scholarly journals Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

2020 ◽  
Author(s):  
Silvia Llonch ◽  
Montserrat Barragán ◽  
Paula Nieto ◽  
Anna Mallol ◽  
Marc Elosua-Bayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Chromosome Segregation ◽  
Maternal Age ◽  
Maturation Stage ◽  
P Value ◽  
Rna Seq ◽  
Transcript Levels ◽  
Human Oocytes ◽  
The Impact

AbstractStudy questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)?Summary answerWhile the oocytes’ transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age.Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32).Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2™ medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant.Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age.Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes.The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts.Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes.Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”.The authors have no conflict of interest to declare.

scholarly journals Single cell profiling of transcriptomic changes during in vitro maturation of human oocytes

2021 ◽  
Author(s):  
Hiroki Takeuchi ◽  
Mari Yamamoto ◽  
Megumi Fukui ◽  
Tadashi Maezawa ◽  
Mikiko Nishioka ◽  
...  
Keyword(s):  
Single Cell ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Maturation Stage ◽  
Developmental Potential ◽  
Human Oocytes ◽  
Transcriptomic Changes

Abstract In vitro maturation of human oocytes is widely used for infertility treatment. However, the success rate of maturation varies depending on patients and molecular mechanisms underlying successful maturation remain unclear. Especially, gene expression profiles of oocytes at each maturation stage need to be revealed to understand the differential developmental abilities of oocytes. Here, we show transcriptomes of human oocytes during in vitro maturation by single cell RNA-seq analyses. Hundreds of transcripts dynamically altered their expression, and we identify molecular pathways and upstream regulators that may govern oocyte maturation. Furthermore, oocytes that are delayed in their maturation show distinct transcriptomes. Finally, we reveal genes whose transcripts are enriched in each maturation stage and that can be used for selecting an oocyte with a high developmental potential. Taken together, our work uncovers transcriptomic changes during human oocyte maturation and provides a molecular insight into the differential developmental potential of each oocyte.

scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

DEFINING ELIGIBILITY CRITERIA FOR FUNDING POLICIES AROUND IN VITRO FERTILIZATION

2015 ◽  
Vol 31 (6) ◽  
pp. 426-433 ◽  
Author(s):  
Devidas Menon ◽  
Alexa A. Nardelli ◽  
Tarek Motan ◽  
Kristin Klein ◽  
Tania Stafinski
Keyword(s):  
Maternal Age ◽  
Smoking Status ◽  
Cochrane Collaboration ◽  
Assessment Tools ◽  
Paternal Age ◽  
Eligibility Criteria ◽  
Vitro Fertilization ◽  
Funding Policies ◽  
The Impact

Objectives: This review aims to assess the state of the science around the potential impact of certain patient characteristics on the safety and effectiveness of in vitro fertilization (IVF).Methods: Following Cochrane Collaboration guidelines and the PRISMA statement, a comprehensive systematic review of reviews and recent primary studies examining the impact of paternal age and maternal age, smoking, and body mass index (BMI) on the safety and effectiveness of IVF was performed. Papers, published between January 2007 and June 2014, were independently reviewed and critically appraised by two researchers using published quality assessment tools for reviews and primary studies. Due to heterogeneity across papers (different study designs and patient selection criteria), a qualitative analysis of extracted information was performed.Results: Seventeen papers (ten systematic reviews and seven primary studies) were included. They comprised evidence from retrospective observational studies in which maternal age, BMI, and smoking status were explored as part of secondary analyses of larger studies. The majority of papers found that the likelihood of achieving a pregnancy was lower among women who were >40 years, had a BMI ≥ 25 and smoked. Advanced maternal age and BMI were also associated with higher rates of preterm birth and low birth weight.Conclusions: Based on available evidence, it may be appropriate to consider “maternal age” and “morbid obesity” in public funding policies that aim to maximize the effectiveness of IVF. However, given inconsistencies in the effect of smoking across different pregnancy-related outcomes, support for incorporating it into funding conditions appears weak.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

P–182 The mechanism of mouse embryo hatching and the impact of laser drilling the zona pellucida: an RNA sequencing study

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Liu ◽  
C Jones ◽  
K Coward
Keyword(s):  
Treatment Group ◽  
Mouse Embryo ◽  
Mouse Embryos ◽  
Z Score ◽  
Rna Seq ◽  
Metabolism Pathway ◽  
Hatching Process ◽  
The Impact

Abstract Study question What is the mechanism of embryo hatching? Will laser-assisted zona pellucida (ZP) drilling alter the embryonic transcriptome? Summary answer Hatching is an ATP-dependent process. Hatching is also associated with Rho-mediated signaling. Laser-assisted ZP drilling might cause alternation in embryo metabolism. What is known already Embryo hatching is a vital process for early embryo development and implantation. Animal data suggests that hatching is the result of multiple factors, such as mechanical pressure, protease activation, and the regulation of maternal secretions. However, little is known about the regulatory signaling mechanisms and the molecules involved. In addition, despite the extensive use of laser-assisted ZP drilling in the clinic, the safety profile of this technique at molecular level is very sparse. The impact of this technique on the embryonic transcriptome has not been studied systematically. Study design, size, duration Eighty mouse embryos were randomly divided into a laser ZP drilling group (n = 40) and an untreated group (n = 40). After treatment, embryos were cultured in vitro for two days. Then, hatching blastocyst (n = 8) and pre-hatching blastocyst (n = 8) from the untreated group, and the hatching blastocyst from the treatment group (n = 8) were processed for RNA sequencing (RNA-seq). Participants/materials, setting, methods Cryopreserved 8-cell stage mouse embryos (B6C3F1 × B6D2F1) were thawed, and a laser was used to drill the embryo ZP in the treatment group. Next, the treated and untreated embryos were individually cultured in vitro to the E4.5 blastocyst stage. The resulting blastocysts were lysed individually and used for subsequent cDNA library preparation and RNA-seq. Following data quality control and alignment, the RNA-seq data were processed for differentially expressed gene analysis and downstream functional analysis. Main results and the role of chance According to the RNA-seq data, 275 differentially expressed genes (DEGs) (230 up-regulated and 45 down-regulated, adjusted P &lt; 0.05) were identified when comparing hatching and pre-hatching blastocysts in the control groups. Analysis suggested that the trophectoderm is the primary cell type involved in hatching, and revealed the potential molecules causing increased blastocyst hydrostatic pressure (Aqp3 and Cldn4). Functional enrichment analysis suggested that ATP metabolism and protein synthesis were activated in hatching blastocysts. DEGs were found to be significantly enriched in several gene ontology terms, particularly in terms of the organization of the cytoskeleton and actin polymerisation (P &lt; 0.0001). Furthermore, according to QIAGEN ingenuity pathway analysis results, Rho signaling was implicated in blastocyst hatching (Actb, Arpc2, Cfl1, Myl6, Pfn1, Rnd3, Septin9, z-score=2.65, P &lt; 0.0001). Moreover, the potential role of hormones (estrogen (z-score=2.24) and prolactin (z-score=2.4)) and growth factors (AGT (z-score=2.41) and FGF2 (z-score=2.213)) were implicated in the hatching process as indicated by the upstream regulator analysis. By comparing the transcriptome between laser-treated and untreated hatching blastocysts, 47 DEGs were identified (adjusted P &lt; 0.05) following laser-assisted ZP drilling. These genes were enriched in metabolism-related pathways (P &lt; 0.05), including the lipid metabolism pathway (Mvd, Mvk, Aacs, Gsk3a, Pik3c2a, Aldh9a1) and the xenobiotic metabolism pathway (Aldh18a1, Aldh9a1, Keap1, and Pik3c2a). Limitations, reasons for caution Findings in mouse embryos may not be fully representative of human embryos. Furthermore, the mechanism of hatching revealed here might only reflect the hatching process of embryos in vitro. Further studies are now necessary to confirm these findings in different conditions and species to determine their clinical significance. Wider implications of the findings: Our study profiled the mouse embryo transcriptome during in vitro hatching, identified potential key genes and mechanisms for future study. In addition, for the first time, we revealed the impact of laser-assisted ZP drilling on the transcriptome, this may help us to assess and improve the existing technique. Trial registration number Not applicable

Abstract A10: Using single-cell RNA sequencing to assess the impact of pancreatic oncogenic Kras on macrophage gene expression in vitro

2019 ◽  
Author(s):  
Katelyn Donahue ◽  
Yaqing Zhang ◽  
Veerin Sirihorachai ◽  
Stephanie The ◽  
Arvind Rao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cell Rna Sequencing ◽  
The Impact

scholarly journals Single cell multiomic analysis of T cell exhaustion in vitro

2019 ◽  
Author(s):  
Mirko Corselli ◽  
Suraj Saksena ◽  
Margaret Nakamoto ◽  
Woodrow E. Lomas ◽  
Ian Taylor ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
T Cell Exhaustion ◽  
Chronic Stimulation ◽  
Cell Exhaustion ◽  
Car T ◽  
The Impact

AbstractA key step in the clinical production of CAR-T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be chronically stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 39 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study – with unprecedented depth - how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naïve to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

scholarly journals Uncoupling p53 from an Embryonic Regulome Exhausts Quiescent CML Stem Cells through Inhibition of a HIF1alpha Molecular Program

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1541-1541
Author(s):  
Mary T Scott ◽  
Wei Liu ◽  
Rebecca Mitchell ◽  
Cassie Clarke ◽  
Hassan Almasoudi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Research Funding ◽  
Embryonic Stem ◽  
Rna Seq ◽  
Combination Drug ◽  
Human Cd34 ◽  
Molecular Features ◽  
Cd34 Cells

Abstract Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML). By integrating known ES regulatory circuitry with transcriptomics datasets, including deep single-cell RNA-seq profiling of 15,670 LSC from five patients with CML, we identified a core ESC regulome in the LSC containing 1243 genes. The significant majority of this regulome (1102 genes) was up-regulated in cycling LSC, whilst quiescent LSC showed up-regulation of a characteristic set of 101 genes, unique to cells with high ESC identity and with regulatory circuitry enriched for c-Myc and Nanog modules. Membership of the ESC regulome included the TP53 gene which was transcriptionally repressed and detected at a lower frequency in quiescent LSC compared to cycling ones (11.8% vs 43.6%). We also demonstrated that tyrosine kinase inhibitors (TKI) repress the ESC regulome and TP53 expression in LSC, suggesting that the regulome safeguards against high levels of TP53 expression, thus promoting survival of quiescent LSC in the presence of TKI. We hypothesized that overcoming the influence of the regulome on TP53 expression would provide an opportunity to eradicate quiescent LSC. To this end, we used an MDM2 inhibitor (MDM2i), RG7388 (idasanutlin) or RG7112, to stabilize the p53 protein, examining its potential in combination with nilotinib (NIL) to eradicate CML LSC in vitro and in vivo, with RG7388 being the most optimized and furthest in development. The combination of NIL plus MDM2i in vitro was more effective at targeted LSC from primary patient samples than NIL treatment alone, as evidenced by reduced CFC and LTC-IC outputs (p&lt;0.05, 0.01 respectively). Intriguingly, the combination of NIL plus MDM2i did not result in significant reductions in the number of LSC compared to NIL only, when we quantified them at the end of drug treatments in pre-clinical mouse models. Instead, we observed a functional decline of the LSC as evidenced by diminished engraftment potential in 2 o recipient mice (p&lt;0.05; SCLtTA x BCR-ABL1 transgenic model) or diminished colony-plating potential (p&lt;0.05). This was followed by near complete depletion of the LSC population (p&lt;0.05) 28 days after cessation of combination drug treatment (patient-derived xenografts/PDX in immunocompromised mice). In order to understand the molecular events underpinning these drug effects on LSC, we performed RNA-seq analysis of drug-treated CD34 + cells in vitro (bulk cells), or of human CD34 + cells obtained from PDX (single cell RNA-seq). CD34 + cells treated with NIL plus MDM2i in vitro showed evidence of increased p53 stabilization and activation of p53 target genes, and this was accompanied by repression of the ESC regulome beyond that normally observed with NIL only. Similarly, in PDX we observed increased repression of the ESC regulome in human CD34 + cells exposed to the combination of NIL plus MDM2i that included repression of HIF1alpha and a signature of genes required for cellular adaptations to hypoxia, and growth factor-mediated resistance to TKI therapy. Further, single cell analysis of differentiated human CD45 + cells from our PDX model, provided compelling evidence that acquisition of this repressive signature in the LSC, through combined NIL plus MDM2i treatment, re-wires them towards a basophilic fate, consistent with functional exhaustion of the LSC compartment. In conclusion, we have identified an ESC regulome in CML LSC and demonstrate that a combination of a TKI plus an MDM2i leads to p53 upregulation which antagonizes this regulome, providing a highly effective strategy to target near complete loss of functional LSC in pre-clinical models. Our study has revealed a new therapeutic paradigm to examine in other cancer stem cell populations that utilize ESC regulatory programs. Disclosures Higgins: Roche/Genentech: Current Employment, Current equity holder in publicly-traded company. Copland: Astellas: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Jazz: Honoraria, Speakers Bureau.

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