Time-resolved systems immunology reveals a late juncture linked to fatal COVID-19.

COVID-19 exhibits extensive patient-to-patient heterogeneity. To link immune response variation to disease severity and outcome over time, we longitudinally assessed circulating proteins as well as 188 surface protein markers, transcriptome, and T cell receptor sequence simultaneously in single peripheral immune cells from COVID-19 patients. Conditional-independence network analysis revealed primary correlates of disease severity, including gene expression signatures of apoptosis in plasmacytoid dendritic cells and attenuated inflammation but increased fatty acid metabolism in CD56dimCD16hi NK cells linked positively to circulating interleukin (IL)-15. CD8+ T cell activation was apparent without signs of exhaustion. Although cellular inflammation was depressed in severe patients early after hospitalization, it became elevated by days 17-23 post symptom onset, suggestive of a late wave of inflammatory responses. Furthermore, circulating protein trajectories at this time were divergent between and predictive of recovery versus fatal outcomes. Our findings stress the importance of timing in the analysis, clinical monitoring, and therapeutic intervention of COVID-19.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.

iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

Human CCR6+ Th Cells Show Both an Extended Stable Gradient of Th17 Activity and Imprinted Plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Laser Processing of Crumpled Porous Graphene/MXene Nanocomposites for a Standalone Gas Sensing System.

Integrating wearable gas sensors with energy harvesting and storage devices can create self-powered systems for continuous monitoring of gaseous molecules. However, the development is still limited by complex fabrication processes, poor stretchability, and sensitivity. Herein, we report the low-cost and scalable laser scribing of crumpled graphene/MXenes nanocomposite foams to combine stretchable self-charging power units with gas sensors for a fully integrated standalone gas sensing system. The crumpled nanocomposite designed in island-bridge device architecture allows the integrated self-charging unit to efficiently harvest kinetic energy from body movements into stable power with adjustable voltage/current outputs. Meanwhile, given the stretchable gas sensor with a large response of ∼1% ppm-1 and an ultralow detection limit of ∼5 ppb to NO2/NH3, the integrated system provides real-time monitoring of the exhaled human breath and the local air quality. The innovations in materials and structural designs pave the way for the future development of wearable electronics.

Inflammatory and interferon gene expression signatures in patients with mitochondrial disease.

BACKGROUND: People with mitochondrial disease (MtD) are susceptible to metabolic decompensation and neurological symptom progression in response to an infection. Increasing evidence suggests that mitochondrial dysfunction may cause chronic inflammation, which may promote hyper-responsiveness to pathogens and neurodegeneration. We sought to examine transcriptional changes between MtD patients and healthy controls to identify common gene signatures of immune dysregulation in MtD. METHODS: We collected whole blood from a cohort of MtD patients and healthy controls and performed RNAseq to examine transcriptomic differences. We performed GSEA analyses to compare our findings against existing studies to identify commonly dysregulated pathways. RESULTS: Gene sets involved in inflammatory signaling, including type I interferons, interleukin-1ß and antiviral responses, are enriched in MtD patients compared to controls. Monocyte and dendritic cell gene clusters are also enriched in MtD patients, while T cell and B cell gene sets are negatively enriched. The enrichment of antiviral response corresponds with an independent set of MELAS patients, and two mouse models of mtDNA dysfunction. CONCLUSIONS: Through the convergence of our results, we demonstrate translational evidence of systemic peripheral inflammation arising from MtD, predominantly through antiviral response gene sets. This provides key evidence linking mitochondrial dysfunction to inflammation, which may contribute to the pathogenesis of primary MtD and other chronic inflammatory disorders associated with mitochondrial dysfunction.

Strategies to avoid internal carotid artery injury in "sandwich" atlantoaxial dislocation patients during surgery.

PURPOSE: To elucidate the anatomic relationship between the internal carotid artery (ICA) and the bony structures of the craniovertebral junction among "sandwich" atlantoaxial dislocation (AAD) patients, and to analyze the risks of injury during surgical procedures. METHODS: The distance from the medial wall of ICA to the midsagittal plane (D1), the shortest distance between the ICA wall and the anterior cortex of the lateral mass of atlas (LMA) (D2) on the most caudal and cranial levels of LMA and the angle (A) between the sagittal plane passing through the screw entry point of C1 lateral mass(C1LM) screw and the medial tangent line of the vessel passing through the entry point were measured. Besides, the location of ICA in front of the atlantoaxial vertebra was divided into 4 categories (Z1-Z4). RESULTS: There was a statistically difference between the male and female patients regarding D1, and the difference between D2 at level a and level b as well as angle A between the left and right sides were statistically different (p < 0.05). Ninety-two ICAs (57.5%) were anteriorly located in Z3, 50 (31.3%) were located in Z4, 17 were located in Z2, and only one ICA was located in Z1 in all 80 patients. CONCLUSIONS: In "sandwich" AAD patients, particular attention should be paid to excessively medialized ICA to avoid ICA injury during trans-oral procedures, and the risk of injuring the ICA with more cranially and medially angulated C1LM screw placement was relatively less during posterior fixation procedures. A novel classification of ICA location was used to describe the relationship between ICA and LMA.

Peroxisome proliferator activated receptor-γ agonist pioglitazone improves vascular and metabolic dysfunction in systemic lupus erythematosus.

OBJECTIVES: Premature cardiovascular events in systemic lupus erythematosus (SLE) contribute to morbidity and mortality, with no effective preventive strategies described to date. Immune dysregulation and metabolic disturbances appear to play prominent roles in the induction of vascular disease in SLE. The peroxisome proliferator activated receptor-gamma agonist pioglitazone (PGZ suppresses vascular damage and immune dysregulation in murine lupus and improves endothelial dysfunction in other inflammatory diseases. We hypothesised that PGZ could improve vascular dysfunction and cardiometabolic parameters in SLE. METHODS: Eighty SLE subjects with mild to severe disease activity were randomised to a sequence of PGZ followed by placebo for 3 months, or vice versa, in a double-blind, cross-over design with a 2-month wash-out period. Primary endpoints were parameters of endothelial function and arterial inflammation, measured by multimodal assessments. Additional outcome measures of disease activity, neutrophil dysregulation, metabolic disturbances and gene expression studies were performed. RESULTS: Seventy-two subjects completed the study. PGZ was associated with a significant reduction in Cardio-Ankle Vascular Index (a measure of arterial stiffness) compared with placebo. Various metabolic parameters improved with PGZ, including insulin resistance and lipoprotein profiles. Circulating neutrophil extracellular trap levels also significantly decreased with PGZ compared with placebo. Most adverse events experienced while on PGZ were mild and resolved with reduction in PGZ dose. CONCLUSION: PGZ was well tolerated and induced significant improvement in vascular stiffness and cardiometabolic parameters in SLE. The results suggest that PGZ should be further explored as a modulator of cardiovascular disease risk in SLE. TRIAL REGISTRATION NUMBER: NCT02338999.

Colchicine's effects on metabolic and inflammatory molecules in adults with obesity and metabolic syndrome: results from a pilot randomized controlled trial.

OBJECTIVE: Recent clinical trials have demonstrated that colchicine may have metabolic and cardiovascular and benefits in at-risk patients; however, the mechanisms through which colchicine may improve outcomes are still unclear. We sought to examine colchicine's effects on circulating inflammatory and metabolic molecules in adults with obesity and metabolic syndrome (MetS). METHODS: Blood samples were collected pre- and post-intervention during a double-blind randomized controlled trial in which 40 adults with obesity and MetS were randomized to colchicine 0.6 mg or placebo twice-daily for 3 months. Serum samples were analyzed for 1305 circulating factors using the SomaScan Platform. The Benjamini-Hochberg procedure was used to adjust the false discovery rate (FDR) for multiple testing. RESULTS: At baseline, age (48.0 ± 13.8 vs. 44.7 ± 10.3 years) and BMI (39.8 ± 6.4 vs. 41.8 ± 8.2 kg/m2) were not different between groups. After controlling for the FDR, 34 molecules were significantly changed by colchicine. Colchicine decreased concentrations of multiple inflammatory molecules, including C-reactive protein, interleukin 6, and resistin, in addition to vascular-related proteins (e.g., oxidized low-density lipoprotein receptor, phosphodiesterase 5A). Conversely, relative to placebo, colchicine significantly increased concentrations of eight molecules including secreted factors associated with metabolism and anti-thrombosis. CONCLUSIONS: In adults with obesity, colchicine significantly affected concentrations of proteins involved in the innate immune system, endothelial function and atherosclerosis, uncovering new mechanisms behind its cardiometabolic effects. Further research is warranted to investigate whether colchicine's IL-6 suppressive effects may be beneficial in COVID-19.

Conventional Co-Housing Modulates Murine Gut Microbiota and Hematopoietic Gene Expression.

Specific-pathogen-free (SPF) mice have improved hematopoietic characteristics relative to germ-free mice, however, it is not clear whether improvements in hematopoietic traits will continue when the level of microorganism exposure is further increased. We co-housed SPF C57BL/6 mice in a conventional facility (CVT) and found a significant increase in gut microbiota diversity along with increased levels of myeloid cells and T cells, especially effector memory T cells. Through single cell RNA sequencing of sorted KL (c-Kit+Lin-) cells, we imputed a decline in long-term hematopoietic stem cells and an increase in granulocyte-monocyte progenitors in CVT mice with up-regulation of genes associated with cell survival. Bone marrow transplantation through competitive repopulation revealed a significant increase in KSL (c-Kit+Sca-1+Lin-) cell reconstitution in recipients of CVT donor cells which occurred when donors were co-housed for both one and twelve months. However, there was minimal to no gain in mature blood cell engraftment in recipients of CVT donor cells relative to those receiving SPF donor cells. We conclude that co-housing SPF mice with mice born in a conventional facility increased gut microbiota diversity, augmented myeloid cell production and T cell activation, stimulated KSL cell reconstitution, and altered hematopoietic gene expression.

[Surgical intervention in adult varicocele: An update].

Varicocele is one of the common causes of infertility in adult males. Surgical intervention can improve the semen quality of some of the patients and significantly increase the pregnancy rate, but fails to benefit them all. Therefore, it is of great practical significance to identify the patients who can significantly benefit from surgical interventions to maximize the effectiveness of surgery and avoid medical waste. This article summarizes the recent progress in surgical indications and strategies for varicocele in adult males, and provides some reference for the management of the disease.

[Vascular damage-related risk factors for erectile dysfunction in patients with type 2 diabetes mellitus: Analysis based on a nomogram model].

OBJECTIVE: To analyze vascular damage-related risk factors for ED in patients with type 2 diabetes mellitus (DM) and develop a nomogram for the prediction of the factors. METHODS: A total of 181 patients with type 2 DM were included for sexual function assessment, and the clinical data on vascular damage were retrieved from the patients system. After preprocessing, the data were described by the number and percentage of different types of cases and subjected to statistical analysis with the R software. The Lasso regression model was used to optimize feature selection. On the premise of the sample size required for logistic regression analysis according to the number of events per variable, multivariable logistic regression analysis was performed on the selected variables and a nomogram was developed for diabetes-induced erectile dysfunction (DIED). Then, the performance of the nomogram was evaluated with respect to its calibration, discrimination and clinical utility using Harrell's concordance index (C-index), the calibration plot and decision curve analysis, as well as bootstrapping for internal validation. RESULTS: ED was diagnosed in 90 (49.7%) of the 181 patients. The risk factors subjected to logistic regression analysis included the duration of DM (OR = 4.440, 95% CI: 1.594－13.105; OR = 7.667, 95% CI: 1.444－48.733), status of carotid intima-media thickness (c-IMT) (OR = 3.767, 95% CI: 1.194－12.691), diabetic retinopathy (DR) (OR = 5.382, 95% CI: 1.373－28.301), diabetic kidney disease (DKD) (OR = 4.959, 95% CI: 1.156－27.728), low-density lipoprotein cholesterol (LDL-C) (OR = 8.210, 95% CI: 2.027－43.507), red blood cell distribution width (RDW) (OR = 2.418, 95% CI: 1.021－5.826), and plasma fibrinogen (Fbg) (OR = 4.649, 95% CI: 2.001－11.339). The C-index of the DIED model was 0.911 (95% CI: 0.869－0.954). The curve representing the performance of the nomogram fit in well with that representing a perfect prediction by the calibration plot. Decision curve analysis indicated that the nomogram was clinically useful for predicting DIED in the type 2 DM patients at the possibility threshold of 6% to 93%. CONCLUSIONS: A nomogram was preliminarily developed for predicting the risk of DIED in type 2 DM patients with respect to the seven independent influencing factors, including the duration of DM, status of c-IMT, DR, DKD, LDL-C, RDW, and Fbg.

[Gene characteristics and changing trend of neonatal thalassemia in Dongguan, China].

OBJECTIVE: To study the gene distribution characteristics of neonatal thalassemia in Dongguan, China and the changing trend of the gene distribution characteristics of neonates with thalassemia in Dongguan in 2014-2018. METHODS: A retrospective analysis was performed for the data on neonatal thalassemia screening from the Dongguan Neonatal Disease Screening System between January 2014 and December 2018. A total of 616 718 neonates were enrolled who were born in Dongguan. RESULTS: Among the 616 718 neonates, 52 308 were positive for primary screening, 10 366 were recalled, 8 576 underwent genetic diagnosis, and 6 432 were confirmed with thalassemia by genetic diagnosis. The carrying rates of thalassemia genes in 2014-2018 were 5.81%, 5.47%, 5.96%, 6.91%, and 7.90% respectively, and showed an upward trend (P<0.001). The positive rates of neonatal thalassemia screening in 2014-2018 were 9.12%, 8.34%, 7.54%, 8.13%, and 9.32% respectively (P<0.001). The positive rates of genetic diagnosis of neonatal thalassemia in 2014-2018 were 0.89%, 1.11%, 1.24%, 0.90%, and 1.09% respectively (P<0.001). In 2014-2018, 5 098 cases of α-thalassemia were detected, accounting for 79.26% of all cases, and 1 230 cases of ß-thalassemia were detected, accounting for 19.12% of all cases. The detection rate of α-thalassemia was significantly higher than that of ß-thalassemia in each year (P<0.001). In 2014-2018, static α-thalassemia, mild α-thalassemia, and mild ß-thalassemia were the main types observed in neonates. CONCLUSIONS: Most of the neonates with thalassemia have α-thalassemia in Dongguan, with static α-thalassemia and mild α-thalassemia as the main types. The carrying rate of thalassemia genes keeps increasing in neonates in Dongguan, and the prevention and treatment of thalassemia is still challenging.

Over-expression of PD-1 Does Not Predict Leukemic Relapse after Allogeneic Stem Cell Transplantation.

Blockade of the T-cell exhaustion marker PD-1 to re-energize the immune response is emerging as a promising cancer treatment. Relapse of hematologic malignancy after allogeneic stem cell transplantation limits the success of this approach, and PD-1 blockade may hold therapeutic promise. However, PD-1 expression and its relationship with post-transplant relapse is poorly described. Because the donor immunity is activated by alloresponses, PD-1 expression may differ from nontransplanted individuals, and PD-1 blockade could risk graft-versus-host disease. Here we analyzed T-cell exhaustion marker kinetics and their relationship with leukemia relapse in 85 patients undergoing myeloablative T-cell-depleted HLA-matched stem cell transplantation. At a median follow-up of 3.5 years, 35 (44%) patients relapsed. PD-1 expression in CD4 and CD8 T cells was comparably elevated in relapsed and nonrelapsed cohorts. Helios+ regulatory T cells and CD8 effector memory cells at day 30 emerged as independent predictors of relapse. Although leukemia antigen-specific T cells did not overexpress PD-1, single-cell analysis revealed LAG3 and TIM3 overexpression at relapse. These findings indicate that PD-1 is an unreliable marker for leukemia-specific T-cell exhaustion in relapsing patients but implies other exhaustion markers and suppressor cells as relapse biomarkers.

Single cell sequencing reveals gene expression signatures associated with bone marrow stromal cell subpopulations and time in culture.

BACKGROUND: Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to better characterize BMSC subpopulations. METHODS: Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq. RESULTS: Transcriptome analysis by microarray and RNA-Seq of unseparated BMSCs from passages 2, 4, 6, 8, 9 and 10 yielded similar results; both data sets grouped passages 4 and 6 and passages 9 and 10 together and genes differentially expressed among these early and late passage BMSCs were similar. 3D Diffusion map visualization of single BMSCs from passages 3, 4, 6, 8 and 9 clustered passages 3 and 9 into two distinct groups, but there was considerable overlap for passages 4, 6 and 8 cells. Markers for early passage, FGFR2, and late passage BMSCs, PLAT, were able to identify three subpopulations within passage 3 BMSCs; one that expressed high levels of FGFR2 and low levels of PLAT; one that expressed low levels of FGFR2 and high levels of PLAT and one that expressed intermediate levels of FGFR2 and low levels of PLAT. CONCLUSIONS: Single BMSCs can be separated by microfluidics and their transcriptome analyzed by next generation sequencing. Single cell analysis of early passage BMSCs identified a subpopulation of cells expressing high levels of FGFR2 that might include skeletal stem cells.

Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells.

Cancer cells frequently exhibit chromosomal abnormalities. Specific cytogenetic aberrations often are predictors of outcome, especially in hematologic neoplasms, such as monosomy 7 in myeloid malignancies. The functional consequences of aneuploidy at the cellular level are difficult to assess because of a lack of convenient markers to distinguish abnormal from diploid cells. We performed single-cell RNA sequencing (scRNA-seq) to study hematopoietic stem and progenitor cells from the bone marrow of 4 healthy donors and 5 patients with bone marrow failure and chromosome gain or loss. In total, transcriptome sequences were obtained from 391 control cells and 588 cells from patients. We characterized normal hematopoiesis as binary differentiation from stem cells to erythroid and myeloid-lymphoid pathways. Aneuploid cells were distinguished from diploid cells in patient samples by computational analyses of read fractions and gene expression of individual chromosomes. We confirmed assignment of aneuploidy to individual cells quantitatively, by copy-number variation, and qualitatively, by loss of heterozygosity. When we projected patients' single cells onto the map of normal hematopoiesis, diverse patterns were observed, broadly reflecting clinical phenotypes. Patients' monosomy 7 cells showed downregulation of genes involved in immune response and DNA damage checkpoint and apoptosis pathways, which may contribute to the clonal expansion of monosomy 7 cells with accumulated gene mutations. scRNA-seq is a powerful technique through which to infer the functional consequences of chromosome gain and loss and explore gene targets for directed therapy.

Long noncoding RNAs of single hematopoietic stem and progenitor cells in healthy and dysplastic human bone marrow.

Long noncoding RNAs (lncRNAs) are regulators of cell differentiation and development. The lncRNA transcriptome in human hematopoietic stem and progenitor cells is not comprehensively defined. We investigated lncRNAs in 979 human bone marrow-derived CD34+ cells by single cell RNA sequencing followed by de novo transcriptome reconstruction. We identified 3,173 lncRNAs in total, among which 2,365 were previously unknown, and we characterized lncRNA stem, differentiation, and maturation signatures. lncRNA expression exhibited high cell-to-cell variation, which was only apparent in single cell analysis. lncRNA expression followed a lineage-specific and highly dynamic pattern during early hematopoiesis. lncRNAs in hematopoietic cells closely correlated with protein-coding genes of known functions in the regulation of hematopoiesis and cell fate decisions, and the potential regulatory roles of lncRNAs in hematopoiesis were imputed by projection from protein-coding genes with a "guilt-by-association" approach. We characterized lncRNAs preferentially expressed in hematopoietic stem cells and in various downstream differentiated lineage progenitors. We also profiled lncRNA expression in single cells from patients with myelodysplastic syndromes and in aneuploid cells in particular. Our study provides a global view of lncRNAs in human hematopoietic stem and progenitor cells. We observed a highly ordered pattern of lncRNA expression and participation in regulation of early hematopoiesis, and coordinate aberrant messenger RNA and lncRNA transcriptomes in dysplastic hematopoiesis. (Registered at clinicaltrials.gov with identifiers: 00001620, 00001397).

PBMC fixation and processing for Chromium single-cell RNA sequencing.

BACKGROUND: Interest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells. METHODS: The methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform. RESULTS: When methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones. CONCLUSIONS: We expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.