High-resolution Gene Expression Analyses Research Articles

The Future of Cardiology: Integrating Single-Cell Transcriptomics with Multi-Omics for Enhanced Cardiac Disease Insights.

Recent advancements in single-cell transcriptome sequencing (scRNA-seq) have revolutionized our understanding of cellular heterogeneity in cardiovascular diseases, enabling the identification of novel therapeutic targets. This technology allows for high-resolution analysis of gene expression at the single-cell level, revealing the complex dynamics of human heart cell development and the diverse roles of cardiac cell types in health and disease. Despite its transformative potential, current applications of scRNA-seq face limitations, including challenges in data integration and the need for comprehensive multi-omic approaches to fully elucidate the mechanisms underlying cardiovascular pathologies. This review highlights the significant insights gained from scRNA-seq studies in the mammalian heart, emphasizing the importance of integrating spatial transcriptomics and other omics technologies to enhance our understanding of cardiac biology. Furthermore, it addresses the critical research gaps in the field, particularly in the context of personalized medicine and the need for improved methodologies to analyze rare cell populations. By exploring these challenges and opportunities, this review aims to pave the way for innovative diagnostic and therapeutic strategies that can ultimately improve outcomes for patients with cardiovascular diseases.

Gastruloids Gain Muscle: Somite Formation in Embryo-Like Structures.

Gastruloids are embryo-like structures that display key features of post-implantation embryonic development, yet whether they fully recapitulate invivo embryogenesis remains unsolved. Recently in Nature, van den Brink etal. (2020) performed high-resolution gene expression analysis to identify significant similarities between mouse gastruloids and embryos in positional lineage segregation and somite formation.

An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples

Establishing clinically relevant single-cell (SC) transcriptomic workflows from cryopreserved tissue is essential to move this emerging immune monitoring technology from the bench to the bedside. Improper sample preparation leads to detrimental cascades, resulting in loss of precious time, money and finally compromised data. There is an urgent need to establish protocols specifically designed to overcome the inevitable variations in sample quality resulting from uncontrollable factors in a clinical setting. Here, we explore sample preparation techniques relevant to a range of clinically relevant scenarios, where SC gene expression and repertoire analysis are applied to a cryopreserved sample derived from a small amount of blood, with unknown or partially known preservation history. We compare a total of ten cell-counting, viability-improvement, and lymphocyte-enrichment methods to highlight a number of unexpected findings. Trypan blue-based automated counters, typically recommended for single-cell sample quantitation, consistently overestimate viability. Advanced sample clean-up procedures significantly impact total cell yield, while only modestly increasing viability. Finally, while pre-enrichment of B cells from whole peripheral blood mononuclear cells (PBMCs) results in the most reliable BCR repertoire data, comparable T-cell enrichment strategies distort the ratio of CD4+ and CD8+ cells. Furthermore, we provide high-resolution analysis of gene expression and clonotype repertoire of different B cell subtypes. Together these observations provide both qualitative and quantitative sample preparation guidelines that increase the chances of obtaining high-quality single-cell transcriptomic and repertoire data from human PBMCs in a variety of clinical settings.

Neuronal Subtype Generation During Postnatal Olfactory Bulb Neurogenesis

In the perinatal and adult forebrain, regionalized neural stem cells lining the ventricular walls produce different types of olfactory bulb interneurons. Although these postnatal stem cells are lineage related to their embryonic counterparts that produce, for example, cortical, septal, and striatal neurons, their output at the level of neuronal phenotype changes dramatically. Tiveron et al. investigated the molecular determinants underlying stem cell regionalization and the gene expression changes inducing the shift from embryonic to adult neuron production. High-resolution gene expression analyses of different lineages revealed that the zinc finger proteins, Zic1 and Zic2, are postnatally induced in the dorsal olfactory bulb neuron lineage. Functional studies demonstrated that these factors confer a GABAergic and calretinin-positive phenotype to neural stem cells while repressing dopaminergic fate. Based on these findings, we discuss the molecular mechanisms that allow acquisition of new traits during the transition from embryonic to adult neurogenesis. We focus on the involvement of epigenetic marks and emphasize why the identification of master transcription factors, that instruct the fate of postnatally generated neurons, can help in deciphering the mechanisms driving fate transition from embryonic to adult neuron production.

High-resolution gene expression analysis of the developing mouse kidney defines novel cellular compartments within the nephron progenitor population.

The functional unit of the kidney is the nephron. During its organogenesis, the mammalian metanephric kidney generates thousands of nephrons over a protracted period of fetal life. All nephrons are derived from a population of self-renewing multi-potent progenitor cells, termed the cap mesenchyme. However, our understanding of the molecular and cellular mechanisms underlying nephron development is at an early stage. In order to identify factors involved in nephrogenesis, we performed a high-resolution, spatial profiling of a number of transcriptional regulators expressed within the cap mesenchyme and early developing nephron. Our results demonstrate novel, stereotypic, spatially defined cellular sub-domains within the cap mesenchyme, which may, in part, reflect induction of nephron precursors. These results suggest a hitherto unappreciated complexity of cell states that accompany the assembly of the metanephric kidney, likely reflecting diverse regulatory actions such as the maintenance and induction of nephron progenitors.

Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues.

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non-specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high-resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC:uidA, rolC:iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.