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Primary NEAT RNA forms sharp foci in HSCs and progenitors. Confocal microscopy analysis of Neat1_1 and Neat1_2 using FISH, in representative primary cell types through hematopoiesis, HSC, MPP, CMP, GMP, MEP, granulocyte, B cell. Neat1_1 (yellow) and Neat1_2 (red) are superimposed over nuclei stained with DAPI (blue). Scale bar denotes 10 μ.m.
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Hematopoietic Stem Cells (HSCs) generate blood and immune cells through a hierarchical process of differentiation. Genes that regulate this process are of great interest for understanding normal and also malignant hematopoiesis. Surprisingly, however, very little is known about long-non-coding RNAs (lncRNA) in HSCs. Neat1 is a lncRNA that plays a m...
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... Among the top 20 most upregulated genes in the delayed aging clusters ( Fig. 2A), many are transcriptional regulators, including Hlf, Mecom, Nfat5, and Mllt3, and long non-coding RNAs, including Malat1, Neat1 and Linc-pint [45][46][47][48][49][50][51]. In particular, the long non-coding RNA Linc-pint is associated with healthy aging in mice, and knockout mice exhibit signs of premature aging in multiple tissues [52]. ...
The age-associated decline in immunity manifests as imbalanced adaptive and innate immune cells, which originate from the aging of the stem cells that sustain their regeneration. Aging variation across individuals is well recognized, but its mechanism remains unclear. Here, we used high-throughput single-cell technologies to compare mice of the same chronological age that exhibited early or delayed immune aging phenotypes. We found that some hematopoietic stem cells (HSCs) in early aging mice upregulated genes related to aging, myeloid differentiation, and stem cell proliferation. Delayed aging was instead associated with genes involved in stem cell regulation and the response to external signals. These molecular changes align with shifts in HSC function. We found that the lineage biases of 30% to 40% of the HSC clones shifted with age. Moreover, their lineage biases shifted in opposite directions in mice exhibiting an early or delayed aging phenotype. In early aging mice, the HSC lineage bias shifted toward the myeloid lineage, driving the aging phenotype. In delayed aging mice, HSC lineage bias shifted toward the lymphoid lineage, effectively counteracting aging progression. Furthermore, the anti-aging HSC clones did not increase lymphoid production but instead decreased myeloid production. Additionally, we systematically quantified the frequency of various changes in HSC differentiation and their roles in driving the immune aging phenotype. Taken together, our findings suggest that temporal variation in the aging of immune cell regeneration among individuals primarily arises from differences in the myelopoiesis of a distinct subset of HSCs. Therefore, interventions to delay aging may be possible by targeting a subset of stem cells.
... NONO displays aggregated clusters throughout hematopoiesis, including B-cell lymphopoiesis, 36 which indicates that NONO might be a potential regulator of the B-cell development and function. In the current study, we generated mice with global deletion of NONO. ...
... As for differentiated hematopoietic cells, B cells maintain relatively higher expression levels of NONO. 36 Our present study demonstrated that NONO is expressed throughout the B-cell development stages and plays an import role in B cell development. Both male and female NONO deficiency impaired early B-cell development at the pro-to pre-B-cell transition stage. ...
The paraspeckle protein NONO is a multifunctional nuclear protein participating in the regulation of transcriptional regulation, mRNA splicing and DNA repair. However, whether NONO plays a role in lymphopoiesis is not known. In this study, we generated mice with global deletion of NONO and bone marrow (BM) chimeric mice in which NONO is deleted in all of mature B cells. We found that the global deletion of NONO in mice did not affect T‐cell development but impaired early B‐cell development in BM at pro‐ to pre‐B‐cell transition stage and B‐cell maturation in the spleen. Studies of BM chimeric mice demonstrated that the impaired B‐cell development in NONO‐deficient mice is B‐cell‐intrinsic. NONO‐deficient B cells displayed normal BCR‐induced cell proliferation but increased BCR‐induced cell apoptosis. Moreover, we found that NONO deficiency impaired BCR‐induced activation of ERK, AKT, and NF‐κB pathways in B cells, and altered BCR‐induced gene expression profile. Thus, NONO plays a critical role in B‐cell development and BCR‐induced B‐cell activation.
... Interestingly, we also found several lncRNAs, H19, NEAT1 and MEG3 which were crucial for normal and malignant hematopoiesis were up-regulated by ingenol. For instance, Nuclear paraspeckle assembly transcript 1 (NEAT1) was highly expressed in HSC, progenitors and immune cells, which is vital for hematopoietic differentiation [67]. NEAT1 knockdown decreases MK differentiation accompanied with decreased CD41/CD42b expression, platelet-like particle (PLP) activity, and IL-8 level in Meg-01 cells [34]. ...
Thrombocytopenia, a most common complication of radiotherapy and chemotherapy, is an important cause of morbidity and mortality in cancer patients. However, there are still no approved agents for the treatment of radiation- and chemotherapy-induced thrombocytopenia (RIT and CIT, respectively). In this study, a drug screening model for predicting compounds with activity in promoting megakaryocyte (MK) differentiation and platelet production was established based on machine learning (ML), and a natural product ingenol was predicted as a potential active compound. Then, in vitro experiments showed that ingenol significantly promoted MK differentiation in K562 and HEL cells. Furthermore, a RIT mice model and c-MPL knock-out (c-MPL-/-) mice constructed by CRISPR/Cas9 technology were used to assess the therapeutic action of ingenol on thrombocytopenia. The results showed that ingenol accelerated megakaryopoiesis and thrombopoiesis both in RIT mice and c-MPL-/- mice. Next, RNA-sequencing (RNA-seq) was carried out to analyze the gene expression profile induced by ingenol during MK differentiation. Finally, through experimental verifications, we demonstrated that the activation of PI3K/Akt signaling pathway was involved in ingenol-induced MK differentiation. Blocking PI3K/Akt signaling pathway abolished the promotion of ingenol on MK differentiation. Nevertheless, inhibition of TPO/c-MPL signaling pathway could not suppress ingenol-induced MK differentiation. In conclusion, our study builds a drug screening model to discover active compounds against thrombocytopenia, reveals the critical roles of ingenol in promoting MK differentiation and platelet production, and provides a promising avenue for the treatment of RIT.
... According to OpenArray results, LINC01268, HOXB-AS3, MEG3 and TCL6 transcripts were selected since deregulated in CD34+ stem/progenitor cells; further lncRNAs have been selected for their associations with myeloid differentiation and deregulation in blood neoplasms [20,[27][28][29][30][31][32][33][34]. Plasma levels of thirteen lncRNAs were evaluated in 65 HDs and 143 MF patients (n = 97 PMF, n = 25 PET-MF, n = 21 PPV-MF) using real time qRT-PCR. ...
Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients’ cohort, it could be used for further studies to design an updated classification model for MF patients.
... For GMP we found reduced expression of long non-coding RNA Neat1 at all time points except day 12 (D3vsD0: logFC = − 0.73, p = 2.1 × 10 -7 ; D6vsD0: logFC = − 0.66, p = 1.3 × 10 -5 ; D9vsD0: logFC = − 0.71, p = 2.4 × 10 -10 ). Neat1 is highly expressed in hematopoietic stem and progenitor cells 24 and has previously been described as a tumor suppressor in hematological malignancies 25,26 . In summary, we found that most cell types had reduced gene expression during disease progression, contrasting with pro-and pre-B cells which showed the strongest initial response to disease, with more overexpressed than underexpressed genes. ...
The bone marrow microenvironment (BMM) plays a key role in leukemia progression, but its molecular complexity in pre-B cell acute lymphoblastic leukemia (B-ALL), the most common cancer in children, remains poorly understood. To gain further insight, we used single-cell RNA sequencing to characterize the kinetics of the murine BMM during B-ALL progression. Normal pro- and pre-B cells were found to be the most affected at the earliest stages of disease and this was associated with changes in expression of genes regulated by the AP1-transcription factor complex and regulatory factors NELFE, MYC and BCL11A. Granulocyte–macrophage progenitors show reduced expression of the tumor suppressor long non-coding RNA Neat1 and disruptions in the rate of transcription. Intercellular communication networks revealed monocyte-dendritic precursors to be consistently active during B-ALL progression, with enriched processes including cytokine-mediated signaling pathway, neutrophil-mediated immunity and regulation of cell migration and proliferation. In addition, we confirmed that the hematopoietic stem and progenitor cell compartment was perturbed during leukemogenesis. These findings extend our understanding of the complexity of changes and molecular interactions among the normal cells of the BMM during B-ALL progression.
... NEAT1 has been shown to be highly expressed in hematopoietic stem cells, progenitors, and immune cells. Terminally differentiated cell and post-mitotic cells such as granulocytes express less NEAT1, which suggests that NEAT1 may play an important role in hematopoietic differentiation (Fallik et al., 2017). However, the regulation of NEAT1 on megakaryocyte differentiation has not been reported so far. ...
Platelets are derived from megakaryocytes and play an important role in blood coagulation. By using high throughput sequencing, we have found that the long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) is abundant in platelets (GEO ID: 200097348). However, little is known about its role in regulating megakaryocyte differentiation and platelet activity. This study aims to clarify the effect of NEAT1 on MEG-01 differentiation and platelet-like particle (PLP) activity. NEAT1 in MEG-01 cells was knocked down by siRNA transfection. The adhesion of MEG-01 and PLP to collagen-coated coverslips was observed under a fluorescence microscope. Flow cytometry was used to investigate cell apoptosis, cell cycle, the levels of D41/CD42b on MEG-01 cells and CD62P on PLPs. Quantitative real-time polymerase chain reaction was used to detect NEAT1 and IL-8 expression levels. Western blot was used to measure the protein levels of Bcl-2, Bax, cleaved caspase-3, and IL-8. RNA-binding protein immunoprecipitation was used to detect the interaction of NEAT1 and splicing factor proline/glutamine-rich (SFPQ). Results showed that NEAT1 knockdown decreased the adhesion ability of thrombin-stimulated MEG-01 and PLP. The expression of CD62P on PLPs and CD41/CD42b on MEG-01 cells was inhibited by NEAT1 knockdown. In addition, NEAT1 knockdown inhibited cell apoptosis with increased Bcl2/Bax ratio and decreased cleaved caspase-3, and reduced the percentage of cells in the G0/G1 phase. Meanwhile, NEAT1 knockdown inhibited the expression of IL-8. A strong interaction of NEAT1 and SFPQ, a transcriptional repressor of IL-8, was identified. NEAT1 knockdown reduced the interaction between SFPQ and NEAT1.The results suggest that lncRNA NEAT1 knockdown decreases MEG-01 differentiation, PLP activity, and IL-8 level. The results also indicate that the regulation of NEAT1 on IL-8 may be realized via a direct interaction between NEAT1 and SFPQ.
... In NB4 cells, in vitro silencing of NEAT1 blocks ATRA-induced differentiation [93]. Consistently, NEAT1 is highly expressed in stem and progenitor cells and significantly reduced in granulocytes [94]. Recently, Zhao et al. also demonstrated in vitro that NEAT1 competitively binds miR-23a-3p to regulate SMC1A expression in AML that consequently inhibits AML cell proliferation and induces cell-cycle arrest and apoptosis [95]. ...
Acute Myeloid Leukemia (AML) is the most common form of leukemia in adults with an incidence of 4.3 per 100,000 cases per year. Historically, the identification of genetic alterations in AML focused on protein-coding genes to provide biomarkers and to understand the molecular complexity of AML. Despite these findings and because of the heterogeneity of this disease, questions as to the molecular mechanisms underlying AML development and progression remained unsolved. Recently, transcriptome-wide profiling approaches have uncovered a large family of long noncoding RNAs (lncRNAs). Larger than 200 nucleotides and with no apparent protein coding potential, lncRNAs could unveil a new set of players in AML development. Originally considered as dark matter, lncRNAs have critical roles to play in the different steps of gene expression and thus affect cellular homeostasis including proliferation, survival, differentiation, migration or genomic stability. Consequently, lncRNAs are found to be differentially expressed in tumors, notably in AML, and linked to the transformation of healthy cells into leukemic cells. In this review, we aim to summarize the knowledge concerning lncRNAs functions and implications in AML, with a particular emphasis on their prognostic and therapeutic potential.
... The function of paraspeckles has been studied in recent years, defining them as a subnuclear organelle for the storage and processing of RNA and the retention of hyperedited messenger RNA, relating them to the stress response, circadian rhythm and viral infection; they have even been related to cell differentiation [13,18,29,49]. The histone locus body is a nuclear domain enriched in the transcription factors needed for the expression and processing of the pre-messenger RNA in replication-dependent histone genes in close proximity to Corpusculum convolutum. ...
Background:
Nuclear bodies (NB) are membrane-less subnuclear organelles that perform important functions in the cell, such as transcription, RNA splicing, processing and transport of ribosomal pre-RNA, epigenetic regulation, and others. The aim of the work was to analyse the classification of NB in the Terminologia Histologica (TH) and biological and bibliographical databases.
Materials and methods:
The semantic structure of the Nucleoplasm section in the TH was analysed and unsystematic bibliographical search was made in the PubMed, SciELO, EMBASE databases and European Bioinformatics Institute (EMBL-EBI) biology database to identify which structures are classified as NB.
Results:
It was found that the terms Corpusculum convolutum, Macula interchromatinea and Corpusculum PML are not correctly classified in the TH, since they are subordinated under the term Chromatinum and not under Corpusculum nucleare. The bibliography consulted showed that 100%, 92.6% and 81.5% of articles mentioned Corpusculum convolutum, Macula interchromatinea and Corpusculum PML, respectively as nuclear bodies.
Conclusions:
It is suggested to relocate the terms Corpusculum convolutum, Macula interchromatinea and Corpusculum PML with the name of Corpusculum nucleare and the incorporation of two new entities to the Histological Terminology according to the information collected: paraspeckles and histone locus body.
Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous and infiltrating IVD cell populations, as well as the potential of resident mesenchymal stem cells (MSCs) for tissue repair. Control and Injured (needle puncture) tail IVDs were extracted from 12 week old female C57BL/6 mice 7 days post injury and clustering analyses, gene ontology, and pseudotime trajectory analyses were used to determine transcriptomic divergences in the cells of the injured IVD, while immunofluorescence was utilized to determine mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that were IVD tissue specific, immune, or vascular cells. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18 ⁺ Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology of DEGs suggested that the most upregulated biological pathways were angiogenesis and T cell related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that cells were driven towards increased cell differentiation due to IVD injury in all IVD tissue clusters except for Krt18 ⁺ NP which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more IVD differentiated cells profiles with injury and localized distinctly within the IVD. This study strengthens the understanding of heterogeneous IVD cell populations response to injury and identifies targetable MSC populations for future IVD repair studies.
Lay Summary
The intervertebral disc (IVD) is a spinal joint that accumulates damage with age but has limited tissue repair capabilities. IVD damage progresses into degeneration, and IVD degeneration is a leading cause of lower back pain. There are no effective therapies to treat IVD degeneration, but understanding the cell populations that change and respond to injury will uncover targets to restore IVD function. Mesenchymal stem cells (MSCs) are cells within the IVD that can potentially replenish the cells lost after IVD damage. To identify the cell populations of the IVD and how they change with injury, we performed single cell RNA sequencing of IVD tissue 7 days post injury and analyzed the differences in gene regulation. We identified diverse cells populations such as IVD specific tissues, immune cells, vascular cells, and MSCs. We discovered the presence of Saa2 and Grem1 expressing MSCs that become less stem cell-like and express higher levels of IVD gene markers after injury. We also determined that Saa2 and Grem1 have slightly different expression patterns in IVD tissues, and this expression becomes reduced after injury. These MSCs could be used in future stem cell therapies to prevent IVD degeneration.
Abstract Figure
Graphical Abstract
Isolation of mouse coccygeal intervertebral discs for scRNASeq analyses
One goal of regenerative medicine is to rejuvenate tissues and extend lifespan by restoring the function of endogenous aged stem cells. However, evidence that somatic stem cells can be targeted in vivo to extend lifespan is still lacking. Here, we demonstrate that after a short systemic treatment with a specific inhibitor of the small RhoGTPase Cdc42 (CASIN), transplanting aged hematopoietic stem cells (HSCs) from treated mice is sufficient to extend the healthspan and lifespan of aged immunocompromised mice without additional treatment. In detail, we show that systemic CASIN treatment improves strength and endurance of aged mice by increasing the myogenic regenerative potential of aged skeletal muscle stem cells. Further, we show that CASIN modifies niche localization and H4K16ac polarity of HSCs in vivo. Single-cell profiling reveals changes in HSC transcriptome, which underlie enhanced lymphoid and regenerative capacity in serial transplantation assays. Overall, we provide proof-of-concept evidence that a short systemic treatment to decrease Cdc42 activity improves the regenerative capacity of different endogenous aged stem cells in vivo, and that rejuvenated HSCs exert a broad systemic effect sufficient to extend murine health- and lifespan.
