The RNA binding proteins LARP4A and LARP4B promote sarcoma and carcinoma growth and metastasis.

RNA-binding proteins (RBPs) are emerging as important regulators of cancer pathogenesis. We reveal that the RBPs LARP4A and LARP4B are differentially overexpressed in osteosarcoma and osteosarcoma lung metastases, as well as in prostate cancer. Depletion of LARP4A and LARP4B reduced tumor growth and metastatic spread in xenografts, as well as inhibiting cell proliferation, motility, and migration. Transcriptomic profiling and high-content multiparametric analyses unveiled a central role for LARP4B, but not LARP4A, in regulating cell cycle progression in osteosarcoma and prostate cancer cells, potentially through modulating key cell cycle proteins such as Cyclins B1 and E2, Aurora B, and E2F1. This first systematic comparison between LARP4A and LARP4B assigns new pro-tumorigenic functions to LARP4A and LARP4B in bone and prostate cancer, highlighting their similarities while also indicating distinct functional differences. Uncovering clear biological roles for these paralogous proteins provides new avenues for identifying tissue-specific targets and potential druggable intervention.

Gradients of glucose metabolism regulate morphogen signalling required for specifying tonotopic organisation in the chicken cochlea.

In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along a proximal-to-distal long (tonotopic) axis. Acquisition of unique morphologies at the appropriate position along the chick cochlea, the basilar papilla, requires that nascent HCs determine their tonotopic positions during development. The complex signalling within the auditory organ between a developing HC and its local niche along the cochlea is poorly understood. Using a combination of live imaging and NAD(P)H fluorescence lifetime imaging microscopy, we reveal that there is a gradient in the cellular balance between glycolysis and the pentose phosphate pathway in developing HCs along the tonotopic axis. Perturbing this balance by inhibiting different branches of cytosolic glucose catabolism disrupts developmental morphogen signalling and abolishes the normal tonotopic gradient in HC morphology. These findings highlight a causal link between graded morphogen signalling and metabolic reprogramming in specifying the tonotopic identity of developing HCs.

Cellular interactions in the pituitary stem cell niche.

Stem cells in the anterior pituitary gland can give rise to all resident endocrine cells and are integral components for the appropriate development and subsequent maintenance of the organ. Located in discreet niches within the gland, stem cells are involved in bi-directional signalling with their surrounding neighbours, interactions which underpin pituitary gland homeostasis and response to organ challenge or physiological demand. In this review we highlight core signalling pathways that steer pituitary progenitors towards specific endocrine fate decisions throughout development. We further elaborate on those which are conserved in the stem cell niche postnatally, including WNT, YAP/TAZ and Notch signalling. Furthermore, we have collated a directory of single cell RNA sequencing studies carried out on pituitaries across multiple organisms, which have the potential to provide a vast database to study stem cell niche components in an unbiased manner. Reviewing published data, we highlight that stem cells are one of the main signalling hubs within the anterior pituitary. In future, coupling single cell sequencing approaches with genetic manipulation tools in vivo, will enable elucidation of how previously understudied signalling pathways function within the anterior pituitary stem cell niche.

Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.

Single Cell RNA-Seq: Cell Isolation and Data Analysis.

Single-cell RNA-sequencing technologies have revolutionized the way that researchers can interrogate cellular relationships and the level of detail by which tissue architecture can be characterized. Multiple cell capturing methods have been developed that, when coupled to next-generation sequencing, can yield cell-to-cell specific information regarding gene expression profiles. One of the commonalities between all of the cell capturing techniques to succeed is the necessity to submit samples with a high cell viability. In addition, these cells should have undergone minimal processing to limit induced stress responses so that their transcriptomes, when sequenced, closely reflect their transcriptomes in vivo at the time of isolation. Below we present a streamlined protocol to isolate fresh cells from tissues in vivo. We also share extensive notes to highlight considerations researchers should take into account before beginning their cell isolation protocol.

Defining human mesenchymal and epithelial heterogeneity in response to oral inflammatory disease.

Human oral soft tissues provide the first barrier of defence against chronic inflammatory disease and hold a remarkable scarless wounding phenotype. Tissue homeostasis requires coordinated actions of epithelial, mesenchymal, and immune cells. However, the extent of heterogeneity within the human oral mucosa and how tissue cell types are affected during the course of disease progression is unknown. Using single-cell transcriptome profiling we reveal a striking remodelling of the epithelial and mesenchymal niches with a decrease in functional populations that are linked to the aetiology of the disease. Analysis of ligand-receptor interaction pairs identify potential intercellular hubs driving the inflammatory component of the disease. Our work establishes a reference map of the human oral mucosa in health and disease, and a framework for the development of new therapeutic strategies.

Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells.

In response to physiological demand, the pituitary gland generates new hormone-secreting cells from committed progenitor cells throughout life. It remains unclear to what extent pituitary stem cells (PSCs), which uniquely express SOX2, contribute to pituitary growth and renewal. Moreover, neither the signals that drive proliferation nor their sources have been elucidated. We have used genetic approaches in the mouse, showing that the WNT pathway is essential for proliferation of all lineages in the gland. We reveal that SOX2+ stem cells are a key source of WNT ligands. By blocking secretion of WNTs from SOX2+ PSCs in vivo, we demonstrate that proliferation of neighbouring committed progenitor cells declines, demonstrating that progenitor multiplication depends on the paracrine WNT secretion from SOX2+ PSCs. Our results indicate that stem cells can hold additional roles in tissue expansion and homeostasis, acting as paracrine signalling centres to coordinate the proliferation of neighbouring cells.

Macrophage modulation of dental pulp stem cell activity during tertiary dentinogenesis.

The interaction between immune cells and stem cells is important during tissue repair. Macrophages have been described as being crucial for limb regeneration and in certain circumstances have been shown to affect stem cell differentiation in vivo. Dentine is susceptible to damage as a result of caries, pulp infection and inflammation all of which are major problems in tooth restoration. Characterising the interplay between immune cells and stem cells is crucial to understand how to improve natural repair mechanisms. In this study, we used an in vivo damage model, associated with a macrophage and neutrophil depletion model to investigate the role of immune cells in reparative dentine formation. In addition, we investigated the effect of elevating the Wnt/ß-catenin pathway to understand how this might regulate macrophages and impact upon Wnt receiving pulp stem cells during repair. Our results show that macrophages are required for dental pulp stem cell activation and appropriate reparative dentine formation. In addition, pharmacological stimulation of the Wnt/ß-catenin pathway via GSK-3ß inhibitor small molecules polarises macrophages to an anti-inflammatory state faster than inert calcium silicate-based materials thereby accelerating stem cell activation and repair. Wnt/ß-catenin signalling thus has a dual role in promoting reparative dentine formation by activating pulp stem cells and promoting an anti-inflammatory macrophage response.

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

Epigenetic mechanisms driving lineage commitment in mesenchymal stem cells.

The increasing application of approaches that allow tracing of individual cells over time, together with transcriptomic and epigenomic analyses is changing the way resident stromal stem cells (mesenchymal stem cells) are viewed. Rather than being a defined, homogeneous cell population as described following in vitro expansion, in vivo, these cells are highly programmed according to their resident tissue location. This programming is evidenced by different epigenetic landscapes and gene transcription signatures in cells before any in vitro expansion. This has potentially profound implications for the heterotypic use of these cells in therapeutic tissue engineering applications.

Molecular Programming of Perivascular Stem Cell Precursors.

Pericytes have been shown to act as precursors of resident adult stem cells in stromal tissues in vivo. When expanded in vitro these cells are capable of giving rise to multiple mesenchymal cell types, irrespective of their tissue of origin. This phenomenon of multi-lineage differentiation is only observed in culture, whereas in vivo, stromal stem cell differentiation is restricted to tissue-specific cell types. An important unanswered question is how a single, widely distributed cell type (a pericyte) gives rise to stem cells with tissue-specific functions and attributes. Using a combination of transcriptomics and epigenomics we have compared the molecular status of two populations of stromal stem cell precursors. Using a LacZ transgene insertion that is expressed in pericytes but not in stem cells, we were able to compare pericyte populations from two different tissues, mouse incisors and bone marrow. Pericytes, freshly isolated from mouse incisors and bone marrow, exhibited transcriptomes and epigenetic landscapes that were extensively different, reflecting their tissue of origin and future in vivo differentiation potential. Dspp, an odontoblast differentiation gene, as well as additional odontogenic genes, are shown to be expressed in dental pulp-derived pericytes. These genetic loci are also decorated with histone modifications indicative of a transcriptionally active chromatin state. In bone marrow pericytes, a major osteogenic differentiation gene, Runx2, is not expressed but is marked by both active and repressive histones and therefore primed to be expressed. Polycomb repressor complex 1 analysis showed that key genes involved in the induction of adipogenesis, chondrogenesis, and myogenesis are targeted by Ring1b and therefore stably repressed. This indicates that pericyte populations are molecularly obstructed from differentiating down certain lineages in vivo. Stem Cells 2018;36:1890-15.