N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

Development and validation of broad-spectrum magnetic particle labelling processes for cell therapy manufacturing.

BACKGROUND: Stem cells are increasingly seen as a solution for many health challenges for an ageing population. However, their potential benefits in the clinic are currently curtailed by technical challenges such as high cell dose requirements and point of care delivery, which pose sourcing and logistics challenges. Cell manufacturing solutions are currently in development to address the supply issue, and ancillary technologies such as nanoparticle-based labelling are being developed to improve stem cell delivery and enable post-treatment follow-up. METHODS: The application of magnetic particle (MP) labelling to potentially scalable cell manufacturing processes was investigated in a range of therapeutically relevant cells, including mesenchymal stromal cells (MSC), cardiomyocytes (CMC) and neural progenitor cells (ReN). The efficiency and the biological effect of particle labelling were analysed using fluorescent imaging and cellular assays. RESULTS: Flow cytometry and fluorescent microscopy confirmed efficient labelling of monolayer cultures. Viability was shown to be retained post labelling for all three cell types. MSC and CMC demonstrated higher tolerance to MP doses up to 100× the standard concentration. This approach was also successful for MP labelling of suspension cultures, demonstrating efficient MP uptake within 3 h, while cell viability was unaffected by this suspension labelling process. Furthermore, a procedure to enable the storing of MP-labelled cell populations to facilitate cold chain transport to the site of clinical use was investigated. When MP-labelled cells were stored in hypothermic conditions using HypoThermosol solution for 24 h, cell viability and differentiation potential were retained post storage for ReN, MSC and beating CMC. CONCLUSIONS: Our results show that a generic MP labelling strategy was successfully developed for a range of clinically relevant cell populations, in both monolayer and suspension cultures. MP-labelled cell populations were able to undergo transient low-temperature storage whilst maintaining functional capacity in vitro. These results suggest that this MP labelling approach can be integrated into cell manufacturing and cold chain transport processes required for future cell therapy approaches.

Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Current status of drug screening and disease modelling in human pluripotent stem cells.

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost.