Developmental self-reactivity determines pathogenic Tc17 differentiation potential of naive CD8+ T cells in murine models of inflammation.

The differentiation of naive CD8+ T cells into effector cells is important for establishing immunity. However, the effect of heterogeneous naive CD8+ T cell populations is not fully understood. Here, we demonstrate that steady-state naive CD8+ T cells are composed of functionally heterogeneous subpopulations that differ in their ability to differentiate into type 17 cytotoxic effector cells (Tc17) in a context of murine inflammatory disease models, such as inflammatory bowel disease and graft-versus-host disease. The differential ability of Tc17 differentiation is not related to T-cell receptor (TCR) diversity and antigen specificity but is inversely correlated with self-reactivity acquired during development. Mechanistically, this phenomenon is linked to differential levels of intrinsic TCR sensitivity and basal Suppressor of Mothers Against Decapentaplegic 3 (SMAD3) expression, generating a wide spectrum of Tc17 differentiation potential within naive CD8+ T cell populations. These findings suggest that developmental self-reactivity can determine the fate of naive CD8+ T cells to generate functionally distinct effector populations and achieve immense diversity and complexity in antigen-specific T-cell immune responses.

Identification of DNA methylation biomarkers for evaluating cardiovascular disease risk from epigenome profiles altered by low-dose ionizing radiation.

BACKGROUND: Environmental exposure, medical diagnostic and therapeutic applications, and industrial utilization of radionuclides have prompted a growing focus on the risks associated with low-dose radiation (< 100 mGy). Current evidence suggests that such radiation can induce epigenetic changes. Nevertheless, whether exposure to low-dose radiation can disrupt endothelial cell function at the molecular level is unclear. Because endothelial cells play crucial roles in cardiovascular health and disease, we aimed to investigate whether low-dose radiation could lead to differential DNA methylation patterns at the genomic level in endothelial cell (EC) lines. METHODS: We screened for changes in DNA methylation patterns in primary human aortic (HAECs) and coronary artery endothelial cells following exposure to low-dose ionizing radiation. Using a subset of genes altered via DNA methylation by low-dose irradiation, we performed gene ontology (GO) analysis to predict the possible biological network mediating the effect of low-dose radiation. In addition, we performed comprehensive validation using methylation and gene expression analyses, and ChIP assay to identify useful biomarkers among candidate genes for use in detecting low-dose radiation exposure in human primary normal ECs. RESULTS: Low-dose radiation is sufficient to induce global DNA methylation alterations in normal EC lines. GO analysis demonstrated that these hyper- or hypo-methylated genes were linked to diverse biological pathways. Our findings indicated a robust correlation between promoter hypermethylation and transcriptional downregulation of four genes (PGRMC1, UNC119B, RERE, and FNDC3B) in response to low-dose ionizing radiation in HAECs. CONCLUSIONS: Based on these findings, the identified genes can serve as potential DNA methylation biomarkers for the assessment of cardiovascular risk upon exposure to low-dose radiation.

miR-199a and miR-199b facilitate diffuse gastric cancer progression by targeting Frizzled-6.

Pathological markers that can monitor the progression of gastric cancer (GC) may facilitate the diagnosis and treatment of patients with diffuse GC (DGC). To identify microRNAs (miRNAs) that can differentiate between early and advanced DGC in the gastric mucosa, miRNA expression profiling was performed using the NanoString nCounter method in human DGC tumors. Ectopic expression of miR-199a and miR-199b (miR-199a/b) in SNU601 human GC cells accelerated the growth rate, viability, and motility of cancer cells and increased the tumor volume and weight in a mouse xenograft model. To study their clinicopathological roles in patients with GC, miR-199a/b levels were measured in human GC tumor samples using in situ hybridization. High miR-199a/b expression level was associated with enhanced lymphovascular invasion, advanced T stage, and lymph-node metastasis. Using the 3'-untranslated region (UTR) luciferase assay, Frizzled-6 (FZD6) was confirmed to be a direct target of miR-199a/b in GC cells. siRNA-mediated depletion of FZD6 enhanced the motility of SNU601 cells, and addback of FZD6 restored cancer cell motility stimulated by miR-199a/b. In conclusion, miR-199a/b promotes DGC progression by targeting FZD6, implying that miR-199a/b can be used as prognostic and diagnostic biomarkers for the disease.

Integrative mapping of the dog epigenome: Reference annotation for comparative intertissue and cross-species studies.

Dogs have become a valuable model in exploring multifaceted diseases and biology relevant to human health. Despite large-scale dog genome projects producing high-quality draft references, a comprehensive annotation of functional elements is still lacking. We addressed this through integrative next-generation sequencing of transcriptomes paired with five histone marks and DNA methylome profiling across 11 tissue types, deciphering the dog's epigenetic code by defining distinct chromatin states, super-enhancer, and methylome landscapes, and thus showed that these regions are associated with a wide range of biological functions and cell/tissue identity. In addition, we confirmed that the phenotype-associated variants are enriched in tissue-specific regulatory regions and, therefore, the tissue of origin of the variants can be traced. Ultimately, we delineated conserved and dynamic epigenomic changes at the tissue- and species-specific resolutions. Our study provides an epigenomic blueprint of the dog that can be used for comparative biology and medical research.

Terminal fucosylation of haptoglobin in cancer-derived exosomes during cholangiocarcinoma progression.

Background: Cholangiocarcinoma (CCA) is a silent tumor with a high mortality rate due to the difficulty of early diagnosis and prediction of recurrence even after timely surgery. Serologic cancer biomarkers have been used in clinical practice, but their low specificity and sensitivity have been problematic. In this study, we aimed to identify CCA-specific glycan epitopes that can be used for diagnosis and to elucidate the mechanisms by which glycosylation is altered with tumor progression. Methods: The serum of patients with various cancers was fractioned into membrane-bound and soluble components using serial ultracentrifugation. Lectin blotting was conducted to evaluate glycosylation. Proteins having altered glycosylation were identified using proteomic analysis and further confirmed using immunoblotting analysis. We performed HPLC, gene analysis, real-time cargo tracking, and immunohistochemistry to determine the origin of CCA glycosylation and its underlying mechanisms. Extracellular vesicles (EV) were isolated from the sera of 62 patients with CCA at different clinical stages and inflammatory conditions and used for glycan analysis to assess their clinical significance. Results: The results reveal that glycosylation patterns between soluble and membrane-bound fractions differ significantly even when obtained from the same donor. Notably, glycans with α1-3/4 fucose and ß1-6GlcNAc branched structures increase specifically in membrane-bound fractions of CCA. Mechanically, it is primarily due to ß-haptoglobin (ß-Hp) originating from CCA expressing fucosyltransferase-3/4 (FUT 3/4) and N-acetylglucosaminyltransferase-V (MGAT5). Newly synthesized ß-Hp is loaded into EVs in early endosomes via a KFERQ-like motif and then secreted from CCA cells to induce tumor progression. In contrast, ß-Hp expressed by hepatocytes is secreted in a soluble form that does not affect CCA progression. Moreover, evaluation of EV glycosylation in CCA patients shows that fucosylation level of EV-Hp gradually increases with tumor progression and decreases markedly when the tumors are eliminated by surgery. Conclusion: This study suggests that terminal fucosylation of Hp in cancer-derived exosomes can be a novel glycan marker for diagnosis and prognosis of CCA. These findings highlight the potential of glycan analysis in different fractions of serum for biomarker discover for other diseases. Further research is needed to understand the role of fucosylated EVs on CCA progression.

RNA Editing Enzyme ADAR1 Suppresses the Mobility of Cancer Cells via ARPIN.

Deamination of adenine or cytosine in RNA, called RNA editing, is a constitutively active and common modification. The primary role of RNA editing is tagging RNA right after its synthesis so that the endogenous RNA is recognized as self and distinguished from exogenous RNA, such as viral RNA. In addition to this primary function, the direct or indirect effects on gene expression can be utilized in cancer where a high level of RNA editing activity persists. This report identified actin-related protein 2/3 complex inhibitor (ARPIN) as a target of ADAR1 in breast cancer cells. Our comparative RNA sequencing analysis in MCF7 cells revealed that the expression of ARPIN was decreased upon ADAR1 depletion with altered editing on its 3'UTR. However, the expression changes of ARPIN were not dependent on 3'UTR editing but relied on three microRNAs acting on ARPIN. As a result, we found that the migration and invasion of cancer cells were profoundly increased by ADAR1 depletion, and this cellular phenotype was reversed by the exogenous ARPIN expression. Altogether, our data suggest that ADAR1 suppresses breast cancer cell mobility via the upregulation of ARPIN.

Photobiomodulation improves the synapses and cognitive function and ameliorates epileptic seizure by inhibiting downregulation of Nlgn3.

BACKGROUND: Temporal lobe epilepsy (TLE) remains one of the most drug-resistant focal epilepsies. Glutamate excitotoxicity and neuroinflammation which leads to loss of synaptic proteins and neuronal death appear to represent a pathogen that characterizes the neurobiology of TLE. Photobiomodulation (PBM) is a rapidly growing therapy for the attenuation of neuronal degeneration harboring non-invasiveness benefits. However, the detailed effects of PBM on excitotoxicity or neuroinflammation remain unclear. We investigated whether tPBM exerts neuroprotective effects on hippocampal neurons in epilepsy mouse model by regulating synapse and synapse-related genes. METHODS: In an in vitro study, we performed imaging analysis and western blot in primary hippocampal neurons from embryonic (E17) rat pups. In an in vivo study, RNA sequencing was performed to identify the gene regulatory by PBM. Histological stain and immunohistochemistry analyses were used to assess synaptic connections, neuroinflammation and neuronal survival. Behavioral tests were used to evaluate the effects of PBM on cognitive functions. RESULTS: PBM was upregulated synaptic connections in an in vitro. In addition, it was confirmed that transcranial PBM reduced synaptic degeneration, neuronal apoptosis, and neuroinflammation in an in vivo. These effects of PBM were supported by RNA sequencing results showing the relation of PBM with gene regulatory networks of neuronal functions. Specifically, Nlgn3 showed increase after PBM and silencing the Nlgn3 reversed the positive effect of PBM in in vitro. Lastly, behavioral alterations including hypoactivity, anxiety and impaired memory were recovered along with the reduction of seizure score in PBM-treated mice. CONCLUSIONS: Our findings demonstrate that PBM attenuates epileptic excitotoxicity, neurodegeneration and cognitive decline induced by TLE through inhibition of the Nlgn3 gene decrease induced by excitotoxicity.

Hierarchical cell-type identifier accurately distinguishes immune-cell subtypes enabling precise profiling of tissue microenvironment with single-cell RNA-sequencing.

Single-cell RNA-seq enabled in-depth study on tissue micro-environment and immune-profiling, where a crucial step is to annotate cell identity. Immune cells play key roles in many diseases, whereas their activities are hard to track due to their diverse and highly variable nature. Existing cell-type identifiers had limited performance for this purpose. We present HiCAT, a hierarchical, marker-based cell-type identifier utilising gene set analysis for statistical scoring for given markers. It features successive identification of major-type, minor-type and subsets utilising subset markers structured in a three-level taxonomy tree. Comparison with manual annotation and pairwise match test showed HiCAT outperforms others in major- and minor-type identification. For subsets, we qualitatively evaluated the marker expression profile demonstrating that HiCAT provide the clearest immune-cell landscape. HiCAT was also used for immune-cell profiling in ulcerative colitis and discovered distinct features of the disease in macrophage and T-cell subsets that could not be identified previously.

ADAR1-dependent miR-3144-3p editing simultaneously induces MSI2 expression and suppresses SLC38A4 expression in liver cancer.

Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), has been implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are largely unknown. Our multistage hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 was the most profoundly dysregulated gene among RNA-editing enzyme family members in liver cancer. Targeted inactivation of ADAR1 inhibited the in vitro tumorigenesis of liver cancer cells. An integrative computational analyses of RNA-edited hotspots and the known editing frequency of miRNAs suggested that the miRNA miR-3144-3p was edited by ADAR1 during liver cancer progression. Specifically, ADAR1 promoted A-to-I editing of canonical miR-3144-3p to replace the adenosine at Position 3 in the seed region with a guanine (ED_miR-3144-3p(3_A < G)) in liver cancer cells. We then demonstrated that Musashi RNA-binding protein 2 (MSI2) was a specific target of miR-3144-3p and that MSI2 overexpression was due to excessive ADAR1-dependent over-editing of canonical miR-3144-3p in liver cancer. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as a specific gene target of ED_miR-3144-3p(3_A < G). The ectopic expression of both ADAR1 and the ED_miR-3144-3p(3_A < G) mimic enhanced mitotic activities, and ADAR1 suppressed SLC38A4 expression in liver cancer cells. Treatments with mouse-specific ADAR1-, MSI2-siRNA-, or SLC38A4-expressing plasmids suppressed tumorigenesis and tumor growth in a mouse model of spontaneous liver cancer. Our findings suggest that the aberrant regulation of ADAR1 augments oncogenic MSI2 effects by excessively editing canonical miR-3144-3p and that the resultant ED_miR-3144-3p(3_A < G) simultaneously suppresses tumor suppressor SLC38A4 expression, contributing to hepatocellular carcinogenesis.

Hypomethylation in MTNR1B: a novel epigenetic marker for atherosclerosis profiling using stenosis radiophenotype and blood inflammatory cells.

BACKGROUND: Changes in gene-specific promoter methylation may result from aging and environmental influences. Atherosclerosis is associated with aging and environmental effects. Thus, promoter methylation profiling may be used as an epigenetic tool to evaluate the impact of aging and the environment on atherosclerosis development. However, gene-specific methylation changes are currently inadequate epigenetic markers for predicting atherosclerosis and cardiovascular disease pathogenesis. RESULTS: We profiled and validated changes in gene-specific promoter methylation associated with atherosclerosis using stenosis radiophenotypes of cranial vessels and blood inflammatory cells rather than direct sampling of atherosclerotic plaques. First, we profiled gene-specific promoter methylation changes using digital restriction enzyme analysis of methylation (DREAM) sequencing in peripheral blood mononuclear cells from eight samples each of cranial vessels with and without severe-stenosis radiophenotypes. Using DREAM sequencing profiling, 11 tags were detected in the promoter regions of the ACVR1C, ADCK5, EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PACSIN3, PAX8-AS1, TLDC1, and ZNF7 genes. Using methylation evaluation, we found that EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PAX8-AS1, and TLDC1 showed > 5% promoter methylation in non-plaque intima, atherosclerotic vascular tissues, and buffy coats. Using logistic regression analysis, we identified hypomethylation of MTNR1B as an independent variable for the stenosis radiophenotype prediction model by combining it with traditional atherosclerosis risk factors including age, hypertension history, and increases in creatinine, lipoprotein (a), and homocysteine. We performed fivefold cross-validation of the prediction model using 384 patients with ischemic stroke (50 [13%] no-stenosis and 334 [87%] > 1 stenosis radiophenotype). For the cross-validation, the training dataset included 70% of the dataset. The prediction model showed an accuracy of 0.887, specificity to predict stenosis radiophenotype of 0.940, sensitivity to predict no-stenosis radiophenotype of 0.533, and area under receiver operating characteristic curve of 0.877 to predict stenosis radiophenotype from the test dataset including 30% of the dataset. CONCLUSIONS: We identified and validated MTNR1B hypomethylation as an epigenetic marker to predict cranial vessel atherosclerosis using stenosis radiophenotypes and blood inflammatory cells rather than direct atherosclerotic plaque sampling.

RANKL-responsive epigenetic mechanism reprograms macrophages into bone-resorbing osteoclasts.

Monocyte/macrophage lineage cells are highly plastic and can differentiate into various cells under different environmental stimuli. Bone-resorbing osteoclasts are derived from the monocyte/macrophage lineage in response to receptor activator of NF-κB ligand (RANKL). However, the epigenetic signature contributing to the fate commitment of monocyte/macrophage lineage differentiation into human osteoclasts is largely unknown. In this study, we identified RANKL-responsive human osteoclast-specific superenhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) by integrating data obtained from ChIP-seq, ATAC-seq, nuclear RNA-seq and PRO-seq analyses. RANKL induced the formation of 200 SEs, which are large clusters of enhancers, while suppressing 148 SEs in macrophages. RANKL-responsive SEs were strongly correlated with genes in the osteoclastogenic program and were selectively increased in human osteoclasts but marginally presented in osteoblasts, CD4+ T cells, and CD34+ cells. In addition to the major transcription factors identified in osteoclasts, we found that BATF binding motifs were highly enriched in RANKL-responsive SEs. The depletion of BATF1/3 inhibited RANKL-induced osteoclast differentiation. Furthermore, we found increased chromatin accessibility in SE regions, where RNA polymerase II was significantly recruited to induce the extragenic transcription of SE-eRNAs, in human osteoclasts. Knocking down SE-eRNAs in the vicinity of the NFATc1 gene diminished the expression of NFATc1, a major regulator of osteoclasts, and osteoclast differentiation. Inhibiting BET proteins suppressed the formation of some RANKL-responsive SEs and NFATc1-associated SEs, and the expression of SE-eRNA:NFATc1. Moreover, SE-eRNA:NFATc1 was highly expressed in the synovial macrophages of rheumatoid arthritis patients exhibiting high-osteoclastogenic potential. Our genome-wide analysis revealed RANKL-inducible SEs and SE-eRNAs as osteoclast-specific signatures, which may contribute to the development of osteoclast-specific therapeutic interventions.

Esr1 but Not CYP19A1 Overexpression in Mammary Epithelial Cells during Reproductive Senescence Induces Pregnancy-Like Proliferative Mammary Disease Responsive to Anti-Hormonals.

Molecular-level analyses of breast carcinogenesis benefit from vivo disease models. Estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) overexpression targeted to mammary epithelial cells in genetically engineered mouse models induces largely similar rates of proliferative mammary disease in prereproductive senescent mice. Herein, with natural reproductive senescence, Esr1 overexpression compared with CYP19A1 overexpression resulted in significantly higher rates of preneoplasia and cancer. Before reproductive senescence, Esr1, but not CYP19A1, overexpressing mice are tamoxifen resistant. However, during reproductive senescence, Esr1 mice exhibited responsiveness. Both Esr1 and CYP19A1 are responsive to letrozole before and after reproductive senescence. Gene Set Enrichment Analyses of RNA-sequencing data sets showed that higher disease rates in Esr1 mice were accompanied by significantly higher expression of cell proliferation genes, including members of prognostic platforms for women with early-stage hormone receptor-positive disease. Tamoxifen and letrozole exposure induced down-regulation of these genes and resolved differences between the two models. Both Esr1 and CYP19A1 overexpression induced abnormal developmental patterns of pregnancy-like gene expression. This resolved with progression through reproductive senescence in CYP19A1 mice, but was more persistent in Esr1 mice, resolving only with tamoxifen and letrozole exposure. In summary, genetically engineered mouse models of Esr1 and CYP19A1 overexpression revealed a diversion of disease processes resulting from the two distinct molecular pathophysiological mammary gland-targeted intrusions into estrogen signaling during reproductive senescence.

Overexpression of Estrogen Receptor α in Mammary Glands of Aging Mice Is Associated with a Proliferative Risk Signature and Generation of Estrogen Receptor α-Positive Mammary Adenocarcinomas.

Age is a risk factor for human estrogen receptor-positive breast cancer, with highest prevalence following menopause. While transcriptome risk profiling is available for human breast cancers, it is not yet developed for prognostication for primary or secondary breast cancer development utilizing at-risk breast tissue. Both estrogen receptor α (ER) and aromatase overexpression have been linked to human breast cancer. Herein, conditional genetically engineered mouse models of estrogen receptor 1 (Esr1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) were used to show that induction of Esr1 overexpression just before or with reproductive senescence and maintained through age 30 months resulted in significantly higher prevalence of estrogen receptor-positive adenocarcinomas than CYP19A1 overexpression. All adenocarcinomas tested showed high percentages of ER+ cells. Mammary cancer development was preceded by a persistent proliferative transcriptome risk signature initiated within 1 week of transgene induction that showed parallels to the Prosigna/Prediction Analysis of Microarray 50 human prognostic signature for early-stage human ER+ breast cancer. CYP19A1 mice also developed ER+ mammary cancers, but histology was more divided between adenocarcinoma and adenosquamous, with one ER- adenocarcinoma. Results demonstrate that, like humans, generation of ER+ adenocarcinoma in mice was facilitated by aging mice past the age of reproductive senescence. Esr1 overexpression was associated with a proliferative estrogen pathway-linked signature that preceded appearance of ER+ mammary adenocarcinomas.

Activation of OSM-STAT3 Epigenetically Regulates Tumor-Promoting Transcriptional Programs in Cervical Cancer.

Despite improvements in preventative strategies, such as regular screenings with Pap tests and human papillomavirus (HPV) tests as well as HPV vaccinations, effective treatment for advanced cervical cancer remains poor. Deregulation of STAT3 is an oncogenic factor that promotes tumorigenesis and epithelial-to-mesenchymal transition (EMT) in various cancers. Oncostatin M (OSM), a pleiotropic cytokine, induces STAT3 activation, exacerbating cervical cancer. However, the mechanism by which the OSM-STAT3 axis epigenetically regulates tumor-progression-related genes in cervical cancer is not well understood. Here, we show that OSM-mediated STAT3 activation promotes pro-tumorigenic gene expression programs, with chromatin remodeling in cervical cancer. Reanalysis of scRNA-seq data performed in cervical cancer uncovered an interaction between the oncostatin M receptor (OSMR) on tumor cells and OSM induced by tumor-associated macrophages (TAMs). Our gene expression profiling (bulk RNA-seq) shows that OSM-induced genes were involved in hypoxia, wound healing, and angiogenesis, which were significantly inhibited by SD-36, a STAT3-selective degrader. Additionally, ATAC-seq experiments revealed that STAT3 binding motifs were preferentially enriched in open chromatin regions of the OSM-STAT3-regulated genes. Among the 50 candidate genes that were regulated epigenetically through the OSM-STAT3 axis, we found that the expression levels of NDRG1, HK2, PLOD2, and NPC1 were significantly correlated with those of OSMR and STAT3 in three independent cervical cancer cohorts. Also, higher expression levels of these genes are significantly associated with poor prognosis in cervical cancer patients. Collectively, our findings demonstrate that the OSM-STAT3 signaling pathway regulates crucial transcriptomic programs through epigenetic changes and that selective inhibition of STAT3 may be a novel therapeutic strategy for patients with advanced cervical cancer.

MicroRNA-769-3p Acts as a Prognostic Factor in Oral Squamous Cell Cancer by Modulating Stromal Genes.

miR-769-3p expression is suppressed in the stromal subtype of head and neck squamous cell carcinoma (HNSCC); however, its role in stromal HNSCC has not been fully elucidated. To investigate the biological relevance of miR-769-3p in the stromal phenotype, we established oral squamous cell cancer (OSCC) cell lines, namely CAL27, HSC3, and YD8, overexpressing miR-769-3p. miR-769-3p expression was positively and negatively correlated with interferon-gamma-related genes and MYC target gene sets, respectively. miR-769-3p decreased OSCC cell migration and invasion as well as mesenchymal marker expression and increased epithelial marker expression. Moreover, miR-769-3p enhanced OSCC cell sensitivity to 5-fluorouracil. High miR-769-3p expression was associated with good prognosis of HNSCC patients. Collectively, these results suggest that miR-769-3p suppression enhances stromal gene expression and promotes the epithelial-to-mesenchymal transition. Therefore, miR-769-3p may be a potential biomarker of the miRNA phenotype in OSCC patients.

Chemically-induced osteogenic cells for bone tissue engineering and disease modeling.

Cell reprogramming can satisfy the demands of obtaining specific cell types for applications such as tissue regeneration and disease modeling. Here we report the reprogramming of human fibroblasts to produce chemically-induced osteogenic cells (ciOG), and explore the potential uses of ciOG in bone repair and disease treatment. A chemical cocktail of RepSox, forskolin, and phenamil was used for osteogenic induction of fibroblasts by activation of RUNX2 expression. Following a maturation, the cells differentiated toward an osteoblast phenotype that produced mineralized nodules. Bulk and single-cell RNA sequencing identified a distinct ciOG population. ciOG formed mineralized tissue in an ectopic site of immunodeficiency mice, unlike the original fibroblasts. Osteogenic reprogramming was modulated under engineered culture substrates. When generated on a nanofiber substrate ciOG accelerated bone matrix formation in a calvarial defect, indicating that the engineered biomaterial promotes the osteogenic capacity of ciOG in vivo. Furthermore, the ciOG platform recapitulated the genetic bone diseases Proteus syndrome and osteogenesis imperfecta, allowing candidate drug testing. The reprogramming of human fibroblasts into osteogenic cells with a chemical cocktail thus provides a source of specialized cells for use in bone tissue engineering and disease modeling.

Identification and Characterization of Cancer-Associated Fibroblast Subpopulations in Lung Adenocarcinoma.

Cancer-associated fibroblasts (CAFs) reside within the tumor microenvironment, facilitating cancer progression and metastasis via direct and indirect interactions with cancer cells and other stromal cell types. CAFs are composed of heterogeneous subpopulations of activated fibroblasts, including myofibroblastic, inflammatory, and immunosuppressive CAFs. In this study, we sought to identify subpopulations of CAFs isolated from human lung adenocarcinomas and describe their transcriptomic and functional characteristics through single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatics analyses. Cell trajectory analysis of combined total and THY1 + CAFs revealed two branching points with five distinct branches. Based on Gene Ontology analysis, we denoted Branch 1 as "immunosuppressive", Branch 2 as "neoantigen presenting", Branch 4 as "myofibroblastic", and Branch 5 as "proliferative" CAFs. We selected representative branch-specific markers and measured their expression levels in total and THY1 + CAFs. We also investigated the effects of these markers on CAF activity under coculture with lung cancer cells. This study describes novel subpopulations of CAFs in lung adenocarcinoma, highlighting their potential value as therapeutic targets.

MarkerCount: A stable, count-based cell type identifier for single-cell RNA-seq experiments.

Cell type identification is a key step toward downstream analysis of single cell RNA-seq experiments. Although the primary objective is to identify known cell populations, good identifiers should also recognize unknown clusters which may represent a previously unidentified subpopulation of a known cell type or tumor cells of an unknown phenotype. Herein, we present MarkerCount, which utilizes the number of expressed markers, regardless of their expression level. MarkerCount works in both reference- and marker-based mode, where the latter utilizes existing lists of markers, while the former uses a pre-annotated dataset to find markers to be used for cell type identification. In both modes, MarkerCount first utilizes the "marker count" to identify cell populations and, after rejecting uncertain cells, reassigns cell type and/or makes corrections in cluster-basis. The performance of MarkerCount was evaluated and compared with existing identifiers, both marker- and reference-based, that can be customized using publicly available datasets and marker databases. The results show that MarkerCount performs better in the identification of known populations as well as of unknown ones, when compared to other reference- and marker-based cell type identifiers for most of the datasets analyzed.