Single-cell resolution characterization of myeloid-derived cell states with implication in cancer outcome.

Tumor-associated myeloid-derived cells (MDCs) significantly impact cancer prognosis and treatment responses due to their remarkable plasticity and tumorigenic behaviors. Here, we integrate single-cell RNA-sequencing data from different cancer types, identifying 29 MDC subpopulations within the tumor microenvironment. Our analysis reveals abnormally expanded MDC subpopulations across various tumors and distinguishes cell states that have often been grouped together, such as TREM2+ and FOLR2+ subpopulations. Using deconvolution approaches, we identify five subpopulations as independent prognostic markers, including states co-expressing TREM2 and PD-1, and FOLR2 and PDL-2. Additionally, TREM2 alone does not reliably predict cancer prognosis, as other TREM2+ macrophages show varied associations with prognosis depending on local cues. Validation in independent cohorts confirms that FOLR2-expressing macrophages correlate with poor clinical outcomes in ovarian and triple-negative breast cancers. This comprehensive MDC atlas offers valuable insights and a foundation for futher analyses, advancing strategies for treating solid cancers.

Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy.

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are an attractive model to study the relationship between signaling and cell fates. Cultured mouse ESCs can exist in multiple states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. The signaling mechanisms regulating the totipotent state and coexistence of these states are poorly understood. Here we identify bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. However, we discover that BMP's role is constrained by the cross-activation of FGF, NODAL, and WNT pathways. We exploit this finding to enhance the proportion of totipotent cells by rationally inhibiting the cross-activated pathways. Single-cell mRNA sequencing reveals that induction of the totipotent state is accompanied by suppression of primed and primitive endoderm states. Furthermore, reprogrammed totipotent cells we generate in culture resemble totipotent cells of preimplantation embryo. Our findings reveal a BMP signaling mechanism regulating both the totipotent state and heterogeneity of ESCs.

EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαß T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy.

Hypoxic, glycolytic metabolism is a vulnerability of B-acute lymphoblastic leukemia-initiating cells.

High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.

Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-Cbx2 axis.

Hematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7's control of hematopoietic maturation. We find that juvenile Cbx2-/- hematopoietic tissues show impairment of B-lymphopoiesis, a precocious adult-like myeloid bias, and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a mechanism of regulation of HSPC output via chromatin modification as a function of age with potential impact on age-biased pediatric and adult blood disorders.

CellComm infers cellular crosstalk that drives haematopoietic stem and progenitor cell development.

Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell-cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta-gonad-mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community.

Bioprinting of Adult Dorsal Root Ganglion (DRG) Neurons Using Laser-Induced Side Transfer (LIST).

Cell bioprinting technologies aim to fabricate tissuelike constructs by delivering biomaterials layer-by-layer. Bioprinted constructs can reduce the use of animals in drug development and hold promise for addressing the shortage of organs for transplants. Here, we sought to validate the feasibility of bioprinting primary adult sensory neurons using a newly developed laser-assisted cell bioprinting technology, known as Laser-Induced Side Transfer (LIST). We used dorsal root ganglion neurons (DRG; cell bodies of somatosensory neurons) to prepare our bioink. DRG-laden- droplets were printed on fibrin-coated coverslips and their viability, calcium kinetics, neuropeptides release, and neurite outgrowth were measured. The transcriptome of the neurons was sequenced. We found that LIST-printed neurons maintain high viability (Printed: 86%, Control: 87% on average) and their capacity to release neuropeptides (Printed CGRP: 130 pg/mL, Control CGRP: 146 pg/mL). In addition, LIST-printed neurons do not show differences in the expressed genes compared to control neurons. However, in printed neurons, we found compromised neurite outgrowth and lower sensitivity to the ligand of the TRPV1 channel, capsaicin. In conclusion, LIST-printed neurons maintain high viability and marginal functionality losses. Overall, this work paves the way for bioprinting functional 2D neuron assays.

Sequential regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium by Ezh1/2.

Across species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1+/- cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources.

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.

Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System.

Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.

Trajectory Algorithms to Infer Stem Cell Fate Decisions.

Single-cell trajectory analysis is an active research area in single-cell genomics aiming at developing sophisticated algorithms to reconstruct complex cell-state transition trajectories. Here, we present a step-by-step protocol to use CellRouter, a multifaceted single-cell analysis platform that integrates subpopulation identification, gene regulatory networks, and trajectory inference to precisely and flexibly reconstruct complex single-cell trajectories. Subpopulations are either user-defined or identified by a graph-clustering approach in which a k-nearest neighbor graph (kNN) is created from cell-to-cell distances in a low-dimensional embedding. Edges in this graph are weighted by network similarity metrics (e.g., Jaccard index) to robustly encode phenotypic relatedness, creating a representation of single-cell transcriptomes suitable for community detection algorithms to identify clusters of densely connected cells. This subpopulation structure represents a map of putative cell-state transitions. CellRouter implements a flow network algorithm to explore this map and reconstruct cell-state transitions in complex single-cell, multidimensional omics datasets. We describe a step-by-step application of CellRouter to hematopoietic stem and progenitor cell differentiation toward four major lineages-erythrocytes, megakaryocytes, monocytes, and granulocytes-to demonstrate key components of CellRouter for single-cell trajectory analysis.

The developmental stage of the hematopoietic niche regulates lineage in MLL-rearranged leukemia.

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

The Lin28/let-7 Pathway Regulates the Mammalian Caudal Body Axis Elongation Program.

The heterochronic genes Lin28a/b and let-7 regulate invertebrate development, but their functions in patterning the mammalian body plan remain unexplored. Here, we describe how Lin28/let-7 influence caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a strikingly increased caudal vertebrae number and tail bud cell proliferation, whereas its knockout did the opposite. Lin28a overexpression downregulated the neural marker Sox2, causing a pro-mesodermal phenotype with a decreased proportion of neural tissue relative to nascent mesoderm. Manipulating Lin28a and let-7 led to opposite effects, and manipulating Lin28a's paralog, LIN28B caused similar yet distinct phenotypes. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan. We propose that the Lin28/let-7 pathway controls the pool of caudal progenitors during tail development, promoting their self-renewal and balancing neural versus mesodermal cell fate decisions.

Reconstruction of complex single-cell trajectories using CellRouter.

A better understanding of the cell-fate transitions that occur in complex cellular ecosystems in normal development and disease could inform cell engineering efforts and lead to improved therapies. However, a major challenge is to simultaneously identify new cell states, and their transitions, to elucidate the gene expression dynamics governing cell-type diversification. Here, we present CellRouter, a multifaceted single-cell analysis platform that identifies complex cell-state transition trajectories by using flow networks to explore the subpopulation structure of multi-dimensional, single-cell omics data. We demonstrate its versatility by applying CellRouter to single-cell RNA sequencing data sets to reconstruct cell-state transition trajectories during hematopoietic stem and progenitor cell (HSPC) differentiation to the erythroid, myeloid and lymphoid lineages, as well as during re-specification of cell identity by cellular reprogramming of monocytes and B-cells to HSPCs. CellRouter opens previously undescribed paths for in-depth characterization of complex cellular ecosystems and establishment of enhanced cell engineering approaches.

TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor κB p65: Single Nucleotide Polymorphism and Estrogen Dependence.

T-cell leukemia 1A (TCL1A) single-nucleotide polymorphisms (SNPs) have been associated with aromatase inhibitor-induced musculoskeletal adverse events. We previously demonstrated that TCL1A is inducible by estradiol (E2) and plays a critical role in the regulation of cytokines, chemokines, and Toll-like receptors in a TCL1A SNP genotype and estrogen-dependent fashion. Furthermore, TCLIA SNP-dependent expression phenotypes can be "reversed" by exposure to selective estrogen receptor modulators such as 4-hydroxytamoxifen (4OH-TAM). The present study was designed to comprehensively characterize the role of TCL1A in transcriptional regulation across the genome by performing RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays with lymphoblastoid cell lines. RNA-seq identified 357 genes that were regulated in a TCL1A SNP- and E2-dependent fashion with expression patterns that were 4OH-TAM reversible. ChIP-seq for the same cells identified 57 TCL1A binding sites that could be regulated by E2 in a SNP-dependent fashion. Even more striking, nuclear factor-κB (NF-κB) p65 bound to those same DNA regions. In summary, TCL1A is a novel transcription factor with expression that is regulated in a SNP- and E2-dependent fashion-a pattern of expression that can be reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq results suggest that TCL1A also acts as a transcriptional coregulator with NF-κB p65, an important immune system transcription factor.

Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.

LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis.

A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dßh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

Interaction of tau with the RNA-Binding Protein TIA1 Regulates tau Pathophysiology and Toxicity.

Dendritic mislocalization of microtubule associated protein tau is a hallmark of tauopathies, but the role of dendritic tau is unknown. We now report that tau interacts with the RNA-binding protein (RBP) TIA1 in brain tissue, and we present the brain-protein interactome network for TIA1. Analysis of the TIA1 interactome in brain tissue from wild-type (WT) and tau knockout mice demonstrates that tau is required for normal interactions of TIA1 with proteins linked to RNA metabolism, including ribosomal proteins and RBPs. Expression studies show that tau regulates the distribution of TIA1, and tau accelerates stress granule (SG) formation. Conversely, TIA1 knockdown or knockout inhibits tau misfolding and associated toxicity in cultured hippocampal neurons, while overexpressing TIA1 induces tau misfolding and stimulates neurodegeneration. Pharmacological interventions that prevent SG formation also inhibit tau pathophysiology. These studies suggest that the pathophysiology of tauopathy requires an intimate interaction with RNA-binding proteins.