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Opinion piece Cite this article: Whiting P, Kerby J, Coffey P, da Cruz L, McKernan R. 2015 Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic. Phil. Trans. R. Soc. B 370: 20140375. http://dx.doi.org/10.1098/rstb.2014.0375 Accepted: 17 July 2015 One contribution of 13 to a discussion meeting issue ‘Cells: from Robert Hooke to cell therapy—a 350 year journey’. Subject Areas: cellular biology Keywords: pluripotent stem cell, regenerative medicine, retina Author for correspondence: Paul Whiting e-mail: [email protected]

Progressing a human embryonic stemcell-based regenerative medicine therapy towards the clinic Paul Whiting1, Julie Kerby1, Peter Coffey2, Lyndon da Cruz2 and Ruth McKernan1 1

Pfizer Neusentis, Granta Park, Cambridge CB21 6GS, UK The London Project to Cure Blindness, Division of ORBIT, Institute of Ophthalmology, University College London, London EC1V 9EL, UK 2

Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. Here, with a focus upon retinal disease, we discuss the current status of the development of such therapies. We also highlight some of our own experiences of progressing a retinal pigment epithelium cell replacement therapy towards the clinic.

1. Introduction In 1998, the publication of the generation of human embryonic stem cell (hESC) lines [1] contained the statement ‘These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine’. Progress towards the utilization of this technology for regenerative medicine therapies has perhaps not been as rapid, or indeed, as straightforward, as hoped or expected at the time. The California-based biotech company Geron was at the vanguard with an hESC-derived oligodendrocyte progenitor cell product, GRNOPC1, as a treatment for spinal cord injury [2]. As part of a phase 1 safety study, the first patient was dosed around 13 years after the initial hESC line derivation paper. Unfortunately, only five subjects were dosed before the trial was halted [3], though for strategic rather than safety reasons. Interestingly, Asterias Biotherapeutics has now licensed the technology and is re-initiating a phase 1 clinical trial (https://clinicaltrials.gov/ct2/show/NCT02302157?term=Asterias&rank=1). At the end of 2014, the first hESC-derived cardiac cell implant was performed by Prof. Menasche (INSERM), in a patient with heart failure (http://presse-inserm.fr/en/stem-cell-therapy-for-heart-failure-first-implantof-cardiac-cells-derived-from-human-embryonic-stem-cells/17502/). Another company at the forefront of developing an hESC-based therapy is Advanced Cell Technology, now known as Ocata Therapeutics. Indeed, their hESC-derived retinal pigment epithelial (RPE) cell therapy product MA09-hRPE has been dosed to 18 subjects in Europe and the USA with either Stargardt’s disease or age-related macular degeneration (AMD), and shown to be generally safe and well tolerated [4]. A recent report has also described the dosing of four Asian patients with MA09-hRPE [5]. Several other hESC-derived RPE therapies are also moving towards the clinic, including those from the California-based team led by Clegg Hinton and Humayun (https://www.cirm.ca.gov/our-progress/ awards/stem-cell-based-treatment-strategy-age-related-macular-degenerationamd), Cell Cure Neurosciences (https://clinicaltrials.gov/ct2/show/NCT 02286089?term=opregen&rank=1) and ourselves (https://clinicaltrials.gov/ct2/ show/NCT016 91261?term=pfizer+amd&rank=17). It is also important to note the landmark clinical study in Japan using induced pluripotent stem cell (IPSC)-derived RPE [6].

& 2015 The Author(s) Published by the Royal Society. All rights reserved.

2. Why start with the eye?

3. Why start with retinal pigment epithelium as a cell replacement therapy? For any therapeutic approach, having a strong rationale based on good preclinical and clinical data is essential in order to maximize the chance of success in the patient. RPE cell replacement therapy for AMD has an extensive literature outlining both preclinical and clinical outcomes on which to base future stem cell therapies. AMD presents in two forms, the wet and the dry. Wet AMD is characterized by neovascularization, retinal bleeding and secondary scarring ultimately leading to RPE and photoreceptor loss. Dry AMD is characterized initially by the formation of drusen deposits with later RPE degeneration and photoreceptor loss [9]. AMD is considered to be a disease of RPE dysfunction and degeneration [10]. The RPE cell monolayer sits as a polarized epithelial sheet situated between the choroidal

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The eye is probably the favourite organ for the development of cell-based therapies. If one includes not only hESC-derived RPE cell replacement therapies mentioned above, but also neural stem cell and mesenchymal stem cell type therapies, there are at least four products that have been evaluated in patients, and perhaps another dozen that are in preclinical development. From a practical perspective, there are a number of reasons. First, safety: the ability to deliver the cells directly into the eye, which is an encapsulated organ, minimizes systemic exposure of the patient to the cells; using ophthalmological methods and imaging technologies, it is possible to non-invasively monitor site of delivery on an ongoing basis; in the event of an aberrant incident (e.g. formation of a teratoma), it is possible to intervene, e.g. via laser ablation or as a worst case removal of the eye. Second, the size of the organ is such that a relatively small number of cells are required (in contrast, for instance, to replacing liver cell function), which is advantageous both from the manufacturing perspective and when considering the cost of goods. Third, the eye is an ideal organ for demonstrating both mechanism of action and efficacy—again, ophthalmological imaging techniques can be used to monitor the presence and viability of the transplanted cells, to monitor the phenotype of the cells (e.g. are the transplanted RPE still pigmented) and to monitor the function of the key transducing cells of the retina, the photoreceptors. Finally, the posterior chamber of the eye is considered to be immune privileged under normal non-disease circumstances [7] and indeed RPE cells themselves are thought to play a critical role in maintaining that environment [8]. If this immune privilege were indeed present, this would clearly be advantageous in the context of an allogeneic cell therapy but remains a point of contention, particularly if one considers the context of the disease state into which the cells are being delivered, which may well mean that any immune privilege is breached.

layer of blood vessels and the photoreceptors. The RPE cell monolayer plays a multifaceted and critical role in retinal function [11]: trophic factor support for photoreceptors and choroid; phagocytosis of shed photoreceptor outer segments; controlling nutrient supply to and from subretinal space and blood; the re-isomerization of all-trans retinal (essential for the visual cycle); the maintenance of immune privilege of posterior chamber. Its dysfunction or loss, as in AMD, leads to photoreceptor dysfunction and degeneration, with the subsequent loss of vision. A classical rodent model of retinal dystrophy, the Royal College of Surgeons (RCS) rat has been used by a number of different laboratories to support the efficacy of RPE cell replacement therapy. This animal has a mutation in the MERTK gene leading to loss of RPE. Delivery of RPE cells (or indeed other cell types; [12]) has been shown to delay the loss of vision [10]. While the relevance of the RCS rat to AMD is debatable, these data do provide a proof of principle regarding the use of exogenous RPE to replace endogenous dysfunctional cells. Further support for the rationale comes from the clinic, in the use of two surgical techniques: macular translocation and autologous RPE-choroid graft, to treat AMD. These approaches are based upon the transfer of healthy RPE-choroid from an unaffected part of the eye to the macular [13,14], replacing diseased RPE. In some patients, though certainly not all, an improvement in vision was seen, maintained over a number of years [15]. The demanding surgery (and the associated adverse events), together with the incomplete clinical response has meant that this is generally not considered a viable approach. However, it does support the hypothesis of an RPE cell replacement therapy for treating AMD. An interesting question arises when one considers whether it is necessary to transplant RPE as a polarized monolayer, or whether a suspension of cells (i.e. an RPE monolayer enzymatically dissociated into single cells) will suffice. In practical terms alone, manufacturing and delivering a cell suspension is far more straightforward than using a cell monolayer. Certainly, the preclinical data using the RCS rat (discussed above) would suggest that an RPE cell suspension would suffice. However, it is also clear that a fully functional RPE cell is only obtained as a monolayer of polarized cells linked by tight junctions [16,17]. Upon dissociation to a single cell suspension, RPE cells undergo an epithelial–mesenchymal transition. While at high cell densities they are able to redifferentiate to RPE, at lower cell densities they maintain a mesenchymal phenotype, lacking any RPE functionality [18,19]. Additionally, RPE monolayers have been shown to be more resistant to oxidative stress-induced cell death [20]. The additional concern is that the Bruchs membrane, upon which the native RPE monolayer sits, is compromised in the aged eye and the disease state, and hESC-derived RPE have been shown to adhere very poorly [20]. Indeed, RPE cells transplanted into preclinical models as a monolayer survive better than those implanted as a dissociated suspension [21]. Finally, delivering a cell suspension will likely lack the precision required to ensure full and even distribution to support the photoreceptor monolayer. Overall, this speaks to the need to deliver the RPE monolayer on an artificial membrane, as is being proposed by both ourselves (figure 1), and others [22]. Despite these data, delivery of RPE as a cell suspension has been the approach taken by Advanced Cell Technology [5], and Cell Cure Neurosciences (https://clinicaltrials.gov/ct2/ show/NCT02286089?term=opregen&rank=1).


In this paper, we discuss our own rationale and strategy for a cell replacement therapy for AMD, and take the opportunity to identify some of the lessons learned as regards the path of a cell replacement regenerative medicine therapy to the clinic.

important to recognize that these initial clinical studies are generally open label, and not statistically powered for efficacy.

vitronectin coating

5. The complexities of developing a cell replacement regenerative medicine therapy

inert membrane

Figure 1. RPE cell replacement therapy. (a) Schematic cross-sectional view; (b) en face view of RPE cells on membrane (the ‘patch’). A final important consideration as to RPE cell replacement therapy is the clinical need and the commercial opportunity. With an ageing population worldwide, the incidence of AMD continues to increase [23]. While currently wet AMD is relatively well managed by anti-vascular endothelial growth factor treatments [24], there is no treatment for dry AMD, which eventually leads to loss of vision resulting in both incapacitation and loss of quality of life. Thus, the patient need is clear.

4. Selecting the right patient population When progressing a novel advanced therapy medicinal product (ATMP) such as a cell replacement therapy into the clinic, choosing the right patient population is critical. The first clinical study has the aim of determining the safety of the product (and any associated intervention, e.g. delivery). As such, the risk– benefit ratio to the subjects must be the key concern. Given that, the initial subjects will almost certainly be patients rather than normal healthy volunteers. Secondary aims of the initial study are likely to be feasibility (e.g. successful delivery of the therapy to the desired site) and proof of mechanism. The latter is a concept borrowed from the world of small molecule drug development, which asks the question of whether the therapeutic agent demonstrates the proposed biological activity at the expected site of action. Broadly speaking, for an RPE cell replacement therapy, this would encompass a demonstration that (i) the cells remain alive, as a monolayer and have an RPE phenotype (extrapolated from confirmation of the maintenance of pigmentation), and (ii) that the cells maintain a viable and functioning photoreceptor layer. As mentioned above, the advantage of the eye is that ophthalmological imaging enables non-invasive monitoring of the transplanted cells and the associated host tissues; this by using, for example, optical coherence tomography, fundus photography to image the back of the eye and microperimetry to correlate the presence of the RPE transplant with the presence of functional photoreceptors and visual sensitivity. Finally, although not the main primary or secondary aim of an initial clinical study, it is also useful if there is a recordable signal of efficacy. For the eye, this is normally the best-corrected visual acuity, as measured by an eye chart. It is of course

Figure 2 indicates some of the activities necessary to bring the RPE cell therapy (shown in figure 1) into an initial clinical study. The breadth of expertise required spans from engineering (design and manufacture of a regulatory compliant surgical delivery device, patch cutter, patch transportation container) through safety assessment/toxicology, cell biology, manufacturing and quality assurance, regulatory affairs, clinical operations, etc. The significant level of interconnectivity and interdependency of the activities is also clear. Below, we discuss some aspects in more detail, highlighting some specific challenges and areas of ambiguity.

6. Assessing the preclinical safety of a human embryonic stem-cell-derived cell replacement therapy Safety is a primary concern for any novel therapeutic, but for a cell replacement therapy ATMP it is the pre-eminent concern. Arguably, the four main risks specifically associated with the pluripotent stem-cell-derived cell therapy product are — the presence of contaminating hESC in the cell therapy product. This would have the potential for teratoma formation upon transplantation. — the presence of tumourogenic cells in the cell therapy product. This could be as a result of the accumulation of chromosomal changes/mutations during the propagation of the cells as part of manufacturing, leading to the potential for tumour formation upon transplantation. — the presence of a contaminating cell population with the potential for deleterious effects upon transplantation. This would particularly be true if the contaminating cells had proliferative potential. — the presence of adventitious agents in the hESC line. Extensive testing for human and animal adventitious agents is required for cell banks and for any human- or animal-derived component. A remaining gap is our ability to test for transmissible spongiform encephalopathies (TSE), particularly following the bovine spongiform encephalitis outbreak in the 1980s. While assays to detect TSE at the required level of sensitivity are under development [25], nothing is available for routine testing and as such currently a risk-assessment-based approach is required to minimize concerns. Mitigating against these risks requires significant time, effort and investment across a number of disciplines: (i) Development and implementation of quality assays for purity/identity and impurity to support the manufacturing process. By definition, for an hESC-derived cell replacement therapy the assay(s) determining purity/identity will need to be bespoke for the particular differentiated cell type. An obvious approach is to use an antibody to a protein marker that identifies to the cell type of interest. For instance, for RPE cells such

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(a) RPE monolayer

development and validation of patch transporter

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development, validation of QC process/release/ comparability assays

GLP pig toxicology GLP mouse teratoma toxicology

development and validation of manufacturing process

GMP engineering run

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investigator brochure IMPD clinical protocol informed consent patient information

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Figure 2. Activities required to progress an RPE cell replacement therapy towards the clinic. The ‘surgical delivery device’ is the instrument required for the subretinal implantation of the patch. The ‘patch cutter’ is the device that cuts the lozenge shaped patch out of the transwell. The ‘patch transporter’ is the container in which the patch will be carried from the GMP manufacturing facility to the surgical suite. ‘GLP’ refers to good laboratory practice; a standard requirement for data used to support a regulatory submission. ‘QC’ refers to quality control. ‘GMP’ refers to good manufacturing practice, a requirement for the manufacture of a material to be used as a therapeutic agent. ‘GMP engineering run’ refers to a trial manufacturing run performed in the GMP facility using protocols to be subsequently employed to support the manufacture of material to be used in the clinical study. The items listed in the blue ellipsoid are the documents necessary to obtain regulatory and ethical approval for a clinical trial; ‘IMPD’ is the Investigational Medicinal Product Dossier, containing information relating to the manufacture of the therapeutic agent under investigation (i.e. in the context of this article, hESC-derived RPE cells on a polyester membrane). markers include pmel17, mertk and cralbp. In terms of impurity, the most likely contaminating cell type of interest will be hESC. There are a number of very well validated markers of hESC, such as TRA-1–60, SSEA3, SSEA4 for an antibodybased assay, or nanog, oct4 for an mRNA-based assay. All of these may be suitable for identifying the presence of residual hESC during the manufacturing process and in the final product. A generic question which arises is—what technology platform should be used for the analysis? This needs to be considered in the context of cost, number of cells required, suitability for a good manufacturing practice (GMP) environment, and whether a population-based assay (e.g. flow cytometry- or plate-based image analysis) or a single cell approach (e.g. conventional immunocytochemistry with manual analysis) is more appropriate. For a therapy at the beginning of its development phase, a ‘low-tech’ approach is probably advisable. A critical question regarding purity and impurity assays is the acceptability criteria. While clearly the precision, sensitivity and robustness of the assay are important factors, fundamentally, it comes down to determining how pure is pure enough? Is greater than 95% purity acceptable, and does it matter what constitutes the other 5% if the product is safe in in vivo toxicology studies? This is not dissimilar from what would be required for the early stage development of a biologic such as a monoclonal antibody. Similarly, what level of sensitivity (i.e. the lower limit of detection) is necessary for an

assay detecting residual hESC? In the real-world situation where GMP compliant technology is constrained and cell supply is limited, most assays will struggle to achieve higher sensitivity than a lower-limit-of-detection of 0.1%. Thus, is the potential presence of one hESC in 1000 cells of the product acceptable? (ii) Analysis of hESC chromosomal/genetic stability. Chromosome instability is a recognized issue for pluripotent stem cells [26]. The most validated and widely accepted methodology for detecting chromosomal abnormalities is metaphase karyology. However, it could be argued that it is a twentiethcentury technology, and in the twenty-first century, we should be using more sensitive techniques such as comparative genome hybridization, chromosome fluorescence in situ hybridization, or indeed genomic sequencing. The danger here of course is that our level of understanding of genomic variation is such that for many chromosomal changes and for most differences at the nucleotide level we are unable to usefully interpret the data; as such, gathering such data serves no useful purpose and indeed has the potential to negatively impact the development of a project. (iii) Preclinical in vitro studies to validate the manufacturing process and demonstrate the absence/lack of survival of hESC. Data can be generated to further allay the fear of residual hESC in the final product. The most obvious approach is to perform so-called spiking experiments, whereby hESC are spiked

Phil. Trans. R. Soc. B 370: 20140375

development and validation of patch cutter

manufacture of hESC and RPE cells for regulatory toxicology

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development and validation of surgical delivery device

manufacture of human fibroblast feeder cell bank

Manufacturing cells in a GMP environment brings with it a new set of challenges compared with generating cells in a research laboratory. The more complex the process, the more

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7. Manufacturing the cell product

challenging it becomes. Thus, a long, multifaceted differentiation protocol with multiple steps and the addition/ removal of multiple exogenous growth factors all add to the challenge and multiply the risk of a problem occurring in the process. Before GMP manufacturing even begins, it will be necessary to source all the required cell culture components to the necessary regulatory compliant grade, together with a risk-based approach, as appropriate; the need for an exotic and expensive growth factor may be inconvenient in the context of a research laboratory, but in the context of sourcing GMP compliant reagents, this is multiplied many fold and may be restrictive. It is also important to consider issues around continuity of supply of material; batch variation, or the need to change suppliers, is undesirable. Finally, if the process is so complex that it is dependent upon a few key staff with the requisite in-depth experience, staff turnover becomes a significant risk to the programme. Thus, the mantra—‘keep it simple’—is important in practical terms. In the context of an RPE cell replacement therapy, we are fortunate enough to have a relatively simple differentiation protocol that relies upon the spontaneous formation of RPE from confluent hESC cultures [17]. Most differentiation protocols are somewhat more complex and technically challenging, as exemplified by the generation of dopaminergic neurons or pancreatic beta cells for example [31,32]. Scale of manufacturing is also an important factor. Before even considering the manufacture of the differentiated cell product, it will be necessary to generate an hESC master cell bank. For an activity which is based very much upon the judgement and experience of the operator, large-scale hESC expansion to a high level of pluripotency within the constraints of GMP is not a cheap or straightforward activity. It is also necessary to factor in the requirement for testing the master cell bank for adventitious agents, both in terms of the significant number of cells required and the cost. In terms of number of differentiated cells required to treat a patient, for an RPE cell therapy, doses of around 1 105 cells per eye are being used ([5] and Whiting et al. 2012, unpublished data), which in principle is relatively small scale and manageable. The dose of oligodendrocyte precursor cells used by Geron in their initial spinal cord injury clinical trial was 2 million cells [2]; in the planned studies by Asterias, the doses will increase to 10 and 20 million (https://clinicaltrials. gov/ct2/show/NCT02302157?term=Asterias&rank=1). Considering, for instance, liver replacement therapies, number of cells could conceivably increase to hundreds of millions per patient, and will bring with it significant challenge. Another realization is that hESC differentiation protocols often take a long time (weeks to months), particularly if they require a ‘maturation’ phase at the end of the differentiation process. The requirement for an extended manufacturing process without an intermediate breakpoint has significant risk that at some point something will go wrong, resulting in loss of the manufacturing campaign and the need to go back to the start of the process. It is also incompatible with the requirement of an annual shutdown and facilities maintenance of a GMP unit. Thus, the inclusion of a step in the manufacturing process that enables a cryopreservation stage is highly desirable. This will most likely be a stage in the differentiation process constituted by a dividing progenitor cell population, as has been used in the generation of cortical neurons [33]. Experience indicates that time spent during the development phase of a manufacturing process optimizing, simplifying and introducing a cryopreservation step will be time well


into the cells at various stages of the manufacturing process, and it is determined whether they are able to survive the culture conditions and manipulations. Given the very demanding cell culture conditions required to maintain pluripotent hESC, it would be surprising if such studies revealed anything other than the rapid demise of the spiked-in hESC. Such experiments have been performed as part of validating an RPE manufacturing process and as expected have demonstrated lack of survival of hESC (Whiting et al. 2012, unpublished data). Of course, as discussed above, the conclusions of such studies are always going to have to be interpreted in the context of the detection limits of the impurity assay. Nevertheless, a conclusion that pluripotent hESC do not survive the manufacturing process represents a further important control step to limit the possibility of contamination of the final product. (iv) Preclinical in vivo studies to demonstrate absence of teratoma or tumour formation, absence of migration of cells away from the site of delivery. The teratoma assay, where cells are injected into an immunodeficient mouse and their ability to form a teratoma is assessed, is considered by many to be the gold standard assay for the demonstration of pluripotency of an hESC line [27]. It is also used as an in vivo toxicology assay, to demonstrate the absence of pluripotent cells in a differentiated cell population [28]. It should be noted that for hESC (as opposed to mouse ESC) it is not actually a very sensitive assay; at least a hundred hESC need to be present to enable the formation of a teratoma, even under optimal conditions [29]. Another important consideration is the site of delivery; teratoma formation does seem to be influenced by the tissue into which the cells are delivered [30]. While the standard sites for delivery are subcutaneous or intramuscular, when a teratoma assay is being performed to evaluate toxicology it may be more appropriate to deliver the cells to the clinically relevant site, e.g. the eye for an RPE cell replacement therapy, as the local environment could conceivably influence teratoma formation. A final question to consider is how many cells should be injected to adequately test the risk of teratoma formation? When toxicology is performed to evaluate the safety of a small molecule drug, doses are chosen so as to give a good margin over the predicted therapeutic dose. When considering a cell replacement therapy this concept of a safety margin becomes more difficult. For instance, if the predicted human dose of cells for an RPE cell replacement therapy is 100 000, what is an appropriate number of cells to dose a mouse in a teratoma, and by what rational basis is it chosen? Practical issues certainly play into the discussion: there may be practical limits to the number/ volume of cells that can be delivered into the clinically relevant site, e.g. the eye of a mouse is small, and so this limits the injection volume to 1 ml and thereby the number of cells to less than 100 000. In summary, the safety assessment of an hESC-derived cell replacement therapy needs to done in the context of a number of considerations and sets of data, both preclinical and clinical, so as to come to a conclusion as to the risk: benefit to the patient.

experience of having to run such a project within a start-up biotech company, or indeed, an academic environment, where this would likely have to be contracted in.

9. Summary 8. Developing a regenerative medicine therapy within pharma: pros and cons

Authors’ contributions. P.W., J.K., P.C., L.d.C. and R.M. all made contributions to the concept and design of this article, the drafting and revision and the approval. Competing interests. P.W., J.K., R.M. are all employees of Pfizer Ltd.

Funding. P.C. is funded by the Medical Research Council, Moorfield’s Eye Hospital BRC, California Institute for Regenerative Medicine.

References 1.

2.

3. 4.

5.

6.

7.

8.

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. 1998 Embryonic stem cell lines derived from human blastocysts. Science 6, 1145– 1147. (doi:10.1126/ science.282.5391.1145) Lebkowski J. 2011 GRNOPC1: the world’s first embryonic stem cell-derived therapy. Interview with Jane Lebkowski. Regen. Med. 6, 11 –13. (doi:10. 2217/rme.11.77) Scott C, Huggett B. 2012 Geron’s quixotic fate. Nat. Biotechnol. 30, 497. (doi:10.1038/nbt.2253) Schwartz SD et al. 2014 Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet 6736, 61 376–61 383. (doi:10.1016/S0140-6736(14) 61376-3) Park K-M, Kim H-J, Lee JH, Choi J, Young Chong S, Shim SH, Del Priore LV, Lanza R. 2015 Treatment of macular degeneration using human embryonic stem cell derived retinal pigment epithelium: preliminary results in Asian patients. Stem Cell Rep. 4, 860–872. (doi:10.1016/j.stemcr.2015.04.005) Reardon S, Cyranoski D. 2014 Japan stem cell trial stirs envy. Nature 513, 287–288. (doi:0.1038/ 513287a) Stein-Streilein J. 2013 Mechanisms of immune privilege in the posterior eye. Int. Rev. Immunol. 32, 42 –56. (doi:10.3109/08830185.2012.740535) Detrick B, Hooks JJ. 2010 Immune regulation in the retina. Immunol. Res. 47, 153 –161. (doi:10.1007/ s12026-009-8146-1)

9.

10.

11.

12.

13.

14.

15.

Cook HL, Patel PJ, Tufail A. 2008 Age-related macular degeneration: diagnosis and management. Br. Med. Bull. 85, 127–149. (doi:10.1093/bmb/ ldn012) da Cruz L, Chen FK, Ahmado A, Greenwood J, Coffey P. 2007 RPE transplantation and its role in retinal disease. Prog. Retin. Eye Res. 26, 598 –635. (doi:10. 1016/j.preteyeres.2007.07.001) Sparrow JR, Hicks D, Hamel CP. 2010 The retinal pigment epithelium in health and disease. Curr. Mol. Med. 10, 802–823. (doi:10.2174/1566524 10793937813) Tzameret A et al. 2014 Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ameliorates retinal degeneration in a rat model of retinal dystrophy. Exp. Eye Res. 118, 135 –144. (doi:10.1016/j.exer.2013.10.023) Chen FK, Uppal GS, MacLaren RE, Coffey PJ, Rubin GS, Tufail A, Aylward GW, Da Cruz L. 2009 Longterm visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration. Clin. Exp. Ophthalmol. 37, 275 –285. (doi:10.1111/j. 1442-9071.2009.01915.x) Chen FK, Patel PJ, Uppal GS, Rubin GS, Coffey PJ, Aylward GW, Da Cruz LA. 2009 Comparison of macular translocation with patch graft in neovascular age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 50, 1848–1855. (doi:10.1167/iovs.08-2845) Muthiah MN, Keane PA, Zhong J, Gias C, Uppal G, Coffey PJ, da Cruz L. 2014 Adaptive optics imaging shows rescue of macula cone photoreceptors.

16.

17.

18.

19.

20.

21.

Ophthalmology 121, 430–431. (doi:10.1016/j. ophtha.2013.10.008) Carr AJ et al. 2009 Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay. Mol. Vis. 15, 283–295. Vugler A et al. 2008 Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp. Neurol. 214, 347–361. (doi:10.1016/j.expneurol. 2008.09.007) Salero E, Blenkinsop TA, Corneo B, Harris A, Rabin D, Stern JH, Temple S. 2012 Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell 10, 88 –95. (doi:10.1016/j.stem.2011.11.018) Choudhary P, Dodsworth B, Sidders B, Gutteridge A, Michaelides C, Duckworth J, Whiting P, Benn C. 2015 A FOXM1 dependent mesenchymal – epithelial transition in retinal pigment epithelium cells. PLoS ONE 10, e0130379. (doi:10.1371/journal.pone. 0130379) Hsiung J, Zhu D, Hinton DR. 2015 Polarized human embryonic stem cell-derived retinal pigment epithelial cell monolayers have higher resistance to oxidative stress-induced cell death than nonpolarized cultures. Stem Cells Transl. Med. 4, 10– 20. (doi:10.5966/sctm.2014-0205) Sugino IK et al. 2011 Comparison of FRPE and human embryonic stem cell-derived RPE behavior on aged human Bruch’s membrane. Invest. Ophthalmol. Vis. Sci. 52, 4979–4997. (doi:10.1167/ iovs.10-5386)

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At first, a large pharmaceutical company may not seem the obvious home for the development of a cell replacement regenerative medicine therapy, and pharma as an industry has not yet committed to this therapeutic modality. Cell replacement regenerative medicines clearly tick the ‘innovation’ box. However, such novel modalities can struggle to fit into a business template designed for small molecules or antibodies. One of the most tangible advantages of being embedded in a pharmaceutical company is the access to know-how. Expertise in regulatory affairs, chemistry, manufacturing and control, previously honed and applied to more conventional biotherapeutics development can be very effectively used to enable a cell replacement therapy programme. Having access to this expertise ‘under one roof’ would clearly contrast to the

Cell replacement regenerative medicine therapies based upon hESC are still a very long way from achieving their potential. Small steps have been taken towards demonstrating safety in humans. However, it is important to recognize that there has yet to be any demonstration of clinical efficacy; this will require larger and robust phase 2 studies. Meanwhile the excitement and expectation, now fed in large part by IPSC technology, continues to grow. At present, however, a number of hurdles need to be overcome before pluripotent stem-cell-based therapies become available to the wider population.

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spent in the long run. Once regulatory approval has been granted for the manufacturing process, while it is possible to introduce subsequent changes into the method through comparability and/or bridging studies, the reality is that you are inevitably somewhat constrained.

30. Cooke MJ, Stojkovic M, Przyborski SA. 2006 Growth of teratomas derived from human pluripotent stem cells is influenced by the graft site. Stem Cells Dev. 15, 254 –259. (doi:10.1089/scd. 2006.15.254) 31. Studer L. 2012 Derivation of dopaminergic neurons from pluripotent stem cells. Prog. Brain Res. 200, 243–263. (doi:10.1016/B978-0-444-595751.00011-9) 32. Pagliuca FW, Millman JR, Gu¨rtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. 2014 Generation of functional human pancreatic b cells in vitro. Cell 159, 428–439. (doi:10.1016/j. cell.2014.09.040) 33. Shi Y, Kirwan P, Livesey FJ. 2012 Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836– 1846. (doi:10.1038/nprot. 2012.116)

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Phil. Trans. R. Soc. B 370: 20140375

26. Na¨rva¨ E et al. 2010 High-resolution DNA analysis of human embryonic stem cell lines reveals cultureinduced copy number changes and loss of heterozygosity. Nat. Biotechnol. 28, 371 –377. (doi:10.1038/nbt.1615) 27. Nelakanti RV, Kooreman NG, Wu JC. 2015 Teratoma formation: a tool for monitoring pluripotency in stem cell research. Curr. Protoc. Stem Cell Biol. 32, 4A.8.1 –4A.8.17. (doi:10.1002/9780470151808. sc04a08s32) 28. Cunningham JJ, Ulbright TM, Pera MF, Looijenga LH. 2012 Lessons from human teratomas to guide development of safe stem cell therapies. Nat. Biotechnol. 30, 849–857. (doi:10.1038/nbt.2329) 29. Gropp M et al. 2012 Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny. PLoS ONE 7, e45532. (doi:10.1371/journal. pone.0045532)


22. Diniz B et al. 2013 Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer. Invest. Ophthalmol. Vis. Sci. 54, 5087–5096. (doi:10.1167/iovs.12-11239) 23. Velez-Montoya R, Oliver SC, Olson JL, Fine SL, QuirozMercado H, Mandava N. 2014 Current knowledge and trends in age-related macular degeneration: genetics, epidemiology, and prevention. Retina 34, 423–441. (doi:10.1097/IAE.0000000000000036) 24. Smith AG, Kaiser PK. 2014 Emerging treatments for wet age-related macular degeneration. Expert Opin. Emerg. Drugs 19, 157– 164. (doi:10.1517/ 14728214.2014.884559) 25. Jackson GS et al. 2014 Population screening for variant Creutzfeldt-Jakob disease using a novel blood test: diagnostic accuracy and feasibility study. JAMA Neurol. 71, 421– 428. (doi:10.1001/ jamaneurol.2013.6001)