Nucleus Of Stem Cells Research Articles

Identification of a compound similar to triiodothyronine in the cells of higher plants

Iodothyronines play an important role in human and animal metabolism; however, their action in plant species is yet to be described. The known beneficial effects of medicinal plants on the function of the thyroid gland suggest the presence of compounds similar to thyroid hormones in plant cells. In this work, we aim to reveal the presence of 3,5,3'-triiodothyronine, the most active representative of iodothyronines, in the cells of higher plants. Using the method of immunoenzyme analysis, it was determined that the roots and stems of bean seedlings contain a compound interacting with a polyclonal serum to 3,5,3'-triiodothyronine. The identified compound is distributed unevenly within the cell: its concentration in the cell nuclei is 7-10 times higher than that in the cytoplasm. Along with bean plants, 8 randomly selected plants belonging to both dicotyledonous and monocotyledonous families were studied. In all the studied species, the presence of an analogue of 3,5,3'-triiodothyronine was determined using the method of immunoenzyme analysis. The use of exogenous iodothyronine - thyroxine (3,5,3',5'-tetraiodothyronine) - for the pre-sowing treatment of wheat seeds reliably improved their germination and led to a significant increase in the root-shoot mass ratio of five-day-old wheat seedlings under the soaking time of 6 and 12 hours. The treatment of wheat seeds with thyroxine led to a significant decrease in the concentration of the identified compound in the stem and root cell nuclei of five-day-old wheat seedlings. The revealed physiological and accumulation effects of exogeus 3,5,3',5' tetraiodothyronine suggest its involvement in gene regulation in the cells of higher plants, similarly to that in animal cells.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

A tube-source X-ray microtomography approach for quantitative 3D microscopy of optically challenging cell-cultured samples

Development and study of cell-cultured constructs, such as tissue-engineering scaffolds or organ-on-a-chip platforms require a comprehensive, representative view on the cells inside the used materials. However, common characteristics of biomedical materials, for example, in porous, fibrous, rough-surfaced, and composite materials, can severely disturb low-energy imaging. In order to image and quantify cell structures in optically challenging samples, we combined labeling, 3D X-ray imaging, and in silico processing into a methodological pipeline. Cell-structure images were acquired by a tube-source X-ray microtomography device and compared to optical references for assessing the visual and quantitative accuracy. The spatial coverage of the X-ray imaging was demonstrated by investigating stem-cell nuclei inside clinically relevant-sized tissue-engineering scaffolds (5x13 mm) that were difficult to examine with the optical methods. Our results highlight the potential of the readily available X-ray microtomography devices that can be used to thoroughly study relative large cell-cultured samples with microscopic 3D accuracy.

The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells

Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/β-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.

The low-expression programming of 11β-HSD2 mediates osteoporosis susceptibility induced by prenatal caffeine exposure in male offspring rats.

Prenatal caffeine exposure (PCE) can cause developmental toxicity of long bones in offspring, but the long-term effects and the underlying mechanism have not been fully clarified. Here, we investigated the effects of PCE peak bone mass accumulation and osteoporosis susceptibility in offspring and its intrauterine programming mechanism. Pregnant Wistar rats were administrated intragastrically with saline or caffeine (120 mg·kg-1 ·day-1 ) on gestational days 9-20. The serum and bone samples were collected from the fetal and postnatal offspring for bone mass, genes expression and corticosterone analysis. Then, rat bone marrow mesenchymal stem cells (BMSCs) were treated with corticosterone in vitro to confirm the molecular mechanism. PCE caused fetal bone dysplasia in male and female offspring. In adulthood, PCE reduced peak bone mass and increased osteoporosis susceptibility in male offspring but not in females. Meanwhile, PCE only decreased the H3K9ac and expression levels of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) before and after birth in the male offspring but not in the females. Moreover, the high level of corticosterone induced by PCE down-regulated the H3K9ac and expression levels of 11β-HSD2 through promoting glucocorticoid receptor (GR; NR3C1) into the nucleus of bone marrow mesenchymal stem cells (BMSCs) and recruiting histone deacetylase 11 (HDAC11) binding to 11β-HSD2 promoter region, which further enhanced the effect of corticosterone on suppressing osteogenic function of BMSCs. PCE caused osteoporosis susceptibility in male adult offspring, which attributed to the low-functional programming of 11β-HSD2 induced by corticosterone via GR/HDAC11 signalling.

Abstract 3784: The metabolic enzyme Hexokinase 2 localizes to the nucleus and regulates stemness in AML through a kinase independent mechanism

Abstract Leukemia stem cells are responsible for the initiation and recurrence of AML. Cell differentiation requires the coordination of metabolic state and gene expression programs. It is known that metabolic intermediates can serve as cofactors in epigenetic modifications in the nucleus. Whether metabolic enzymes can directly localize to the nucleus, influence gene expression and regulate stemness in AML is unknown. To identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells we analyzed stem and bulk fractions of 8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells in the CD34+CD38- fraction. Hexokinase 2 (HK2) was detected in the nuclear fraction of 8227 cells and nuclear HK2 was increased in 8227 stem cells compared to bulk cells. Conversely, the metabolic enzymes aconitase, citrate synthesase, enolase, fumarase, GAPDH, glucose-6-phosphate isomerase, phosphofructokinase, pyruvate kinase and succinate dehydrogenase A were not detectable in the nuclear fraction of either stem or bulk cells. We confirmed that HK2 was present in the nuclear fraction of OCI-AML2, NB4, HL60 and TEX AML cell lines and 7/9 primary patient samples. To determine whether nuclear HK2 was functionally important for AML stem cells, we overexpressed HK2 tagged with a nuclear localizing sequence (PAAKRVKLD). Over-expression of nuclear localized HK2 enhanced clonogenic growth and engraftment into immune deficient mice. Likewise, over-expression of nuclear localized HK2 blocked differentiation after treatment with all-trans retinoic acid (ATRA). Next, we investigated whether the kinase activity of HK2 was necessary for its nuclear effects on AML stemness and differentiation. We generated a kinase dead mutant of HK2 (D209A, D657A) tagged with a nuclear localizing signal (PAAKRVKLD). Over-expression of nuclear-localized kinase dead HK2 exerted a similar phenotype to overexpressed nuclear HK2 without manipulation of the kinase domain as it enhanced clonogenic growth and blocked cell differentiation after ATRA treatment. To understand the mechanism by which nuclear HK2 influenced AML stemness, we identified proteins that interacted with nuclear HK2 using proximity-dependent biotin labeling (BioID) and mass spectrometry. Proteins involved in DNA damage repair and chromatin remodeling were identified, including exonuclease 3′-5′ domain-containing 2 (EXD2), sirtuin 1 (SIRT1), E3 ubiquitin-protein ligase (URB5) and tyrosyl-DNA phosphodiesterase 2 (TDP2). Thus, our data suggest that nuclear HK2 influences DNA repair, which has been linked to stemness and differentiation. In support of this hypothesis, we identified a PAR binding motif in HK2 and over-expression of nuclear HK2 conferred resistance to the PARP inhibitor, olaparib. In summary, HK2 localizes to the nucleus in AML stem cells and maintains stemness. These effects are independent of its kinase activity. Rather, HK2 may regulate DNA damage repair through a PAR dependent mechanism. Thus, we have identified a new role for metabolic enzymes in the regulation of stemness and differentiation. Citation Format: Grace Egan, Geethu E. Thomas, Parasvi S. Patel, Jordan Chin, Aaron Botham, Rose Hurren, Neil MacLean, Razq Hakem, Aaron D. Schimmer. The metabolic enzyme Hexokinase 2 localizes to the nucleus and regulates stemness in AML through a kinase independent mechanism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3784.

Automatic three-dimensional segmentation of mouse embryonic stem cell nuclei by utilising multiple channels of confocal fluorescence images.

Time-lapse confocal fluorescence microscopy images from mouse embryonic stem cells (ESCs) carrying reporter genes, histone H2B-mCherry and Mvh-Venus, have been used to monitor dynamic changes in cellular/differentiation characteristics of live ESCs. Accurate cell nucleus segmentation is required to analyse the ESC dynamics and differentiation at a single cell resolution. Several methods used concavities on nucleus contours to segment overlapping cell nuclei. Our proposed method evaluates not only the concavities but also the size and shape of every 2D nucleus region to determine if any of the strait, extrusion, convexity and large diameter criteria is satisfied to segment overlapping nuclei inside the region. We then use a 3D segmentation method to reconstruct simple, convex, and reasonably sized 3D nuclei along the image stacking direction using the radius and centre of every segmented region in respective microscopy images.To avoid false concavities on nucleus boundaries, fluorescence images of the H2B-mCherry reporter are used for localisation of cell nuclei and Venus fluorescence images are used for determining the cell colony ranges. We use a series of image preprocessing procedures to remove noise outside and inside cell colonies, and in respective nuclei, and to smooth nucleus boundaries based on the colony ranges. We propose dynamic data structures to record every segmented nucleus region and solid in sets (volumes) of 3D confocal images. The experimental results show that the proposed image preprocessing method preserves the areas of mouse ESC nuclei on microscopy images and that the segmentation method effectively segment out every nucleus with a reasonable size and shape. All 3D nuclei in a set (volume) of confocal microscopy images can be accessed by the dynamic data structures for 3D reconstruction. The 3D nuclei in time-lapse confocal microscopy images can be tracked to calculate cell movement and proliferation in consecutive volumes for understanding the dynamics of the differentiation characteristics about ESCs. LAY DESCRIPTION: Embryonic stem cells (ESCs) are considered as an ideal source for basic cell biology study and producing medically useful cells in vitro.This study uses time-lapse confocal fluorescence microscopy images from mouse ESCs carrying reporter gene to monitor dynamic changes in cellular/differentiation characteristics of live ESCs. To automate analyses of ESC differentiation behaviours, accurate cell nucleus segmentation to distinguish respective cells are required. A series of image preprocessing procedures are implemented to remove noise in live-cell fluorescence images but yield overlapping cell nuclei. A segmentation method that evaluates boundary concavities and the size and shape of every nucleus is then used to determine if any of the strait, extrusion, convexity, large and local minimum diameter criteria satisfied to segment overlapping nuclei. We propose a dynamic data structure to record every newly segmented nucleus. The experimental results show that the proposed image preprocessing method preserves the areas of mouse ESC nuclei and that the segmentation method effectively detects overlapping nuclei. All segmented nuclei in confocal images can be accessed using the dynamic data structures to be visualised and manipulated for quantitative analyses of the ESC differentiation behaviours. The manipulation can be tracking of segmented 3D cell nuclei in time-lapse images to calculate their dynamics of differentiation characteristics.

Dynamical reorganization of the pluripotency transcription factors Oct4 and Sox2 during early differentiation of embryonic stem cells

Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more unspecific, interactions with chromatin. Our results reveal that differentiation cues trigger early changes of Oct4 and Sox2 nuclear distributions that also include modifications in TF-chromatin interactions. This dynamical reorganization precedes Oct4 and Sox2 downregulation and may contribute to modulate their function at early differentiation stages.

Chromatin condensation retains the osteogenic transcription factor, RUNX2, in the nucleus of human mesenchymal stem cells

Chromatin condensation retains the osteogenic transcription factor, RUNX2, in the nucleus of human mesenchymal stem cells

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.

Chromatin Compaction, Auxeticity, and the Epigenetic Landscape of Stem Cells

An unusual mechanical behavior in the nuclei of mouse stem cells may be linked to the compaction of chromatin (DNA and the proteins that bind to it), a step toward understanding how and why stem cells become other cell types.

Linking substrate and nucleus via actin cytoskeleton in pluripotency maintenance of mouse embryonic stem cells.

Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process.

CircRNA_0006393 promotes osteogenesis inglucocorticoid‑induced osteoporosis by sponging miR‑145‑5p and upregulating FOXO1.

Glucocorticoids are the most common cause of glucocorticoid‑induced osteoporosis(GIOP). Moreover, the role of circular RNAs (circRNAs) in the regulation of bone metabolism remains unclear. Therefore, in the present study, it was hypothesized that hsa_circ_0006393 may play an important role in GIOP. To investigate the role of circRNAs in GIOP, treatment with dexamethasone or transfection with a vector overexpressing hsa_circ_0006393 were performed using invitro cell and invivo mouse models. Reverse transcription‑quantitative PCR, fluorescence insitu hybridization and western blotting were performed to investigate the function of hsa_circ_0006393 invitro. In addition, the effects of hsa_circ_0006393 on osteogenesis were investigated. Dual‑energy X‑ray absorptiometry analysis was performed to examine the osteogenic potential of hsa_circ_0006393 invivo. Moreover, the mechanism underlying hsa_circ_0006393‑mediated bone metabolism regulation via the microRNA (miR)‑145‑5p/forkhead boxO1 (FOXO1) pathway was investigated. The present results suggested that the expression level of hsa_circ_0006393 was decreased in patients with GIOP. Furthermore, the overexpression of hsa_circ_0006393 increased the expression level of genes associated with osteogenesis. Moreover, hsa_circ_0006393 was identified to be localized mainly in the cytoplasm and nucleus of bone marrow mesenchymal stem cells. miR‑145‑5p was found to be directly targeted by hsa_circ_0006393. Collectively, hsa_circ_0006393 increases the expression levels of osteogenic genes during bone remodeling by sponging miR‑145‑5p and upregulating FOXO1.

Differential distribution of the epigenetic marker 5-hydroxymethylcytosine occurs in hair follicle stem cells during bulge activation.

Hair follicle (HF) cycling is dependent upon activation and differentiation of an epithelial subpopulation of cells with stem-like characteristics. These cells express cytokeratin 15 (CK15) and are sequestered within a specialized niche termed the follicular bulge. The pathways that mediate bulge activation are poorly understood, although growing evidence suggests a role for epigenetic events. Here we investigated murine and human HFs to determine whether a recently described epigenetic hydroxymethylation marker, 5-hmC, known to mediate cell growth and differentiation, may play a role in bulge activation. We found the bulge region of murine HFs to show variable 5-hmC distribution within the nuclei of CK15-positive stem cells during early anagen, a pattern that was not associated with resting stem cells of telogen follicles, which did not express 5-hmC. Moreover, during phases of early anagen that were induced in an organ culture model, spatial alterations in bulge stem cell 5-hmC reactivity, as assessed by dual labeling, were noted. These preliminary findings suggest that 5-hmC may play a dynamic role in bulge activation during anagen growth, and provide a foundation for further experimental inquiry into epigenomic regulation of HF stem cells.

Nucleus-localized adiponectin is survival gatekeeper through miR-214-mediated AIFM2 regulation.

Adiponectin secreted from adipocytes into plasma has anti-aging, anti-obesity and anti-inflammation effects. Here, we detected intracellular adiponectin localized in the nuclei of human and mouse pluripotent stem cells, mouse germ cells and some somatic cells. Nucleus-localized (Nu) adiponectin protein is characterized by an N-terminal truncated monomer form in a native state, compared with intact multimer forms of cytoplasm-localized (Cy) adiponectin protein. Doxycycline-induced over-expression of ADIPONECTIN caused cell death in human and mouse Nu-type pluripotent stem cells. Genome-wide gene expression analysis indicated that apoptosis by ADIPONECTIN over-expression was induced in accompany with upregulation of AIFM2 and MEG3. Upregulation of AIFM2 and MEG3 and down-regulation of miR-214-3p verified by qPCR analyses after ADIPONECTIN over-expression indicated that the MEG3/miR-214/AIFM2 pathway played a role in the apoptotic cell death of pluripotent cells. Adiponectin-induced cell death was rescued by the treatment with miR-214-3p mimic. Global data analysis shows that Nu adiponectin has a role in microRNA-mediated post-transcription regulation, cell-cell interactions and chromatin remodeling as a survival gatekeeper.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness.We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples.We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A).The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus.Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation.In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. [Display omitted] DisclosuresSchimmer:Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.

Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet.In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. DisclosuresNo relevant conflicts of interest to declare.

BICD1 mediates HIF1\u03b1 nuclear translocation in mesenchymal stem cells during hypoxia adaptation

Hypoxia inducible factor 1α (HIF1α) is a master regulator leading to metabolic adaptation, an essential physiological process to maintain the survival of stem cells under hypoxia. However, it is poorly understood how HIF1α translocates into the nucleus in stem cells under hypoxia. Here, we investigated the role of a motor adaptor protein Bicaudal D homolog 1 (BICD1) in dynein-mediated HIF1α nuclear translocation and the effect of BICD1 regulation on hypoxia adaptation and its therapeutic potential on human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). In our results, silencing of BICD1 but not BICD2 abolished HIF1α nuclear translocation and its activity. BICD1 overexpression further enhanced hypoxia-induced HIF1α nuclear translocation. Hypoxia stimulated direct bindings of HIF1α to BICD1 and the intermediate chain of dynein (Dynein IC), which was abolished by BICD1 silencing. Akt inhibition reduced the binding of BICD1 to HIF1α and nuclear translocation of HIF1α. Conversely, Akt activation or GSK3β silencing further enhanced the hypoxia-induced HIF1α nuclear translocation. Furthermore, BICD1 silencing abolished hypoxia-induced glycolytic reprogramming and increased mitochondrial ROS accumulation and apoptosis in UCB-MSCs under hypoxia. In the mouse skin wound healing model, the transplanted cell survival and skin wound healing capacities of hypoxia-pretreated UCB-MSCs were reduced by BICD1 silencing and further increased by GSK3β silencing. In conclusion, we demonstrated that BICD1-induced HIF1α nuclear translocation is critical for hypoxia adaptation, which determines the regenerative potential of UCB-MSCs.

Argonaute-miRNA Complexes Silence Target mRNAs in the Nucleus of Mammalian Stem Cells

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.

Development of a Novel Orthogonal Double Gradient for High‐Throughput Screening of Mesenchymal Stem Cells–Materials Interaction

AbstractSurface gradients provide a powerful platform to accelerate multiscale design by efficiently studying of material–cell interactions to ultimately enhance function of synthetic clinical biomaterials. Herein, a novel orthogonal double gradient is reported in which surface stiffness and wettability vary independently and continuously in perpendicular directions, providing unique combinations of stiffness and wettability over a broad range (stiffness: 6–89 MPa; water contact angle: 29°–90°). It is found that mesenchymal stem cell behavior is nonlinearly regulated by surface stiffness and wettability. These combined stiffness and wettability properties of a material significantly affect stem cell adhesion, spreading, nucleus size, and vinculin expression. This high‐throughput screening system enables elucidation of the relationships between biointerface properties and biological behavior, and thereby serves a potential tool for accelerating the development of high‐performance biomaterials.