Final Report Summary - BEST-STEM CELLS (Biochemically equivalent substitutive technology for stem cells)
Executive summary:
The biochemically equivalent substitutive technology (BEST) for stem cells project was a focused small-medium-scale collaborative research project (ID 223410) addressing the FP7-HEALTH-2007-B call fiche (1.4.7) for development of stem cell culture conditions. The rationale for the project was that improvement of the efficacy and safety of human stem cell culture for research, industrial and clinical applications requires the reduction or elimination of current reliance on animal cell and tissue derived biological reagents (i.e. extracellular matrix, blood serum / serum fractions) which can be a source of variance and pathogens. The goal of the project was thus to develop new knowledge and reagents for human stem cell culture. Research was predominantly focused on pluripotent human embryonic stem cell (hESC) culture but also addressed multipotent human mesenchymal stem cell (hMSC) culture, using an hESC-derived mesenchymal progenitor (hES-MP) as a model system for the latter to overcome variability normally associated with adult tissue derived hMSC. The consortium was composed of 8 academic and commercial partners across 5 European Union (EU) Member States, namely the University of Edinburgh (UEDIN), Roslin cells (RC) and Glycomar (GLYCO) in the United Kingdom (UK); INSERM and CNRS in France; University of Bonn (UBONN) in Germany; Bioneer (BIO) in Denmark, and University of Liege (ULg) in Belgium. The contact details for the list of beneficiaries is provided in Final Report Annex I, Figure 1.
The principle achievements of the project included the:
(1) Discovery of chemical polymers substrates supporting that attachment and growth of hESC and hMSC, from which cells could be gently dissociated by lowering of ambient temperature. These provide an alternative to current requirements for animal tissue derived substrates, and harsher methods of cell dissociation with a potential to damage cells including treatment with cell protein digesting enzymes, chemicals or mechanical (WP1.1: UEDIN).
(2) Discovery and validation of chemical small molecules to substitute or reduce current requirements for protein growth factors and serum supplements for growth of hESC and hMSC, respectively (WP1.2: UEDIN).
(3) Development of recombinant high-affinity and / or membrane permeant proteins affecting hESC survival and growth and validation of the significance of the pathways being targeted (WP 1.3 and 1.4: CNRS, UBONN, INSERM, Bioneer).
(4) Development of an aseptic serum free cryopreservation protocol for hESC and identification of non-vertebrate of origin cryoprotective agents (WP1.5: ULg, GLYCO).
(5) Provision of training in research quality assurance to provide consortium partners with improved awareness of what is required to translate basic research into good manufacturing practice (GMP) (WP0.2: RCs).
(6) Public dissemination of BEST-STEM CELL consortium principles and outputs (WP0.1: All).
Project context and objectives:
The founding principle of the BEST-STEM CELLs project is that the future safety and efficacy of stem cell based therapies is critically dependent on the development of BEST for cell culture. This is necessary to reduce or obviate the current dependence that all stem cell types and their derivatives have on vertebrate-animal cell and tissue derived biological reagents (i.e. extracellular matrix preparations, blood serum / serum fractions) for their growth, differentiation and storage. These reagents can contribute to variations in stem cell behaviour through their own variability in purity and bioactivity. They also pose serious risks to transplant recipients and through them, the general population, for transmission of known and unknown adventitious pathogens. Clinical delivery of stem cell based therapies ultimately also requires compliance with evolving standards for GMP and regulatory governance. These significantly prolong the time it takes for research gains at the bench to be translated to bedside clinical treatments. In principle, this can be improved by early promotion of research quality assurance and assessment of methods and tool translatability to GMP standards. The mission of the BEST-STEM CELLS project is thus to develop and warrant as translatable to GMP standards, strategies and tools that will yield safe and efficacious BEST for stem cell culture.
Central to safety and efficacy is the extent to which the reagents used to cultivate and store stem cells can be biochemically defined, and thus free from unknown contaminants and variables inherent in biological reagents. It is also important that new methods and tools have no residual undesirable effects (such as for example induction of genetic instability), and that ultimately they can be automated so as to minimise skilled operator dependence. Of the stem cell types available for research and therapeutic use, pluripotent human embryo stem cells (hESCs) and those artificially induced into this state (i.e. human-induced pluripotent stem cells (hiPSC)) pose both the greatest challenges and rewards. Their reward lies in their unparalleled growth and differentiation potential. These ultimately could provide the quantities of cells that are likely to be required to treat most diseases, as well as offer effective treatment for conditions which presently are either not or are inadequately being addressed by adult tissue derived cells. hMSCs, normally sourced from diverse adult tissues such as bone marrow, placenta and fat constitute another stem cell type for which there is significant clinical interest, either as a multipotent stem cell source of bone and cartilage to rebuild these tissues or as immune modulating cells. In comparison with hESC and hIPSC these are mortal (i.e. will not grow indefinitely) and their properties are less well defined and variable in relation to donor age, health, tissue of origin and time in culture. A renewable and standardisable supply of hMSC-like cells can be derived from human pluripotent stem cells, thereby facilitating discovery of supportive reagents.
Thus, the main objectives for the project were to develop new knowledge and reagents for human stem cell culture and storage, focusing on pluripotent and multipotent hESC and hMSCs, respectively. To represent the latter the project relied on a hESC-derived mesenchymal progenitor (hES-MP). Specifically, project work packages sought to:
(a) discover chemically defined polymer substrates that could support adhesion and growth (WP1.1);
(b) discover chemically defined small molecules that could substitute, reduce or augment current reliance on protein growth factors or serum as culture supplements (WP1.2);
(c) develop and evaluate new high affinity and/or membrane permeable recombinant proteins for controlling biological mechanisms controlling pluripotent stem cell growth (WPs 1.3 1.4);
(e) develop an aseptic serum free cryopreservation protocol and identify sustainable marine invertebrate derived cryoprotectants supporting human pluripotent stem cell cryopreservation (WP1.5);
(f) provide training in quality assurance and gap assess (i.e. identify faults, omissions and outstanding issues associated with) methods and tools developed by the BEST-STEM CELLS project in order to comply with future GMP regulatory requirements, most notably the EU Tissues and Cells Directive (WP0.2);
(g) publically disseminate knowledge / awareness of BEST-STEM CELLS research outputs (WP0.1).
Project Results:
Polymer substrates supporting human stem cell culture
BEST Stem cells research at UEDIN has identified and validated a family of polymers based on 2-(diethylamino)ethyl acrylate (HG9, 20, 21) which support hESC renewal and permit gentle, chemically defined cell passaging achieved by reduction of temperature from 37 to 15 degrees of Celsius for 15 minutes followed by gentle pipetting of the medium (Final Report Annex I, Figure 2; for Video see Final Report Annex III). One member of this hydrogel family (HG21) was demonstrated to be capable of supporting continuous long-term (> 6 months) growth and maintenance of a pluripotent stem cell phenotype validated using 3 independent hESC lines (RCM1, RH1 and H9). This was achieved in a biologically defined animal serum-free medium. This work has been published in Nature Communications (Zhang et al., 2013). Seeking to improve the screening throughput further UEDIN has also established a super high-density array enabling concurrent evaluation of 7 000 different polymers in a single cellular-based screen (Final Report Annex I, Figure 3). Polymers identified in this screen were validated for their capacity to support extended growth of hESC whose passage is mediated enzymatically. At time of writing the manuscript describing these results has been submitted for publication.
HG21 epitomises the kind of output the BEST Stem cell project was intended to yield. It constitutes a chemically defined alternative to currently used, undefined biological substrates and provides a flexible and scalable approach for improving the definition, efficacy and safety of pluripotent human stem cell culture systems for research, industrial and clinical applications. Since the initiation of the BEST-STEM CELLS project a number of polymer and peptide-polymer substrates have been reported with a capacity to sustain hESC (Mei et al., 2010; Melkoumian et al., 2010; Vila-Diaz et al., 2010). Limitations in these advances have included variation in cell line responsiveness and/or requirements for feeder cell conditioning of media or coating of surfaces with serum or serum proteins. Critically, for all substrates reported to date cell dissociation at passaging has required one or more treatments involving mechanical scraping or colony picking, proteolytic enzymatic digestion, or chemically mediated chelation of divalent cations (e.g. calcium and magnesium). While mechanical dissociation is laborious and not readily scalable, enzymatic and chemical treatments can damage cells by removal of important surface proteins or ions (e.g. calcium).
The HG21 polymer has the potential to support hiPSCs both in the course of reprogramming and for established cell lines. In the context of the former it could improve the efficiency of generating hiPSC by providing a means to selectively passage reprogrammed cells. Experiments validating its application to hiPSC generation and culture are ongoing.
Parallel effort in the BEST-STEM CELLS project at UEDIN also sought to identify chemically defined substrates supportive of hMSC growth and potency. These cells, normally sourced from adult tissues such as bone marrow and fat, can adhere to polystyrene plastic surfaces of tissue culture-ware without any additional substrates, although such attachment is non-selective, permits spontaneous differentiation and supports only limited growth. Despite the apparent lack of specificity implied by substrate-independent attachment of hMSC to polystyrene plastic, mesenchymal stem cell growth, function and differentiation is influenced by cell interaction with materials. This includes interactions with specific biochemical molecules (i.e. micro-contact printed patterns of fibronectin, modified or additional functional groups (i.e. alcohol and amino groups), substrate stiffness and topography (Benoit et al., 2008; Curran et al., 2006; Engler et al., 2006; Kilian et al., 2010; McMurray et al., 2011).
Using an hESC-derived mesenchymal progenitor (hES-MP), as a surrogate for human adult tissue derived MSC whose properties can vary between donors, and tissue source, BEST-STEM CELLS research at UEDIN has identified a finite set of 5 polymers supporting a mesenchymal stem cell phenotype and growth following successive cell passaging, a subset of which are thermomodulatable enabling gentle release at passaging. At time of writing the capacity of these to support long term growth and differentiation potential of both hES-MP and adult adipose derived MSC was being evaluated. Research completed to date has validated that they are more selective of an MSC phenotype. Thus, they provide a means to enrich for these cells, which can be exploited to prepare a more homogenous population for research or clinical applications.
Small molecules supporting human pluri- and multi-potency
UEDIN has discovered multiple small molecules that substitute for the absence of either or both basic Fibroblast Growth Factor (bFGF) and transforming growth factor-beta (TGFb) in the context of hESC growth. The first of these substitutes for the former through modulation of calcium signaling (Ermakov et al., 2012). Others have been confirmed to phosphorylate protein kinases (ERK, SMAD) which are normally activated by bFGF and TGF-beta or LIF (STAT-3). Additionally, a complementary project supported in part by funding for the BEST-STEM CELLS project has validated an affinity targetable controlled release approach to delivery of growth factors in the culture of pluripotent stem cell cells using a biodegradable nanoparticle platform (Corradetti et al., 2012). In a concurrently published paper this platform has also been validated for delivery of small molecules (Park et al., 2012). The affinity targeted and controlled release capacities of this platform provide for a reduction in the requirement for costly growth factors by a factor of over 10^4. It also provides targeted cells with a selective growth advantage by minimising the potential for bio-active factors to support or have competing effects on diverging (i.e. differentiating) cell populations.
Using the aforementioned hESC-derived mesenchymal progenitor (hES-MP), UEDIN has also identified a finite set of small molecules which augment growth (12) and expression (5) of MSC cells expressing the canonical MSC marker, STRO-1. While this research did not progress sufficiently to understand the biological significance of these effects or their mechanism, it provides leads for future studies and targets for modulation of both MSC expansion and function. As regards the latter all of the small molecules that enhanced STRO-1 expression have been reported to modulate cell-signalling pathways directly or indirectly in immune modulation and inflammation, an important non-stem cell function attributed to MSC. Thus, small molecules identified in this project may not only serve to promote the expansion of MSC when presented as a media supplement, they could also serve to augment the therapeutic properties of these cells following transplantation, which could be accomplished through their encapsulation and affinity-targeted controlled release in biodegradable particles as noted in the aforementioned complementary research on pluripotent stem cells.
Recombinant protein technology for pluripotent human stem cells
BEST-STEM CELLS consortium partners at UKB (UBONN), BIO and CNRS-ENS have used recombinant protein technology to develop high affinity and membrane permeant proteins providing novel opportunities to control pluripotent human stem cell induction, renewal and viability.
At CNRS-ENS, peptide aptamer technology providing high-affinity ligands for manipulation of biological processes has yielded novel probes that are extremely toxic to embryonic stem cells, and as such could be used to remove them in the event that they contaminate a population of differentiated derivatives. A novel approach for batchwise identification of both the target of a peptide aptamer and the aptamer itself has been developed, as well as novel forms of membrane permeant peptide aptamers through collaborative research with UKB. This underpins ongoing an future collaborative research beyond BEST-STEM CELLS between these partners and with UEDIN, to evaluate these reagents in the context of controlling pluripotent stem cell induction and growth. Notably with novel peptide aptamers that activate calcineurin (Dibenedetto et al., 2013).
Expertise in recombinant protein production and high-affinity and membrane proteins at Bioneer and UKB were also combined in the BEST Stem cells project to develop and apply membrane permeant versions of pluripotency associated factors, notably a membrane permeant version of one of the key transcription factors regulating pluripotent stem cell renewal, Nanog. This was evaluated in the context of controlling pluripotent stem cell renewal and to induction of a pluripotent stem cell phenotype (see below).
Aseptic serum free cryopreservation
At ULg, the BEST-STEM CELLS project has yielded the development of an aseptic, serum-free vitrification method for hESC. With GLYCO it has also identified two candidate microalgae derived polysaccharides that are non-toxic as assessed using established embryo and embryonic stem cell assays. These constitute new approaches and reagents for cryopreservation likely to be of broad utility for eukaryotic cells in general.
Novel insight regarding control of pluripotent human stem cells
Importantly, the novel reagents and tools developed in the BEST-STEM CELLS project have also led to important insights as to the mechanisms controlling a pluripotent phenotype in human cells. A pluripotent state is traditionally defined as the ability of a stem cell to give rise to all of the cells of the body. In a mouse model this can be verified by transplantation of cells into a preimplantation embryo and having them contribute to all tissues in the resulting animal including the gonads (testis and ovaries) in the form of sperm and eggs with a competence to form viable offspring, or alternatively differentiation of these cell types ex vivo. Studies in the mouse have identified three prospective sources of pluripotent stem cells, the inner cell mass of a preimplantation embryo, classically referred to as embryonic stem cells and successive sub-populations of derivative epiblast and primordial germ cells. These cells vary in a number of ways, with the former conceptually distinguished from the latter as constituting a more naive vs primed state.
At the present time, the developmental state of human and primate embryonic stem cells resembles that of mouse epiblast stem cells (EpiSC) including morphology, low clonogenicity, growth factor requirements, and overall expression profiles. They are genomically unstable (Taapken et al., 2011), and gene targeting by homologous recombination in hESCs has proven difficult (De Los Angeles et al., 2012). Whereas mEpiSCs can be efficiently reverted into mESCs, the conversion of hESCs from a primed into a bona fide naive state has proven difficult and transient (De Los Angeles et al., 2012). In culture human pluripotent stem cells (whether hESC or iPSC) and mEpiSCs are cultured in the presence of basic FGF and TGFb / Activin, while naive mESCs are best maintained in the presence of LIF and inhibitors to GSK and MEK (2i) (Nichols and Smith, 2009). Switching of media results in differentiation or death of the corresponding cell types. Naive and primed cells are thus regulated by distinct signaling pathways. A major question in the field is why hESCs or hIPSCs cannot be derived in the presence of same growth factors and pathway regulators as mESCs.
BEST-STEM CELLS research at INSERM, has yielded one possible explanation for why human cells respond differently to LIF and this is that they appear to fail to activate the target genes of STAT3, a transcription factor normally recruited and activated upon LIF stimulation. The capacity is partially restored if hESCs are engineered to express a ligand-dependent form of STAT3 that bypasses the LIF receptor. The combination of LIF and ligand-dependent STAT3 allows hESC to escape from bFGF dependency, and to enter a new state closer to the naive pluripotent state. These findings raise the question of whether manipulation of the LIF-STAT3 pathway together with other factors, such as for example small molecules which modulate calcium signalling, and / or supportive polymer substrates as discovered by UEDIN can be used to generate bona fide naive pluripotent stem cells in human.
One of the transcription factors which is pivitol to maintenance of naive and primate states of pluripotency is Nanog. Using membrane permeant versions of Nanog UKB has demonstrated its importance for promotion of self-renewal and inhibiting differentiation to an endodermal lineage, and its independence from signaling pathways that have been implicated. Additionally, the consequence of introducing Nanog into differentiated primary and immortalised cells alone was evaluated. These studies have also demonstrated how protein transduction is an attractive tool to understand protein function without genetic manipulation.
Promotion of research quality
Lastly, the BEST-STEM CELLS project sought to promote an awareness of quality assurance in research and stem cell manufacturing within the consortium which is normally lacking within academic research institutions, due to lack of understanding and infrastructure. RCs trained consortium partners in the principles of quality assurance and gap assessed for quality and prospective requirements for translation of research to manufacturing standards the operating practices and research outputs of consortium partner laboratories. The latter focused on the most advanced of the BEST-STEM CELLS research outputs, HG21, the thermomodulatable hydrogel supporting long-term hESC renewal and pluripotency.
References
(1) Benoit, D. S. W., Schwartz, M. P., Durney, A. R. & Anseth, K. S. Small functional groups for controlled differentiation of hydrogel-encapsulated hMSCs. Nature Mater. 7, 816-823 (2008).
(2) Corradetti B, Freile P, Pells S, Fahmy T, De Sousa PA. Embryonic stem cell renewal mediated by affinity targeted paracrine stimulation. Biomaterials, Oct, 33 (28): 6634-43. (2012).
(3) Curran, J. M., Chen, R. & Hunt, J. A. The guidance of hMSC differentiation in vitro by controlled modifications to the cell substrate. Biomaterials 27, 4783-4793 (2006).
(4) De Los Angeles, A., Loh, Y.H. Tesar, P.J. and Daley, G.Q. Accessing naive human pluripotency. Curr Opin Genet Dev 22, 111 (2012).
(5) Dibenedetto S., C. D., Stebe, P.N. Baumlé V, Léault, J. Terreux, R., Bickle M, De Chassey, Mikaelian, IJ, Colas, P., Spichty M., Zoli M., Rudkin B.B. (2013). Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers. Mol. Cell. Proteomics, (EPub ahead of Print).
(6) Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677-689 (2006).
(7) Ermakov A, Pells S, Freile P, Ganeva V, Wildenhain J, Bradley M, Pawson A, Millar R, De Sousa PA. A role for intracellular calcium downstream of G-protein signaling in maintenance of undifferentiated hESCs. Stem Cell Research 9(3):171-184 (2012).
(8) McMurray RJ, Gadegaard N, Tsimbouri PM, Burgess KV, McNamara LE, Tare R, Murawski K, Kingham E, Oreffo RO, Dalby MJ. Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat Mater. 17;10(8):637-44 (2011).
(9) Kilian, K. A., Bugarija, B., Lahn, B. T. & Mrksich, M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl Acad. Sci. USA 107, 4872-4877 (2010). (10) Mei, Y. et al. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat. Mater. 9, 768-778 (2010).
(11) Melkoumian, Z. et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of hESCs. Nat. Biotechnol. 28, 606-610 (2010).
(12) Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487-492.
(13) Park J, Wrzesinski SH, Stern E, Look M, Criscione J, Ragheb R, Jay SM, Demento SL, Agawu A, Licona Limon P, Ferrandino AF, Gonzalez D, Habermann A, Flavell RA, Fahmy TM. Combination delivery of TGFb inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy. Nat Mater. Oct;11(10):895-905 (2012).
(14) Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of hESCs. Nat. Biotechnol. 28, 581-583 (2010).
(15) Zhang R, Mjoseng HK, Hoeve MA, Bauer NG, Pells S, Besseling R, Velugotla S, Tourniaire G, Kishen REB, Tsenkina Y, Armit C, Duffy CRE, Helfen M, Edenhofer F, De Sousa PA* and Bradley M* A thermoresponsive chemically-defined hydrogel for long term culture of hESCs Nature Communications 4: 1335. doi: 10.1038/ncomms2341. (2013).
Potential Impact:
The BEST-STEM CELLs project has yielded new and improved technology for the cultivation of human stem cells of embryonic and adult origin. At time of writing it had yielded the following tangible outputs:
(1) Training and professional development
Over the course of project, BEST Stem cells has supported 57 people with a marginal female gender bias (female / male: 31 / 26) for durations of 1 to 42 months. Of these 32 were experienced Doctor of Philosophy (PhD) researchers (12:20), 21 were experienced non-PhD researchers (16:5) and 4 were PhD students (3:1). Consortium scientists received training in quality assurance in research provided by RCs, as well as introductory lectures in the diverse methodologies and technologies which each consortium partner had expertise in. This included recombinant protein technologies (Bioneer, UKB, CNR-ENS), live visible reporting systems as applied to pluripotent stem cells (INSERM), polymer chemistry and screening (UEDIN), glycobiology (GLYCO) and cryopreservation (ULg). In the closing public dissemination meeting, consortium partners and members of the public were also introduced to the expertise of the invited speakers on manufacturing science (Prof. David Williams, University of Loughborough), social value systems and regulatory policy development (Prof. Joyce Tait, University of Edinburgh), and immunotherapy and biomedical engineering of nanotechnology for affinity targeted and controlled drug release (Prof. Tarek Fahmy, Yale University).
(2) Public dissemination
The consortium has collectively participated in public launch and closing meetings. The launch of the project coincided with the first general assembly meeting held in Lyon, France on 29 and 30 October 2009. The launch was communicated by a press release that received regional press coverage in the UK and France (Final Report Annex I, Figure 4, 5). The public launch of the project also coincided with the establishment of the project website: http://www.beststemcells.ed.ac.uk. In the course of the project partners communicated ongoing research progress at international and national meetings, invited lectures and public forums, cumulatively totalling 61 engagements in 10 countries including the UK, France, Germany, Belgium, USA, Canada, Singapore, Japan and China. These addressed audiences ranging from 20 to 500 delegates, cumulatively totaling approximately 5000 people. Press releases were issued in association with high profile accomplishments, most notably the communication of the hESC supportive thermomodulatable hydrogels. Lastly the consortium held a public dissemination meeting in Edinburgh on 22 October 2012, attended by over 60 participants at which partners presented project outputs and expertise together with invited national and international scientists presenting complementary research in keynote addresses (See Final Report Annex II, BEST SC Public Dissemination meeting Programme OCT2012).
Publications
There have been 3 publications, in Cell Stem Cell (Impact Factor, IF, 25), Nature Communications (IF 7.9) and Stem Cell Research (IF 5.3) that were a direct output of the research. Indirect outputs include one publication in Biomaterials (IF 7.9) and another accepted in press in Molecular and Cellular Proteomics (IF 7.9). These emanated from three consortium partners UKB, UEDIN, and CNRS-ENS (Their et al., 2012; Zhang et al., 2013; Ermakov et al., 2012; Corradetti et al., 2012; Dibenedetto et al., 2013). Additionally at least another 5 manuscripts have been submitted or will be submitted within 2013 from INSERM (1), UKB (1), and UEDIN (3).
Intellectual property
There have been two patents filed from UEDIN pertaining to the polymer and small molecule research. One has been commercially licensed. The other has been optioned to a commercial company for time limited consideration. Another 10 invention disclosures are being prepared or have been submitted by partners at UEDIN, ULg, CNRS-ENS/INSERM, UKB and BIONEER for institutional evaluation for suitability for filing with national patent offices. This intellectual property covers the four domains of research proposed in the project pertaining to polymer substrates, small molecules, recombinant protein technology and marine organism derived products in the context of stem cell growth and cryopreservation. The prospective application and exploitation of these outputs is as follows:
(i) Polymer substrates supporting human stem cell culture
UEDIN has filed intellectual property pertaining to the nature and use of a chemically defined thermomodulatable polymer hydrogel with high affinity for and support of long-term growth of pluripotent human stem cells (GB1110042.7 WO2012/172291). This has been licensed to a UK materials company. The exploitable product resulting from this research will take the form of polymer coated tissue culture plastic ware or micro-carriers (ie. Beads) for purposes of achieving scalable growth of pluripotent human stem cells for research, industrial screening and therapeutic applications. Additional IP is anticipated in relation to polymers supporting hMSC phenotype, growth and multi-potency. The application of these will depend on what aspects of hMSC specific polymers support. This may range from selective enrichment of cells isolated from primary tissue, differentiated from pluripotent stem cells through to support of scalable growth as a coating on established plasticware or micro-carriers.
(ii) Small molecules supporting human pluri- and multi-potency
UEDIN has also filed intellectual property pertaining to small molecule substitutes for bFGF mediated signalling in the context of pluripotent human stem cell growth that has been optioned by an international market leading company specialising in stem cell culture media and reagents. Additional intellectual property is anticipated in relation to SM supporting both human pluripotent and multipotent stem cell culture. These will be applied in the context of proprietary media formulations supporting stem cell culture. As small molecules will be cheaper to manufacture than recombinant proteins or serum, this should help reduce the cost media for research, industrial and clinical applications. Resulting media formulations should also be less variable and less likely to be contaminated by pathogens that can inadvertently contaminate a biological product either during its production or extraction from a tissue or animal.
(iii) Recombinant protein technology for control of a pluripotent human stem cell phenotype
The BEST-STEM CELLS consortium partners at UKB (UBONN), BIO and CNRS-ENS have used recombinant protein technology to develop high affinity and membrane permeant proteins providing novel opportunities to control pluripotent human stem cell induction, renewal and viability. These inventions have been institutionally disclosed for assessment of suitability for filing with regional patent offices as novel intellectual property. It is envisaged that these biomolecules could be licensed for distribution as reagents for use in academic and industrial research and screening or for therapeutic applications.
(iv) Aseptic serum free cryopreservation of hESCs
Marine organism derived molecules supplied by GLYCO and evaluated by ULg in the context of pluripotent stem cell cryopreservation may serve as part of an aseptic biochemically defined method and kit for eurkaryotic cell, particularly human pluripotent stem cell, vitrification. Funding to progress this aspiration has been obtained by ULg. This follows from recently published parallel effort by this group demonstrating that vitrification is superior to slow freezing in lowering the intracellular concentration of cryoprotectants despite exposure to higher concentration of cryoprotectant solutions (Vanderzwalmen et al., 2013).
Ethical considerations
The BEST-STEM CELLS project was executed in a compliance with the general ethical principles and specific requirements defined by the member states in which the research was conducted, specifically as pertains to the use of human embryo derived stem cells and animals. As regards to former:
- The research was necessary and supported the development of treatments that could be treatable with adult tissue derived cells.
- The research was carried out strictly within all relevant legislations and after having obtained approval from the concerned local ethics committees, relevant national regulatory bodies, and the European Commission.
- All relevant national and European legislation as well as international conventions and declarations were respected.
- Where required, local, regional or national ethics approval will have been obtained before the research to which it relates is carried out.
- Advisors had been included in the Advisory Panel qualified to monitor the ethical implications of the work and to make recommendations and develop guidelines on how to carry out the proposed research in full compliance with ethical principles if necessary.
- The project did not use cells derived from human embryos which were created explicitly for research or from human embryos created by means of somatic cell nuclear transfer.
Over five years on from the development of the capacity to induce a pluripotent stem cell-like state in human cells (Okita et al., 2007) it is now abundantly clear that although these cells closely resemble embryonic stem cells they are in fact a distinct entity which cannot entirely substitute for embryo derived stem cells. Notably there will be subject to different influences affection genomic stability (e. genomic copy number variation) relating to the source material from which they originate or the reprogramming process (Carey et al., 2011). They also possess a residual epigenetic memory of reprogrammed cell origin and with that a distinct metabolic profile both of which can impinge on their undifferentiated properties and differentiation potential (Cheng et al., 2012; Meissen et al., 2012). This will have the potential to undermine their utility in research, industrial screening and therapeutic applications, or at the very least impede the translatability of knowledge developed through research on hESCs. While induced pluripotent stem cells will continue to be perceived by many opposed to embryonic stem cells, as more ethically favourable it remains the case that they cannot substitute entirely for them and carry other risks. From a research perspective this includes the possibility that knowledge gained from differentiated cells derived from them has been influenced by the reprogramming process and is thus not representative of adult cells. Comparison of differentiated derivatives derived from them with those derived from established embryonic stem cells could help promote confidence in their use. From a therapeutic perspective there is also a greater risk of adventitious pathogen transmission originating from an adult cell donor for pathogens that could not be readily detected or screened for. These would originate from environmental exposures associated with donor life history. A preimplantation embryo from which embryonic stem cells could be derived would be comparatively protected from such exposures, as it never would have established a direct connection with the maternal blood supply. Lastly, there is the ethical challenge of safeguarding an adult tissue donor from information or liabilities arising from research on or clinical application of induced pluripotent stem cell derived products. These do not exist in the case of an embryonic stem cell line for which there is no sentient source exists.
Collectively, this substantiates the merits and continued relevance of the BEST-STEM CELLS research outputs. These outputs will serve to underpin ongoing international commitment to the use of hESCs in research, industrial and clinical applications and underpin parallel and growing developments of induced pluripotent stem cells in these contexts as well.
References
(1) Carey BW, Markoulaki S, Hanna JH, Faddah DA, Buganim Y, Kim J, Ganz K, Steine EJ, Cassady JP, Creyghton MP, Welstead GG, Gao Q, Jaenisch R. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell. 2;9(6):588-98. (2011).
(2) Cheng L, Hansen NF, Zhao L, Du Y, Zou C, Donovan FX, Chou BK, Zhou G, Li S, Dowey SN, Ye Z; NISC Comparative Sequencing Program, Chandrasekharappa SC, Yang H, Mullikin JC, Liu PP. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell. 10(3):337-44 (2012).
(3) Corradetti B, Freile P, Pells S, Fahmy T, De Sousa PA. Embryonic stem cell renewal mediated by affinity targeted paracrine stimulation. Biomaterials, Oct, 33 (28): 6634-43. (2012).
(4) Dibenedetto S, Cluet D, Stebe PN, Baumlé V, Léault J, Terreux R, Bickle M, De Chassey, B, Mikaelian, I, Colas, P, Spichty M, Zoli M, Rudkin BB. Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers. Mol. Cell Proteomics, in press (2013).
(5) Ermakov A, Pells S, Freile P, Ganeva V, Wildenhain J, Bradley M, Pawson A, Millar R, De Sousa PA. A role for intracellular calcium downstream of G-protein signaling in maintenance of undifferentiated hESCs. Stem Cell Research 9(3):171-184 (2012).
(6) Meissen JK, Yuen BT, Kind T, Riggs JW, Barupal DK, Knoeplfler PS, Fiehn O. Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.PLoS One. 7(10):e46770. doi: 10.1371/journal.pone.0046770. Epub 2012 Oct 15. (2012).
(7) Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 19;448(7151):313-7. (2007).
(8) Thier, M., P. Worsdorfer, Y.B. Lakes, R. Gorris, S. Herms, T. Opitz, D. Seiferling, T. Quandel, P. Hoffmann, M.M. Nothen, O. Brustle, and F. Edenhofer. 2012. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 10:473-9.
(9) Vanderzwalmen P, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S, Zech N, Ectors FJ. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentrations of cryoprotectant solutions. Hum Reprod Advance. Doi:10.1093/humrep/det107. (2013).
(10) Zhang R, Mjoseng HK, Hoeve MA, Bauer NG, Pells S, Besseling R, Velugotla S, Tourniaire G, Kishen REB, Tsenkina Y, Armit C, Duffy CRE, Helfen M, Edenhofer F, De Sousa PA* and Bradley M* A thermoresponsive chemically-defined hydrogel for long term culture of hESCs Nature Communications 4: 1335. doi: 10.1038/ncomms2341 (2013).
Contact details: Dr Paul De Sousa
BEST-SC Project Coordinator
MRC Centre for Regenerative Medicine, University of Edinburgh
49 Little France Crescent, Edinburgh EH16 4SB
Tel: +44-013-1242-6646, Fax: +44-013-1242-6201
email: paul.desousa@ed.ac.uk
List of websites: http://www.beststemcells.ed.ac.uk
The biochemically equivalent substitutive technology (BEST) for stem cells project was a focused small-medium-scale collaborative research project (ID 223410) addressing the FP7-HEALTH-2007-B call fiche (1.4.7) for development of stem cell culture conditions. The rationale for the project was that improvement of the efficacy and safety of human stem cell culture for research, industrial and clinical applications requires the reduction or elimination of current reliance on animal cell and tissue derived biological reagents (i.e. extracellular matrix, blood serum / serum fractions) which can be a source of variance and pathogens. The goal of the project was thus to develop new knowledge and reagents for human stem cell culture. Research was predominantly focused on pluripotent human embryonic stem cell (hESC) culture but also addressed multipotent human mesenchymal stem cell (hMSC) culture, using an hESC-derived mesenchymal progenitor (hES-MP) as a model system for the latter to overcome variability normally associated with adult tissue derived hMSC. The consortium was composed of 8 academic and commercial partners across 5 European Union (EU) Member States, namely the University of Edinburgh (UEDIN), Roslin cells (RC) and Glycomar (GLYCO) in the United Kingdom (UK); INSERM and CNRS in France; University of Bonn (UBONN) in Germany; Bioneer (BIO) in Denmark, and University of Liege (ULg) in Belgium. The contact details for the list of beneficiaries is provided in Final Report Annex I, Figure 1.
The principle achievements of the project included the:
(1) Discovery of chemical polymers substrates supporting that attachment and growth of hESC and hMSC, from which cells could be gently dissociated by lowering of ambient temperature. These provide an alternative to current requirements for animal tissue derived substrates, and harsher methods of cell dissociation with a potential to damage cells including treatment with cell protein digesting enzymes, chemicals or mechanical (WP1.1: UEDIN).
(2) Discovery and validation of chemical small molecules to substitute or reduce current requirements for protein growth factors and serum supplements for growth of hESC and hMSC, respectively (WP1.2: UEDIN).
(3) Development of recombinant high-affinity and / or membrane permeant proteins affecting hESC survival and growth and validation of the significance of the pathways being targeted (WP 1.3 and 1.4: CNRS, UBONN, INSERM, Bioneer).
(4) Development of an aseptic serum free cryopreservation protocol for hESC and identification of non-vertebrate of origin cryoprotective agents (WP1.5: ULg, GLYCO).
(5) Provision of training in research quality assurance to provide consortium partners with improved awareness of what is required to translate basic research into good manufacturing practice (GMP) (WP0.2: RCs).
(6) Public dissemination of BEST-STEM CELL consortium principles and outputs (WP0.1: All).
Project context and objectives:
The founding principle of the BEST-STEM CELLs project is that the future safety and efficacy of stem cell based therapies is critically dependent on the development of BEST for cell culture. This is necessary to reduce or obviate the current dependence that all stem cell types and their derivatives have on vertebrate-animal cell and tissue derived biological reagents (i.e. extracellular matrix preparations, blood serum / serum fractions) for their growth, differentiation and storage. These reagents can contribute to variations in stem cell behaviour through their own variability in purity and bioactivity. They also pose serious risks to transplant recipients and through them, the general population, for transmission of known and unknown adventitious pathogens. Clinical delivery of stem cell based therapies ultimately also requires compliance with evolving standards for GMP and regulatory governance. These significantly prolong the time it takes for research gains at the bench to be translated to bedside clinical treatments. In principle, this can be improved by early promotion of research quality assurance and assessment of methods and tool translatability to GMP standards. The mission of the BEST-STEM CELLS project is thus to develop and warrant as translatable to GMP standards, strategies and tools that will yield safe and efficacious BEST for stem cell culture.
Central to safety and efficacy is the extent to which the reagents used to cultivate and store stem cells can be biochemically defined, and thus free from unknown contaminants and variables inherent in biological reagents. It is also important that new methods and tools have no residual undesirable effects (such as for example induction of genetic instability), and that ultimately they can be automated so as to minimise skilled operator dependence. Of the stem cell types available for research and therapeutic use, pluripotent human embryo stem cells (hESCs) and those artificially induced into this state (i.e. human-induced pluripotent stem cells (hiPSC)) pose both the greatest challenges and rewards. Their reward lies in their unparalleled growth and differentiation potential. These ultimately could provide the quantities of cells that are likely to be required to treat most diseases, as well as offer effective treatment for conditions which presently are either not or are inadequately being addressed by adult tissue derived cells. hMSCs, normally sourced from diverse adult tissues such as bone marrow, placenta and fat constitute another stem cell type for which there is significant clinical interest, either as a multipotent stem cell source of bone and cartilage to rebuild these tissues or as immune modulating cells. In comparison with hESC and hIPSC these are mortal (i.e. will not grow indefinitely) and their properties are less well defined and variable in relation to donor age, health, tissue of origin and time in culture. A renewable and standardisable supply of hMSC-like cells can be derived from human pluripotent stem cells, thereby facilitating discovery of supportive reagents.
Thus, the main objectives for the project were to develop new knowledge and reagents for human stem cell culture and storage, focusing on pluripotent and multipotent hESC and hMSCs, respectively. To represent the latter the project relied on a hESC-derived mesenchymal progenitor (hES-MP). Specifically, project work packages sought to:
(a) discover chemically defined polymer substrates that could support adhesion and growth (WP1.1);
(b) discover chemically defined small molecules that could substitute, reduce or augment current reliance on protein growth factors or serum as culture supplements (WP1.2);
(c) develop and evaluate new high affinity and/or membrane permeable recombinant proteins for controlling biological mechanisms controlling pluripotent stem cell growth (WPs 1.3 1.4);
(e) develop an aseptic serum free cryopreservation protocol and identify sustainable marine invertebrate derived cryoprotectants supporting human pluripotent stem cell cryopreservation (WP1.5);
(f) provide training in quality assurance and gap assess (i.e. identify faults, omissions and outstanding issues associated with) methods and tools developed by the BEST-STEM CELLS project in order to comply with future GMP regulatory requirements, most notably the EU Tissues and Cells Directive (WP0.2);
(g) publically disseminate knowledge / awareness of BEST-STEM CELLS research outputs (WP0.1).
Project Results:
Polymer substrates supporting human stem cell culture
BEST Stem cells research at UEDIN has identified and validated a family of polymers based on 2-(diethylamino)ethyl acrylate (HG9, 20, 21) which support hESC renewal and permit gentle, chemically defined cell passaging achieved by reduction of temperature from 37 to 15 degrees of Celsius for 15 minutes followed by gentle pipetting of the medium (Final Report Annex I, Figure 2; for Video see Final Report Annex III). One member of this hydrogel family (HG21) was demonstrated to be capable of supporting continuous long-term (> 6 months) growth and maintenance of a pluripotent stem cell phenotype validated using 3 independent hESC lines (RCM1, RH1 and H9). This was achieved in a biologically defined animal serum-free medium. This work has been published in Nature Communications (Zhang et al., 2013). Seeking to improve the screening throughput further UEDIN has also established a super high-density array enabling concurrent evaluation of 7 000 different polymers in a single cellular-based screen (Final Report Annex I, Figure 3). Polymers identified in this screen were validated for their capacity to support extended growth of hESC whose passage is mediated enzymatically. At time of writing the manuscript describing these results has been submitted for publication.
HG21 epitomises the kind of output the BEST Stem cell project was intended to yield. It constitutes a chemically defined alternative to currently used, undefined biological substrates and provides a flexible and scalable approach for improving the definition, efficacy and safety of pluripotent human stem cell culture systems for research, industrial and clinical applications. Since the initiation of the BEST-STEM CELLS project a number of polymer and peptide-polymer substrates have been reported with a capacity to sustain hESC (Mei et al., 2010; Melkoumian et al., 2010; Vila-Diaz et al., 2010). Limitations in these advances have included variation in cell line responsiveness and/or requirements for feeder cell conditioning of media or coating of surfaces with serum or serum proteins. Critically, for all substrates reported to date cell dissociation at passaging has required one or more treatments involving mechanical scraping or colony picking, proteolytic enzymatic digestion, or chemically mediated chelation of divalent cations (e.g. calcium and magnesium). While mechanical dissociation is laborious and not readily scalable, enzymatic and chemical treatments can damage cells by removal of important surface proteins or ions (e.g. calcium).
The HG21 polymer has the potential to support hiPSCs both in the course of reprogramming and for established cell lines. In the context of the former it could improve the efficiency of generating hiPSC by providing a means to selectively passage reprogrammed cells. Experiments validating its application to hiPSC generation and culture are ongoing.
Parallel effort in the BEST-STEM CELLS project at UEDIN also sought to identify chemically defined substrates supportive of hMSC growth and potency. These cells, normally sourced from adult tissues such as bone marrow and fat, can adhere to polystyrene plastic surfaces of tissue culture-ware without any additional substrates, although such attachment is non-selective, permits spontaneous differentiation and supports only limited growth. Despite the apparent lack of specificity implied by substrate-independent attachment of hMSC to polystyrene plastic, mesenchymal stem cell growth, function and differentiation is influenced by cell interaction with materials. This includes interactions with specific biochemical molecules (i.e. micro-contact printed patterns of fibronectin, modified or additional functional groups (i.e. alcohol and amino groups), substrate stiffness and topography (Benoit et al., 2008; Curran et al., 2006; Engler et al., 2006; Kilian et al., 2010; McMurray et al., 2011).
Using an hESC-derived mesenchymal progenitor (hES-MP), as a surrogate for human adult tissue derived MSC whose properties can vary between donors, and tissue source, BEST-STEM CELLS research at UEDIN has identified a finite set of 5 polymers supporting a mesenchymal stem cell phenotype and growth following successive cell passaging, a subset of which are thermomodulatable enabling gentle release at passaging. At time of writing the capacity of these to support long term growth and differentiation potential of both hES-MP and adult adipose derived MSC was being evaluated. Research completed to date has validated that they are more selective of an MSC phenotype. Thus, they provide a means to enrich for these cells, which can be exploited to prepare a more homogenous population for research or clinical applications.
Small molecules supporting human pluri- and multi-potency
UEDIN has discovered multiple small molecules that substitute for the absence of either or both basic Fibroblast Growth Factor (bFGF) and transforming growth factor-beta (TGFb) in the context of hESC growth. The first of these substitutes for the former through modulation of calcium signaling (Ermakov et al., 2012). Others have been confirmed to phosphorylate protein kinases (ERK, SMAD) which are normally activated by bFGF and TGF-beta or LIF (STAT-3). Additionally, a complementary project supported in part by funding for the BEST-STEM CELLS project has validated an affinity targetable controlled release approach to delivery of growth factors in the culture of pluripotent stem cell cells using a biodegradable nanoparticle platform (Corradetti et al., 2012). In a concurrently published paper this platform has also been validated for delivery of small molecules (Park et al., 2012). The affinity targeted and controlled release capacities of this platform provide for a reduction in the requirement for costly growth factors by a factor of over 10^4. It also provides targeted cells with a selective growth advantage by minimising the potential for bio-active factors to support or have competing effects on diverging (i.e. differentiating) cell populations.
Using the aforementioned hESC-derived mesenchymal progenitor (hES-MP), UEDIN has also identified a finite set of small molecules which augment growth (12) and expression (5) of MSC cells expressing the canonical MSC marker, STRO-1. While this research did not progress sufficiently to understand the biological significance of these effects or their mechanism, it provides leads for future studies and targets for modulation of both MSC expansion and function. As regards the latter all of the small molecules that enhanced STRO-1 expression have been reported to modulate cell-signalling pathways directly or indirectly in immune modulation and inflammation, an important non-stem cell function attributed to MSC. Thus, small molecules identified in this project may not only serve to promote the expansion of MSC when presented as a media supplement, they could also serve to augment the therapeutic properties of these cells following transplantation, which could be accomplished through their encapsulation and affinity-targeted controlled release in biodegradable particles as noted in the aforementioned complementary research on pluripotent stem cells.
Recombinant protein technology for pluripotent human stem cells
BEST-STEM CELLS consortium partners at UKB (UBONN), BIO and CNRS-ENS have used recombinant protein technology to develop high affinity and membrane permeant proteins providing novel opportunities to control pluripotent human stem cell induction, renewal and viability.
At CNRS-ENS, peptide aptamer technology providing high-affinity ligands for manipulation of biological processes has yielded novel probes that are extremely toxic to embryonic stem cells, and as such could be used to remove them in the event that they contaminate a population of differentiated derivatives. A novel approach for batchwise identification of both the target of a peptide aptamer and the aptamer itself has been developed, as well as novel forms of membrane permeant peptide aptamers through collaborative research with UKB. This underpins ongoing an future collaborative research beyond BEST-STEM CELLS between these partners and with UEDIN, to evaluate these reagents in the context of controlling pluripotent stem cell induction and growth. Notably with novel peptide aptamers that activate calcineurin (Dibenedetto et al., 2013).
Expertise in recombinant protein production and high-affinity and membrane proteins at Bioneer and UKB were also combined in the BEST Stem cells project to develop and apply membrane permeant versions of pluripotency associated factors, notably a membrane permeant version of one of the key transcription factors regulating pluripotent stem cell renewal, Nanog. This was evaluated in the context of controlling pluripotent stem cell renewal and to induction of a pluripotent stem cell phenotype (see below).
Aseptic serum free cryopreservation
At ULg, the BEST-STEM CELLS project has yielded the development of an aseptic, serum-free vitrification method for hESC. With GLYCO it has also identified two candidate microalgae derived polysaccharides that are non-toxic as assessed using established embryo and embryonic stem cell assays. These constitute new approaches and reagents for cryopreservation likely to be of broad utility for eukaryotic cells in general.
Novel insight regarding control of pluripotent human stem cells
Importantly, the novel reagents and tools developed in the BEST-STEM CELLS project have also led to important insights as to the mechanisms controlling a pluripotent phenotype in human cells. A pluripotent state is traditionally defined as the ability of a stem cell to give rise to all of the cells of the body. In a mouse model this can be verified by transplantation of cells into a preimplantation embryo and having them contribute to all tissues in the resulting animal including the gonads (testis and ovaries) in the form of sperm and eggs with a competence to form viable offspring, or alternatively differentiation of these cell types ex vivo. Studies in the mouse have identified three prospective sources of pluripotent stem cells, the inner cell mass of a preimplantation embryo, classically referred to as embryonic stem cells and successive sub-populations of derivative epiblast and primordial germ cells. These cells vary in a number of ways, with the former conceptually distinguished from the latter as constituting a more naive vs primed state.
At the present time, the developmental state of human and primate embryonic stem cells resembles that of mouse epiblast stem cells (EpiSC) including morphology, low clonogenicity, growth factor requirements, and overall expression profiles. They are genomically unstable (Taapken et al., 2011), and gene targeting by homologous recombination in hESCs has proven difficult (De Los Angeles et al., 2012). Whereas mEpiSCs can be efficiently reverted into mESCs, the conversion of hESCs from a primed into a bona fide naive state has proven difficult and transient (De Los Angeles et al., 2012). In culture human pluripotent stem cells (whether hESC or iPSC) and mEpiSCs are cultured in the presence of basic FGF and TGFb / Activin, while naive mESCs are best maintained in the presence of LIF and inhibitors to GSK and MEK (2i) (Nichols and Smith, 2009). Switching of media results in differentiation or death of the corresponding cell types. Naive and primed cells are thus regulated by distinct signaling pathways. A major question in the field is why hESCs or hIPSCs cannot be derived in the presence of same growth factors and pathway regulators as mESCs.
BEST-STEM CELLS research at INSERM, has yielded one possible explanation for why human cells respond differently to LIF and this is that they appear to fail to activate the target genes of STAT3, a transcription factor normally recruited and activated upon LIF stimulation. The capacity is partially restored if hESCs are engineered to express a ligand-dependent form of STAT3 that bypasses the LIF receptor. The combination of LIF and ligand-dependent STAT3 allows hESC to escape from bFGF dependency, and to enter a new state closer to the naive pluripotent state. These findings raise the question of whether manipulation of the LIF-STAT3 pathway together with other factors, such as for example small molecules which modulate calcium signalling, and / or supportive polymer substrates as discovered by UEDIN can be used to generate bona fide naive pluripotent stem cells in human.
One of the transcription factors which is pivitol to maintenance of naive and primate states of pluripotency is Nanog. Using membrane permeant versions of Nanog UKB has demonstrated its importance for promotion of self-renewal and inhibiting differentiation to an endodermal lineage, and its independence from signaling pathways that have been implicated. Additionally, the consequence of introducing Nanog into differentiated primary and immortalised cells alone was evaluated. These studies have also demonstrated how protein transduction is an attractive tool to understand protein function without genetic manipulation.
Promotion of research quality
Lastly, the BEST-STEM CELLS project sought to promote an awareness of quality assurance in research and stem cell manufacturing within the consortium which is normally lacking within academic research institutions, due to lack of understanding and infrastructure. RCs trained consortium partners in the principles of quality assurance and gap assessed for quality and prospective requirements for translation of research to manufacturing standards the operating practices and research outputs of consortium partner laboratories. The latter focused on the most advanced of the BEST-STEM CELLS research outputs, HG21, the thermomodulatable hydrogel supporting long-term hESC renewal and pluripotency.
References
(1) Benoit, D. S. W., Schwartz, M. P., Durney, A. R. & Anseth, K. S. Small functional groups for controlled differentiation of hydrogel-encapsulated hMSCs. Nature Mater. 7, 816-823 (2008).
(2) Corradetti B, Freile P, Pells S, Fahmy T, De Sousa PA. Embryonic stem cell renewal mediated by affinity targeted paracrine stimulation. Biomaterials, Oct, 33 (28): 6634-43. (2012).
(3) Curran, J. M., Chen, R. & Hunt, J. A. The guidance of hMSC differentiation in vitro by controlled modifications to the cell substrate. Biomaterials 27, 4783-4793 (2006).
(4) De Los Angeles, A., Loh, Y.H. Tesar, P.J. and Daley, G.Q. Accessing naive human pluripotency. Curr Opin Genet Dev 22, 111 (2012).
(5) Dibenedetto S., C. D., Stebe, P.N. Baumlé V, Léault, J. Terreux, R., Bickle M, De Chassey, Mikaelian, IJ, Colas, P., Spichty M., Zoli M., Rudkin B.B. (2013). Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers. Mol. Cell. Proteomics, (EPub ahead of Print).
(6) Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677-689 (2006).
(7) Ermakov A, Pells S, Freile P, Ganeva V, Wildenhain J, Bradley M, Pawson A, Millar R, De Sousa PA. A role for intracellular calcium downstream of G-protein signaling in maintenance of undifferentiated hESCs. Stem Cell Research 9(3):171-184 (2012).
(8) McMurray RJ, Gadegaard N, Tsimbouri PM, Burgess KV, McNamara LE, Tare R, Murawski K, Kingham E, Oreffo RO, Dalby MJ. Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat Mater. 17;10(8):637-44 (2011).
(9) Kilian, K. A., Bugarija, B., Lahn, B. T. & Mrksich, M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl Acad. Sci. USA 107, 4872-4877 (2010). (10) Mei, Y. et al. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat. Mater. 9, 768-778 (2010).
(11) Melkoumian, Z. et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of hESCs. Nat. Biotechnol. 28, 606-610 (2010).
(12) Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487-492.
(13) Park J, Wrzesinski SH, Stern E, Look M, Criscione J, Ragheb R, Jay SM, Demento SL, Agawu A, Licona Limon P, Ferrandino AF, Gonzalez D, Habermann A, Flavell RA, Fahmy TM. Combination delivery of TGFb inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy. Nat Mater. Oct;11(10):895-905 (2012).
(14) Villa-Diaz, L.G. et al. Synthetic polymer coatings for long-term growth of hESCs. Nat. Biotechnol. 28, 581-583 (2010).
(15) Zhang R, Mjoseng HK, Hoeve MA, Bauer NG, Pells S, Besseling R, Velugotla S, Tourniaire G, Kishen REB, Tsenkina Y, Armit C, Duffy CRE, Helfen M, Edenhofer F, De Sousa PA* and Bradley M* A thermoresponsive chemically-defined hydrogel for long term culture of hESCs Nature Communications 4: 1335. doi: 10.1038/ncomms2341. (2013).
Potential Impact:
The BEST-STEM CELLs project has yielded new and improved technology for the cultivation of human stem cells of embryonic and adult origin. At time of writing it had yielded the following tangible outputs:
(1) Training and professional development
Over the course of project, BEST Stem cells has supported 57 people with a marginal female gender bias (female / male: 31 / 26) for durations of 1 to 42 months. Of these 32 were experienced Doctor of Philosophy (PhD) researchers (12:20), 21 were experienced non-PhD researchers (16:5) and 4 were PhD students (3:1). Consortium scientists received training in quality assurance in research provided by RCs, as well as introductory lectures in the diverse methodologies and technologies which each consortium partner had expertise in. This included recombinant protein technologies (Bioneer, UKB, CNR-ENS), live visible reporting systems as applied to pluripotent stem cells (INSERM), polymer chemistry and screening (UEDIN), glycobiology (GLYCO) and cryopreservation (ULg). In the closing public dissemination meeting, consortium partners and members of the public were also introduced to the expertise of the invited speakers on manufacturing science (Prof. David Williams, University of Loughborough), social value systems and regulatory policy development (Prof. Joyce Tait, University of Edinburgh), and immunotherapy and biomedical engineering of nanotechnology for affinity targeted and controlled drug release (Prof. Tarek Fahmy, Yale University).
(2) Public dissemination
The consortium has collectively participated in public launch and closing meetings. The launch of the project coincided with the first general assembly meeting held in Lyon, France on 29 and 30 October 2009. The launch was communicated by a press release that received regional press coverage in the UK and France (Final Report Annex I, Figure 4, 5). The public launch of the project also coincided with the establishment of the project website: http://www.beststemcells.ed.ac.uk. In the course of the project partners communicated ongoing research progress at international and national meetings, invited lectures and public forums, cumulatively totalling 61 engagements in 10 countries including the UK, France, Germany, Belgium, USA, Canada, Singapore, Japan and China. These addressed audiences ranging from 20 to 500 delegates, cumulatively totaling approximately 5000 people. Press releases were issued in association with high profile accomplishments, most notably the communication of the hESC supportive thermomodulatable hydrogels. Lastly the consortium held a public dissemination meeting in Edinburgh on 22 October 2012, attended by over 60 participants at which partners presented project outputs and expertise together with invited national and international scientists presenting complementary research in keynote addresses (See Final Report Annex II, BEST SC Public Dissemination meeting Programme OCT2012).
Publications
There have been 3 publications, in Cell Stem Cell (Impact Factor, IF, 25), Nature Communications (IF 7.9) and Stem Cell Research (IF 5.3) that were a direct output of the research. Indirect outputs include one publication in Biomaterials (IF 7.9) and another accepted in press in Molecular and Cellular Proteomics (IF 7.9). These emanated from three consortium partners UKB, UEDIN, and CNRS-ENS (Their et al., 2012; Zhang et al., 2013; Ermakov et al., 2012; Corradetti et al., 2012; Dibenedetto et al., 2013). Additionally at least another 5 manuscripts have been submitted or will be submitted within 2013 from INSERM (1), UKB (1), and UEDIN (3).
Intellectual property
There have been two patents filed from UEDIN pertaining to the polymer and small molecule research. One has been commercially licensed. The other has been optioned to a commercial company for time limited consideration. Another 10 invention disclosures are being prepared or have been submitted by partners at UEDIN, ULg, CNRS-ENS/INSERM, UKB and BIONEER for institutional evaluation for suitability for filing with national patent offices. This intellectual property covers the four domains of research proposed in the project pertaining to polymer substrates, small molecules, recombinant protein technology and marine organism derived products in the context of stem cell growth and cryopreservation. The prospective application and exploitation of these outputs is as follows:
(i) Polymer substrates supporting human stem cell culture
UEDIN has filed intellectual property pertaining to the nature and use of a chemically defined thermomodulatable polymer hydrogel with high affinity for and support of long-term growth of pluripotent human stem cells (GB1110042.7 WO2012/172291). This has been licensed to a UK materials company. The exploitable product resulting from this research will take the form of polymer coated tissue culture plastic ware or micro-carriers (ie. Beads) for purposes of achieving scalable growth of pluripotent human stem cells for research, industrial screening and therapeutic applications. Additional IP is anticipated in relation to polymers supporting hMSC phenotype, growth and multi-potency. The application of these will depend on what aspects of hMSC specific polymers support. This may range from selective enrichment of cells isolated from primary tissue, differentiated from pluripotent stem cells through to support of scalable growth as a coating on established plasticware or micro-carriers.
(ii) Small molecules supporting human pluri- and multi-potency
UEDIN has also filed intellectual property pertaining to small molecule substitutes for bFGF mediated signalling in the context of pluripotent human stem cell growth that has been optioned by an international market leading company specialising in stem cell culture media and reagents. Additional intellectual property is anticipated in relation to SM supporting both human pluripotent and multipotent stem cell culture. These will be applied in the context of proprietary media formulations supporting stem cell culture. As small molecules will be cheaper to manufacture than recombinant proteins or serum, this should help reduce the cost media for research, industrial and clinical applications. Resulting media formulations should also be less variable and less likely to be contaminated by pathogens that can inadvertently contaminate a biological product either during its production or extraction from a tissue or animal.
(iii) Recombinant protein technology for control of a pluripotent human stem cell phenotype
The BEST-STEM CELLS consortium partners at UKB (UBONN), BIO and CNRS-ENS have used recombinant protein technology to develop high affinity and membrane permeant proteins providing novel opportunities to control pluripotent human stem cell induction, renewal and viability. These inventions have been institutionally disclosed for assessment of suitability for filing with regional patent offices as novel intellectual property. It is envisaged that these biomolecules could be licensed for distribution as reagents for use in academic and industrial research and screening or for therapeutic applications.
(iv) Aseptic serum free cryopreservation of hESCs
Marine organism derived molecules supplied by GLYCO and evaluated by ULg in the context of pluripotent stem cell cryopreservation may serve as part of an aseptic biochemically defined method and kit for eurkaryotic cell, particularly human pluripotent stem cell, vitrification. Funding to progress this aspiration has been obtained by ULg. This follows from recently published parallel effort by this group demonstrating that vitrification is superior to slow freezing in lowering the intracellular concentration of cryoprotectants despite exposure to higher concentration of cryoprotectant solutions (Vanderzwalmen et al., 2013).
Ethical considerations
The BEST-STEM CELLS project was executed in a compliance with the general ethical principles and specific requirements defined by the member states in which the research was conducted, specifically as pertains to the use of human embryo derived stem cells and animals. As regards to former:
- The research was necessary and supported the development of treatments that could be treatable with adult tissue derived cells.
- The research was carried out strictly within all relevant legislations and after having obtained approval from the concerned local ethics committees, relevant national regulatory bodies, and the European Commission.
- All relevant national and European legislation as well as international conventions and declarations were respected.
- Where required, local, regional or national ethics approval will have been obtained before the research to which it relates is carried out.
- Advisors had been included in the Advisory Panel qualified to monitor the ethical implications of the work and to make recommendations and develop guidelines on how to carry out the proposed research in full compliance with ethical principles if necessary.
- The project did not use cells derived from human embryos which were created explicitly for research or from human embryos created by means of somatic cell nuclear transfer.
Over five years on from the development of the capacity to induce a pluripotent stem cell-like state in human cells (Okita et al., 2007) it is now abundantly clear that although these cells closely resemble embryonic stem cells they are in fact a distinct entity which cannot entirely substitute for embryo derived stem cells. Notably there will be subject to different influences affection genomic stability (e. genomic copy number variation) relating to the source material from which they originate or the reprogramming process (Carey et al., 2011). They also possess a residual epigenetic memory of reprogrammed cell origin and with that a distinct metabolic profile both of which can impinge on their undifferentiated properties and differentiation potential (Cheng et al., 2012; Meissen et al., 2012). This will have the potential to undermine their utility in research, industrial screening and therapeutic applications, or at the very least impede the translatability of knowledge developed through research on hESCs. While induced pluripotent stem cells will continue to be perceived by many opposed to embryonic stem cells, as more ethically favourable it remains the case that they cannot substitute entirely for them and carry other risks. From a research perspective this includes the possibility that knowledge gained from differentiated cells derived from them has been influenced by the reprogramming process and is thus not representative of adult cells. Comparison of differentiated derivatives derived from them with those derived from established embryonic stem cells could help promote confidence in their use. From a therapeutic perspective there is also a greater risk of adventitious pathogen transmission originating from an adult cell donor for pathogens that could not be readily detected or screened for. These would originate from environmental exposures associated with donor life history. A preimplantation embryo from which embryonic stem cells could be derived would be comparatively protected from such exposures, as it never would have established a direct connection with the maternal blood supply. Lastly, there is the ethical challenge of safeguarding an adult tissue donor from information or liabilities arising from research on or clinical application of induced pluripotent stem cell derived products. These do not exist in the case of an embryonic stem cell line for which there is no sentient source exists.
Collectively, this substantiates the merits and continued relevance of the BEST-STEM CELLS research outputs. These outputs will serve to underpin ongoing international commitment to the use of hESCs in research, industrial and clinical applications and underpin parallel and growing developments of induced pluripotent stem cells in these contexts as well.
References
(1) Carey BW, Markoulaki S, Hanna JH, Faddah DA, Buganim Y, Kim J, Ganz K, Steine EJ, Cassady JP, Creyghton MP, Welstead GG, Gao Q, Jaenisch R. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell. 2;9(6):588-98. (2011).
(2) Cheng L, Hansen NF, Zhao L, Du Y, Zou C, Donovan FX, Chou BK, Zhou G, Li S, Dowey SN, Ye Z; NISC Comparative Sequencing Program, Chandrasekharappa SC, Yang H, Mullikin JC, Liu PP. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell. 10(3):337-44 (2012).
(3) Corradetti B, Freile P, Pells S, Fahmy T, De Sousa PA. Embryonic stem cell renewal mediated by affinity targeted paracrine stimulation. Biomaterials, Oct, 33 (28): 6634-43. (2012).
(4) Dibenedetto S, Cluet D, Stebe PN, Baumlé V, Léault J, Terreux R, Bickle M, De Chassey, B, Mikaelian, I, Colas, P, Spichty M, Zoli M, Rudkin BB. Calcineurin A vs NS5A-TP2/HDDC2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers. Mol. Cell Proteomics, in press (2013).
(5) Ermakov A, Pells S, Freile P, Ganeva V, Wildenhain J, Bradley M, Pawson A, Millar R, De Sousa PA. A role for intracellular calcium downstream of G-protein signaling in maintenance of undifferentiated hESCs. Stem Cell Research 9(3):171-184 (2012).
(6) Meissen JK, Yuen BT, Kind T, Riggs JW, Barupal DK, Knoeplfler PS, Fiehn O. Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.PLoS One. 7(10):e46770. doi: 10.1371/journal.pone.0046770. Epub 2012 Oct 15. (2012).
(7) Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 19;448(7151):313-7. (2007).
(8) Thier, M., P. Worsdorfer, Y.B. Lakes, R. Gorris, S. Herms, T. Opitz, D. Seiferling, T. Quandel, P. Hoffmann, M.M. Nothen, O. Brustle, and F. Edenhofer. 2012. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 10:473-9.
(9) Vanderzwalmen P, Connan D, Grobet L, Wirleitner B, Remy B, Vanderzwalmen S, Zech N, Ectors FJ. Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentrations of cryoprotectant solutions. Hum Reprod Advance. Doi:10.1093/humrep/det107. (2013).
(10) Zhang R, Mjoseng HK, Hoeve MA, Bauer NG, Pells S, Besseling R, Velugotla S, Tourniaire G, Kishen REB, Tsenkina Y, Armit C, Duffy CRE, Helfen M, Edenhofer F, De Sousa PA* and Bradley M* A thermoresponsive chemically-defined hydrogel for long term culture of hESCs Nature Communications 4: 1335. doi: 10.1038/ncomms2341 (2013).
Contact details: Dr Paul De Sousa
BEST-SC Project Coordinator
MRC Centre for Regenerative Medicine, University of Edinburgh
49 Little France Crescent, Edinburgh EH16 4SB
Tel: +44-013-1242-6646, Fax: +44-013-1242-6201
email: paul.desousa@ed.ac.uk
List of websites: http://www.beststemcells.ed.ac.uk
