Progenitor Gene Research Articles

Abstract 210: Reprogramming Mouse Fibroblasts Into Cardiac Progenitor Cells Using Crispr Technique Targeting Endogenic Genes

Background and Objective: CRISPR tools that allow for precise manipulation of individual loci have not been used in generation of i nduced c ardiac p rogenitor c ells ( iCPC s). This study was designed to determine the feasibility and effectiveness of reprogramming fibroblasts into iCPC using CRISPR activation (CRISPRa) system. Methods: Tail-tip fibroblasts (TTFs) were isolated from Nkx2-5 cardiac enhancer GFP reporter mice. A gRNA pool targeting 17 progenitor genes was synthesized and transduced with dCas9-VP64 lentivirus into TTFs ( Fig.1A ). The phenotype of iCPCs was then characterized by immunostaining and FACS of progenitor markers. Finally, the cardiac-lineage differentiation potential of iCPCs was determined by immunostaining and electrophysiological assay under defined induction mediums. Results: iCPCs with GFP expression were formed in TTFs after transduction of CRISPRa targeting Isl1, Gata4, Baf60c, Tbx5 and Nkx2-5 (Fig.1B), while GFP was not activated by control virus. Cardiac progenitor markers were activated in iCPCs as shown by immunostaining (Fig.1C). The generation efficiency of Flk1-postive iCPCs induced by CRISPRa was ~60% as showed by FACS. iCPCs can be differentiated into cardiomyocytes as identified by immunostaining of cardiac-specific markers (Fig.1D). The iCPC-derived cardiomyocytes displayed spontaneous beating and showed cardiac action potentials (Fig.1E). Conclusion: The CRISPRa system is an efficient and specific way to generate iCPCs, which could provide a novel source of cells for cardiac regenerative medicine. Conversion of fibroblasts into cardiac progenitors poses a novel therapeutic option for repair of myocardial infarction.

KLF3 Mediates Epidermal Differentiation through the Epigenomic Writer CBP.

SummaryImpairments in the differentiation process can lead to skin diseases that can afflict ∼20% of the population. Thus, it is of utmost importance to understand the factors that promote the differentiation process. Here we identify the transcription factor KLF3 as a regulator of epidermal differentiation. Knockdown of KLF3 results in reduced differentiation gene expression and increased cell cycle gene expression. Over half of KLF3's genomic binding sites occur at active enhancers. KLF3 binds to active enhancers proximal to differentiation genes that are dependent upon KLF3 for expression. KLF3's genomic binding sites also highly overlaps with CBP, a histone acetyltransferase necessary for activating enhancers. Depletion of KLF3 causes reduced CBP localization at enhancers proximal to differentiation gene clusters, which leads to loss of enhancer activation but not priming. Our results suggest that KLF3 is necessary to recruit CBP to activate enhancers and drive epidermal differentiation gene expression.

Redundant and additive functions of the four Lef/Tcf transcription factors in lung epithelial progenitors

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.

Ancient genes can be served as pan-cancer diagnostic and prognostic biomarkers.

One important challenge for cancer is efficient biomarkers monitoring its formation and developments remain greatly limited. Although the accumulated big omics data provide great opportunities to the above purpose, the biomarkers identified by the data‐driven strategy often do not work well in new datasets, which is one of the main bottlenecks limiting their utilities. Given that atavistic phenotype is generally observed in cancer cells, we have been suggested that the activity of progenitor genes in tumour could serve as an efficient cancer biomarker. For doing so, we first curated 77 progenitor genes and then proposed a quantitative score to evaluate cancer progenitorness. After applying progenitorness score to ~ 22 000 samples, 33 types of cancers from 81 datasets, this method generally performs well in the diagnosis, prognosis and therapy monitoring of cancers. This study proposed a potential pan‐cancer biomarker and revealed a significant role of atavism in the formation and development of cancers.

The SQSTM1-NUP214 fusion protein interacts with Crm1, activates Hoxa and Meis1 genes, and drives leukemogenesis in mice.

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.

Dnmt3a loss and Idh2 neomorphic mutations mutually potentiate malignant hematopoiesis

AbstractMutations in the epigenetic regulators DNMT3A and IDH1/2 co-occur in patients with acute myeloid leukemia and lymphoma. In this study, these 2 epigenetic mutations cooperated to induce leukemia. Leukemia-initiating cells from Dnmt3a−/− mice that express an IDH2 neomorphic mutant have a megakaryocyte-erythroid progenitor–like immunophenotype, activate a stem-cell–like gene signature, and repress differentiated progenitor genes. We observed an epigenomic dysregulation with the gain of repressive H3K9 trimethylation and loss of H3K9 acetylation in diseased mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). HDAC inhibitors rapidly reversed the H3K9 methylation/acetylation imbalance in diseased mouse HSPCs while reducing the leukemia burden. In addition, using targeted metabolomic profiling for the first time in mouse leukemia models, we also showed that prostaglandin E2 is overproduced in double-mutant HSPCs, rendering them sensitive to prostaglandin synthesis inhibition. These data revealed that Dnmt3a and Idh2 mutations are synergistic events in leukemogenesis and that HSPCs carrying both mutations are sensitive to induced differentiation by the inhibition of both prostaglandin synthesis and HDAC, which may reveal new therapeutic opportunities for patients carrying IDH1/2 mutations.

Identification of FMR1-regulated molecular networks in human neurodevelopment.

RNA-binding proteins (RNA-BPs) play critical roles in development and disease to regulate gene expression. However, genome-wide identification of their targets in primary human cells has been challenging. Here, we applied a modified CLIP-seq strategy to identify genome-wide targets of the FMRP translational regulator 1 (FMR1), a brain-enriched RNA-BP, whose deficiency leads to Fragile X Syndrome (FXS), the most prevalent inherited intellectual disability. We identified FMR1 targets in human dorsal and ventral forebrain neural progenitors and excitatory and inhibitory neurons differentiated from human pluripotent stem cells. In parallel, we measured the transcriptomes of the same four cell types upon FMR1 gene deletion. We discovered that FMR1 preferentially binds long transcripts in human neural cells. FMR1 targets include genes unique to human neural cells and associated with clinical phenotypes of FXS and autism. Integrative network analysis using graph diffusion and multitask clustering of FMR1 CLIP-seq and transcriptional targets reveals critical pathways regulated by FMR1 in human neural development. Our results demonstrate that FMR1 regulates a common set of targets among different neural cell types but also operates in a cell type–specific manner targeting distinct sets of genes in human excitatory and inhibitory neural progenitors and neurons. By defining molecular subnetworks and validating specific high-priority genes, we identify novel components of the FMR1 regulation program. Our results provide new insights into gene regulation by a critical neuronal RNA-BP in human neurodevelopment.

Live bird markets as evolutionary epicentres of H9N2 low pathogenicity avian influenza viruses in Korea

ABSTRACT Live bird markets (LBMs) in Korea have been recognized as a reservoir, amplifier, and source of avian influenza viruses (AIVs); however, little was known about the role of LBMs in the epidemiology of AIVs in Korea until recently. Through 10 years of surveillance (2006–2016) we have isolated and sequenced H9N2 viruses in Korean LBMs. To understand how H9N2 evolves and spreads in Korea, a statistical Bayesian phylogenetic model was used. Phylogenetic analysis suggests that three separate introductions of progenitor gene pools, Korean domestic duck-origin and two wild aquatic bird-origin AIVs, contributed to the generation of the five genotypes of H9N2 viruses in Korea. Phylogenetic reconstruction of ecological states infer that the LBMs are where chickens become infected with the virus, with domestic ducks playing a major role in the transmission and evolution of the H9N2 viruses. Three increases in the genetic diversity of H9N2 viruses were observed and coincided with transitions in host species and the locations (domestic farm, LBM, slaughterhouse, and wild aquatic bird habitat) where the viruses were isolated, accompanying genetic reassortment. Following the introduction of a wild aquatic bird-origin AIVs in 2008, six genes of the Korean lineage H9N2 virus were replaced with genes originating from wild aquatic birds, and viruses with this new genotype became predominant in Korean LBMs.

PRDM1 controls the sequential activation of neural, neural crest and sensory progenitor determinants.

During early embryogenesis, the ectoderm is rapidly subdivided into neural, neural crest and sensory progenitors. How the onset of lineage determinants and the loss of pluripotency markers are temporally and spatially coordinated in vivo is still debated. Here, we identify a crucial role for the transcription factor PRDM1 in the orderly transition from epiblast to defined neural lineages in chick. PRDM1 is initially expressed broadly in the entire epiblast, but becomes gradually restricted as cell fates are specified. We find that PRDM1 is required for the loss of some pluripotency markers and the onset of neural, neural crest and sensory progenitor specifier genes. PRDM1 directly activates their expression by binding to their promoter regions and recruiting the histone demethylase Kdm4a to remove repressive histone marks. However, once neural lineage determinants become expressed, they in turn repress PRDM1, whereas prolonged PRDM1 expression inhibits neural, neural crest and sensory progenitor genes, suggesting that its downregulation is necessary for cells to maintain their identity. Therefore, PRDM1 plays multiple roles during ectodermal cell fate allocation.

Renewed proliferation in adult mouse cochlea and regeneration of hair cells

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.

Differential Inhibition of Sox10 Functions by Notch-Hes Pathway.

In the developing central nervous system, the terminal differentiation of oligodendrocytes (OLs) is regulated by both extrinsic and intrinsic factors. Recent studies have suggested that the Notch-Hes signaling pathway influences the maturation of oligodendrocytes in culture and during development. However, the specific Notch receptors and their downstream effectors Hes genes that are involved in oligodendrocyte maturation have not been investigated systematically. In this study, we showed that Notch1 and Notch3 are expressed in oligodendrocyte precursor cells (OPCs) during gliogenesis, and Hes5 is the major Notch downstream transcription factor that is transiently expressed in OPCs. Overexpression of Notch intracellular domain (NICD) and Hes5 proteins in embryonic chicken spinal cord suppressed both the endogenous and Sox10-induced Mbp gene expression. Unexpectedly, overexpression of NICD/Hes5 did not inhibit Sox10 induction of Olig2 expression and Myrf induced Mbp expression, suggesting the differential inhibitory effects of NICD/Hes5 signaling on Sox10 activation of myelin-related genes and early progenitor genes.

Marcks Is a Critical Downstream Mediator of IL-1-Driven AML Progression

Background: Whole-genome sequencing and expression studies have revealed significant heterogeneity in the molecular abnormalities driving AML. Selective inhibitors have been developed for many of the pathways influenced by these genetic alterations, but successful translation of these agents into the clinic is limited by both disease heterogeneity and drug resistance. Targeting of inflammatory pathways to block leukemia progression and eliminate leukemic clones is an emerging concept in AML therapy. We have shown that elevated levels of pro-inflammatory cytokine, interleukin-1 (IL-1), in AML microenvironment enhances the growth of leukemic progenitors in variety of genetic subtypes while inhibiting the normal progenitors' growth. To reveal molecular mechanisms underlying such paradoxical effect, we performed RNA-seq analysis on AML and healthy progenitors post IL-1 stimulation. We found myristoylated alanine-rich C-kinase substrate (MARCKS) is one of the most differentially expressed genes in AML progenitors compared to healthy progenitors. MARCKS is a major substrate of protein kinase C, and plays a crucial role in cell survival, migration, and cell cycle progression. Increased MARCKS expression promotes metastasis in solid tumors and inhibiting its activation is being proposed as a therapeutic strategy. However, its role in AML has not yet been investigated. Here, we show a crucial role of MARCKS activation in IL-1-mediated leukemia progression. Method and Results: Using the RNA-seq gene expression data of 451 primary AML patient samples (Tyner et al., Nature 2018), we tested the correlation of MARCKS with IL1R1 receptor expression in AML primary samples and found it to be positively correlated (r = 0.45, p < 0.0001). The correlation was regardless of sex, age, and mutation status. Using q-PCR and western blot analysis, we showed that MARCKS expression, protein level, and its activation (phosphorylation) are elevated in AML samples at basal level and after IL-1 stimulation when compared to the healthy progenitors (~3 fold change). These results validated our transcriptome data and suggested an important role for MARCKS in IL-1-mediated AML progression. To identify the functional significance of MARCKS in AML, we used two independent doxycycline inducible shRNAs to knockdown MARCKS in AML cell lines (MOLM-14 and THP-1). Our data show that MARCKS depletion in AML cells reduces the cell viability overtime to 40%, cell growth to 4 fold, and colony formation ability to 2 fold. Mechanistically, the knockdown of MARCKS in AML cells decreased SKP2 and increased p27 protein levels, suggesting MARCKS regulates cell cycle progression in these cells. We xenografted MOLM-14 cells expressing MARCKS shRNA into NSG mice by tail vein injections and induced the knockdown in vivo by feeding mice doxycycline containing chow. The bone marrow and spleen cells were analyzed by flow cytometry for human and mouse cell markers approximately 3 weeks post-treatment. We observed that the knockdown of MARCKS decreased the leukemia burden in xenograft model as observed by ~80% reduction in human leukemia cells in the bone marrow, ~40% reduction in human leukemia cells in spleen, and ~50% reduction in spleen size compared to the controls, suggesting MARCKS has a critical role in leukemia progression. Conclusion: MARCKS is over-expressed and -activated in various AML genetic subtypes. IL-1 stimulation of AML progenitors increases MARCKS phosphorylation. MARCKS promotes AML progression by increasing the cellular growth, survival, and cell cycle progression of leukemic cells. These results suggest that MARCKS may serve as marker for IL-1 mediated inflammatory stress and offers a route for new targeted therapy. Disclosures No relevant conflicts of interest to declare.

The SQSTM1-NUP214 Fusion Protein Cooperates with Crm1 to Activate Hoxa Genes and Drives Leukemogenesis in Mice

Background: The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the NUP phenylalanine-glycine (FG) repeat motifs that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we studied the SQSTM1-NUP214 fusion. Methods: We synthesized a SQSTM1-NUP214 fusion protein which retains only a short C-terminal portion of NUP214 containing FG motifs that mediate interaction with Crm1, and then introduced point mutations targeting these FG motifs (SQSTM1-NUP214FGmut). We compared the activity of these two fusion proteins using co-immunoprecipitation with CRM1, methylcellulose colony assays, murine transplantation, RT-qPCR, and chromatin immunoprecipitation. Results: We found that the ability of the SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Further, mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was greatly diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice (6/6), whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia (0/7). These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impairing the interaction of SQSTM1-NUP214 with Crm1 reduced binding of the fusion protein to Hoxa and Meis1 loci. Conclusions: These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes. Disclosures No relevant conflicts of interest to declare.

Single-Cell RNA-Seq to Assess Differential Responses to Tnfα in Human Hematopoietic Stem and Progenitor Cells in Myeloproliferative Neoplasm

Somatic mutations in hematopoietic stem and progenitor cells (HSPCs) leading to constitutive activation of thrombopoietin receptor signaling result in myeloproliferative neoplasms (MPN). The most common mutation found in MPN patients occurs in the Janus kinase 2 gene (JAK2V617F). We have previously found that JAK2V617F hematopoietic progenitors are resistant to tumor necrosis factor alpha (TNFα), however this mechanism is not well defined. We hypothesize that resistance to TNFα in JAK2V617F hematopoietic stem and progenitors is a driver of the competitive advantage over non-malignant clones. Here, we used droplet-based single-cell RNA-sequencing to investigate transcriptional profiling in primary human HSPCs. First, we harvested white blood cells from fresh bone marrow aspirates from one MPN patient (Polycythemia Vera with 71% JAK2V617F allele burden) as well as one unaffected individual then sorted Lin-/CD34+/CD38- hematopoietic progenitors. Immediately following sorting, half of the cells were stimulated with TNFα for 4 hours while the other half of cells were used as unstimulated controls. We then utilized the 10X Chromium platform to generate single-cell droplets for the 8,129 total cells from the unaffected individual and 33,299 total cells from the MPN patient. We ran alignment using the CellRanger pipeline then performed analysis using the Seurat package in R. Expression profiles of untreated HSPCs in both normal and MPN cells revealed high expression of genes involved in important pathways for hematopoiesis (Polycomb repressive complexes, chromatin regulation, the ubiquitin proteasome system etc.). Expression of CD34 was confirmed in both MPN and non-MPN cells, though CD34 expression was reduced following TNFα stimulation. Expression of stem (i.e. THY1, ITGA6) and progenitor (i.e. PTPRC) genes were detected within both individuals, which highlights the heterogeneity within Lineage-/CD34+/CD38- cells. Following stimulation with TNFα, we observed expression of genes in canonical pathways downstream of TNF including NF-κB, Mitogen-Activated Protein Kinase (MAPK), and Transforming Growth Factor Beta (TGFβ). Indeed, we observed a baseline level of expression of TGFβ-related genes in both normal and MPN cells. Upon inflammatory stimulation, normal HSPCs upregulated SMAD expression which are involved in the TGFβ pathway. Strikingly, we did not observe an increase in SMAD expression in MPN cells following TNF. This suggests a dampened response via the TGFβ pathway to TNF in MPN cells. Additionally, we found that TNF-stimulated HPSCs from the unaffected individual expressed canonical genes of the TNF pathway that encode for chemokines, cytokines, transcription factors and negative feedback regulators. In normal TNF-stimulated cells, we identified highly expressed genes involved in the caspase cascade, suggesting a robust apoptotic response in normal HPSCs. However, there was a lower expression of caspases in stimulated MPN cells, suggesting a dampened apoptotic response to TNF. One observation that was unique to TNF-stimulated cells from the MPN individual was the expression of glycoproteins involved in angiogenesis and platelet aggregation. Taken together, these data serve as a proof of principle for transcriptional profiling of primary human hematopoietic stem and progenitor cells and that this cell population rapidly and robustly alter their gene expression program upon TNFα stimulation. In conclusion, we show that HSPCs from an MPN patient exhibit a dampened response to TNF compared to normal HSPCs. Specifically, we observed a lower expression of genes involved with apoptosis and TGFβ signaling in MPN cells compared to normal cells following TNF stimulation. The finding of a dampened apoptotic response to TNF is consistent with the hypothesis that JAK2V617F cells gain a selective advantage over normal cells under inflammatory stress. To our knowledge, this is the first report of single-cell RNA-seq analysis on primary human HSPCs following FACS and inflammatory stimulation. Disclosures Fleischman: incyte: Speakers Bureau.

Induction of developmental toxicity and cardiotoxicity in zebrafish embryos/larvae by acetyl-11-keto-β-boswellic acid (AKBA) through oxidative stress

Acetyl-11-keto-β-boswellic acid (AKBA), a triterpenoid from Boswellia serrate, is regarded as an angiogenesis inhibitor. However, its toxicity is unknown. The aim of this study was to examine its developmental toxicity and cardiotoxicity. A developmental toxicity assay in zebrafish embryos/larvae from 4 to 96 hours post-fertilization (hpf) was performed and a cardiotoxicity assay was designed from 48 to 72 hpf. Markers of oxidative stress and related genes were selected to access the possible mechanisms. According to the results, AKBA induced pericardium edema, yolk-sac edema, abnormal melanin, spinal curvature, hatching inhibition and shortened body length. Further, increased SV-BA distance, reduced heart rate, increased pericardium area and decreased blood flow velocity were detected in AKBA treated groups. The inhibition of cardiac progenitor gene expression, such as Nkx2.5 and Gata4, may be related to cardiotoxicity. The activities of antioxidant enzymes were decreased and the content of MDA was increased. In addition, AKBA treatment decreased the expression levels of Mn-Sod, Cat, and Gpx. These results suggested that AKBA induced developmental toxicity and cardiotoxicity through oxidative stress. As far as we know, this is the first report on the toxicity of AKBA. It reminds us to pay attention to developmental toxicity and cardiotoxicity of AKBA.

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA.

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.

Single-Cell Analysis Reveals Regulatory Gene Expression Dynamics Leading to Lineage Commitment in Early T Cell Development.

Intrathymic Tcell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing Tcell genes, but the underlying steps have remained obscure. Single-cellprofiling was used to define the order of regulatory changes, employing single-cell RNA sequencing (scRNA-seq) for full-transcriptome analysis, plus sequential multiplexed single-molecule fluorescent in situ hybridization (seqFISH) to quantitate functionally important transcripts in intrathymic precursors. Single-cell cloning verified high Tcell precursor frequency among theimmunophenotypically defined "early Tcell precursor" (ETP) population; a discrete committed granulocyte precursor subset was also distinguished. We established regulatory phenotypes of sequential ETP subsets, confirmed initial co-expression of progenitor with Tcell specification genes, defined stage-specific relationships between cell cycle and differentiation, and generated a pseudotime model from ETP to T lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to Tcell specification.

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of γ-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Mitogenic and progenitor gene programmes in single pilocytic astrocytoma cells

Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) programme that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway.

1014 - THE PRC2 CO-FACTOR JARID2 IS A KEY REGULATOR OF NORMAL AND NEOPLASTIC HEMATOPOIESIS

The property of self-renewal sustains the hematopoietic stem cell (HSC) pool for the lifetime of an organism. While tremendous progress has been made into understanding specific genes and pathways that regulate HSC self-renewal, the molecular mechanisms by which self-renewal is repressed in downstream hematopoietic progenitors is largely unknown. Understanding the mechanisms that restrict self-renewal potential in hematopoietic progenitors may allow augmentation of limited sources of HSCs to improve bone marrow transplantation, or enhance self-renewal potential of progenitor cells in diseases associated with HSC depletion such as aplastic anemia. Polycomb repressive complexes are epigenetic regulators that control multiple aspects of stem cell fate. PRC2 is responsible for the establishment of repressive chromatin mark H3K27me3. While the importance of core PRC2 components in hematopoiesis is established, the function of PRC2 co-factors remains less defined. We recently identified a crucial role for the PRC2 co-factor JARID2 in normal and malignant hematopoiesis. We show that JARID2 is required for epigenetic repression of self-renewal genes in multipotent progenitors (MPPs). Loss of JARID2 leads to acquisition of self-renewal in MPPs, associated with de-repression of self-renewal genes. In malignant hematopoiesis, deletion of JARID2 lead to accelerated development of MPN or transformation to sAML, depending on the genetic and development context. Our data demonstrate that Jarid2 restricts self-renewal capacity in multipotent progenitors and is a tumor suppressor in chronic myeloid neoplasms. Ongoing studies aim to define how JARID2 expression is regulated in normal development and hematopoietic transformation, how JARID2/PRC2 target specificity is achieved in hematopoietic progenitor cells, and if JARID2 has non-canonical (PRC2-indpendent) functions in hematopoiesis.