Emma Stephenson

Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions

BACKGROUND AIMS Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. METHODS, RESULTS AND CONCLUSIONS Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntingtons disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions.

Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product–free environment

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3–4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product–free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.

International community consensus standard for reporting derivation of human embryonic stem cell lines

The development and implementation of new methods in human embryonic stem cell (hESC) research is fraught with difficulties, not least of which is the highly variable reporting of the number and quality of embryos used to derive hESC lines. Without a clear minimum information convention among the derivation teams, understanding and comparing derivation methods and their potential impact on the resulting stem cell line will continue to be extremely difficult. In order to address this issue, we consulted international teams regarding the implementation of a minimum information convention for derivation with the aim of universal use, data collection and central analysis, followed by a multi-author publication. The responses demonstrated overwhelming support for such a system; over 90% of the respondents agreed that a universal standard for reporting the derivation of hESC lines was essential as part of the international effort to advance the field efficiently, and over 87% plan to use this standard and share collected data in Spring 2008 for central analysis and public dissemination. A number of future steps are planned in order to ensure that this standard evolves with the field and remains relevant and up-to-date. Our aim is to incorporate these data within existing international initiatives aimed at improving derivation standards. This article is an open-access publication in order to make the convention freely available to the international community and encourage universal participation.

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells

The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

Proposal for a universal minimum information convention for the reporting on the derivation of human embryonic stem cell lines

The implementation of novel methodologyacross the human embryonic stem cell (hESC)field is fraught with difficulties, not least ofwhich is the highly variable reporting of thenumber and quality of embryos used to deriveeach new hESC line. It is almost impossible toascertain from the literatu re a true efficiency forderiving a hESC line. Until there is a clear min-imum information (MI) convention amongstthe derivation teams, understanding and com-paring novel derivation methodologies andtheir potential impact on the resulting stem cellline will continue to be impossible or at bestextremely difficult. In this editorial, we high-light the variation in data published and voicesuggestions as to how the reporting can becomestandardized and so facilitate progress inhuman embryonic stem cell derivation. Theauthors request that interested parties commenton the system proposed, in order that a consen-sus article can be published in approximately4 months time.There is no universally agreed method forreporting the quality and type of embryo usedas starting material for hESC line derivation.Whilst many groups have published their hESCderivation data and protocols, few have statedin their manuscripts the complete essentialunderlying metadata with respect to theirsource material to enable reliable comparisonsto be made. However, it is beginning to beacknowledged that this lack of an accepted andestablished reporting system results in severalhurdles for stem cell researchers. In clinical-assisted reproduction, standards for embryo andblastocyst quality have been devised that corre-late with likelihood of implantation, and mightbeneficially be applied to stem cell derivation.Many advanced fields of biology have bene-fited greatly from the creation of their ownbespoke reporting standards. These evolvingstandards have enabled all the researchers in aparticular field to make the most of oneanother’s data. Successful examples include theStandard Metabolic Reporting StructuresWorking Group

Strategy for the creation of clinical grade hESC line banks that HLA-match a target population.

Here, we describe a pre‐derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence‐specific primers (PCR‐SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre‐derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.

Promises and challenges of the first clinical-grade induced pluripotent stem cell bank

It has only been approximately 6 years since Shinya Yamanaka, a Japanese scientist from Kyoto University, found a way of reverting differentiated cells from an adult into an embryoniclike pluripotent state [1]. These cells, termed induced pluripotent stem (iPS) cells are as powerful as human embryonic stem (hES) cells; they can be expanded infinitely, and upon differentiation provide an unlimited number of cells for autografts or close HLA-matched recipients, completely avoiding or significantly reducing the host immune response and need for immunosuppression. Importantly, they can be obtained by simple, minimally invasive procedures, such as phlebotomy or hair plucking, which are free from the ethical issues that plague the hES cell field. For his revolutionary work, Yamanaka was awarded the Nobel Prize in 2012. Following this major breakthrough in regenerative medicine everybody, including government funding agencies, jumped on the iPS cell bandwagon. In July 2011, the Innovative Medicines Initiative, Europe’s largest public–private initiative, launched a call for the generation of banks of iPS cells for drug discovery and safety assessment. The California Institute for Regenerative Medicine is intending to build a bank containing several thousand iPS cell lines for distribution to researchers, while the UK has its own plans too. However, iPS cell-based therapy was not considered in any of these cases. Most of the lines will be derived from people with various diseases in order to study disease pathophysiology. Yet, in spite of insufficient knowledge about the nature of these cells, unresolved technical issues, labor-intensive methodology and prohibitive costs, Yamanaka, supported by the Japanese government, recently revealed plans to build a bank of iPS cells for therapeutic use [2]. This bank of 75 iPS cell lines with carefully selected HLA haplotypes would match nearly 80% of

Generation of KCL038 clinical grade human embryonic stem cell line

The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene.

The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL040 clinical grade human embryonic stem cell line.

The KCL040 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.