Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Human Primary Airway Basal Cells Display a Continuum of Molecular Phases from Health to Disease in Chronic Obstructive Pulmonary Disease.

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.

Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.

Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA- fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a Lin-CD34+CD38-/lowCD45RA-cKIT-CD26+ population as a potential therapeutic target for improved therapy response.

Single-cell multiomics of human fetal hematopoiesis define a developmental-specific population and a fetal signature.

Knowledge of human fetal blood development and how it differs from adult blood is highly relevant to our understanding of congenital blood and immune disorders and childhood leukemia, of which the latter can originate in utero. Blood formation occurs in waves that overlap in time and space, adding to heterogeneity, which necessitates single-cell approaches. Here, a combined single-cell immunophenotypic and transcriptional map of first trimester primitive blood development is presented. Using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), the molecular profile of established immunophenotype-gated progenitors was analyzed in the fetal liver (FL). Classical markers for hematopoietic stem cells (HSCs), such as CD90 and CD49F, were largely preserved, whereas CD135 (FLT3) and CD123 (IL3R) had a ubiquitous expression pattern capturing heterogenous populations. Direct molecular comparison with an adult bone marrow data set revealed that the HSC state was less frequent in FL, whereas cells with a lymphomyeloid signature were more abundant. An erythromyeloid-primed multipotent progenitor cluster was identified, potentially representing a transient, fetal-specific population. Furthermore, differentially expressed genes between fetal and adult counterparts were specifically analyzed, and a fetal core signature was identified. The core gene set could separate subgroups of acute lymphoblastic leukemia by age, suggesting that a fetal program may be partially retained in specific subgroups of pediatric leukemia. Our detailed single-cell map presented herein emphasizes molecular and immunophenotypic differences between fetal and adult blood cells, which are of significance for future studies of pediatric leukemia and blood development in general.

Concurrent stem- and lineage-affiliated chromatin programs precede hematopoietic lineage restriction.

The emerging notion of hematopoietic stem and progenitor cells (HSPCs) as a low-primed cloud without sharply demarcated gene expression programs raises the question on how cellular-fate options emerge and at which stem-like stage lineage priming is initiated. Here, we investigate single-cell chromatin accessibility of Lineage-, cKit+, and Sca1+ (LSK) HSPCs spanning the early differentiation landscape. Application of a signal-processing algorithm to detect transition points corresponding to massive alterations in accessibility of 571 transcription factor motifs reveals a population of LSK FMS-like tyrosine kinase 3 (Flt3)intCD9high cells that concurrently display stem-like and lineage-affiliated chromatin signatures, pointing to a simultaneous gain of both lympho-myeloid and megakaryocyte-erythroid programs. Molecularly and functionally, these cells position between stem cells and committed progenitors and display multi-lineage capacity in vitro and in vivo but lack self-renewal activity. This integrative molecular analysis resolves the heterogeneity of cells along hematopoietic differentiation and permits investigation of chromatin-mediated transition between multipotency and lineage restriction.

Defining the Emerging Blood System During Development at Single-Cell Resolution.

Developmental hematopoiesis differs from adult and is far less described. In the developing embryo, waves of lineage-restricted blood precede the ultimate emergence of definitive hematopoietic stem cells (dHSCs) capable of maintaining hematopoiesis throughout life. During the last two decades, the advent of single-cell genomics has provided tools to circumvent previously impeding characteristics of embryonic hematopoiesis, such as cell heterogeneity and rare cell states, allowing for definition of lineage trajectories, cellular hierarchies, and cell-type specification. The field has rapidly advanced from microfluidic platforms and targeted gene expression analysis, to high throughput unbiased single-cell transcriptomic profiling, single-cell chromatin analysis, and cell tracing-offering a plethora of tools to resolve important questions within hematopoietic development. Here, we describe how these technologies have been implemented to address a wide range of aspects of embryonic hematopoiesis ranging from the gene regulatory network of dHSC formation via endothelial to hematopoietic transition (EHT) and how EHT can be recapitulated in vitro, to hematopoietic trajectories and cell fate decisions. Together, these studies have important relevance for regenerative medicine and for our understanding of genetic blood disorders and childhood leukemias.

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations. In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types. In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing.

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.

Dissection of progenitor compartments resolves developmental trajectories in B-lymphopoiesis.

To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19+ progenitor compartment.

Defining the Minimal Factors Required for Erythropoiesis through Direct Lineage Conversion.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.