Single Cell RNA Analysis Research Articles

Glycogen accumulation, central carbon metabolism, and aging of hematopoietic stem and progenitor cells

Inspired by recent proteomic data demonstrating the upregulation of carbon and glycogen metabolism in aging human hematopoietic stem and progenitor cells (HPCs, CD34+ cells), this report addresses whether this is caused by elevated glycolysis of the HPCs on a per cell basis, or by a subpopulation that has become more glycolytic. The average glycogen content in individual CD34+ cells from older subjects (> 50 years) was 3.5 times higher and more heterogeneous compared to younger subjects (< 35 years). Representative glycolytic enzyme activities in HPCs confirmed a significant increase in glycolysis in older subjects. The HPCs from older subjects can be fractionated into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity, while the subset with a high GU capacity could scarcely be detected in younger subjects. Thus, we conclude that upregulated glycolysis in aging HPCs is caused by the expansion of a more glycolytic HPC subset. Since single-cell RNA analysis has also demonstrated that this subpopulation is linked to myeloid differentiation and increased proliferation, isolation and mechanistic characterization of this subpopulation can be utilized to elucidate specific targets for therapeutic interventions to restore the lineage balance of aging HPCs.

Recent advances and future perspectives in vector-omics.

We have reviewed recent progress and the remaining challenges in vector-omics. We have highlighted several technologies and applications that facilitate novel biological insights beyond achieving a reference-quality genome assembly. Among other topics, we have discussed the applications of chromatin conformation capture, chromatin accessibility assays, optical mapping, full-length RNA sequencing, single cell RNA analysis, proteomics, and population genomics. We anticipate that we will witness a great expansion in vector-omics research not only in its application in a broad range of species, but also its ability to uncover novel genetic elements and tackle previously inaccessible regions of the genome. It is our hope that the continued innovation in device portability, cost reduction, and informatics support will in the foreseeable future bring vector-omics to every vector laboratory and field station in the world, which will have an unparalleled impact on basic research and the control of vector-borne infectious diseases.

Parkinson’s disease over the last 20 years – new concepts and developments

Much has changed in our understanding and approach to Parkinson’s Disease (PD). This in part is because of new scientific methods such as the discovery of how to make induced pluripotent stem cells (iPSCs); the development of improved gene editing techniques such as CRISPR/Cas9 and the ability to undertake single cell RNA analyses. However, much of our new understanding comes directly from observations made in the clinic and related biomaterials. In this short review I will highlight some of this.

The Transcription Factor RORα Preserves ILC3 Lineage Identity and Function during Chronic Intestinal Infection.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

TMOD-26. MODELING GLIOBLASTOMA BY IMPLANTATION OF INTACT PATIENT-DERIVED ORGANOIDS INTO RODENT BRAINS

Abstract Glioblastoma multiforme (GBM) is the most common primary and aggressive brain tumors in adults with extremely poor prognosis and limited treatment options. A major hallmark of GBM is the rapid and diffused infiltration of tumor cells into the surrounding healthy tissue that contribute to tumor recurrence and therapeutic resistance. However, existing in vitro cell culture or in vivo xenograft models inadequately recapitulate the inter-tumoral and intra-tumoral heterogeneity which are key features of GBM. For example, common oncogenic drivers of GBM such as epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation do not persist in traditional in vitro models due to selection pressures, thus requires exogenous overexpression. Alternatively, EGFR statuses can be maintained in xenografted mice, but implantation of the primary GBM cells into the flank is required to first establish the tumor prior to secondary injection into the brains. Recently, we have established a novel protocol for culturing GBM tissue as organoids (GBOs) directly from patient tumor resection that retain many distinct cell populations in vitro with high fidelity evidenced by histological, whole-exome, bulk and single cell RNA analyses. Compared to prolonged generation time of previously established in vitro and xenograft models, our methodology is robust for generating GBOs within 1–2 weeks from initial resection. In addition, these GBOs can be readily xenografted into the adult mouse brains as an intact organoid, exhibit rapid and aggressive infiltration phenotypes, and maintains driver mutation EGFRviii within as little as one month. Consequently, they can be used to test in vivo treatment efficacies in a timely fashion. The presence of diverse cell types in this GBO model offers a promising platform for not only understanding of tumor biology, but also more strategic development of new therapies.

435 Human group 2 innate lymphoid cells differentiate into interleukin-17A producing cells in psoriasis

The IL-23/IL-17A axis is critical in the defense against extracellular pathogens as well as the development of several autoimmune diseases including psoriasis. Here we focused on the capability of human innate lymphoid cell (ILC) subsets to produce IL-17A. Initial experiments revealed unexpectedly that purified dermis-derived group 2 ILCs (ILC2s), but not ILC3s, were induced to produce this cytokine after co-culture with Candida albicans-exposed undivided dermal cells. The CRTH2+ ILC2s transdifferentiated towards IL-17A producing NKp44– ILC3-like cells. This feature was also found in ILC2s derived from peripheral blood or cord blood and appeared to be driven by IL-1β, IL-23 and TGF-β and coincided with up-regulation of RORγt and reciprocal down-regulation of GATA3. Flow cytometry, RNA expression microarray, and unbiased single-cell RNA analyses disclosed a c-Kit (CD117)+ CCR6+ ILC2 subpopulation having some features in common with ILC3s, including expression of RORγt which accommodates IL-17A production in response to IL-1β and IL-23. This ILC2 subset also expressed the skin homing receptor CCR10. The c-Kit– ILC2s were also able to produce IL-17A but required during stimulation with IL-1β and IL-23 the additional presence of TGF-β, which promoted the upregulation of IL23R, CCR6 and c-Kit in these cells. The switch into IL-17A producing ILC3-like cells could be reversed by IL-4. The plasticity process we describe here has clinical relevance as we show that the proportion of IL-17A producing ILC3s is increased at the expense of ILC2s within the lesional skin of psoriatic patients as compared to healthy control skin. We identify ILC2s as a novel cellular source of IL-17A, providing a novel therapeutic target to ameliorate IL-17A pathogenicity.

Proteomics and single-cell RNA analysis of Akap4-knockout mice model confirm indispensable role of Akap4 in spermatogenesis

Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa.

Neural stem cells: origin, heterogeneity and regulation in the adult mammalian brain.

In the adult rodent brain, neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), which are specialized niches in which young neurons for the olfactory bulb (OB) and hippocampus, respectively, are generated. Recent studies have significantly modified earlier views on the mechanisms of NSC self-renewal and neurogenesis in the adult brain. Here, we discuss the molecular control, heterogeneity, regional specification and cell division modes of V-SVZ NSCs, and draw comparisons with NSCs in the SGZ. We highlight how V-SVZ NSCs are regulated by local signals from their immediate neighbors, as well as by neurotransmitters and factors that are secreted by distant neurons, the choroid plexus and vasculature. We also review recent advances in single cell RNA analyses that reveal the complexity of adult neurogenesis. These findings set the stage for a better understanding of adult neurogenesis, a process that one day may inspire new approaches to brain repair.

Single cell RNA analysis identifies cellular heterogeneity and adaptive responses of the lung at birth

The respiratory system undergoes a diversity of structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify the heterogeneity of pulmonary cell types and dynamic changes in gene expression mediating adaptation to respiration, here we perform single cell RNA analyses of mouse lung on postnatal day 1. Using an iterative cell type identification strategy we unbiasedly identify the heterogeneity of murine pulmonary cell types. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore we compare temporal changes in RNA expression patterns before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, including activation of cell stress and the unfolded protein response during perinatal adaptation of the lung. The present data provide a single cell view of the adaptation to air breathing after birth.

Endocardial cell heterogeneity at the origin of valvular cell clones

Valves are affected in 1/3 of cardiac congenital diseases. Cardiac valves derive from the process of endothelial-mesenchymal transition (EMT) of endocardial cells in the atrioventricular canal (AVC) and in the outflow tract of embryonic mouse hearts at E9.5 and E10.5, respectively. The origin and fate of endocardial cells as well as how EMT is specifically triggered in a subset of the latter are still poorly understood. To determine the extent of endocardial cells and valvular cells heterogeneity and in turn to assess whether endocardial cells are already committed to a valvular specific cell type. Clonal analysis: a genetic labeling of endocardial valve progenitors was performed using Brainbow and CAGERT2Cre mouse strains. Single cell sequencing: Tie2Cre/− male was bred with RosatdTomato females. AVC cells were dissected out from E9.5 embryos. Mitral valve leaflets were dissected out from both E16.5 and P0 mice. All cells were enzymatically dissociated and tomato+ cells sorted out by FACS and used for single cell RNA sequencing using the 10X genomics technology. Preliminary results point to a cell heterogeneity in each cell population so far investigated. A full panel of both clonal analysis and single cell RNA analysis will be presented.

Hypothalamic progesterone receptor-A mediates gonadotropin surges, self priming and receptivity in estrogen-primed female mice

Ovarian progesterone (Prog) is an essential steroid hormone for the secretion of GnRH and reproductive behavior. It exerts primary effects through the progesterone receptor (PR). When analyzed separately in vitro, PR isoforms (PR-A, PR-B) display striking differences in transcriptional activity. The present study was undertaken to determine the in vivo impact of each isoform on hypothalamic function in female mice with ablation of a single isoform, either PR-A or PR-B. To this end, we used single-cell RNA analyses, reverse transcriptase real-time (q)PCR mRNA analyses of punched-out tissue, immunohistochemistry, and reproductive behavior. We provide evidence for the requirement of PR-A in individual ventrolateral ventromedial nucleus (vlVMN) neurons for Prog-facilitated proceptive and receptive behaviors in estrogen benzoate (EB)-primed females and the reciprocal male interactions. We clarify histological and molecular mechanisms of PR isoform activity by showing that (1) PR-A is predominant in individual vlVMN neurons controlling female lordosis circuitry, whilst (2) PR-B is predominant in those VMN subdivisions that provide for amplification of PR-A activity. We go on to demonstrate that PR-A is dominant in the anteroventral periventricular nucleus but not the arcuate nucleus that feed fibers into and around the VMN. In the medial preoptic area, high levels of GnRH RNA in EB-primed PR-A-expressing mice were seen coincident with increased plasma LH levels. Two consecutive GnRH pulses enhanced LH only in primed PR-A-expressing females. In all, the findings are consistent with the hypothesis that hypothalamic PR-A-mediated genomic activities result in reproductive behavior coordinated with ovulation.

Single-cell gene expression analysis: implications for neurodegenerative and neuropsychiatric disorders.

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively.

Nicotinic receptor subunits alpha 3, alpha 4, and beta 2 and high affinity nicotine binding sites are expressed by P19 embryonal cells.

Controlled exposure to retinoic acid (RA) induces the murine embryonal carcinoma cell line P19S18O1A1 (P19) to differentiate into a variety of cell types. One of the cell types exhibits neuronal-like morphology and expresses neuronal markers including neurofilament proteins, glutamate receptors, and the cholinergic enzymes choline acetyl-transferase and acetylcholinesterase. In this study we use Northern blot analysis, double-label immunocytochemistry, and single cell RNA analysis using polymerase chain reaction to show that RA-treated P19 cells with neuronal-like morphology also express neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3, alpha 4, and beta 2. Greater than 80% of RA-treated P19 cells with a neuronal-like phenotype express nAChR alpha 4 subunit transcripts and both alpha 4 and beta 2 protein. The RA-induced expression of alpha 3 transcripts accounts for a comparably small number of nAChR-containing cells (< 20%) of which half coexpress alpha 4 transcripts. Expression of high-levels of alpha 4 RNA is dependent upon both cell-cell contact and RA exposure. The appearance of nAChR subunits also coincides with RA-induced expression of high affinity [3H]-nicotine binding receptors. The P19 cell line offers an inducible neuronal cell system to study mammalian neuronal nicotinic receptor expression and the development of high affinity nicotinic binding sites similar to those expressed in the mammalian central nervous system.

A randomised trial comparing endometrial resection and abdominal hysterectomy for the treatment of menorrhagia.

To determine the advantages and disadvantages of endometrial resection and abdominal hysterectomy for the surgical treatment of women with menorrhagia. Randomised study of two treatment groups with a minimum follow up of nine months. Royal Berkshire Hospital, Reading. 51 of 78 menorrhagic women without pelvic pathology who were on the waiting list for abdominal hysterectomy. Endometrial resection or abdominal hysterectomy (according to randomisation). Endometrial resections were performed by an experienced hysteroscopic surgeon; hysterectomies were performed by two other gynaecological surgeons. Length of operating time, hospitalisation, recovery; cost of surgery; short term results of endometrial resection. Operating time was shorter for endometrial resection (median 30 (range 20-47) minutes) than for hysterectomy (50 (39-74) minutes). The hospital stay for endometrial resection (median 1 (range 1-3) days) was less than for hysterectomy (7 (5-12) days). Recovery after endometrial resection (median 16 (range 5-62) days) was shorter than after hysterectomy (58 (11-125) days). The cost was 407 pounds for endometrial resection and 1270 pounds for abdominal hysterectomy. Four women (16%) who did not have an acceptable improvement in symptoms after endometrial resection had repeat resections. No woman has required hysterectomy during a mean follow up of one year. For women with menorrhagia who have no pelvic pathology endometrial resection is a useful alternative to abdominal hysterectomy, with many short term benefits. Larger numbers and a longer follow up are needed to estimate the incidence of complications and the long term efficacy of endometrial resection.