Boon-Seng Soh

Mitochondrial diseases such as mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) manifest due to mutations in the mitochondrial genome. This leads to defects in mitochondrial respiration and ATP generation, affecting normal cellular function. Recently, it has been shown that the transfer of healthy donor mitochondria into recipient cells, termed “mitotherapy”, has been successful in boosting recipient cell metabolism and function. However, the mechanistic route by which mitotherapy promotes cellular function is uncertain. Here we demonstrate in vitro uptake of exogenous donor mitochondria and expression of the donor mitochondrial genes, resulting in metabolic and functional improvements in MELAS endothelial cells. Improvements were evident at both early and prolonged time points following treatment using healthy, functioning mitochondria, in contrast to the treatment of MELAS mutant mitochondria or sonicated healthy mitochondria. Transcriptomic analysis suggests that the improved cellular energetics enabled an upregulation of translation machinery, potentially counteracting the translational deficiencies characteristic of MELAS. Importantly, the treated healthy wild-type mitochondria are retained in recipient MELAS endothelial cells, decreasing their mutant mitochondrial DNA load, termed heteroplasmy. These findings suggest a novel route in tackling diseases that stem from mitochondrial dysfunctions.

The ubiquity of polystyrene nanoplastics (PS-NPs) in our environment raises substantial concerns about their potential impact on human health. Recent studies have shown that PS-NPs cross the blood-brain-barrier and accumulate in the central nervous system (CNS), raising the concerns on the causal role of PS-NP exposure and neurodegenerative diseases. In this study, we utilized human-induced pluripotent stem cell-derived neurons to examine the effects of PS-NPs on neuronal function and health. Our results revealed that PS-NPs penetrate neuronal cells in a size-dependent manner, and are bound by various cellular proteins, including TDP43, a key protein implicated in amyotrophic lateral sclerosis (ALS). Interestingly, CK1 and GSK3β kinases that are known to phosphorylate TDP-43 were found to be associated with PS-NPs. This observation suggests that PS-NPs may play a role in facilitating conditions that lead to TDP43 phosphorylation. We further demonstrate that exposure to healthy motor neurons with PS-NPs resulted in ALS-like phenotypes, characterized by hyperphosphorylated TDP-43, disrupted neuronal morphology, impaired mitochondrial respiration, and accelerated motor neuron death. These findings suggest that PS-NPs contribute to the pathogenesis of neurodegenerative diseases such as ALS and highlight the urgent need for strategies to limit human exposure to nanoplastics.

Mechanical stress within organoids is a pivotal indicator in disease modeling and pharmacokinetics, yet current tools lack the ability to rapidly and dynamically screen these mechanics. Here, we introduce biocompatible and compressible hollow microlasers that realize all-optical assessment of cellular stress within organoids. The laser spectroscopy yields identification of cellular deformation at the nanometer scale, corresponding to tens of pascals stress sensitivity. The compressibility enables the investigation of the isotropic component, which is the fundamental mechanics of multicellular models. By integrating with a microwell array, we demonstrate the high-throughput screening of mechanical cues in tumoroids, establishing a platform for mechano-responsive drug screening. Furthermore, we showcase the monitoring and mapping of dynamic contractile stress within human embryonic stem cell-derived cardiac organoids, revealing the internal mechanical inhomogeneity within a single organoid. This method eliminates time-consuming scanning and sample damage, providing insights into organoid mechanobiology.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs’ ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Background: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications. Methods: By exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent. Results: In this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs. Conclusion: Our study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

Plastic has become an ubiquitous environmental pollutant and nanoplastics (NPs) that are within the size range of 1nm to 1000nm could form upon weathering. Considering its sheer size, NPs are speculated to be more hazardous than their larger counterparts. Despite the growing concern, there is still limited understanding on the effects of NPs on human heart. Therefore, we aim to utilise human embryonic stem cells-derived-cardiomyocytes (hESC-CMs) to investigate the effects related to the uptake and accumulation of NPs in human heart. Firstly, more mature CMs were generated to better recapitulate the effects of NPs in adulthood. NPs effects were then elucidated over 3, 5 and 7 days of NPs treatment. The size-dependent uptake and accumulation of NPs was then established in CMs. Generally, oxidative stress and endoplasmic reticulum stress was upregulated in CMs in a dose-dependent manner. On the other hand, a rise in apoptosis was noted on all timepoints and was significant on day 7. Correspondingly, arrhythmia was also induced by day 7 of NPs treatment. Overall, our findings suggested that an exposure and accumulation of nanoplastics within hESC-CMs leads to oxidative stress, endoplasmic reticulum stress and decreased cell viability, resulting in an arrhythmic phenotype.