Single-cell mRNA Analysis Research Articles

Understanding mechanisms of JAK1 inhibition on synovial fibroblasts using combinatorial approaches of bulk and single cell RNAseq analyses.

The aim of these studies was to characterise the molecular effects of a tool JAK1 inhibitor on cultured primary fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) through both total and individual cell analysis. RA-FLS cultures from 6 (Bulk RNA-seq) or 4 (ScRNA-seq) donors were pre-treated with various concentrations (100 nM and 1μM) of ABT-317 with/without exposure to 25% SEB-conditioned PBMC medium to mimic the RA inflammatory milieu. Cells were subjected to both bulk RNA-seq (36 libraries) and single cell RNA-seq (scRNA-seq; 24 libraries) to identify biological processes impacted by CM and ABT-317 treatments. In our bulk RNA-seq analysis, a total of 2,605 differentially expressed genes (DEGs) were identified between CM-stimulation and unstimulated groups, while 1,122 DEGs were found between ABT-317 1μM and DMSO in CM-stimulated groups using thresholds of log2 (fold change) ≥ |0.58| and FDR ≤ 10%. Both bulk and single cell mRNA analysis of RA-FLS treated with a combination of CM and ABT-317 demonstrated the expected changes in inflammatory pathways such as interferon and IL-6 signalling. However, other non-inflammation associated pathways were also altered by ABT-317. In addition, the single cell analysis highlighted that FLS segregate into distinctive clusters upon combination CM and ABT-317 treatment, suggesting JAK inhibition can drive RA-FLS into multiple heterogenous cell populations. Interestingly, one of the unique RA-FLS clusters that emerged from the CM and ABT-317 treatment showed matrix metalloproteinase-3 (MMP3)high expression as well as several gene signatures that are not found in any other ABT-317 derived clusters. JAK inhibition with ABT-317 is effective at globally inhibiting CM-induced pro- and non-inflammatory pathways in FLS cultures, but also results in several distinct fibroblast populations with unique gene-associated pathways. This study advances the molecular understanding of JAK1 inhibitor effects on fibroblasts that may contribute to clinical efficacy.

Single-Cell RNA Sequencing Reveals Alterations in Patient Immune Cells with Pulmonary Long COVID-19 Complications.

Since the emergence of the COVID-19 pandemic, the effects of SARS-CoV-2 have been extensively researched. While much is already known about the acute phase of the infection, increasing attention has turned to the prolonged symptoms experienced by a subset of individuals, commonly referred to as long COVID-19 patients. This study aims to delve deeper into the immune landscape of patients with prolonged symptoms by implementing single-cell mRNA analysis. A 71-year-old COVID-19 patient presenting with persistent viral pneumonia was recruited, and peripheral blood samples were taken at 3 and 2 years post-acute infection onset. Patients and control peripheral blood mononuclear cells (PBMCs) were isolated and single-cell sequenced. Immune cell population identification was carried out using the ScType script. Three months post-COVID-19 patients' PBMCs contained a significantly larger immature neutrophil population compared to 2-year and control samples. However, the neutrophil balance shifted towards a more mature profile after 18 months. In addition, a notable increase in the CD8+ NKT-like cells could be observed in the 3-month patient sample as compared to the later one and control. The subsequent change in these cell populations over time may be an indicator of an ongoing failure to clear the SARS-CoV-2 infection and, thus, lead to chronic COVID-19 complications.

The prognostic and biology of tumour-infiltrating lymphocytes in the immunotherapy of cancer.

Tumour immunotherapy has achieved remarkable clinical success in many different types of cancer in the past two decades. The outcome of immune checkpoint inhibitors in cancer patients has been linked to the quality and magnitude of T cell, NK cell, and more recently, B cell within the tumour microenvironment, suggesting that the immune landscape of a tumour is highly connected to patient response and prognosis. It is critical to understanding tumour immune microenvironments for identifying immune modifiers of cancer progression and developing cancer immunotherapies. The infiltration of solid tumours by immune cells with anti-tumour activity is both a strong prognostic factor and a therapeutic goal. Recent approaches and applications of new technologies, especially single-cell mRNA analysis in dissecting tumour microenvironments have brought important insights into the biology of tumour-infiltrating immune cells, revealed a remarkable degree of cellular heterogeneity and distinct patterns of immune response. In this review, we willdiscuss recent advances in the understanding of tumour infiltrated lymphocytes, their prognostic benefit, and predictive value for immunotherapy.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Precise identification of cancer cells from allelic imbalances in single cell transcriptomes

A fundamental step of tumour single cell mRNA analysis is separating cancer and non-cancer cells. We show that the common approach to separation, using shifts in average expression, can lead to erroneous biological conclusions. By contrast, allelic imbalances representing copy number changes directly detect the cancer genotype and accurately separate cancer from non-cancer cells. Our findings provide a definitive approach to identifying cancer cells from single cell mRNA sequencing data.

Single-cell mRNA analysis of colon tissue during DSS colitis in CX3CR1cre/+EP4fl/fl mice suggests PGE2 signaling via EP4 promotes survival of protective macrophages

Abstract Prostaglandin E2 signaling through its receptor EP4 on macrophages (MPs) protects against DSS-induced colitis, however the mechanisms involved are not clear. Here we perform single cell RNA sequencing on the colons of CX3CR1cre/+EP4fl/fl(Cre+) mice and CX3CR1+/+EP4fl/fl(Cre−) littermate controls at days 0, 3 and 7 following DSS treatment. We identified monocyte (MO), MP, DC1, DC2 and pre-DC populations, with MPs clustering into Hes1hi, Mrc1hi Siglec1(CD169)hi, Retnlahi, and Il1r2hiCd80hi populations. With colitis there was a dramatic increase in the proportions of inflammatory MOs and a loss of all mature MPs, along with an increase in cDC2 but not cDC1 in both mouse strains. However, in Cre+ mice, which develop worse disease with universal GI bleeding, the loss of Mrc1hi Siglec1hi MPs was accelerated together with an increased proportion of Il1r2hiCd80hi MPs compared to Cre− littermates. Analysis of DEGs showed the Il1r2hiCd80hi MP cluster expresses high levels of leukocyte chemoattractants, suggesting that PGE2 may prevent leukocyte recruitment during inflammation. Cre+ mice also had fewer Retnlahi macrophages than Cre− mice both at days 0 and 7 post DSS. Genes highly expressed in the Retnlahi cluster are associated with wound healing and maintenance of vascular integrity indicating their loss could account for the enhanced GI bleeding in the inflamed Cre+ mice. This is the first detailed scmRNA analysis of immune cell populations involved in DSS colitis. While additional investigation is necessary, the data suggest a role for PGE2 signaling via EP4 on MPs in preventing excessive inflammatory cell infiltration, decreasing vascular permeability, and contributing to tissue repair by maintaining protective MP populations.

Abstract P353: Adipose-derived Stem Cells Contribute To Vascular Remodeling Via Clec11a + Subpopulation

Introduction: Recent researches identified the existence of perivascular adipose-derived stem cells (ADSCs) which could differentiate into vascular lineages and participate in vascular remodeling. Single-cell mRNA analysis revealed cellular heterogeneity of subcutaneous ADSCs in respect to cell clustering and cell differentiation. However, such analysis of perivascular ADSCs has not been investigated at a single-cell level. Hypothesis: There is a significant difference among perivascular ADSCs subpopulations in respect to vascular-lineage differentiation. Methods: We performed droplet-based single-cell profiling of subcutaneous and perivascular adipose stromal cells and compared ADSCs regarding their heterogeneity, gene ontology, and cell fate trajectory by applying single-cell analysis as well as in vitro and in vivo assays. Results: Single-cell analysis uncovered 4 perivascular ADSCs subpopulations including Dpp4+ , Col4a2+ / Icam1+ , Clec11a+ / Cpe+ and Sult1e1+ cells. Notably, the Clec11a + subpopulation comprised the bulk of perivascular ADSCs, while was hardly presented in subcutaneous ADSCs. Further gene-set enrichment analysis suggested Clec11a + ADSCs were potentially involved with TGF-β signaling pathways and pseudotemporal analysis predicted that Clec11a + subpopulation lay at the end of the differential trajectory towards smooth muscle cells (SMCs). In vitro assays displayed that perivascular ADSCs could differentiate into SMCs via CLEC11A regulation when treated by TGF-β1. To further elucidate the role of the Clec11a + subpopulation in SMCs differentiation, we labeled CLEC11A+ and CLEC11A- perivascular ADSCs by lentivirus transfection and isolated them by FACS assay. CLEC11A+ cells showed the greater capability of SMCs differentiation in response to TGF-β1 in vitro and enhanced neointima formation when transplanted to the adventitial side of guidewire injured arteries. Conclusions: The present study depicted the unique heterogeneity of perivascular ADSCs and the novel role of the Clec11a + subpopulation, providing a supplement for the relationship between perivascular ADSCs and vascular SMCs.

Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning

T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

Abstract 15355: Interleukin-1beta Inhibition Attenuates Accelerated Atherosclerosis in Mice With Tet2 Loss of Function in a Sex-dependent Fashion

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) describes somatic mutations of bone marrow derived blood cells in the absence of overt hematological disease. CHIP correlates with aging and atherosclerotic cardiovascular diseases in humans. Tet2 mutations, common in CHIP, augment atherosclerosis. Tet2 -deficient macrophages over-express Interleukin-(IL)1β mRNA in response to LDL. IL-1β neutralization reduces recurrent events in patients post myocardial infarction with residual inflammatory risk.We previously showed that IL-1β neutralization reduces evolution of established mouse atherosclerosis. Hypothesis: This study tested the hypothesis that IL-1β neutralization will reduce early accelerated atherosclerosis caused by Tet2 deficiency in hematopoietic cells. Methods: We transplanted lethally irradiated LDL-R -/- atherosclerotic prone mice with either Tet2 +/+ or Tet2 - /- bone marrow cells. Chimeric mice consumed a western diet and received a selective anti-mouse IL-1β monoclonal antibody or isotype-matched control. After nine weeks of treatment, we analyzed atherosclerotic lesions morphologically and by single cell mRNA analysis. Results: IL-1β neutralization reduced accelerated atherosclerosis associated with Tet2 deficiency in female but not in male mice (Table). Single cell RNA-sequencing analysis of the dissociated aortic arch revealed differences in macrophage expression of genes involved in innate immune response interleukin signaling pathways between male and female mice. Conclusions: This unexpected sex-dependence of accelerated atherogenesis in Tet2 - /- bone-marrow mice on IL-1β signaling lays the ground for further mechanistic exploration.

The differential contribution of pacemaker neurons to synaptic transmission in the pyloric network of the Jonah crab, Cancer borealis.

Many neurons receive synchronous input from heterogeneous presynaptic neurons with distinct properties. An instructive example is the crustacean stomatogastric pyloric circuit pacemaker group, consisting of the anterior burster (AB) and pyloric dilator (PD) neurons, which are active synchronously and exert a combined synaptic action on most pyloric follower neurons. Previous studies in lobster have indicated that AB is glutamatergic, whereas PD is cholinergic. However, although the stomatogastric system of the crab Cancer borealis has become a preferred system for exploration of cellular and synaptic basis of circuit dynamics, the pacemaker synaptic output has not been carefully analyzed in this species. We examined the synaptic properties of these neurons using a combination of single-cell mRNA analysis, electrophysiology, and pharmacology. The crab PD neuron expresses high levels of choline acetyltransferase and the vesicular acetylcholine transporter mRNAs, hallmarks of cholinergic neurons. In contrast, the AB neuron expresses neither cholinergic marker but expresses high levels of vesicular glutamate transporter mRNA, consistent with a glutamatergic phenotype. Notably, in the combined synapses to follower neurons, 70-75% of the total current was blocked by putative glutamatergic blockers, but short-term synaptic plasticity remained unchanged, and although the total pacemaker current in two follower neuron types was different, this difference did not contribute to the phasing of the follower neurons. These findings provide a guide for similar explorations of heterogeneous synaptic connections in other systems and a baseline in this system for the exploration of the differential influence of neuromodulators.NEW & NOTEWORTHY The pacemaker-driven pyloric circuit of the Jonah crab stomatogastric nervous system is a well-studied model system for exploring circuit dynamics and neuromodulation, yet the understanding of the synaptic properties of the two pacemaker neuron types is based on older analyses in other species. We use single-cell PCR and electrophysiology to explore the neurotransmitters used by the pacemaker neurons and their distinct contribution to the combined synaptic potentials.

Single-cell mRNA analysis of colon phagocyte heterogeneity identifies two major macrophage developmental pathways

Abstract Tissue macrophages (MPs) develop distinctive functions due to unique transcriptional programs driven by local environmental ques. In the intestine, it is known that MPs are heterogeneous cells that differentiate from blood monocytes and embryonic precursors, and are essential for homeostasis with the vast microbiome through their ability to kill invading microbes, clear apoptotic cells, and produce regulatory cytokines. However, the full extent of MP heterogeneity, their developmental relationships, and how the intestinal microbiota affects MP generation is not known. Here, we performed single cell RNA sequencing of colonic myeloid cells from specific pathogen free (SPF) and germ free (GF) C57BL/6 mice and found extensive heterogeneity of colon MP, with at least six different identifiable populations, with less heterogeneity of dendritic cells (DCs). Unsupervised modeling of developmental pathways predicted two major trajectories from common CCR2+precursors resulting in CD11c+CD9+MMRintCD121b+and CD11c−CD9intMMRhiCD121b−MP populations. The generation of both mature populations of colon MPs was impaired in germ-free mice, which coincided with the appearance of a dominant unique population of MPs, whereas the generation of DC populations in GF mice were unaffected or increased. Furthermore, the two major MP populations were clearly distinct from other populations regarding their gene expression profile, localization within the lamina propria, and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations, highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.

KCNQ/M-channels regulate mouse vagal bronchopulmonary C-fiber excitability and cough sensitivity.

Increased airway vagal sensory C-fiber activity contributes to the symptoms of inflammatory airway diseases. The KCNQ/Kv7/M-channel is a well-known determinant of neuronal excitability, yet whether it regulates the activity of vagal bronchopulmonary C-fibers and airway reflex sensitivity remains unknown. Here we addressed this issue using single-cell RT-PCR, patch clamp technique, extracellular recording of single vagal nerve fibers innervating the mouse lungs, and telemetric recording of cough in free-moving mice. Single-cell mRNA analysis and biophysical properties of M-current (IM) suggest that KCNQ3/Kv7.3 is the major M-channel subunit in mouse nodose neurons. The M-channel opener retigabine negatively shifted the voltage-dependent activation of IM, leading to membrane hyperpolarization, increased rheobase, and suppression of both evoked and spontaneous action potential (AP) firing in nodose neurons in an M-channel inhibitor XE991-sensitive manner. Retigabine also markedly suppressed the α,β-methylene ATP-induced AP firing in nodose C-fiber terminals innervating the mouse lungs, and coughing evoked by irritant gases in awake mice. In conclusion, KCNQ/M-channels play a role in regulating the excitability of vagal airway C-fibers at both the cell soma and nerve terminals. Drugs that open M-channels in airway sensory afferents may relieve the sufferings associated with pulmonary inflammatory diseases such as chronic coughing.

Leveraging Transcriptional Dynamics to Improve BRAF Inhibitor Responses in Melanoma

Melanoma is known to be a heterogeneous tumor, but the impact of this heterogeneity upon therapeutic response is not well understood. In the current study, we used single cell mRNA analysis and our Single Cell HETerogeneity (SinCHET) platform to define the transcriptional diversity of melanoma and its dynamic response to both BRAF inhibitor therapy and treatment holidays. Our analyses showed melanoma cell lines and patient specimens to be composed of >3 co-existent, transcriptionally distinct states. Among these, State #1 had increased expression of cyclin D1, ERBB3, STAT3/5, MITF and I²-catenin and lower expression of c-JUN and RTKs such as Axl and EGFR. State #2 was characterized by higher expression of ERBB3, Axl and the transcription factor c-JUN. State #3 was characterized by high Axl, c-JUN, E2F1, WEE1, c-MET and EGFR expression and lower expression of MITF, ERBB3 and SMAD9. The cell state composition was dynamically regulated in response to BRAF inhibitor therapy, with State #1 declining and States #2 and #3 increasing in response to drug. The percentage of cells in State #1 recovered following a drug holiday, allowing for a successful therapy rechallenge. Other melanomas that lacked State #1 had varying degrees of intrinsic drug resistance and were not amenable to rechallenge. We next leveraged the differences in fitness between the different transcriptional states in the absence and presence of drug to develop a mathematical model that optimized therapy schedules to retain the drug sensitive population. In vivo validation of the mathematical model demonstrated that the personalized adaptive dosing schedules were better at suppressing tumor growth than either continuous or fixed intermittent BRAF inhibitor schedules. Together our studies provide the first preclinical evidence that transcriptional heterogeneity at the single cell level predicts for the initial sensitivity to BRAF inhibitor therapy. We further demonstrate that manipulating transcriptional heterogeneity through personalized adaptive therapy schedules that can delay the time to resistance. Funding Statement: Supported by SPORE grant P50 CA168536, R21 CA198550, R21 CA216756 (to KSMS) K99 CA226679 (to IS), Moffitt Cancer Center PSOC, U54 CA193489 (EK & AA) and the Cancer Center Support Grant P30 CA076292 from the NIH (NCI/NIH). We also acknowledge the support Miami Center for AIDS Research (CFAR) at the University of Miami Miller School of Medicine funded by P30AI073961. Declaration of Interests: We have no competing interest to declare. Ethics Approval Statement: All animal experiments were carried out in compliance with ethical regulations and protocols approved by the University of South Florida Institutional Animal Care and Use Committee.

Abstract 150: Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system

Abstract Advances in single cell RNA quantification techniques have enabled comprehensive study of subpopulations of cells within a heterogeneous population. The application of single cell quantification techniques to oncology is helping to elucidate the complex variability in genetic and epigenetic interactions that occur within tumor cells and their microenvironment. However, current single cell RNA-sequencing methods are limited by their reliance on costly infrastructure and laborious experimental protocols. We developed the GemCode Platform, which combines microfluidics with molecular barcoding and custom bioinformatics software to enable 3’ mRNA counting from thousands of single cells. Here we utilized the GemCode Platform to profile primary cells from healthy donors and cancer patients. Cell lines and cancer samples were obtained from commercial sources. Single cells, reagents and a single gel bead containing barcoded oligonucleotides were encapsulated into picoliter-sized droplets using the 10X Genomics GemCode Platform. The platform achieved extremely high cell loading efficiency (> 50%), enabling the creation of libraries from precious samples. Lysis and barcoded reverse transcription of RNAs from single cells were performed inside each droplet. High quality next generation sequencing libraries were finished in a single bulk reaction. The GemCode software suite was utilized for processing, interactive analysis and visualization of single cell gene expression data. We demonstrated single cell behavior through mouse- and human cell mixing experiments with a low doublet rate of <1%, making the platform suitable for profiling of rare cancer cell populations. We profiled >40,000 peripheral blood mononuclear cells from healthy donors and detected all major subpopulations (i.e., B cells, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, monocytes) in similar proportions to those previously reported in the literature. Notably, the high-throughput nature of the platform enabled resolution of finer sub-structures such as CD4+ effector memory cells and CD4+ central memory cells. Experiments comparing cells isolated from patients with hematologic malignancies (such as CLL, AML and CML) with whole bone marrow from healthy donors further demonstrate the power of single cell profiling for characterizing disease-associated changes in complex tissues. We demonstrate the ability to perform high-throughput gene expression profiling of mRNAs in single cells. The high-throughput platform enables detection of rare cells in a heterogeneous tumor population. Moreover, efficient cell loading enables analysis of clinically relevant sample types with limited cell input. An integrated single cell mRNA analysis will lead to novel insights into the molecular characteristics of individual cancer cells and provide targets for therapeutic intervention. Citation Format: Grace X.y. Zheng, Tarjei Mikkelsen, Jessica Terry, Phillip Belgrader, Paul Ryvkin, Ryan Wilson, Tobias D. Wheeler, Zachary Bent, Geoff McDermott, Solongo Ziraldo, Alexander Wong, Michael Schnall-Levin, Ben Hindson. Single cell mRNA quantification from 1000s of cells in healthy and malignant tumor samples using a high-throughput droplet-based system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 150.

MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cells

MECP2 mutations cause the X-linked neurodevelopmental disorder Rett Syndrome (RTT) by consistently altering the protein encoded by the MECP2e1 alternative transcript. While mutations that simultaneously affect both MECP2e1 and MECP2e2 isoforms have been widely studied, the consequence of MECP2e1 deficiency on human neurons remains unknown. Here we report the first isoform-specific patient induced pluripotent stem cell (iPSC) model of RTT. RTTe1 patient iPS cell-derived neurons retain an inactive X-chromosome and express only the mutant allele. Single-cell mRNA analysis demonstrated they have a molecular signature of cortical neurons. Mutant neurons exhibited a decrease in soma size, reduced dendritic complexity and decreased cell capacitance, consistent with impaired neuronal maturation. The soma size phenotype was rescued cell-autonomously by MECP2e1 transduction in a level-dependent manner but not by MECP2e2 gene transfer. Importantly, MECP2e1 mutant neurons showed a dysfunction in action potential generation, voltage-gated Na+ currents, and miniature excitatory synaptic current frequency and amplitude. We conclude that MECP2e1 mutation affects soma size, information encoding properties and synaptic connectivity in human neurons that are defective in RTT.

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture.

The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

Cryopreserved Human B Cells as an Alternative Source for Single Cell mRNA Analysis

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO]-stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the 'house-keeping gene' glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells.

In situ-RT and immunolaser microdissection for mRNA analysis of individual cells isolated from epilepsy-associated glioneuronal tumors

Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy (n=6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription (in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short- vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance (glyceraldehyde dehydrogenase; GAPDH), medium-abundance (glial fibrillary acidic protein; GFAP), or low abundance (neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types (myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.

1] Preparation of cDNA from single cells and subcellular regions

Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.

Activation of cAMP-dependent C1- currents in guinea-pig paneth cells without relevant evidence for CFTR expression.

1. To determine whether Paneth cells exhibit functional expression of cAMP-activated Cl- currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole-cell patch clamp and single-cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea-pig small intestine. 2. Prominent activation of Cl- currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP-activated Cl- current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A. 3. Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl- channel, such as the ohmic current-voltage relationship, the anion selectivity with a Type III sequence (Br- > Cl- > I- >> F- >= gluconate-), I--induced blockage, insensitivity to a stilbene-derivative Cl- channel blocker, and sensitivity to a carboxylate analogue Cl- channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl- channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl- currents exhibited inactivation kinetics. 4. Expression of CFTR mRNA could not be detected by RT-PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole-crypt level. The presence of a small number of CFTR-expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry. 5. Taken together, it appears that guinea-pig Paneth cells functionally express cAMP-activated Cl- conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.