scRNA-seq for Microcephaly Research [I]: Single-Cell Droplet Encapsulation, mRNA Capture, and cDNA Synthesis.

Single-cell RNA sequencing (scRNA-seq) allows for the transcriptomic profiling of a sample tissue with single-cell resolution. The concept of scRNA-seq builds on traditional, "bulk" RNA-seq by recording and preserving the cellular origin of each transcript throughout library preparation. Here we describe an adaptation of the Drop-Seq method (Macosko et al. Cell 161, 1202-1214, 2015), in which nanoliter-scale droplets are used to physically separate dissociated cells, while a cell-specific DNA barcode is simultaneously introduced. Following barcoding, cDNAs can be mixed and pooled while retaining the identity of the cell of origin. The benefit of the Drop-Seq approach is high throughput from relatively small samples of tissue. The method described here is appropriate for processing an input of as few as 150,000 cells, with a final yield of as many as 5000 single-cell transcripts captured.

scRNA-seq for Microcephaly Research [II]: Preparation of Single-Cell cDNA Libraries.

Droplet-based single-cell RNA-seq (scRNA-seq) requires the addition of bar codes that mark the cell and transcript of origin. Cell-specific bar codes, typically added by cDNA elongation on beads, label each transcript derived from an individual cell. Transcript-specific bar codes serve as unique molecular identifiers and allow for the maintenance of transcript proportions after PCR-based amplification of cDNA. This chapter provides methods for generating bar-coded scRNA-seq libraries after droplet encapsulation.

scRNA-seq for Microcephaly Research [III]: Computational Analysis of scRNA-seq Data.

Single-cell transcriptomic analysis (scRNA-seq) can enable researchers to explore the gene expression patterns of thousands of individual cells simultaneously. Processing the complex data generated by scRNA-seq requires specialized computational tools. This chapter focuses on the analytical aspect of scRNA-seq workflow, with a focus on resolving biological signals from large-scale scRNA-seq data produced by the Drop-Seq platform.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Comprehensive multi-omics single-cell data integration reveals greater heterogeneity in the human immune system.

Single-cell transcriptomics enables the definition of diverse human immune cell types across multiple tissues and disease contexts. Further deeper biological understanding requires comprehensive integration of multiple single-cell omics (transcriptomic, proteomic, and cell-receptor repertoire). To improve the identification of diverse cell types and the accuracy of cell-type classification in multi-omics single-cell datasets, we developed SuPERR, a novel analysis workflow to increase the resolution and accuracy of clustering and allow for the discovery of previously hidden cell subsets. In addition, SuPERR accurately removes cell doublets and prevents widespread cell-type misclassification by incorporating information from cell-surface proteins and immunoglobulin transcript counts. This approach uniquely improves the identification of heterogeneous cell types and states in the human immune system, including rare subsets of antibody-secreting cells in the bone marrow.

Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.

Protracted yet Coordinated Differentiation of Long-Lived SARS-CoV-2-Specific CD8+ T Cells during Convalescence.

CD8+ T cells can potentiate long-lived immunity against COVID-19. We screened longitudinally-sampled convalescent human donors against SARS-CoV-2 tetramers and identified a participant with an immunodominant response against residues 322 to 311 of nucleocapsid (Nuc322-331), a peptide conserved in all variants of concern reported to date. We conducted 38-parameter cytometry by time of flight on tetramer-identified Nuc322-331-specific CD8+ T cells and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins, and took 32 serological measurements. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and Ab pillars of adaptive immunity. Over the approximately six month period of convalescence monitored, we observed a slow and progressive decrease in the activation state and polyfunctionality of Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph node-homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence into a state characteristic of long-lived, self-renewing memory.

Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence.

CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322-331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38-parameter CyTOF phenotyping on tetramer-identified Nuc322-331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS-CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a ~6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.

Cryptic developmental events determine medulloblastoma radiosensitivity and cellular heterogeneity without altering transcriptomic profile.

It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.

Antiapoptotic Bcl-2 family proteins BCL-xL and MCL-1 integrate neural progenitor survival and proliferation during postnatal cerebellar neurogenesis.

The tendency of brain cells to undergo apoptosis in response to exogenous events varies across neural development, with apoptotic threshold dependent on proliferation state. Proliferative neural progenitors show a low threshold for apoptosis, while terminally differentiated neurons are relatively refractory. To define the mechanisms linking proliferation and apoptotic threshold, we examined the effect of conditionally deleting Bcl2l1, the gene that codes the antiapoptotic protein BCL-xL, in cerebellar granule neuron progenitors (CGNPs), and of co-deleting Bcl2l1 homologs, antiapoptotic Mcl-1, or pro-apoptotic Bax. We found that cerebella in conditional Bcl2l1-deleted (Bcl-xLcKO) mice were severely hypoplastic due to the increased apoptosis of CGNPs and their differentiated progeny, the cerebellar granule neurons (CGNs). Apoptosis was highest as Bcl-xLcKO CGNPs exited the cell cycle to initiate differentiation, with proliferating Bcl-xLcKO CGNPs relatively less affected. Despite the overall reduction in cerebellar growth, SHH-dependent proliferation was prolonged in Bcl-xLcKO mice, as more CGNPs remained proliferative in the second postnatal week. Co-deletion of Bax rescued the Bcl-xLcKO phenotype, while co-deletion of Mcl-1 enhanced the phenotype. These findings show that CGNPs require BCL-xL to regulate BAX-dependent apoptosis, and that this role can be partially compensated by MCL-1. Our data further show that BCL-xL expression regulates MCL-1 abundance in CGNPs, and suggest that excessive MCL-1 in Bcl-xLcKO mice prolongs CGNP proliferation by binding SUFU, resulting in increased SHH pathway activation. Accordingly, we propose that BCL-xL and MCL-1 interact with each other and with developmental mechanisms that regulate proliferation, to adjust the apoptotic threshold as CGNPs progress through postnatal neurogenesis to CGNs.

Projected t-SNE for batch correction.

MOTIVATION: Low-dimensional representations of high-dimensional data are routinely employed in biomedical research to visualize, interpret and communicate results from different pipelines. In this article, we propose a novel procedure to directly estimate t-SNE embeddings that are not driven by batch effects. Without correction, interesting structure in the data can be obscured by batch effects. The proposed algorithm can therefore significantly aid visualization of high-dimensional data. RESULTS: The proposed methods are based on linear algebra and constrained optimization, leading to efficient algorithms and fast computation in many high-dimensional settings. Results on artificial single-cell transcription profiling data show that the proposed procedure successfully removes multiple batch effects from t-SNE embeddings, while retaining fundamental information on cell types. When applied to single-cell gene expression data to investigate mouse medulloblastoma, the proposed method successfully removes batches related with mice identifiers and the date of the experiment, while preserving clusters of oligodendrocytes, astrocytes, and endothelial cells and microglia, which are expected to lie in the stroma within or adjacent to the tumours. AVAILABILITY AND IMPLEMENTATION: Source code implementing the proposed approach is available as an R package at https://github.com/emanuelealiverti/BC_tSNE, including a tutorial to reproduce the simulation studies. CONTACT: aliverti@stat.unipd.it.

scRNA-seq in medulloblastoma shows cellular heterogeneity and lineage expansion support resistance to SHH inhibitor therapy.

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.