Cell Lines as Biological Models: Practical Steps for More Reliable Research.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM).

Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM.

Generation of TWO G51D SNCA missense mutation iPSC lines (CRICKi011-A, CRICKi012-A) from two individuals at risk of Parkinson's disease.

Mutations or multiplications of the SNCA (Synuclein Alpha) gene cause rare autosomal dominant Parkinson's disease (PD). The SNCA G51D missense mutation is associated with a synucleinopathy that shares PD and multiple system atrophy (MSA) characteristics. We generated induced pluripotent stem cell (iPSC) lines from two individuals with SNCA G51D missense mutations at risk of PD. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. The resulting human iPSCs displayed normal morphology, expressed markers associated with pluripotency, and differentiated into the three germ layers. The iPSC lines could facilitate disease-modelling and therapy development studies for synucleinopathies.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Toxoplasma-proximal and distal control by GBPs in human macrophages.

Human guanylate binding proteins (GBPs) are key players of interferon-gamma (IFNγ)-induced cell intrinsic defense mechanisms targeting intracellular pathogens. In this study, we combine the well-established Toxoplasmagondii infection model with three in vitro macrophage culture systems to delineate the contribution of individual GBP family members to control this apicomplexan parasite. Use of high-throughput imaging assays and genome engineering allowed us to define a role for GBP1, 2 and 5 in parasite infection control. While GBP1 performs a pathogen-proximal, parasiticidal and growth-restricting function through accumulation at the parasitophorous vacuole of intracellular Toxoplasma, GBP2 and GBP5 perform a pathogen-distal, growth-restricting role. We further find that mutants of the GTPase or isoprenylation site of GBP1/2/5 affect their normal function in Toxoplasma control by leading to mis-localization of the proteins.

Generation of FOUR iPSC lines (CRICKi004-A; CRICKi005-A; CRICKi006-A, CRICKi007-A) from Spinal muscle atrophy patients with lower extremity dominant (SMALED) phenotype.

Spinal muscular atrophy with lower extremity dominant (SMALED) is a hereditary neuromuscular disorder characterized by degeneration of spinal cord motor neurons resulting in lower limbs muscle weakness and paralysis. Mutations in DYNC1H1, which encodes BICD2, a multifunctional adaptor for microtubule motor proteins, cause the disorder. Here, we generated four induced pluripotent stem cell (iPSC) lines from patients with SMALED. Dermal fibroblasts were obtained from the MRC neuromuscular disease biobank and reprogrammed using non-integrating mRNA-based protocol. Characterization of the four iPSC lines included karyotyping and Sanger sequencing, while the expression of associated markers confirmed pluripotency and differentiation potential.

Rostrocaudal patterning and neural crest differentiation of human pre-neural spinal cord progenitors in vitro.

The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by WNT/fibroblast growth factor (FGF) signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which compromises production of the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity for 7-10 passages, dividing to self-renew and to make neural crest progenitors, while gradually adopting a more posterior identity by activating colinear HOX gene expression. The HOX clock can be halted through GDF11-mediated signal inhibition to produce a PNP and NC population with a thoracic identity that can be maintained for up to 30 passages.

The International Stem Cell Banking Initiative (ISCBI).

The International Stem Cell Banking Initiative(ISCBI) was started in 2007 to bring together the leading stem cell banks distributing human pluripotent stem cell (hPSC) lines for research and development, to discuss best practice across a range of issues from donor consent to delivery of cells for use in research, diagnostics and cell-based medicines. ISCBI holds workshops around the world and on-line and regularly publishes summaries of discussions and consensus amongst experts in stem cell biology, biobanking technology, regulation and policy making. To date, experts from more than 28 countries have contributed to ISCBI activities which are frequently run in collaboration with other stem cell organisations and has co-ordinated closely with the International Stem Cell Initiative and the hPSCreg European Commission funded database of hPSC lines and clincal trials.

Generation of an iPSC line (CRICKi001-A) from an individual with a germline SMARCA4 missense mutation and autism spectrum disorder.

Germline missense mutations in the BAF swi/snf chromatin remodeling subunit SMARCA4 are associated with neurodevelopmental disorders, including Coffin Siris Syndrome (CSS). Here, we generated an induced pluripotent stem cell line from a male patient with atypical CSS features and a de novo heterozygous missense mutation in the SMARCA4 gene (c.3607C>T, p.(Arg1203Cys)). Hair root derived keratinocytes were reprogrammed using non-integrative Sendai virus vector delivery of pluripotency factors. iPSCs generated display normal morphology and molecular karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Acquisition and Reception of Primary Tissues, Cells, or Other Biological Specimens.

The use and banking of biological material for research or clinical application is a well-established practice. The material can be of human or non-human origin. The processes involved in this type of activity, from the sourcing to receipt of materials, require adherence to a set of best practice principles that assure the ethical and legal procurement, traceability, and quality of materials.

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

The development of 'feeder' cells for the preparation of clinical grade hES cell lines: challenges and solutions.

The development of human embryonic stem cell (hESC) lines for research and therapy is hampered by the need to improve the basic methodologies for cell culture expansion. In most current methods hESC lines are cultured on a mouse or human feeder cell layer which appears to be the most reliable way to maintain cells stably in the undifferentiated state. However, co-culture introduces complications for studying stem cell biology and the delivery of safe therapies for the future. This article reviews the specific risks associated with any proposed clinical use of feeder cells of mouse origin and compares these with the benefits and risks of using human feeder cells. The further work required to establish clinical grade feeder cell lines for hESC line culture is significant and costly. Much work is being done to find feeder-free culture systems but these are at an early stage of development and there may be consequences that affect the value of the hESCs for research and development. These challenges should be viewed in the context of the huge amount of work that will be required over many years to develop robust differentiation protocols and establish fully defined procedures and adequate safety data for embryonic stem cell products.