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.


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

Sally Temple - Cells, Leadership & Audacious Innovation

The eye is often referred to as the window into the soul. Whether that is true or not depends I suppose on who’s doing the viewing, as the subjective interpretation invariably dictates the meaning. This however in science can be balanced via rigorous protocols of evidence based assessment in a clinical setting and peer review. The subjective becomes objective and the perspective become clearer. In the stem cell field some of the earliest approaches to regenerative medicine have been in the CNS, with a number of high profile clinical stage multi and pluripotent trials. Those in the clinic with ongoing trials are reporting promising indications of disease stability and restorative potential. More data is required but the overall momentum moving forward portends to a variety of treatment methodologies from a number of cell sources. An active area of CNS clinical research has been for the retina, where there are unmet medical conditions in need of new effective solutions for low vision & blinding diseases. Early attempts to restore retinal function via the transplantation of donated adult and fetal retinal tissue and cells were deemed inefficient and lacked solid efficacy data. Those experiments however have paved the way for the current focus on using more developed & novel multipotent cells, as well as from pluripotent sources. One such retinal program is being led by Dr. Sally Temple of the Neural Stem Cell Institute based on a unique population of adult retinal stem cells. I sat down with Sally at ISSCR 2015 and it's fitting she rounds out the Interview segments from Sweden as she is ISSCR's President Elect now and is looking to the future, as we all are, with high expectations and great promise to meet those tangible opportunities head on.

The Neural Stem Cell Institute (NSCI) is home to some of the most interesting work in stem cell technology today. Its origin as a research hub for neural cell investigation lies with Sally's history and her pursuit and discovery of the first CNS stem cells in the mouse. As with a number of other leading scientists she started with uncovering complex neural biological systems and the mechanistic pathways of cell constructs of the CNS, which included the eye. She is the recipient of the MacArthur "Genius" Award and a highly respected leader in the field.

NSCI is funded by the NY State NYSTEM along with donations as a non-profit and is based in upstate NY - near Albany, in a town called Rensselaer. It holds a foundational patent estate to an adult stem cell discovery that now forms the lead translational focus of the institute - an adult retinal stem cell in the RPE layer which can be sourced from donor tissue and expanded to therapeutic doses. This same cell can also form a variety of other cell types via a biological trans-differentiation pathway called the EMT into bone, fat and cartilage. The team has published a number of high profile papers (eg 1,2,3) on the science which underpin the clinical translation work. Their projects have many prestigious collaborators, including the Kellogg Eye Center and Mount Sinai, amongst others.

The eye is uniquely interconnected as a sensory organ, yet accessible, which has made it a natural target for NSCI to lead off with. The program is earmarked for a clinical trial in the not too distant future. RPE cell transplants are a hot cell therapy area. A number of groups are in clinical trials using various different sources and application methods using RPEs - notably Ocata, Riken, Coffey/Pfizer & BioTime/CellCure. Some groups are also in the clinic using different retinal cells, while others still are in various pre-clinical stages of development. All this attention and focus on the eye is for a good reason - it's accessible and in-vivo activity can be observed in detail. However, most importantly the momentum is building as the data reported to-date is showing safety & potential efficacy.  

Sally's team at NSCI includes her co-founder & partner Jeffrey Stern, a retinal surgeon, and a notable listing of well respected scientists and researchers, including: Chris Fasano, a leading member of the investigator team, who is also known for producing the official ISSCR Stem Cell Podcast with Yosif Ganat.

The work at the institute is not solely eye cell centric, as you will see when exploring the various sub-sections of the research going on there. The basic theme throughout is indeed neural and CNS in general.

Stem Cell Podcast w/ Sally Temple

The adult stem cell discovery that Sally, Jeff and collaborators at their NSCI uncovered has resonated throughout the community. It's simplicity is captivating and it's implications far reaching. The very nature of regeneration and the body's own capacity to heal itself is powerful stuff. That is what we all wish for, methods by which we can assist our own abilities in all manners throughout our lives - why not also with our own health.

Yet, there is still a basic question to be resolved - if there are these cell populations in our organs & tissue, just waiting for those cues, can we indeed awaken our "Inner Salamander?"

I hope you find the interview transcript below informative. I have great admiration for innovators and no more so that those that fight for patient solutions in a not-for-profit foundation. 

Good luck Sally, Jeff and all the team in my home State!



M - Can you explain your discovery of an adult retinal stem cell and use of that for research and therapeutics.

ST - The idea for discovery research and looking for retinal cells that might have regenerative potential I have to attribute to my husband Jeff Stern. He started in basic research and decided he wanted to work with people and went to medical school. He ended up coming back into the field of ophthalmology but with that research mindset. We of course talked over the years about neural stem cells and the discovery of tissues that you’d think don’t have regenerative potential but actually do.

M - Stimulated to have that potential?

ST - That’s the point. I firmly believe that we have that ability and if we can simulate it we can.

M - Somewhat like the salamander effect?

ST - Exactly. So Jeff put in recently for the Audacious Goals competition of the National Eye Institute. All the applications were anonymous so no one knew who submitted what and it was open to everyone, worldwide. They picked 10 winners and one of those was Jeff’s project. He was picked for “Reawakening your Inner Salamander” to take advantage of that.

M - I like it. I used a salamander image a little while ago - it’s a poignant reference.

ST . It is and of course the salamander RPE can regenerate and make the entire neural retina. So if you remove the photoreceptors and you remove the neural retina entirely in salamanders the RPE cells will change, proliferate and then make new retina cells.

M - Was this an area of study for you and your husband?

ST - We were aware of that because when Jeff worked in vision doing physiology, the physics of electrical physiology, he worked with salamanders and so he was very familiar with the regenerative literature and we thought let’s look in the human eye for a stem cell and if so could it be activated. We did experiments to establish these stem cells in the human system. We grew them in clones so we could watch an individual cell and see how many progeny it could make.

M - From what source?

ST - We took it from human cadaver tissue and we removed the retina and took the RPE, which you could obtain very cleanly. We removed the anterior portion of the eye, which people have said may contain proliferative cells in perhaps a ciliary margin.

M - The Canadians?

ST - Yes, Derek van der Kooy and Vincent Tropepe. In some animals there’s a ciliary margin but it’s not as clear where the ciliary margin is in humans but just in case we removed the anterior portion. We wanted to look within the RPE and we wanted to make sure we knew what cell type we were looking at. We cloned them and made movies of them. We took them from the eye and demonstrated that only a sub-population, less that 10% and in some preparations only 3% of the RPE cells will divide extensively.

M - Do you believe in-vivo they do that on a regular basis or are they stopped?

VPR - Fibroblastic Scar (UCL image)
ST - In-vivo people have found it very hard to see any proliferative cells but there are circumstances in which the RPE is thought to proliferate. Unfortunately under certain pathological circumstances you will see the RPE layer migrate through the retina and out into the vitreous and proliferate through creating these awful contractile membranes which will pull the retina off. That type of epiretinal membrane formation is quite common.

M - Almost like a mutated cell process.

ST - It’s like some of the cells are undergoing some of the transformative processes of the EMT state. So we knew there were circumstances under which some of the RPE can proliferate in-vivo. Perhaps sometimes this can be beneficial. Maybe they could proliferate a little bit and help the retina recover from damage.

M - They already do a lot of work.

ST - Yes, the RPE are amazing. They’re such a humble little cell but if they die the retina dies. That’s how important they are. They are important for the blood retina barrier, fluid balance, cytokine protection, phagocytosis and more. So we found a sub-population of the cells will self-renew extensively making hundreds and thousands of cell progeny from one cell.

[discussion break]

M - To recap there are adult retina cells that can proliferate as evidenced by the EMT phenomena and that if stimulated can be a source of retinal tissue

ST - Exactly, so the idea is there’s a sub-population that can be activated to proliferate. If those are the cells that contribute to those abnormal masses we don’t know for sure but what we do know is that the cells can proliferate. We can take a single cell and make numerous progeny. We can split those prodigy up so now we have clones of those originals that you can then put in different media.

M - More so than what was achieved with fetal cells?

ST - I don’t know if they cloned a single fetal cell. These are adult cells that we cloned out. From a single cell we get a clone and put it in different media conditions and we have shown that the same cell that can give RPE can also produce fat, cartilage and bone.

M - Along the MSC line?

ST - Yes so RPE can make MSCs and can undergo EMT. We think that could be a good model for the epiretinal formation. We don’t know if it’s the originating cell in-vivo but it can do that. It was a surprise because you wouldn’t have thought of a CNS cell giving rise to MSC progeny.

M - I’ve spoken with Dr. Maher in Barcelona who is pioneering a lot of work on wisdom teeth and he says the same thing in reverse. They can make neural crest cells and other non-MSC cell types, plus the MSC lineages . Does this have to do with the CNS connection also?

ST - The CNS is the brain, the retina and the spinal cord. The neural crest of course is from the dorsal part of the neural tube and its migratory. The cranial neural crest does have progenitors that give rise to bone and cartilage etc. A mixture of cells, as well as neural cells. We know that our cells are CNS cells at the beginning and are probably not going through the neural crest stage. They don’t seem to make sensory neurons and sympathetic neurons etc. We don’t think of changing RPE into neural crest. For some reason the RPE has retained the potential to make MSCs for whatever reason. I can’t explain but that is the case.

M - Have you done genetics on that?

ST - Yes we’re in the process of doing that now and studying this progression into MSCs because we want to understand that so we can prevent, but that happens pathologically. At the same time we know we can take those cells and make beautifully stable RPEs for 2 years in culture.

M - and make a lot of them

ST - Yes a lot of them. From one donor we can make 5 x 10⁸ cells which is a lot. Let’s say a patient age-related macular degeneration may require 50,000 or 100,000, because you’re only covering that tiny macular region, we hoping one donor’s cells will be able to treat hundreds of patients. We have made plans for all the moving parts. You have to get manufacturing and regulatory to approve so we’re not doing it ourselves. We’re using a facility and transferring the technology so we know they are making the highest quality cells.

M - This is an academic institution?

ST - Yes it is. An academic GMP facility at the University of Rochester and they’ve been wonderful. We do a lot of back and forth to make sure the cells are correct.

M - This is sort of a NY project?

ST - It is mostly. It’s funded through the NY State via the NYSTEM program. We wouldn’t have been able to do this without them. They have been tremendous. It’s very expensive. We have got to solve this problem of why it costs so much to do this. They gave us $10.8m over 4 years to do all the preparations for the manufacturing and the efficacy to get to an IND. What we’re hoping once we get through that process is that we can then move into clinical trials.

M - If you can show there’s a signal. I’m not sure if you’ll need to go through a small trial to get to that stage.

ST - Probably a Phase 1. We’re planning about 18 patients. It’s very interesting to be in an area like this. In the beginning people weren’t talking very much about the RPE and then there was a recognition that this would be a great target tissue because it’s the eye and you can actually watch what’s happening once you put the cells in. There are sensitive visual tests.

M - Have you added all those specialists to the team now?

ST - Yes. Jeff of course is a retinal surgeon.

M - Will he be part of the trial?

ST - We think it’s important to be hands off with the safety study, so it will be done independently. That way we have some comfort. We’ll know the cells are safe and there will be no conflict.   

M - Once you publish you can receive credit.

ST - The efficacy data to-date is really exciting.

M - Using the RCS rats?

ST - Yes, the RCS rat. You know people have said with that model anything works. This is not true.

M - You can look at the past examples

ST - We haven’t published yet but what we have shown is that the cells have to be at a particular stage of development.

M - That’s what I’ve been talking about before and with the community here.

ST - Robin Ali?

M - Yes that’s right Robin Ali’s work and clinically with Dr. Lanza’s trials having used cells differentiated to a certain point.

ST - There’s a sweet spot in the developmental profile - the earliest proliferating cells and the latest mature cells don’t work as well as cells in the middle of the process.
M - Yes. It’s important to get there as efficiently as possible, extract, freeze & thaw?

ST - Yes. That sweet spot was a surprise so we’re lining up all these elements to use the cells. I do feel good now having different groups using different sources. Some using iPS, some using ES some putting them on a scaffold and some injecting a suspension - like we plan to do.

M - I spoke with Masayo Takahashi the other day, she’s wonderful, and she was explaining how excited her team was about the progressional steps they’re taking from monolayer to suspension. There’s an acknowledgement that there’s a need for suspension in certain cases.

ST - Oh good. I’m glad she’s doing that.

M - That was very important to hear as I felt there was the advanced stage but there are of course other stages. Robin Ali felt that photoreceptors need to come into play more and importantly so as to restore function & vision. That’s certainly true depending on the disease, state of the eye and point in time. The patient acceptance of surgery along that progression is vital to understand because if you’re looking at 20/40 or 20/80 you're going to have a different opinion than if you were 20/200+ so there is an issue there imo.

ST - So Jeff, if he was here, he would say to you “I’m a retinal surgeon and if there was a non-surgical solution I would prefer it.” That’s why we’re excited by the cell we’ve identified as it’s in our eyes. The RPE is so neat, it’s actually laid down in the embryo so when we look at the eye it’s the black center. Those cells were done when you were in utero and really don’t proliferate very much. So we think that’s one of the reasons we’ve been able to activate them from even a 99 year old. They haven’t been used up. There’s no hayflick limit as they haven’t been dividing and dividing and exhausted. They have preserved their potential to divide. We take them out and put them in culture. These cells from 99 year olds that have not divided for a century will start to divide in 36 hours.

M - Source therefore is not that big an issue for you. Is it the standardization in the manufacturing area that will be a challenge?

ST - Not really, cadaver eyes are readily available because they are already collected for corneal transplants

M - The donor consent forms are already there.

ST - Yes. People are so wonderful in their generosity because these are light & vision saving possibilities. So the cornea is already taken, we take the part that is generally thrown away and utilize that. They’re available and they’re in us. So if they’re there and we could activate them safely for an endogenous repair that would be the goal.

M - Have you seen the BMP4 inhibition study from Derek van der Kooy’s team? He was trying to do something similar. Evidentially there was some form of stop in his cells also and they’re working to find some chemical formula to regulate inhibition but you have to be very specific otherwise they show off target effects.

ST - The RPE cells in-vivo don’t divide very much, if at all, the question is whether this is because of inhibition or the lack of activators. So what we found is we can take growth factors that stimulate the growth in-vitro and put those in the eye of animals and they do activate the cells. We think lack of activators is probably one of the reasons and we can add these. At the same time it’s possible that if you add in something that Derek is describing you get even more activation but I think you have to be very careful. You don’t want too much activation because there would be a concern there you could get a growth.

M - jCyte are doing some interesting work. They’re looking at delivering the factors by way of cells intravitreally.

ST - Yes we’re looking to actually isolate the factors

M - CIRM has funded their program. Henry Klassen is moving it and they’ve got an approved IND for RP [since this interview clinical trial has started - see here].

ST - Yes he’s putting cells in to protect

M - He experimented on different formats and settled on the cells as factor delivery vehicles as his approach first

ST - Good idea

M - There are other possibilities for action via MSCs intravitreally or systemically plus via some of the recent work on neuroprotection using photoreceptor progenitor factors. Is that something similar in how it may work?

ST - Perhaps. I know that Jensen are putting umbilical cord cells under the retina

M - They haven’t been too revealing in terms of data

ST - Right, that’s something I would say is so helpful if we’re working in the same area to share as much as possible because we learn from each other. I don’t know what they’re doing and it’s a highly sensitive area of the body, so safety is a key issue. So to have to inject subretinally once may be ok, but to have to do it repeatedly is a concern. Certainly if you could do an intravitreal injection of an activating factor that has great appeal.

M - Is that where you’d like to get to

ST - Yes. Our animal studies in that area are progressing. We think we have a pipeline developing

M - So the first would be the transplant then during that phase you would develop the concept further?

ST - Yes the endogenous stimulation that’s exactly what we’re doing

M - That’s a plan that will be successful imo - the degree of success is yet to be seen of course but it’s worth every effort.

ST - Of course

M - I noticed you were working with the Israelis on an element of the protocols using NIC expansion. Are you utilizing some of that methodology?

ST - We also grow iPS cells and I think you’re referring to Eyal Banin and Benjamin Rubinoff and the use of NIC. We find it is beneficial yes.

M - It helps with proliferation or how?

ST - Not quite sure what it does to the cells but it helps the differentiation of the cells and they look robust.

M - Do they over mature as a result?

ST - I don’t think so

M - Ok there’s a sweet spot issue that’s important

ST - Yes so that is the case for our adult cells, which I mentioned we’re preparing a paper on.

M - When’s that due out?

ST - Oh yes, I think Richard Davis, who’s working with us on that, would say “ah, the figures are almost all done, writing it up”

M - In the post!

ST - I know, it’s in the post! I’ll will let you know when it’s ready.

M - Thx, would love to read it. I think we’ve covered so much, just a few more notes here. The Allo source, is there a need for immunosuppressants - systemic or local dose? How will that work?

ST - I have to say when doing a clinical trial you have less leeway than you’d imagine on these different details. We’ve been encouraged to do a very strong immunosuppression early on and part of our clinical team includes physicians that specialize in immune issues in the eye. A uveitis specialist. We recruited a very prominent scientist at Mount Sinai, his name is Douglas Jabs. So we’re aware of that. Because the RPE is the blood retina barrier if it is diseased you could get it broken down. So we’re starting with Allo and the tissue is prevalent and available enough that we could HLA match to help reduce the immune issue. Then, given the cells are already in the eye, if the activation product doesn’t work we could probably do an extraction & expansion for an autologous transplant of cells.

M - Would you do an iPS or a retina stem cell sample from the eye?
ST - We’d probably take it directly from the eye

M - Because a skin biopsy or blood sample is easier

ST - It is. We also know we can take these cells from the subretinal fluid. They’re there and it’s probably just going into the subretinal space and sucking some out. The reason we know this is that in certain circumstances they have to take out fluid such as in a retinal detachment and normally that is thrown away. We have a protocol to grow cells from that. It’s small scale and it doesn’t work every time but we’re not actively trying to harvest the cells right now, but in theory it could be done.

M - I’m very happy for you and your team - it’s great work. At the end of the day fighting hard for the future solutions is really all about the next generation and what we can do for our own loved ones who are older. If we can stop this evil circle it will be worth it and try to do it in a way that makes it economically viable. Thank you so much Sally.

ST - Thank you.