Inflammatory recruitment of healthy hematopoietic stem and progenitor cells in the acute myeloid leukemia niche.

Inflammation in the bone marrow (BM) microenvironment is a constitutive component of leukemogenesis in acute myeloid leukemia (AML). Current evidence suggests that both leukemic blasts and stroma secrete proinflammatory factors that actively suppress the function of healthy hematopoietic stem and progenitor cells (HSPCs). HSPCs are also cellular components of the innate immune system, and we reasoned that they may actively propagate the inflammation in the leukemic niche. In two separate congenic models of AML we confirm by evaluation of the BM plasma secretome and HSPC-selective single-cell RNA sequencing (scRNA-Seq) that multipotent progenitors and long-lived stem cells adopt inflammatory gene expression programs, even at low leukemic infiltration of the BM. In particular, we observe interferon gamma (IFN-γ) pathway activation, along with secretion of its chemokine target, CXCL10. We show that AML-derived nanometer-sized extracellular vesicles (EVAML) are sufficient to trigger this inflammatory HSPC response, both in vitro and in vivo. Altogether, our studies indicate that HSPCs are an unrecognized component of the inflammatory adaptation of the BM by leukemic cells. The pro-inflammatory conversion and long-lived presence of HSPCs in the BM along with their regenerative re-expansion during remission may impact clonal selection and disease evolution.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

The Prognostic Potential of Human Prostate Cancer-Associated Macrophage Subtypes as Revealed by Single-Cell Transcriptomics.

Macrophages in the tumor microenvironment are causally linked with prostate cancer development and progression, yet little is known about their composition in neoplastic human tissue. By performing single cell transcriptomic analysis of human prostate cancer resident macrophages, three distinct populations were identified in the diseased prostate. Unexpectedly, no differences were observed between macrophages isolated from the tumorous and nontumorous portions of the prostatectomy specimens. Markers associated with canonical M1 and M2 macrophage phenotypes were identifiable, however these were not the main factors defining unique subtypes. The genes selectively associated with each macrophage cluster were used to develop a gene signature which was highly associated with both recurrence-free and metastasis-free survival. These results highlight the relevance of tissue-specific macrophage subtypes in the tumor microenvironment for prostate cancer progression and demonstrates the utility of profiling single-cell transcriptomics in human tumor samples as a strategy to design gene classifiers for patient prognostication. IMPLICATIONS: The specific macrophage subtypes present in a diseased human prostate have prognostic value, suggesting that the relative proportions of these populations are related to patient outcome. Understanding the relative contributions of these subtypes will not only inform patient prognostication, but will enable personalized immunotherapeutic strategies to increase beneficial populations or reduce detrimental populations.

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

Androgen receptor signalling in macrophages promotes TREM-1-mediated prostate cancer cell line migration and invasion.

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.

Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart.

While the heart regenerates poorly in mammals, efficient heart regeneration occurs in zebrafish. Studies in zebrafish have resulted in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium. To identify which processes occur in proliferating cardiomyocytes we have used a single-cell RNA-sequencing approach. We uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the nonproliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes. Moreover, these cells have reduced expression of mitochondrial genes and reduced mitochondrial activity, while glycolysis gene expression and glucose uptake are increased, indicative for metabolic reprogramming. Furthermore, we find that the metabolic reprogramming of border zone cardiomyocytes is induced by Nrg1/ErbB2 signaling and is important for their proliferation. This mechanism is conserved in murine hearts in which cardiomyocyte proliferation is induced by activating ErbB2 signaling. Together these results demonstrate that glycolysis regulates cardiomyocyte proliferation during heart regeneration.

Cell Type Purification by Single-Cell Transcriptome-Trained Sorting.

Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.

A transcriptomic roadmap to α- and ß-cell differentiation in the embryonic pancreas.

During pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into α-, ß-, γ- and δ-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on α- and ß-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev Pseudotime analysis revealed specific branches for developing α- and ß-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into α- and ß-cells.

Oral Mucosal Organoids as a Potential Platform for Personalized Cancer Therapy.

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs. SIGNIFICANCE: This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813.

Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.

Dermal Condensate Niche Fate Specification Occurs Prior to Formation and Is Placode Progenitor Dependent.

Cell fate transitions are essential for specification of stem cells and their niches, but the precise timing and sequence of molecular events during embryonic development are largely unknown. Here, we identify, with 3D and 4D microscopy, unclustered precursors of dermal condensates (DC), signaling niches for epithelial progenitors in hair placodes. With population-based and single-cell transcriptomics, we define a molecular time-lapse from pre-DC fate specification through DC niche formation and establish the developmental trajectory as the DC lineage emerges from fibroblasts. Co-expression of downregulated fibroblast and upregulated DC genes in niche precursors reveals a transitory molecular state following a proliferation shutdown. Waves of transcription factor and signaling molecule expression then coincide with DC formation. Finally, ablation of epidermal Wnt signaling and placode-derived FGF20 demonstrates their requirement for pre-DC specification. These findings uncover a progenitor-dependent niche precursor fate and the transitory molecular events controlling niche formation and function.

Mapping the physical network of cellular interactions.

A cell's function is influenced by the environment, or niche, in which it resides. Studies of niches usually require assumptions about the cell types present, which impedes the discovery of new cell types or interactions. Here we describe ProximID, an approach for building a cellular network based on physical cell interaction and single-cell mRNA sequencing, and show that it can be used to discover new preferential cellular interactions without prior knowledge of component cell types. ProximID found specific interactions between megakaryocytes and mature neutrophils and between plasma cells and myeloblasts and/or promyelocytes (precursors of neutrophils) in mouse bone marrow, and it identified a Tac1+ enteroendocrine cell-Lgr5+ stem cell interaction in small intestine crypts. This strategy can be used to discover new niches or preferential interactions in a variety of organs.

Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

Circadian networks in human embryonic stem cell-derived cardiomyocytes.

Cell-autonomous circadian oscillations strongly influence tissue physiology and pathophysiology of peripheral organs including the heart, in which the circadian clock is known to determine cardiac metabolism and the outcome of for instance ischemic stress. Human pluripotent stem cells represent a powerful tool to study developmental processes in vitro, but the extent to which human embryonic stem (ES) cell-derived cardiomyocytes establish circadian rhythmicity in the absence of a systemic context is unknown. Here we demonstrate that while undifferentiated human ES cells do not possess an intrinsic functional clock, oscillatory expression of known core clock genes emerges spontaneously during directed cardiac differentiation. We identify a set of clock-controlled output genes that contain an oscillatory network of stress-related transcripts. Furthermore, we demonstrate that this network results in a time-dependent functional response to doxorubicin, a frequently used anti-cancer drug with known cardiotoxic side effects. Taken together, our data provide a framework from which the effect of oscillatory gene expression on cardiomyocyte physiology can be modeled in vitro, and demonstrate the influence of a functional clock on experimental outcome.

Identity and dynamics of mammary stem cells during branching morphogenesis.

During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate with near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term.

A Single-Cell Transcriptome Atlas of the Human Pancreas.

To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data.

Adult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. Knowing the identity of the stem cell is crucial to understanding tissue homeostasis and its aberrations upon disease. Here we present a computational method for the derivation of a lineage tree from single-cell transcriptome data. By exploiting the tree topology and the transcriptome composition, we establish StemID, an algorithm for identifying stem cells among all detectable cell types within a population. We demonstrate that StemID recovers two known adult stem cell populations, Lgr5+ cells in the small intestine and hematopoietic stem cells in the bone marrow. We apply StemID to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. We hope that StemID will accelerate the search for novel stem cells by providing concrete markers for biological follow-up and validation.

Concise review: the dynamics of induced pluripotency and its behavior captured in gene network motifs.

The flexibility of cellular identity is clearly demonstrated by the possibility to reprogram fully differentiated somatic cells to induced pluripotent stem (iPS) cells through forced expression of a set of transcription factors. The generation of iPS cells is of great interest as they provide a tremendous potential for regenerative medicine and an attractive platform to investigate pluripotency. Despite having gathered much attention, the molecular details and responsible gene regulatory networks during the reprogramming process are largely unresolved. In this review, we analyze the sequence and dynamics of reprogramming to construct a timeline of the molecular events taking place during induced pluripotency. We use this timeline as a road map to explore the distinct phases of the reprogramming process and to suggest gene network motifs that are able to describe its systems behavior. We conclude that the gene networks involved in reprogramming comprise several feedforward loops combined with autoregulatory behavior and one or more AND gate motifs that can explain the observed dynamics of induced pluripotency. Our proposed timeline and derived gene network motif behavior could serve as a tool to understand the systems behavior of reprogramming and identify key transitions and/or transcription factors that influence somatic cell reprogramming. Such a systems biology strategy could provide ways to define and explore the use of additional regulatory factors acting at defined gene network motifs to potentially overcome the current challenges of inefficient, slow, and partial somatic cell reprogramming and hence set ground of using iPS cells for clinical and therapeutic use.