Cell Development Pathways Research Articles

Determinism and variability of the morphogenesis pathways

Along with a strict determinism of early embryogenesis in most living organisms, some of them exhibit variability of cell fates and developmental pathways. Here we discuss the phenomena of determinism and variability of developmental pathways, defining its dependence upon cell potency, cell sensitivity to the external signals and cell signaling. We propose a set of conjectures on the phenomenon of variability of developmental pathways, and denote a difference between a normal (local) variability, leading to an invariant final structure (e.g., embryo shape), and fundamental one, which is a switching between different developmental pathways, leading to different possible structures.For illustrating our conjectures, we analyzed early developmental stages of plant embryos with different levels of variability of morphogenesis pathways, and provide a set of computational experiments by Morphogenesis Software.

Agammaglobulinemia: from X-linked to Autosomal Forms of Disease.

Interruptions or alterations in the B cell development pathway can lead to primary B cell immunodeficiency with resultant absence or diminished immunoglobulin production. While the most common cause of congenital agammaglobulinemia is X-linked agammaglobulinemia (XLA), accounting for approximately 85% of cases, other genetic forms of agammaglobulinemia have been identified. Early recognition and diagnosis of these conditions are pivotal for improved outcomes and prevention of sequelae and complications. The diagnosis of XLA is often delayed, and can be missed if patient has a mild phenotype. The lack of correlation between phenotype and genotype in this condition makes management and predicting outcomes quite difficult. In contrast, while less common, autosomal recessive forms of agammaglobulinemia present at younger ages and with typically more severe clinical features resulting in an earlier diagnosis. Some diagnostic innovations, such as KREC level measurements and serum BCMA measurements, may aid in facilitating an earlier identification of agammaglobulinemia leading to prompt treatment. Earlier diagnosis may improve the overall health of patients with XLA.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Hematopoietic stem and progenitor cells are present in healthy gingiva tissue

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.

Potential biomarkers and lncRNA-mRNA regulatory networks in invasive growth hormone-secreting pituitary adenomas.

Growth hormone-secreting pituitary adenomas (GH-PAs) are common subtypes of functional PAs. Invasive GH-PAs play a key role in restricting poor outcomes. The transcriptional changes in GH-PAs were evaluated. In this study, the transcriptome analysis of six different GH-PA samples was performed. The functional roles, co-regulatory network, and chromosome location of differentially expressed (DE) genes in invasive GH-PAs were explored. Bioinformatic analysis revealed 101 DE mRNAs and 70 DE long non-coding RNAs (lncRNAs) between invasive and non-invasive GH-PAs. Functional enrichment analysis showed that epithelial cell differentiation and development pathways were suppressed in invasive GH-PAs, whereas the pathways of olfactory transduction, retinol metabolism, drug metabolism-cytochrome P450, and metabolism of xenobiotics by cytochrome P450 had an active trend. In the protein-protein interaction network, 11 main communities were characterized by cell- adhesion, -motility, and -cycle; transport process; phosphorus and hormone metabolic processes. The SGK1 gene was suggested to play a role in the invasiveness of GH-PAs. Furthermore, the up-regulated genes OR51B6, OR52E4, OR52E8, OR52E6, OR52N2, MAGEA6, MAGEC1, ST8SIA6-AS1, and the down-regulated genes GAD1-AS1 and SPINT1-AS1 were identified in the competing endogenous RNA network. The RT-qPCR results further supported the aberrant expression of those genes. Finally, the enrichment of DE genes in chromosome 11p15 and 12p13 regions were detected. Our findings provide a new perspective for studies evaluating the underlying mechanism of invasive GH-PAs.

TgAP2IX-5 is a key transcriptional regulator of the asexual cell cycle division in Toxoplasma gondii

Apicomplexan parasites have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells (budding) is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown. Using Toxoplasma gondii as a model Apicomplexan, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. Moreover, TgAP2IX-5 acts as a limiting factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways.

MITF functions as a tumor suppressor in non-small cell lung cancer beyond the canonically oncogenic role.

Microphthalamia-associated transcription factor (MITF) is a critical mediator in melanocyte differentiation and exerts oncogenic functions in melanoma progression. However, the role of MITF in non-small cell lung cancer (NSCLC) is still unknown. We found that MITF is dominantly expressed in the low-invasive CL1-0 lung adenocarcinoma cells and paired adjacent normal lung tissues. MITF expression is significantly associated with better overall survival and disease-free survival in NSCLC and serves as an independent prognostic marker. Silencing MITF promotes tumor cell migration, invasion and colony formation in lung adenocarcinoma cells. In xenograft mouse model, MITF knockdown enhances metastasis and tumorigenesis, but decreases angiogenesis in the Matrigel plug assay. Whole transcriptome profiling of the landscape of MITF regulation in lung adenocarcinoma indicates that MITF is involved in cell development, cell cycle, inflammation and WNT signaling pathways. Chromatin immunoprecipitation assays revealed that MITF targets the promoters of FZD7, PTGR1 and ANXA1. Moreover, silencing FZD7 reduces the invasiveness that is promoted by silencing MITF. Strikingly, MITF has significantly inverse correlations with the expression of its downstream genes in lung adenocarcinoma. In summary, we demonstrate the suppressive role of MITF in lung cancer progression, which is opposite to the canonical oncogenic function of MITF in melanoma.

A rank-based marker selection method for high throughput scRNA-seq data

BackgroundHigh throughput microfluidic protocols in single cell RNA sequencing (scRNA-seq) collect mRNA counts from up to one million individual cells in a single experiment; this enables high resolution studies of rare cell types and cell development pathways. Determining small sets of genetic markers that can identify specific cell populations is thus one of the major objectives of computational analysis of mRNA counts data. Many tools have been developed for marker selection on single cell data; most of them, however, are based on complex statistical models and handle the multi-class case in an ad-hoc manner.ResultsWe introduce RankCorr, a fast method with strong mathematical underpinnings that performs multi-class marker selection in an informed manner. RankCorr proceeds by ranking the mRNA counts data before linearly separating the ranked data using a small number of genes. The step of ranking is intuitively natural for scRNA-seq data and provides a non-parametric method for analyzing count data. In addition, we present several performance measures for evaluating the quality of a set of markers when there is no known ground truth. Using these metrics, we compare the performance of RankCorr to a variety of other marker selection methods on an assortment of experimental and synthetic data sets that range in size from several thousand to one million cells.ConclusionsAccording to the metrics introduced in this work, RankCorr is consistently one of most optimal marker selection methods on scRNA-seq data. Most methods show similar overall performance, however; thus, the speed of the algorithm is the most important consideration for large data sets (and comparing the markers selected by several methods can be fruitful). RankCorr is fast enough to easily handle the largest data sets and, as such, it is a useful tool to add into computational pipelines when dealing with high throughput scRNA-seq data. RankCorr software is available for download at https://github.com/ahsv/RankCorrwith extensive documentation.

Dynamical modeling predicts an inflammation-inducible CXCR7+ B cell precursor with potential implications in lymphoid blockage pathologies.

BackgroundThe blockage at the early B lymphoid cell development pathway within the bone marrow is tightly associated with hematopoietic and immune diseases, where the disruption of basal regulatory networks prevents the continuous replenishment of functional B cells. Dynamic computational models may be instrumental for the comprehensive understanding of mechanisms underlying complex differentiation processes and provide novel prediction/intervention platforms to reinvigorate the system.MethodsBy reconstructing a three-module regulatory network including genetic transcription, intracellular transduction, and microenvironment communication, we have investigated the early B lineage cell fate decisions in normal and pathological settings. The early B cell differentiation network was simulated as a Boolean model and then transformed, using fuzzy logic, to a continuous model. We tested null and overexpression mutants to analyze the emergent behavior of the network. Due to its importance in inflammation, we investigated the effect of NFkB induction at different early B cell differentiation stages.ResultsWhile the exhaustive synchronous and asynchronous simulation of the early B cell regulatory network (eBCRN) reproduced the configurations of the hematopoietic progenitors and early B lymphoid precursors of the pathway, its simulation as a continuous model with fuzzy logics suggested a transient IL-7R+ ProB-to-Pre-B subset expressing pre-BCR and a series of dominant B-cell transcriptional factors. This conspicuous differentiating cell population up-regulated CXCR7 and reduced CXCR4 and FoxO1 expression levels. Strikingly, constant but intermediate NFkB signaling at specific B cell differentiation stages allowed stabilization of an aberrant CXCR7+ pre-B like phenotype with apparent affinity to proliferative signals, while under constitutive overactivation of NFkB, such cell phenotype was aberrantly exacerbated from the earliest stage of common lymphoid progenitors. Our mutant models revealed an abnormal delay in the BCR assembly upon NFkB activation, concomitant to sustained Flt3 signaling, down-regulation of Ebf1, Irf4 and Pax5 genes transcription, and reduced Ig recombination, pointing to a potential lineage commitment blockage.DiscussionFor the first time, an inducible CXCR7hi B cell precursor endowed with the potential capability of shifting central lymphoid niches, is inferred by computational modeling. Its phenotype is compatible with that of leukemia-initiating cells and might be the foundation that bridges inflammation with blockage-related malignancies and a wide range of immunological diseases. Besides the predicted differentiation impairment, inflammation-inducible phenotypes open the possibility of newly formed niches colonized by the reported precursor. Thus, emergent bone marrow ecosystems are predicted following a pro-inflammatory induction, that may lead to hematopoietic instability associated to blockage pathologies.

In silico identification of common and specific signatures in coronary heart diseases.

Coronary heart disease (CHD) is on the increase in developing countries, where lifestyle choices such as smoking, bad diet, and no exercise contribute and increase the incidence of high blood pressure and high cholesterol levels to cause CHD. Through utilization of a biomarker-based approach for developing interventions, the aim of the study was to identify differentially expressed genes (DEGs) and their association and impact on various bio-targets. The microarray datasets of both healthy and CHD patients were analyzed to identify the DEGs and their interactions using Gene Ontology, PANTHER, Reactome, and STRING (for the possible associated genes with multiple targets). Our data mining approach suggests that the DEGs were associated with molecular functions, including protein binding (75%) and catalytic activity (56%); biological processes such as cellular process (83%), biological regulation (57%), and metabolic process (44%); and cellular components such as cell (65%) and organelle (58%); as well as other associations including apoptosis, inflammatory, cell development and metabolic pathways. The molecular functions were further analyzed, and protein binding in particular was analyzed using network analysis to determine whether there was a clear association with CHD and disease. The ingenuity pathway analysis revealed pathways related to cell cholesterol biosynthesis, the immune system including cytokinin signaling, in which, the understanding of DEGs is crucial to predict the advancement of preventive strategies. Results of the present study showed that, there is a need to validate the top DEGs to rule out their molecular mechanism in heart failure caused by CHD.

Exosomal MicroRNAs in Military Personnel with Mild Traumatic Brain Injury: Preliminary Results from the Chronic Effects of Neurotrauma Consortium Biomarker Discovery Project.

Chronic symptoms after mild traumatic brain injury (mTBI) are common among veterans and service members, and represent a significant source of morbidity, with those who sustain multiple mTBIs at greatest risk. Exosomal micro-RNAs (miRNAs), mediators of intercellular communication, may be involved in chronic TBI symptom persistence. Exosomal miRNA (exomiR) was extracted from 153 participants enrolled in the Chronic Effect of Neurotrauma Consortium (CENC) longitudinal study (no TBI, n = 35; ≥ 3 mTBIs (rTBI), n = 45; 1-2 mTBIs, n = 73). Analyses were performed with nCounter® Human miRNA Expression Panels and Ingenuity Pathway Analysis (IPA) for identification of gene networks associated with TBI. Generalized linear models were used to analyze the predictive value of exomiR dysregulation and remote neurobehavioral symptoms. Compared with controls, there were 17 dysregulated exomiRs in the entire mTBI group and 32 dysregulated exomiRs in the rTBI group. Two miRNAs, hsa-miR-139-5p and hsa-miR-18a-5p, were significantly differentially expressed in the rTBI and 1-2 mTBI groups. IPA analyses showed that these dysregulated exomiRs correlated with pathways of inflammatory regulation, neurological disease, and cell development. Within the rTBI group, exomiRs correlated with gene activity for hub-genes of tumor protein TP53, insulin-like growth factor 1 receptor, and transforming growth factor beta. TBI history and neurobehavioral symptom survey scores negatively and significantly correlated with hsa-miR-103a-3p expression. Participants with remote mTBI have distinct exomiR profiles, which are significantly linked to inflammatory and neuronal repair pathways. These profiles suggest that analysis of exosomal miRNA expression may provide novel insights into the underlying pathobiology of chronic TBI symptom persistence.

Developmental Programming: Sheep Granulosa and Theca Cell-Specific Transcriptional Regulation by Prenatal Testosterone.

Prenatal testosterone (T)-treated sheep, similar to polycystic ovarian syndrome women, manifest reduced cyclicity, functional hyperandrogenism, and polycystic ovary (PCO) morphology. The PCO morphology results from increased follicular recruitment and persistence of antral follicles, a consequence of reduced follicular growth and atresia, and is driven by cell-specific gene expression changes that are poorly understood. Therefore, using RNA sequencing, cell-specific transcriptional changes were assessed in laser capture microdissection isolated antral follicular granulosa and theca cells from age 21 months control and prenatal T-treated (100 mg intramuscular twice weekly from gestational day 30 to 90; term: 147 days) sheep. In controls, 3494 genes were differentially expressed between cell types with cell signaling, proliferation, extracellular matrix, immune, and tissue development genes enriched in theca; and mitochondrial, chromosomal, RNA, fatty acid, and cell cycle process genes enriched in granulosa cells. Prenatal T treatment 1) increased gene expression of transforming growth factor β receptor 1 and exosome component 9, and decreased BCL6 corepressor like 1, BCL9 like, and MAPK interacting serine/threonine kinase 2 in both cells, 2) induced differential expression of 92 genes that included increased mitochondrial, ribosome biogenesis, ribonucleoprotein, and ubiquitin, and decreased cell development and extracellular matrix-related pathways in granulosa cells, and 3) induced differential expression of 56 genes that included increased noncoding RNA processing, ribosome biogenesis, and mitochondrial matrix, and decreased transcription factor pathways in theca cells. These data indicate that follicular function is affected by genes involved in transforming growth factor signaling, extracellular matrix, mitochondria, epigenetics, and apoptosis both in a common as well as a cell-specific manner and suggest possible mechanistic pathways for prenatal T treatment-induced PCO morphology in sheep.

Immunoglobulin light chain κ precedes λ rearrangement in swine but a majority of λ+ B cells are generated earlier

Developmental pathways for B cell lymphogenesis are sufficiently known only in mice and humans. However, both of these species rearrange immunoglobulin heavy chains (IgH) before light chains (IgL) while IgL precedes IgH rearrangement in swine. We demonstrate here that this reversed order of rearrangements have some concealed consequences: (1) we confirmed that although IgLκ rearrangement is initial, most IgLλ+ B cells are generated earlier and before IgH rearrangements, while most IgLκ+ B cells later and after IgH rearrangements, (2) the second IgLκ rearrangement can occur after IgLλ rearrangement, (3) early formed B cells bear only single in-frame IgH rearrangements, (4) many IgLκ+ B cells carry IgLλ rearrangements that can be productive and occurring on both alleles in one cell, and (5) although VpreB and λ5 genes are present in swine, they are preferentially expressed in non-B cells. In summary, our findings reveal that swine use an alternative B cell developmental pathway as compared to mice and humans.

SITE-1 protease ablation in the osterix-lineage results in bone marrow dysplasia with neutrophilia coupled to osteopenia

SITE-1 protease ablation in the osterix-lineage results in bone marrow dysplasia with neutrophilia coupled to osteopenia

Exploration of Cell Development Pathways through High-Dimensional Single Cell Analysis in Trajectory Space.

SummaryHigh-dimensional single cell profiling coupled with computational modeling is emerging as a powerful tool to elucidate developmental programs directing cell lineages. We introduce tSpace, an algorithm based on the concept of “trajectory space”, in which cells are defined by their distance along nearest neighbor pathways to every other cell in a population. Graphical mapping of cells in trajectory space allows unsupervised reconstruction and exploration of complex developmental sequences. Applied to flow and mass cytometry data, the method faithfully reconstructs thymic T cell development and reveals development and trafficking regulation of tonsillar B cells. Applied to the single cell transcriptome of mouse intestine and C. elegans, the method recapitulates development from intestinal stem cells to specialized epithelial phenotypes more faithfully than existing algorithms and orders C. elegans cells concordantly to the associated embryonic time. tSpace profiling of complex populations is well suited for hypothesis generation in developing cell systems.

Role of lncRNAs in stem cell maintenance and differentiation.

Embryonic Stem cells are widely studied to elucidate the disease and developmental processes because of their capability to differentiate into cells of any lineage, Pervasive transcription is a distinct feature of all multicellular organisms and genomic elements such as enhancers and bidirectional or unidirectional promoters regulate these processes. Thousands of loci in each species produce a class of transcripts called noncoding RNAs (ncRNAs), that are well known for their influential regulatory roles in multiple biological processes including stem cell pluripotency and differentiation. The number of lncRNA species increases in more complex organisms highlighting the importance of RNA-based control in the evolution of multicellular organisms. Over the past decade, numerous studies have shed light on lncRNA biogenesis and functional significance in the cell and the organism. In this review, we focus primarily on lncRNAs affecting the stem cell state and developmental pathways.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

SummaryThe buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

A combinatorial library of biodegradable polyesters enables non-viral gene delivery to post-mitotic human stem cell-derived polarized RPE monolayers.

Safe and effective delivery of DNA to post-mitotic cells, especially highly differentiated cells, remains a challenge despite significant progress in the development of gene delivery tools. Biodegradable polymeric nanoparticles (NPs) offer an array of advantages for gene delivery over viral vectors due to improved safety, carrying capacity, ease of manufacture, and cell-type specificity. Here we demonstrate the use of a high-throughput screening (HTS) platform to synthesize and screen a library of 148 biodegradable polymeric nanoparticles, successfully identifying structures that enable efficient transfection of human pluripotent stem cell differentiated human retinal pigment epithelial (RPE) cells with minimal toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently than leading commercially available transfection reagents. Novel synthetic polymers are described that enable high efficacy non-viral gene delivery to hard-to-transfect polarized human RPE monolayers, enabling gene loss- and gain-of-function studies of cell signaling, developmental, and disease-related pathways. One new synthetic polymer in particular, 3,3'-iminobis(N,N-dimethylpropylamine)-end terminated poly(1,5-pentanediol diacrylate-co-3 amino-1-propanol) (5-3-J12), was found to form self-assembled nanoparticles when mixed with plasmid DNA that transfect a majority of these human post-mitotic cells with minimal cytotoxicity. The platform described here can be utilized as an enabling technology for gene transfer to human primary and stem cell-derived cells, which are often fragile and resistant to conventional gene transfer approaches.

Proteomics analysis of G protein-coupled receptor kinase 4-inhibited cellular growth of HEK293 cells

G protein-coupled receptor kinases (GRKs) are involved in a wide range of cellular physiology and pathological activities by specifically phosphorylating activated G protein-coupled receptors (GPCRs) to terminate GPCR signaling, or through regulating non-GPCR substrates. We recently reported that overexpression of GRK4 halts cell proliferation and induces cellular senescent phenotype in HEK293 cells. In this study, a quantitative proteomic assay was performed to analyze the protein profiles between HEK293 cells expressing and not expressing GRK4. Results revealed 39 upregulated and 59 downregulated differently expressed proteins (DEPs) in a total of 4124 identified proteins. Gene ontology (GO) annotation and functional enrichment revealed that the DEPs were related to metabolic processes regulated by the binding of these RNA/proteins under the biological processes. The Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis showed pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression are involved in. Immunoblotting validation of expression of six key target proteins, CALM1, STAT3, CDK1, CDK6, TOP2A, and GRK4, which speculatively maintain abnormal activity in the above pathways, was consistent with the results of proteomics analysis. Lastly, a biological phenotype assay confirmed that GRK4 promoted HEK293 cell growth blockage and G1/0 arrest. Taken together, this study identified some novel molecules that involve in GRK4 signaling and provided valuable information for further studying the mechanisms underlying GRK4-induced proliferative inhibition. SignificanceA quantitative proteomic assay was performed in HEK293 cells expressing and not expressing GRK4 39 upregulated and 59 downregulated differently expressed proteins (DEPs)were identified.DEPs involved in pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression.Biological phenotype assay confirmed that GRK4 prompted HEK293 cell growth blockage and G1/0 arrest.

Abstract 967: Single-cell transcriptomics uncovers clonal heterogeneity linked to drug response and cellular phenotype in adult brain tumor stem cells

Abstract Brain tumours remain a largely incurable disease, with glioblastoma multiforme (GBM) representing the predominant form of malignancy. GBMs contain a rare population of cells, termed brain tumour stem cells (BTSCs) that drive tumour growth and disease relapse. BTSCs can be isolated and expanded in culture from primary GBMs and retain self-renewal and proliferative capacity in vitro. However, the functional diversity within the BTSC fraction, as well as relationship to the tumour bulk remains unknown. To investigate genotypic and phenotypic states within the stem-like compartment of GBM, we profiled 54,442 patient-derived BTSCs isolated from 25 GBMs using single cell RNA-sequencing (10X Genomics Chromium). Within each BTSC population, we observed diverse transcriptional clonotypes ranging from 1-7 subpopulations per sample. BTSCs grown as adherent monolayers displayed greater heterogeneity (median 4 clusters, range 2-7) compared to sphere-based cultures (median 2 clusters, range 1-3). Across samples, clusters were commonly associated with cell cycling, glial cell development and DNA replication pathways. Within multiple BTSC cultures, we identified transcriptional subclones with variable stemness properties, such as expression of ASCL1, suggesting a hierarchy of differentiation within the tumor-initiating fraction of GBM. Additionally, inference of CNVs from scRNA-seq data revealed subclonal somatic CNVs within BTSCs correspond to distinct transcriptional clonotypes that may partially explain stemness phenotypes and the formation of subpopulations during culture. To nominate targeted and combination therapies against BTSC populations in GBM, we next mapped transcriptomic features of BTSC subpopulations to high-throughput drug-screening data from central nervous system cell lines aggregated by the PharmacoDB platform.This approach nominated FDA-approved therapies that may be effective against subpopulations within each BTSC culture. In some cases, subpopulations were predicted to be sensitive to different drugs, opening a path for testing combination therapies nominated from single cell RNA-seq in vitro. Overall, these data illustrate the extent to which BTSC cultures functionally recapitulate the stem fraction in bulk primary tumours, as well as define networks of self-renewal, therapeutic resistance and targetable vulnerabilities in glioblastoma. Ongoing efforts include single nuclei RNA-seq of primary GBM tumours to enable comparison of transcriptional programs of BTSC cultures with cellular populations found in primary tissues. Citation Format: Laura M. Richards, Owen Whitley, Florence M. Cavalli, Zhaleh Safikhani, Fiona Coutinho, H. Artee Luchman, Benjamin Haibe-Kains, Samuel Weiss, Peter Dirks, Gary Bader, Trevor J. Pugh. Single-cell transcriptomics uncovers clonal heterogeneity linked to drug response and cellular phenotype in adult brain tumor stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 967.