Superior Induced Pluripotent Stem Cell Generation through Phactr3-Driven Mechanomodulation of Both Early and Late Phases of Cell Reprogramming.

Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes.

Advances in single-cell long-read sequencing technologies.

With an increase in accuracy and throughput of long-read sequencing technologies, they are rapidly being assimilated into the single-cell sequencing pipelines. For transcriptome sequencing, these techniques provide RNA isoform-level information in addition to the gene expression profiles. Long-read sequencing technologies not only help in uncovering complex patterns of cell-type specific splicing, but also offer unprecedented insights into the origin of cellular complexity and thus potentially new avenues for drug development. Additionally, single-cell long-read DNA sequencing enables high-quality assemblies, structural variant detection, haplotype phasing, resolving high-complexity regions, and characterization of epigenetic modifications. Given that significant progress has primarily occurred in single-cell RNA isoform sequencing (scRiso-seq), this review will delve into these advancements in depth and highlight the practical considerations and operational challenges, particularly pertaining to downstream analysis. We also aim to offer a concise introduction to complementary technologies for single-cell sequencing of the genome, epigenome and epitranscriptome. We conclude by identifying certain key areas of innovation that may drive these technologies further and foster more widespread application in biomedical science.

A Roadmap for Selecting and Utilizing Optimal Features in scRNA Sequencing Data Analysis for Stem Cell Research: A Comprehensive Review.

Stem cells and the cells they produce are unique because they vary from one cell to another. Traditional methods of studying cells often overlook these differences. However, the development of new technologies for studying individual cells has greatly changed biological research in recent years. Among these innovations, single-cell RNA sequencing (scRNA-seq) stands out. This technique allows scientists to examine the activity of genes in each cell, across thousands or even millions of cells. This makes it possible to understand the diversity of cells, identify new types of cells, and see how cells differ across different tissues, individuals, species, times, and conditions. This paper discusses the importance of scRNA-seq and the computational tools and software that are essential for analyzing the vast amounts of data generated by scRNA-seq studies. Our goal is to provide practical advice for bioinformaticians and biologists who are using scRNA-seq to study stem cells. We offer an overview of the scRNA-seq field, including the tools available, how they can be used, and how to present the results of these studies effectively. Our findings include a detailed overview and classification of tools used in scRNA-seq analysis, based on a review of 2,733 scientific publications. This review is complemented by information from the scRNA-tools database, which lists over 1,400 tools for analyzing scRNA-seq data. This database is an invaluable resource for researchers, offering a wide range of options for analyzing their scRNA-seq data.

Exploring the cell interactome: deciphering relative impacts of cell-cell communication in cell co-culture using a novel microfluidic device.

The human body is made up of approximately 40 trillion cells in close contact, with the cellular density of individual tissues varying from 1 million to 1 billion cells per cubic centimetre. Interactions between different cell types (termed heterotypic) are thus common in vivo. Communication between cells can take the form of direct cell-cell contact mediated by plasma membrane proteins or through paracrine signalling mediated through the release, diffusion, and receipt of soluble factors. There is currently no systematic method to investigate the relative contributions of these mechanisms to cell behaviour. In this paper, we detail the conception, development and validation of a microfluidic device that allows cell-cell contact and paracrine signalling in defined areas and over a variety of biologically relevant length scales, referred to as the interactome-device or 'I-device'. Importantly, by intrinsic device design features, cells in different regions in the device are exposed to four different interaction types, including a) no heterotypic cell interaction, b) only paracrine signalling, c) only cell-cell direct contact, or d) both forms of interaction (paracrine and cell-cell direct contact) together. The device design was validated by both mathematical modelling and experiments. Perfused stem cell culture over the medium term and the formation of direct contact between cells in the culture chambers was confirmed. The I-device offers significant flexibility, being able to be applied to any combination of adherent cells to determine the relative contributions of different communication mechanisms to cellular outcomes.

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.

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells.

The development of skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes is a major endeavor in the field. Human induced pluripotent stem cells (hiPSCs) are successfully used to derive skin cells, but the field is still far from meeting the goal of creating skin containing appendages, such as hair follicles and sweat glands. Here, the goal is to generate skin organoids (SKOs) from human skin fibroblast or placental CD34+ cell-derived hiPSCs. With all three hiPSC lines, complex SKOs with stratified skin layers and pigmented hair follicles are generated with different efficacies. In addition, the hiPSC-derived SKOs develop sebaceous glands, touch-receptive Merkel cells, and more importantly eccrine sweat glands. Together, physiologically relevant skin organoids are developed by direct induction of embryoid body formation, along with simultaneous inactivation of transforming growth factor beta signaling, activation of fibroblast growth factor signaling, and inhibition of bone morphogenetic protein signaling pathways. The skin organoids created in this study can be used as valuable platforms for further research into human skin development, disease modeling, or reconstructive surgeries.

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology.

Aging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments.

Protocol for generating embedding-free brain organoids enriched with oligodendrocytes.

In response to the scarcity of advanced in vitro models dedicated to human CNS white matter research, we present a protocol to generate neuroectoderm-derived embedding-free human brain organoids enriched with oligodendrocytes. We describe steps for neuroectoderm differentiation, development of neural spheroids, and their transferal to Matrigel. We then detail procedures for the development, maturation, and application of oligodendrocyte-enriched brain organoids. The presence of myelin-producing cells makes these organoids useful for studying human white matter diseases, such as leukodystrophy.

CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.

DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.

Generation of a human induced pluripotent stem cell line (UQi001-A-1) edited with the CRISPR-Cas9 system to carry the heterozygous TARDBP c.1144G > A (p.A382T) missense mutation.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the TDP-43 protein is believed to play a central role in disease pathophysiology. Using the CRISPR-Cas9 system, we introduced the heterozygous c.1144G > A (p.A382T) missense mutation in exon 6 of the TARDBP gene into an iPSC line derived from a healthy individual. These edited iPSCs displayed normal cellular morphology, expressed major pluripotency markers, were capable of tri-lineage differentiation, and possessed a normal karyotype.

Embryonic Stem Cell-Derived Neurons as a Model System for Epigenome Maturation during Development.

DNA methylation in neurons is directly linked to neuronal genome regulation and maturation. Unlike other tissues, vertebrate neurons accumulate high levels of atypical DNA methylation in the CH sequence context (mCH) during early postnatal brain development. Here, we investigate to what extent neurons derived in vitro from both mouse and human pluripotent stem cells recapitulate in vivo DNA methylation patterns. While human ESC-derived neurons did not accumulate mCH in either 2D culture or 3D organoid models even after prolonged culture, cortical neurons derived from mouse ESCs acquired in vivo levels of mCH over a similar time period in both primary neuron cultures and in vivo development. mESC-derived neuron mCH deposition was coincident with a transient increase in Dnmt3a, preceded by the postmitotic marker Rbfox3 (NeuN), was enriched at the nuclear lamina, and negatively correlated with gene expression. We further found that methylation patterning subtly differed between in vitro mES-derived and in vivo neurons, suggesting the involvement of additional noncell autonomous processes. Our findings show that mouse ESC-derived neurons, in contrast to those of humans, can recapitulate the unique DNA methylation landscape of adult neurons in vitro over experimentally tractable timeframes, which allows their use as a model system to study epigenome maturation over development.

Noncoding RNA of Zika Virus Affects Interplay between Wnt-Signaling and Pro-Apoptotic Pathways in the Developing Brain Tissue.

Zika virus (ZIKV) has a unique ability among flaviviruses to cross the placental barrier and infect the fetal brain causing severe abnormalities of neurodevelopment known collectively as congenital Zika syndrome. In our recent study, we demonstrated that the viral noncoding RNA (subgenomic flaviviral RNA, sfRNA) of the Zika virus induces apoptosis of neural progenitors and is required for ZIKV pathogenesis in the developing brain. Herein, we expanded on our initial findings and identified biological processes and signaling pathways affected by the production of ZIKV sfRNA in the developing brain tissue. We employed 3D brain organoids generated from induced human pluripotent stem cells (ihPSC) as an ex vivo model of viral infection in the developing brain and utilized wild type (WT) ZIKV (producing sfRNA) and mutant ZIKV (deficient in the production of sfRNA). Global transcriptome profiling by RNA-Seq revealed that the production of sfRNA affects the expression of >1000 genes. We uncovered that in addition to the activation of pro-apoptotic pathways, organoids infected with sfRNA-producing WT, but not sfRNA-deficient mutant ZIKV, which exhibited a strong down-regulation of genes involved in signaling pathways that control neuron differentiation and brain development, indicating the requirement of sfRNA for the suppression of neurodevelopment associated with the ZIKV infection. Using gene set enrichment analysis and gene network reconstruction, we demonstrated that the effect of sfRNA on pathways that control brain development occurs via crosstalk between Wnt-signaling and proapoptotic pathways.

Cellular senescence and premature aging in Down Syndrome.

Down syndrome (DS) is a genetic disorder caused by an extra copy of chromosome 21, resulting in cognitive impairment, physical abnormalities, and an increased risk of age-related co-morbidities. Individuals with DS exhibit accelerated aging, which has been attributed to several cellular mechanisms, including cellular senescence, a state of irreversible cell cycle arrest that is associated with aging and age-related diseases. Emerging evidence suggests that cellular senescence may play a key role in the pathogenesis of DS and the development of age-related disorders in this population. Importantly, cellular senescence may be a potential therapeutic target in alleviating age-related DS pathology. Here, we discuss the importance of focusing on cellular senescence to understand accelerated aging in DS. We review the current state of knowledge regarding cellular senescence and other hallmarks of aging in DS, including its putative contribution to cognitive impairment, multi-organ dysfunction, and premature aging phenotypes.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Zika virus noncoding RNA cooperates with the viral protein NS5 to inhibit STAT1 phosphorylation and facilitate viral pathogenesis.

All flaviviruses, including Zika virus, produce noncoding subgenomic flaviviral RNA (sfRNA), which plays an important role in viral pathogenesis. However, the exact mechanism of how sfRNA enables viral evasion of antiviral response is not well defined. Here, we show that sfRNA is required for transplacental virus dissemination in pregnant mice and subsequent fetal brain infection. We also show that sfRNA promotes apoptosis of neural progenitor cells in human brain organoids, leading to their disintegration. In infected human placental cells, sfRNA inhibits multiple antiviral pathways and promotes apoptosis, with signal transducer and activator of transcription 1 (STAT1) identified as a key shared factor. We further show that the production of sfRNA leads to reduced phosphorylation and nuclear translocation of STAT1 via a mechanism that involves sfRNA binding to and stabilizing viral protein NS5. Our results suggest the cooperation between viral noncoding RNA and a viral protein as a novel strategy for counteracting antiviral responses.

Generation of human induced pluripotential stem cells from individuals with complex heterozygous, isogenic corrected, and homozygous Bloc1s1 mutations.

Genetic studies show that BLOC1S1 modulates mitochondrial and endosome-lysosome function (Wu et al., 2021a). Furthermore, Bloc1s1 mutations are linked to leukodystrophy (Bertoli-Avella et al., 2021). The Vanderver laboratory identified additional individuals with leukodystrophy that harbored either complex heterozygous (Bloc1s1 c.206A > C and c.359G > A), or homozygous (Bloc1s1 c.185 T > C) point mutations. We generated induced pluripotential stem cell (iPSC) lines from these subjects, from parents of the complex heterozygous mutations patient, and from CRISPR isogenic (c.206A > C and c.359G > A) corrected iPSC-line. These complex heterozygous, homozygous, and isogenic-corrected Bloc1s1 lines were phenotypically normal and were capable of differentiation towards the three germ layers.

Generation of iPSC lines from hereditary spastic paraplegia 56 (SPG56) patients and family members carrying CYP2U1 mutations.

Hereditary spastic paraplegia 56 (SPG56) is an extremely rare autosomal recessive disorder caused by mutations in the CYP2U1 gene, involved in fatty acid metabolism. SPG56 causes progressive spasticity in upper and lower limbs, though due to the rarity of this subtype of spastic paraplegia, the molecular causes remain unclear and no treatment or cure exists. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from two unrelated patients with SPG56 and two heterozygous family members. These lines can be used to investigate the mechanisms driving progressive spasticity and evaluate the potential for gene replacement therapies.

Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants.

Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.

The hallmarks of aging in Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is caused by absence of the catalytic activity of ATM, a protein kinase that plays a central role in the DNA damage response, many branches of cellular metabolism, redox and mitochondrial homeostasis, and cell cycle regulation. A-T is a complex disorder characterized mainly by progressive cerebellar degeneration, immunodeficiency, radiation sensitivity, genome instability, and predisposition to cancer. It is increasingly recognized that the premature aging component of A-T is an important driver of this disease, and A-T is therefore an attractive model to study the aging process. This review outlines the current state of knowledge pertaining to the molecular and cellular signatures of aging in A-T and proposes how these new insights can guide novel therapeutic approaches for A-T.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.