advent of gene editing technologies that have the potential to alter the fate of human disease and the burden it represents to society. These policy reviews and the resulting position statements have been brought on by concerns that human gene editing presents a challenge to the perceived boundaries by which scientific discovery and possible therapeutic interventions are applied. 

However, as we have seen knowledge trumps and is critical to all human endeavors, given information is paramount to decision making. Accumulating scientific data in the unknown cause and effect realm of biological systems provides the fundamental opportunity to address the task of solving real world problems. This is the tenant by which translational science has always operated and without which we wouldn't have made profound human advances to our condition.     

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. 

What is apparent now is the scientific community's consensus on the inherent value of lab study of early embryonic state development using genetic tools to advance knowledge through research while adhering to the principals of caution in progressing any attempts to implement the alteration of the germ line for reproductive purposes. This was the Middle Way path.

The US National Academies summation of their International Summit on Gene Editing affirmed the above in early December last year and called for an ongoing forum to further the dialogue on the topic as the science develops with a view to establishing new recommended guidelines if and when appropriate. The fluid nature of the science requires such and was therefore prudent to set this investigative precedent for all respective regulatory bodies to consider, including the US. 

Previously the UK reaffirmed its position as a leading member of the international scientific community by being the first country to consider Mitochondrial DNA replacement therapy for families with genetic disorders wishing to have a baby free of the diseases associated with such inherited problems. Most recently the UK has agreed to allow genetic research on fertilized pre-embryos for infertility studies without intent to implant for reproductive purposes and always adhering to the prior 14 day limit on embryo development. A modification to the UK law was required for the Mitochondrial DNA therapy to proceed to review stage, while the research on early pre-embryos was already allowable under existing legislative framework which required prior approval and strict oversight.

Following on from the UK's position on Mitochondrial DNA therapy the US National Academies earlier this week announced its recommendation on this genetic intervention procedure for germ-line modification application. The result being an additional affirmation of the scientific potential to alleviate disease through continued research and potential use of the technology in the US. Notable was the necessary recommended investigational support for early pre-embryo studies. Current congressional restrictions inhibit actual Federal support for such studies on viable early stage pre-embryos. This may change in the fiscal year 2017 appropriations Bill as the restriction is not permanent. Support for non-viable pre-embryo research was expressly noted in the US National Academies recommendation paper.           

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. 

Coming around full circle, there would be little progress without the support for all forms of scientific innovation.

Advocacy for Cures.

Cheers

Refs:

1. International Summit on Human Gene Editing Washington DC - "On Human Gene Editing: International Summit Statement" (Dec. 3rd 2016)
2. UK "Scientists get 'gene editing' go-ahead" (BBC Feb. 1st 2016)
3. The National Academies "Clinical Investigations of Mitochondrial Replacement Techniques Are ‘Ethically Permissible’ If Significant Conditions Are Met, Says New Report" (Feb. 3rd 2016)
4. California Stem Cell Report items by David Jensen re: CIRM (1,2) & ISSCR (3) (Feb. 5th 2016)
5. Related blog posts: 1, 2, 3, 4

Science Validation & The Rising Sun

Strategy, survival, direction, competition and growth opportunities are but a few fundamental elements in the maze of everyday life all professionals must navigate - no more so than those on the leading edge of managing scientific innovation.

Being at the forefront of change often elicits backdraft currents and polarization of entrenched positions. This is natural and can be seen as a positive reinforcement of the high threshold one must strive to in order to achieve acceptance. A sort of quality assay if you will - one which is analogous to peer review in a business setting.

A lot has been written on the news of the Ocata/Astellas deal regarding the value calculation and little on the actual merits of Validation for Ocata’s science, the step forward in its development plan and the broader implications for the stem cell sector.

This writer has covered Ocata for a long time and can attest to the finer details of the saga that was its Survival and Vindication. A road which has seen its fair share of episodic highlights - perhaps too many.

One could speak at length and talk of the dedicated character of those involved with the science and the constant pressure within & on the company to prove itself in a demanding uncompromising field. One piece termed the company a “lightning rod” - the story has all the hallmarks.

The end of an era? Yes, perhaps it is in many ways. I prefer to see it as the close of the 2nd act in a 3 act structure, where the rain is falling and mingling with the tears.

Lost independence isn’t easy to come to grips with. However, moving on from being a small volatile publicly traded company, with all the influences that entails, to being housed within a protective and nurturing parental structure is a very positive outcome - for the programs and patients in need. 

It signifies so much with regard to the science and efforts to help define the standards of a new treatment methodology in medicine. 

The deal is a solid affirmation from established pharma that the stem cell therapeutic sector is worth banking on. This comes on the heels of other momentum building developments in the space and perhaps is indicative of a growth driven consolidation phase.

There are many worthwhile questions surrounding the announcement and events leading up to the decision to sell that remain, even after recent company disclosures, and one would look to those involved to address them for the record. Lock stock comes to mind?

There is a saying which is often spoken in Catalonia - “the sun always rises” and aims to reinforce and embrace the positive.

Cheers

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.

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:

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

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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

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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.

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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

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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

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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

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.

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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

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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

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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

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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

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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
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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