Analysis of three characterization assays reveals ddPCR of LIN28A as the most sensitive for the detection of residual pluripotent stem cells in cellular therapy products.

BACKGROUND AIMS: With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. DISCUSSION: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.

Generation of human induced pluripotent stem cell lines from sporadic, sporadic frontotemporal dementia, familial SOD1, and familial C9orf72 amyotrophic lateral sclerosis (ALS) patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Clinical heterogeneity and complex genetics pose challenges to understanding disease mechanisms and producing effective cures. To model clinical heterogeneity, we generated human induced pluripotent stem cells (iPSCs) from two sporadic ALS patients (sporadic ALS and sporadic ALS with frontotemporal dementia), two familial ALS patients (familial SOD1 mutation positive and familial C9orf72 repeat expansion positive), and four age- and sex-matched healthy controls. These iPSCs can be used to generate 2D and 3D in vitro models of ALS to investigate mechanisms of disease and screen for therapeutics.

Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution.

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.

Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease.

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.