Transcriptome Profiling Of Cells Research Articles

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with “secretory”, “contractile” and “vascular” transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Computational Design of Synthetic Optical Barcodes in Microdroplets

AbstractBarcodes are useful for identifying objects across time, space, and information modalities. However, materializing and decoding optical and multimodal barcodes on microscopic objects remains difficult despite the increasing need for multiplexed cell analysis. Here, a computational design of randomly combinatorial is presented, yet decodable barcodes in microdroplets. The design is based on a novel Real2Sim2Real framework: it first collects experimental images of optically distinct microparticles, then simulates massive combinatorial images by randomly assembling the imaged particles to train a neural network‐based decoder. It is demonstrated that the decoder, even though trained via simulation, accurately identifies the randomly assembled particles in real hydrogel microdroplets. It also shows that the microdroplets with an additional DNA barcoding functionality are applicable to individually link independently measured microscopic images and transcriptome profiles of pooled single cells.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group > ND group > HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

Defining the Drivers of Idelalisib-Related Early Autoimmune Toxicity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) patients with disease resistant to BTK and BCL-2 inhibitors can potentially be treated with PI3K inhibitors to overcome resistance and improve outcomes. PI3K inhibitors such as idelalisib and duvelisib have shown promising efficacy in CLL patients, but severe toxicities remain a significant hurdle to their implementation in the clinic (PMID: 24615777). By investigating the molecular mechanisms that lead to severe early autoimmune toxicity with idelalisib, we hope to predict or prevent toxicity, thus expanding the use of this efficacious PI3K inhibitor class of drugs. To this end, the Brown lab showed that idelalisib-induced hepatitis is autoimmune. CyTOF analysis in CLL patients with early severe toxicity in a front-line idelalisib clinical trial cohort showed a significant decrease in CD4+ T regulatory cells (Treg) with a relative shift in favor of T effectors in peripheral blood. In addition, the Brown lab showed that the Th17 pathway is more active at baseline, with a further increase in patients with toxicity (PMID: 27247136, 36732326). Since the CYTOF data is restricted to fewer mostly T-cell markers in our studies, an unbiased single cell RNA sequencing (scRNA-seq) approach is warranted to explore the full effects of idelalisib on immune cells and CLL cells in patients with and without autoimmune toxicity. The question remains what molecular mechanisms drive the idelalisib-induced decrease in the Treg population, its effect on natural killer (NK) and CD8 T-cell function, and the activation of the Th17 pathway in CLL patients with early toxicity. We therefore carried out the first systematic effort to understand the mechanisms of idelalisib toxicity in molecular detail using combined scRNA and scTCR-seq. In this study, we profile T-cell enriched PBMCs of matched pre-treatment and on-idelalisib treatment samples from CLL patients with early severe toxicity and without any toxicity (n=4 CLL patients from each group, a total of 16 samples) using 10X single cell transcriptomic and TCR profiling. Raw sequencing data files were processed using the Cell Ranger suite (version 7.1.0) from 10x Genomics. Sample identification based on SNP information was done using the Popscle suite. Seurat package (version 4.9.9.9) was used for the single cell analysis. After filtering cells with fewer than 600 features, less than 1000 UMIs, mitochondrial content higher than 15%, 51,487 cells were left for the downstream analysis. To explore transcriptional heterogeneity and perform initial cell clustering, principal component analysis and nonlinear dimensional reduction using UMAP were applied using 20 dimensions. Leiden algorithm was used for cluster identification. Cell types were identified using Azimuth with the PBMC reference dataset and confirmed with expression plots. The initial observations of the cell proportions show that baseline samples from patients with early idelalisib toxicity have fewer Tregs, NK, and CD4+ cytotoxic T (CTL) cells compared to patients without toxicity. In addition, we observed a decline in Tregs, gamma-delta (γδ) T, and CD4+ CTL cells and a concurrent increase in proliferative CD4+ and CD8+ T-cells and proliferative NK on idelalisib treatment in the early toxicity group. On the contrary, CD4+ CTL and CD4+ T effector memory (TEM) cells are enriched with idelalisib therapy in patients without toxicities. At the same time, more CD4+ T-Cell Memory (TCM), γδ T, CD8+ naive T, CD8+ TCM, and Mucosal-associated invariant T (MAIT) cells are present in pretreatment samples from patients with toxicity compared to patients without toxicity. Furthermore, proliferative T-cells are depleted with idelalisib therapy in patients without toxicity whereas they are enriched in patients with early toxicity. To further characterize idelalisib-induced depletion of Tregs, we reclustered them to identify distinct subgroups and found particular clusters of Treg were depleted in patients with early toxicity. In the non-toxicity group, more diversity of Tregs was identified with depletion of some clusters but also an increase in a cluster. At present, we are integrating the scRNA & scTCR-seq data to characterize the cellular diversity and heterogeneous phenotypic states of the T-cell populations and updated results unraveling molecular mechanisms that drive early severe toxicity in CLL patients with idelalisib treatment will be presented at the meeting.

Unrevealing Hematopoietic Reconstruction Discrepancies after Allo-UCBT and Allo-PBSCT at Single Cell Resolution

Umbilical cord blood (UCB) and G-CSF mobilizing peripheral blood stem cell (G-PBSC) are the main sources of hematopoietic stem cells for transplantation on clinic. Clinical data have shown that hematopoietic reconstruction in allogeneic cord blood transplantation (allo-UCBT) is significantly delayed compared with allogeneic peripheral blood stem cell transplantation (allo-PBSCT), suggesting that the cellular composition and reconstruction dynamics of UCB are significantly different from that of G-PBSC. Following on the heels of our recently study that found HSC/MPPs show differentiation bias to megakaryocytic-erythroid lineage and strong proliferation tendency, and defined a neutrophil progenitor population named S100A hi Neu2 which is inversely associated with the incidence of GVHD after allo-PBSCT (Yingying Huo et al, Science Immunology 2023), we next aimed to analyze the differences of hematopoietic reconstitution between allo-UCBT and allo-PBSCT and the causes of delayed hematopoietic reconstitution after allo-UCBT. In this study, we collected peripheral blood (PB) and bone marrow (BM) samples from 8 patients with bone marrow failure syndrome (BMFs) who received allo-UCBT (n=4) or allo-PBSCT (n=4) at different time points (d14, d21, d30, d60, d90, d180, d365) after transplantation. At the same time, UCB, G-PBSC and BM of normal children were collected as controls. Lin -CD34 + cells from BM, G-PBSC and UCB were separated by FACS for STRT-seq, the total nucleated cells (TNCs) of all samples were isolated for 10X transcriptome sequencing. We used 276628 hematopoietic cells to map the single cell transcriptome profiles of hematopoietic reconstruction by different transplantation methods and annotated 44 cell types. Compared with the G-PBSC, the immunogenicity of HSC/MPPs in Lin -CD34 + cells derived from UCB decreased, the proportion increased, and the differentiation bias of HSC/MPPs to monocytic and erythroid lineage was stronger, but the differentiation ability to myeloid and megakaryocytic lineage was decreased, the proportion of EBM, MEP and CMP decreased. For TNCs, the proportion of T cells and NK cells increased, and NK_CD56dim_CD16 had a stronger immune function. Compared with allo-PBSCT, the differentiation of HSC/MPPs and CMP into neutrophils in the early stage after allo-UCBT was weakened. The proportion of MEP cells decreased, and the differentiation ability of HSC/MPPs to MEP, HSC/MPPs and MEP to Mk was weakened. In summary, we found that the delayed reconstruction of neutrophils and platelets in patients with allo-UCBT are related to the nature of the graft, suggesting the importance of reasonable intervention of HSC derived from UCB to accelerate platelet implantation.

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

Single-Cell Characterization of TP53-Mutated AML Patients Treated with Frontline Azacitidine, Venetoclax, and Magrolimab Reveals Mechanisms of Response and Resistance

Background: TP53 mutations, found in around 20% of AML cases, often lead to therapy resistance and unfavorable outcomes. Recent studies hint at the potential efficacy of CD47, a ‘don't eat me’ signal, in treating TP53-mutated AML. The ongoing clinical trial (NCT04435691) is assessing the efficacy of a triple therapy combo-magrolimab (anti-CD47 mAb), azacitidine, and venetoclax (AVM)-with promising initial results in TP53-mutated AML patients. However, understanding resistance and relapse remains crucial. Methods: We performed paired 5' scRNA and scTCR profiling on 3 healthy donors and 27 samples from 11 treatment-naive TP53-mutated AML patients (median age 63, range 59-73 years; 8 responders (CR/CRi); 3 non-responders (NR)), both pre-AVM (n=11) and post-AVM-point of remission (n=11), relapse (n=2), or primary resistance (n=3). Results: 42,910 AML cells and 137,169 bone marrow microenvironment cells passed quality checks. AML cell identification was confirmed via correlation with clinical flow cytometry, inferred copy number alterations, and immunohistochemistry markers. We leveraged Symphony to phenotypically infer AML cell types based on their transcriptional similarity to normal hematopoietic cells. Overall, samples displayed heterogeneous distribution of cell types at time of diagnosis. However, the 3 samples from the 3 primary resistant patients demonstrated a marked and significant expansion of AML cell proportion exhibiting an erythroid differentiation phenotype post-AVM (Fig. A). Also, the 2 patients who relapsed 4.8- and 7.1-months following AVM demonstrated expansion of erythroid differentiation proportion (Fig. A) and higher LSC17 at time of relapse. This suggests a selection favoring erythroid clones as a mechanism of primary and acquired resistance following AVM therapy. We conducted a pathway enrichment analysis using Hallmark gene sets to identify significantly altered AML-intrinsic biological pathways between responders and non-responders. CR/CRi patient-derived AML cells exhibited an enriched inflammatory response, specifically in TNFα and IFNγ signaling pathways-known CD47 expression regulators-and increased CD47 expression (Fig. B). In contrast, AML cells from non-responsive patients showed an enrichment of oxidative phosphorylation (Fig. B). Of note, phagocytic activity of myeloid cells was high in non-responders suggesting that despite the likely activity of magrolimab, other mechanisms of primary resistance were at play. As anti-CD47-mediated phagocytosis can stimulate antigen presentation and trigger T cell activation, we delved into the T cell subtypes and their corresponding cellular responses. There were not significant differences in the distribution of CD4 and CD8 pretreatment in CR/CRi versus NR patients. However, CD8 dysfunction score was significantly higher in non-responders at baseline. Following therapy, the CD8 dysfunction score was also significantly higher at time of relapse among responders. Further, effector CD8 T cells of NR patients had enrichment for cytotoxicity and TCR signaling pathways, and higher expression of exhaustion-related genes. Conclusions: Through single cell transcriptomic profiling of TP53-mutated AML patients treated with the triplet combination of AVM on protocol we identified erythroid differentiation and oxidative phosphorylation as possible intrinsic resistance mechanisms. Additionally, non-responders exhibited a dysfunctional T cell state prior to treatment, while relapses were associated with development of CD8 dysfunction. Overall, this data provides valuable insight into the mechanisms of response and resistance to magrolimab-based therapy. TCR repertoire and AML-immune cell interaction analyses are ongoing.

Characterizing Inferred Functional and Phenotypical States of Newly Diagnosed Acute Myeloid Leukemia from Single Cell Transcriptomic Profiling

Background: Acute myeloid leukemia (AML) is a heterogeneous disease, marked by leukemia stem cells (LSCs), which significantly contribute to treatment resistance and dismal prognosis. While appreciable efforts have been made to study the diversity of functional and phenotypic states of LSCs, integrating these states from inferred single cell profiling has not been explored. Moreover, the spatial distribution and variability of hierarchical leukemic states within diverse cytogenetic groups, and their potential connection to transcriptional profiles, remain understudied, highlighting a gap in our understanding of this complex disease. Methods: To investigate the heterogeneity across different cytogenetics,we performed single cell RNA profiling (scRNA) of bone marrow (BM) mononulear cells from 20 newly diagnosed adult AML patients with diploid (n=7), del5/5q (n=5), del7/7q (n=5) and double deletion (del5/5q & del7/7q; n=3). A total of 111,130 cells passed quality assessment, of which 56,168 (50.5%) cells were identified as AML cells by integrating flow cytometry, immunohistochemistry, copy number variation, and FISH with the scRNA expression profiles. Functional states of AML cells were predicted by a recently proposed hierarchy of AML LSCs representing distinct maturation states (Zeng et al Nat Med 2022). Phenotypical states were annotated by Symphony using a healthy BM and validated in newly sequenced healthy BM samples. We also applied deconvolution scRNA analysis with spatial GeoMx profiling to identify spatial location of primitive versus differentiated AML cells. Results: Differential gene expression and gene set enrichment analysis (GSEA) revealed transcriptional and biological diversity across cytogenetic groups. Del7/7q cells showed high expression of human leukocyte antigen (HLA) genes and CD74, consistent with enrichment for a inflammatory phenotype revealed by GSEA. Del5/5q group demonstrated enrichment in proliferative pathways and heat shock proteins. Some diploid cells showed high expression of monocytic markers, while the others were highly expressing GMP markers, indicating the co-existence of different subclones. Differentiation states of leukemic cells are reflected in the hierarchy of cellular compositions. There is a diverse spectrum of cell types within each cytogenetic group and each patient. However, AML cells from diploid patients were enriched for GMP-like and monocyte-like cells, while phenotypically HSC-like cells showed an incremental trend in the sequence of diploid, del5/5q, del7/7q, and double deletion (Figure 1A). The inferred functional LSC states in our data also showed diverse distribution across cytogenetic groups, with nondiploid cells harboring more LSC. We also evaluated the biological differences of cytogenetic groups within each phenotypical and functional subset. Our data revealed consistent upregulation of proliferation in del5/5q and inflammation in del7/7q with double deletion preserving both features. However, diploid cells were downregulated in almost all. These data suggest cytogenetics as a major driver of ITH even for phenotypically or functionally similar AML cells. By correlating phenotypical axis with LSC states, we found a shared transcriptional program in LSC-like states across phenotypical axis except for Mono-like and DC-like cells, demonstrating the broad distribution of inferred functional LSC spanning the phenotypical axis from primitive to differentiated cells (Figure 1B). This was also observed in other LSC signatures, suggesting that AML cells with LSC capacity exist across the phenotypic of HSC-like cells to more phenotypically differentiated cells. We then performed spatial deconvolution using GeoMx of CD34 + or CD68 + myeloid areas of interest using the phenotypical signature. GeoMx revealed that primitive cells were more likely to be proximal to bone, while more differentiated states were enriched in distal region from the bone, indicating a spatial migration at time of differentiation. Conclusions: ScRNA profiling of newly diagnosed AML revealed the existence of heterogeneous LSC functional states underlying varying degrees of differentiation. We identified cytogenetics as a major source of ITH. Also, primitive cells were likely to localize to bone regions. These findings provide new insights in developing specific therapeutic strategies to improve clinical outcomes.

Deconvoluting Clonal and Cellular Architecture in IDH-Mutant Acute Myeloid Leukemia

Introduction Mutations in Isocitrate dehydrogenase 1/2 ( IDH) occur in 20% of acute myeloid leukemias (AML) and frequently co-occur with DNMT3A, NPM1, SRSF2, and NRAS. In clonal hematopoiesis (CH), IDH mutations confer high risk of AML transformation. Recently developed methods linking single cell genotype to single cell transcriptomic profiles and cell lineage offer the opportunity to define the transcriptomic consequences of each mutation across leukemic cell lineages and clonal phylogenies. Here, we develop an integrative single cell laboratory and analytical framework to study the genetic, clonal and cellular heterogeneity of IDH mutated CH and AML, across disease stages (diagnosis and relapse) and deliver a high-resolution map of IDH-mutant AML. Methods Bone marrow or peripheral blood mononuclear samples from 15 AML patients, 2 CH individuals, and 12 unaffected age-matched donors were profiled by single cell RNA-sequencing. For the AML cases, a median of 3 samples per patient were profiled under treatment with IDH inhibitor (IDHi) monotherapy (n=5 patients), IDHi in combination with intensive chemotherapy (NCT02632708, n=6) or in combination with hypomethylating agent (n=2). This dataset comprised 259,610 cells across AML (n=190,133), CH (n=16,705) and normal controls (n=52,772). Genotyping of Transcriptomes (Nam et al., 2019) (for IDH1/2, NPM1, SRSF2, and NRAS mutations) across 6 AML patients enabled genotyping 23% of cells at diagnosis for at least one clone-defining mutation. Results Through meta-analysis of 3,653 diagnostic AML samples profiled by a targeted DNA panel (Papaemmanuil et al., 2016; Tazi et al., 2022), we identified DNMT3A, SRSF2 and NPM1 as critical co-operative mutations in IDH-mutant AML. Amongst the 15 AML patients, 6 had IDH1 mutation, 8 had IDH2 mutations, and 1 had both IDH1 and IDH2. The co-occurring mutations in DNMT3A (n=11 patients), NPM1 (n=5), SRSF2 (n=5) reflected the genomic landscape of IDH-mutant AML. At diagnosis, IDH-mutant AML was enriched in immature hematopoietic stem and progenitor-like blasts. Compared to normal hematopoiesis, IDH-mutant AML blasts upregulated inflammatory response markers including TNFɑ/NFκB-signaling and downregulated proliferation-associated pathways including G2M Checkpoint, and MYC and E2F Targets, and DNA damage response. To investigate how co-mutations modify the IDH-mutant phenotype, we established an integrative single-cell profiling approach to assign single cells to distinct phylogenetic subclones and extract clone-specific transcriptional programs. SRSF2 and NPM1 co-mutations modulated the AML blast phenotype, towards erythroid ( SRSF2) or granulomonocytic lineage ( NPM1). NRAS- or SRSF2-mutant subclones defined distinct gene expression-derived clusters and upregulated proliferation and metabolic pathways. Furthermore, NPM1 co-mutated blasts upregulated KMT2A-target genes. Next, we tracked clonal responses to combination IDHi and chemotherapy using GoT and targeted DNA sequencing. In 4 cases, the ancestral clone, defined by IDH and DNMT3A mutations, retained clonal dominance in both mature and immature myeloid compartments at complete response (median 4 weeks). Conversely, subclones defined by mutations in NPM1, NRAS, or SRSF2, were either reduced or not detectable (limit of detection 2%). In two cases, mutations were not detected after therapy. In patients who relapsed after IDHi monotherapy treatment, blasts upregulated stemness, fatty acid metabolism, OxPHOS, and inflammation at relapse compared to diagnosis. Furthermore, MHC Class II was downregulated at relapse, pointing to immune evasion of AML as a potential relapse mechanism. Finally, we investigated the role of IDH2 mutations at CH. IDH-mutant CH-derived monocytes upregulated inflammation-associated pathways, mirroring the phenotypes observed in IDH-mutant AML at diagnosis. Conclusion We defined how IDH mutations and their frequent co-mutations dysregulated hematopoiesis. We leveraged state-of-the-art single cell techniques to assign leukemic cells to phylogenetic subclones and delineated the role of serially acquired co-mutations in shaping lineage bias and transcriptional programs. Furthermore, we defined genetic, clonal and transcriptional changes at three stages of the AML disease process: diagnosis, remission, and relapse under IDH inhibitor therapy.

CNSC-25. NEURAL SIGNATURE OF GBM EXHIBITS SYNAPTOGENIC AND OPC-LIKE FEATURES AND INDEPENDENTLY PREDICTS PATIENT SURVIVAL

Abstract Interactions of neural cells with glioma cells drive tumor growth. In this study, we identified an epigenetically-defined, malignant neural signature of IDH-wildtype glioblastoma (GBM) that significantly influenced patients’ outcome. We used DNA methylation-based reference signatures of neural cells to deconvolve 1.058 primary patient GBM samples. Samples were classified based on the neural reference signature and annotated as low or high neural GBM. A high neural GBM was linked to hypomethylation of CpG sites associated with invasiveness, neuron-to-glioma-interactions, and transsynaptic signaling. Through spatially resolved, single cell transcriptomic and proteomic profiling, we discovered a correlation between the high neural signature and the upregulation of early development programs in OPC and NPC-like cells. These traits were accompanied by diminished immune infiltration and immunological response, further highlighting the distinctive neural-like nature of GBM. High neural GBM demonstrated increased connectivity as evidenced by magnetoencephalography and resting state magnet resonance imaging. Spatiotemporal tumor sampling showed a homogeneous and stable neural signature in newly diagnosed and recurrent GBM. In newly diagnosed GBM, a high neural signature correlated with decreased survival in 306 patients (median OS 15.0 versus 20.0 months, p< 0.001). Patient-derived xenograft models corroborated the aggressive nature of high neural GBM, showing a significantly reduced median OS of 58 days compared to 73 days (p< 0.01). These high neural GBM xenografts also exhibited an elevated tumor burden and an increase in neuron-glioma synapse formation. Consideration of the extent of resection showed a dependency on the neural subgroup as high neural GBM had an increased benefit from a complete contrast enhancing tumor removal when compared to low neural GBM. As a preoperative biomarker, the concentration of BDNF was increased in serum of high neural GBM patients (p < 0.01). Overall, we present an epigenetically-defined malignant neural signature in GBM that is associated with an unfavorable patient survival.

MODL-50. CLONAL EXPANSION OF SPONTANEOUS AND INJECTED TUMORS IN SYNGENEIC MOUSE MODELS OF GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) tumors are highly heterogenous and plastic. To better understand how tumors respond to treatment, it is important to use immunocompetent mouse models. We've created various mouse GBM models with common genetic mutations found in different subtypes of GBM, including classical, proneural, mesenchymal, and BRAF-driven. METHODS Using a combination of CRISPR and PiggyBac Transposon constructs, we injected plasmid mixtures into the ventricles of the mouse brain during the post-natal period. Electroporation was then used to enable the plasmids to enter nearby neural stem cells, which led to the spontaneous formation of tumors (spontaneous model). We collected tumor cells from these models and injected them into the adult mouse brain to create the injected mouse GBM model. Here we use spatial and single cell transcriptome profiling approaches to characterize these models at endpoint. Spatial transcriptome data was collected using the Visium 10X platform and tumor spots were deconvoluted from non-malignant spots using consensus non-negative matrix factorization. Copy number alterations (CNAs) were inferred using non-malignant spots and single-cell clusters as a reference. RESULTS We found that copy number states are largely homogenous within each sample, supporting an early population expansion in vivo. CNAs found in spontaneous tumors are also found in the matched injected tumors, which in turn harbour additional CNAs, suggesting that further selection occurs in vitro. Some of the CNAs include relevant cancer genes, suggesting the observed clonal expansions are potentially driven by additional somatic alterations that cooperate with the engineered driver mutations. Studying these models using orthogonal methods will allow us to discover alterations that synergize with the original drivers and delineate principles of GBM initiation and progression in an immune-competent environment.

Integrative single-cell RNA-seq analysis of vascularized cerebral organoids

BackgroundCerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids.ResultsWe integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells.ConclusionsThis study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.

Abstract 16780: Single Cell Profiles of Human Heart Failure and Myocardial Diseases

Background: Heart failure represents the end-stage of a range of diseases which adversely impact the heart muscle. The extent by which cellular and molecular mechanisms differ between myocardial diseases in heart failure remains incompletely understood. Methods: We performed isolation and droplet-based RNA-sequencing of 394 247 single nuclei from 103 explanted hearts from transplant and ventricular assist device recipients and 7 unused donor hearts without heart disease. After sample and nuclei level quality control, doublet removal and sample integration; principal component analysis and shared nearest neighbor clustering were used to identify distinct cell populations. Cell type composition and transcriptomic profiles were compared across donor hearts and 10 myocardial diseases (hypertrophic, dilated, ischemic, restrictive, arrythmogenic and non-compaction cardiomyopathy; rheumatic and congenital heart disease; cardiac amyloidosis and graft rejection; n=1-45/group). Results: We identified and manually annotated 14 distinct cell populations that were present across controls and myocardial diseases, including transcriptionally diverse subgroups of cardiomyocytes, endothelial cells and pericytes. Compared to controls, we observed disease-specific differences in cell populations with regards to composition, heterogeneity, and specific transcriptional profiles. Conclusions: Our results reveal differences in single cell profiles of individual heart muscle diseases compared to controls without heart disease.

Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum

Toxoplasma gondii and Neospora caninum are two major apicomplexan protozoan parasites with heteroxenous life cycles and worldwide distributions. The transplacental transmission of N. caninum causes bovine abortion, which resulting in serious economic losses to the dairy industry. Although T. gondii was also reported to cause abortions in pregnant woman and small ruminants, scarce cases about the symptom to the host cattle and the causality remains unknown. In this study, transcriptome analysis of Madin Darby bovine kidney (MDBK) cells infected with T. gondii and N. caninum was performed to uncover the differences in susceptibility of cattle to the two parasites. The results showed that 256 and 2225 differentially expressed genes (DEGs) were detected in cells infected with N. caninum and T. gondii, respectively. Moreover, significant biological differences were revealed by the functional analysis including GO and KEGG enrichment. One serpin peptidase inhibitor (SEPRINA14), which is associated with immunosuppression during pregnancy, was found to significantly decrease in cells infected with N. caninum and increase in cells infected with T. gondii-infected cells. Pattern recognition receptors TLR3 and NOD2 were also significantly upregulated in N. caninum-infected MDBK cells, but not in T. gondii. They could induce an increased inflammatory response leading to severe tissue damage. In addition, the interleukin 12 receptor subunit beta 2 (IL12β2), which plays an essential role in Th1 and Th2 cell differentiation and inflammatory bowel disease, was also markedly upregulated in the N. caninum infected cells, which led to an imbalance in the Th1 and Th2 cells by promoting the Th1 cellular response. Altogether, our findings recognized a new understanding on the differences between T. gondii and N. caninum infection of MDBK cells, where SEPRINA14, TLR3, NOD2, and IL12β2 may be the key genes that affect the difference in susceptibility of cattle to T. gondii and N. caninum, especially in pregnant animals. This study provides more clues as to why N. caninum is more likely to cause abortions in cattle.

Stratification of Multiple Myeloma Patients in the Commpass Study By Single Cell Transcriptomics of Plasma Cells Associates with Clinical Outcome

Introduction: There is substantial variation in the prognosis and response to therapy among multiple myeloma (MM) patients. This diversity is influenced by both intrinsic tumor features and the tumor microenvironment. We hypothesize that the clinical outcomes post IMiD and/or PI are linked to molecular subtypes dictated by tumor sample transcriptomics. We previously showed that molecular subtyping based on the CoMMpass bulk RNAseq data has an association with time to second-line therapy (log rank p = 0.0018; Hamidi et al., Blood 140: 7208-7210). Here, we applied the same molecular subtyping schema to single cell RNAseq isolated plasma cell transcriptomic profiles. Methods: Bone marrow biopsies from newly diagnosed MM patients (N = 200) in the MMRF CoMMpass study (NCT01454297) were collected, enriched by CD138 negative bead selection, and scRNAseq was performed by MMRF Immune Profiling Consortium, a collaboration between MMRF and laboratories at Beth Israel Deaconess Medical Center, Icahn School of Medicine at Mt. Sinai, Mayo Clinic, Washington University in St. Louis, and Emory University. After quality control and unsupervised clustering, plasma cells were subset out and pseudo-bulk expression profiles were generated for baseline samples with more than 100 plasma cells (N = 95). Unsupervised machine learning approach based on consensus non-negative matrix factorization (cNMF) was applied to identify the transcriptomics-based molecular subtypes. A random forest model, built from scRNAseq-based molecular subtypes (scNMF), was applied to bulk RNAseq data with a larger sample size (N = 754) to train a predictive molecular subtyping (pNMF). Potential cell-cell communication events based on scRNAseq were also predicted by LIgand-receptor ANalysis frAmework (LIANA). Results: 705,959 single cells passed the quality control and were broadly annotated as T/NK cells, B cells, plasma cells, myeloid cells, erythroid, progenitor, and stromal cells. Consistent with previous reports, CD138 negative bead selection did not completely remove plasma cells, resulting in an average of 18.5% plasma cells (n = 130,213). Subsequent unsupervised clustering of plasma cells exhibited a patient specific clustering pattern, suggestive of the heterogeneity of these tumor-derived cells. Unsupervised NMF on the pseudo-bulk expressions of baseline plasma cells identified five patient subsets. In the same cohort of patients (N = 95), subtypes assigned by the single cell had a better association with time to second-line therapy ( p-value = 0.0027 in scNMF as compared to p = 0.13 in bulk NMF). The median time to 2nd line of 2,443 days in scNMF5.1 versus 585 days in scNMF5.4; and the median time to 2nd line of 1,597 days in NMF5.2 versus 850 days in NMF5.3. A predicted subtyping based on scRNAseq data (pNMF) had a significantly better association with time to second-line therapy (Figure 1A, log rank p = 0.00088), compared to the published molecular subtyping based on bulk RNAseq (log rank p = 0.0018). The median time to 2nd line of 1,342 days in pNMF5.1 versus 597 days in pNMF5.5; compared to the median time to 2nd line of 1,347 days in NMF5.2 versus 850 days in NMF5.3. The best clinical outcome subset pNMF5.1 was characterized with higher levels of IFN pathways (Figure 1B, Hallmark interferon gamma and alpha pathways Wilcoxon p = 0.003 and p = 0.010, respectively) and higher levels of tumor-promoting inflammatory microenvironment (Hallmarks inflammatory response and TNFα signaling via NF-κB p = 0.003 and p = 0.010, respectively). The worst outcome subset pNMF5.5, on the other hand, displayed higher levels of glycolysis, a promoting factor of tumor development ( p < 0.001). Moreover, the total number of cell-cell interactions between the major cell types differed based on scRNAseq subtypes, informing the underlying biology driving the molecular heterogeneity. We identified a positive feedback loop of CXCL12/CXCR4 signaling between stromal and plasma cells, enhancing tumor cell proliferation. Conclusions: The identification of molecular subtypes based on single-cell data through an innovative unsupervised machine learning approach can have significant implications for determining which NDMM patients are likely to respond to established therapies; and the cellular level resolution allows deconvolution of cell-cell signaling, enhancing our understanding of the underlying molecular biology and mechanisms of resistance.

Vesicular Stomatitis Virus Elicits Early Transcriptome Response in Culicoides sonorensis Cells.

Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus.

A cell transcriptomic profile provides insights into adipocytes of porcine mammary gland across development

BackgroundStudying the composition and developmental mechanisms in mammary gland is crucial for healthy growth of newborns. The mammary gland is inherently heterogeneous, and its physiological function dependents on the gene expression of multiple cell types. Most studies focused on epithelial cells, disregarding the role of neighboring adipocytes.ResultsHere, we constructed the largest transcriptomic dataset of porcine mammary gland cells thus far. The dataset captured 126,829 high-quality nuclei from physiological mammary glands across five developmental stages (d 90 of gestation, G90; d 0 after lactation, L0; d 20 after lactation, L20; 2 d post natural involution, PI2; 7 d post natural involution, PI7). Seven cell types were identified, including epithelial cells, adipocytes, endothelial cells, fibroblasts cells, immune cells, myoepithelial cells and precursor cells. Our data indicate that mammary glands at different developmental stages have distinct phenotypic and transcriptional signatures. During late gestation (G90), the differentiation and proliferation of adipocytes were inhibited. Meanwhile, partly epithelial cells were completely differentiated. Pseudo-time analysis showed that epithelial cells undergo three stages to achieve lactation, including cellular differentiation, hormone sensing, and metabolic activation. During lactation (L0 and L20), adipocytes area accounts for less than 0.5% of mammary glands. To maintain their own survival, the adipocyte exhibited a poorly differentiated state and a proliferative capacity. Epithelial cells initiate lactation upon hormonal stimulation. After fulfilling lactation mission, their undergo physiological death under high intensity lactation. Interestingly, the physiological dead cells seem to be actively cleared by immune cells via CCL21-ACKR4 pathway. This biological process may be an important mechanism for maintaining homeostasis of the mammary gland. During natural involution (PI2 and PI7), epithelial cell populations dedifferentiate into mesenchymal stem cells to maintain the lactation potential of mammary glands for the next lactation cycle.ConclusionThe molecular mechanisms of dedifferentiation, proliferation and redifferentiation of adipocytes and epithelial cells were revealed from late pregnancy to natural involution. This cell transcriptomic profile constitutes an essential reference for future studies in the development and remodeling of the mammary gland at different stages.

A branching model of lineage differentiation underpinning the neurogenic potential of enteric glia

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.

The emerging roles of deep crypt secretory cells in colonic physiology.

Deep crypt secretory (DCS) cells are a population of epithelial cells located at the colonic crypt base that share some similarities to Paneth and goblet cells. They were initially defined as c-Kit expressing cells, though subsequent work showed that they are more specifically marked by Reg4 in the murine colon. The best-understood function of DCS cells at present is supporting the stem cell niche by generating Notch and EGF ligands. However, as these cells also express immunoregulatory (e.g., Ccl6) and host defense (e.g., Retnlb) genes, it is likely they have additional functions in maintaining colonic health outside of maintenance of the stem niche. Recent advances in single-cell transcriptomic profiling hint at additional epithelial and immune roles that may exist for these cells and have aided in elucidating their developmental lineage. This review highlights the emerging evidence supporting a crucial role for DCS cells in intestinal physiology, the current understanding of how these cells are regulated, and their potential role(s) in colonic disease.