scholarly journals Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1

2021 ◽  
Author(s):  
Lu Song ◽  
Xinran Tian ◽  
Randy Schekman
Keyword(s):  
Cyclin D1 ◽  
Extracellular Vesicles ◽  
Neural Development ◽  
Neuronal Cells ◽  
Buoyant Density ◽  
Embryonic Stem ◽  
Neural Induction ◽  
Induction Effect ◽  
Differentiated Cells

Extracellular vesicles (EVs) that are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of extracellular vesicles that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation and mESCs differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mouse embryonic stem cells (mESCs). Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that extracellular vesicles regulate neural development through sorting of cyclin D1.

scholarly journals Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Lu Song ◽  
Xinran Tian ◽  
Randy Schekman
Keyword(s):  
Stem Cells ◽  
Cyclin D1 ◽  
Extracellular Vesicles ◽  
Neural Development ◽  
Neuronal Cells ◽  
Buoyant Density ◽  
Embryonic Stem ◽  
Neural Induction ◽  
Induction Effect

Extracellular vesicles (EVs) are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of EVs that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation, and mouse embryonic stem cells (mESCs) differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mESCs. Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that EVs regulate neural development through sorting of cyclin D1.

scholarly journals Small extracellular vesicles encapsulating CCL2 from activated astrocytes induce microglial activation and neuronal apoptosis after traumatic spinal cord injury

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuluo Rong ◽  
Chengyue Ji ◽  
Zhuanghui Wang ◽  
Xuhui Ge ◽  
Jiaxing Wang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Extracellular Vesicles ◽  
Neuronal Apoptosis ◽  
Microglial Activation ◽  
Neuronal Cells ◽  
Cell Communication ◽  
Bioactive Lipids ◽  
Cord Injury

Abstract Background Spinal cord injury (SCI) is a severe traumatic disease which causes high disability and mortality rates. The molecular pathological features after spinal cord injury mainly involve the inflammatory response, microglial and neuronal apoptosis, abnormal proliferation of astrocytes, and the formation of glial scars. However, the microenvironmental changes after spinal cord injury are complex, and the interactions between glial cells and nerve cells remain unclear. Small extracellular vesicles (sEVs) may play a key role in cell communication by transporting RNA, proteins, and bioactive lipids between cells. Few studies have examined the intercellular communication of astrocytes through sEVs after SCI. The inflammatory signal released from astrocytes is known to initiate microglial activation, but its effects on neurons after SCI remain to be further clarified. Methods Electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were applied to characterize sEVs. We examined microglial activation and neuronal apoptosis mediated by astrocyte activation in an experimental model of acute spinal cord injury and in cell culture in vitro. Results Our results indicated that astrocytes activated after spinal cord injury release CCL2, act on microglia and neuronal cells through the sEV pathway, and promote neuronal apoptosis and microglial activation after binding the CCR2. Subsequently, the activated microglia release IL-1β, which acts on neuronal cells, thereby further aggravating their apoptosis. Conclusion This study elucidates that astrocytes interact with microglia and neurons through the sEV pathway after SCI, enriching the mechanism of CCL2 in neuroinflammation and spinal neurodegeneration, and providing a new theoretical basis of CCL2 as a therapeutic target for SCI.

Abstract 34: Human Cardiac Mesenchymal Cell Derived Extracellular Vesicles Promote Angiogenesis Through Angiopoietin-TIE2 Signaling

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Asif Pathan ◽  
Talha Farid ◽  
Abdur Rahman Khan ◽  
Marjan Nasr ◽  
Marcin Wysoczynski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Embryonic Stem ◽  
Tube Formation ◽  
Right Atrial ◽  
Boyden Chamber ◽  
Human Umbilical Cord ◽  
Angiogenic Activity ◽  
Cell Based Therapy

Cell-based therapy is considered a promising approach to treat the damaged heart due to myocardial infarction. Although the mechanisms for their beneficial action are not yet clear, exosome/extracellular vesicles (EVs) secreted by these cells may be involved in their reparative paracrine signaling. Previous studies have suggested that EVs isolated from several cell types (e.g. cardiosphere-derived cells, embryonic stem cell, CD34+ stem cells) induce angiogenic activity both in vitro and in vivo . Here, we investigated whether EVs secreted by adult human cardiac mesenchymal cells (hCMCs) exhibit pro-angiogenic activity, and if so, what signaling molecules are involved in this process. hCMCs were isolated from right atrial appendage of patients undergoing cardiac procedures and were characterized by the expression of classical mesenchymal markers- CD29 (99.1%), CD73 (99.0%), CD90 (20.4%), CD105 (99.3%), CD 31 (16.8%), CD34 (0.9%) and CD45 (0.1%). EVs isolated from serum-free 24-hour hCMC conditioned media using PEG4000-based precipitation technique exhibited two distinct population of particles with size range of 10-60nm and 100-500nm in diameter; expressed characteristic exosomal markers- CD63, HSP70, Flotillin-1 and were negative for cellular organelle markers- calreticulin (ER and apoptotic bodies), prohibitin (mitochondria), GM130 (Golgi), Lamin B (nuclear protein), β-actin (cytoskeleton) and PMP70 (peroxisomes) as determined by immunoblotting. In vitro assays revealed that hCMC EVs promote human umbilical cord endothelial cells (HUVECs) proliferation, transwell migration in Boyden chamber and tube formation on Matrigel, indicative of enhanced angiogenesis. Angiogenic proteomic array identified that angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) proteins are highly enriched in EVs secreted by hCMCs. Furthermore, hCMC EV mediated HUVEC migration and tube formation was inhibited by TIE2 kinase inhibitor. Overall, these findings suggest that ANG-1 and ANG-2 are the key component of hCMC secreted EVs and they promote angiogenesis by activating TIE2 receptor in endothelial cells.

scholarly journals Neural Induction, Neural Fate Stabilization, and Neural Stem Cells

2002 ◽  
Vol 2 ◽  
pp. 1147-1166 ◽  
Author(s):  
Sally A. Moody ◽  
Hyun-Soo Je
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Stem Cells ◽  
Neural Development ◽  
Current Knowledge ◽  
Embryonic Stem ◽  
Neural Induction ◽  
Neural Plate ◽  
Natural Rate ◽  
Neural Fate

The promise of stem cell therapy is expected to greatly benefit the treatment of neurodegenerative diseases. An underlying biological reason for the progressive functional losses associated with these diseases is the extremely low natural rate of self-repair in the nervous system. Although the mature CNS harbors a limited number of self-renewing stem cells, these make a significant contribution to only a few areas of brain. Therefore, it is particularly important to understand how to manipulate embryonic stem cells and adult neural stem cells so their descendants can repopulate and functionally repair damaged brain regions. A large knowledge base has been gathered about the normal processes of neural development. The time has come for this information to be applied to the problems of obtaining sufficient, neurally committed stem cells for clinical use. In this article we review the process of neural induction, by which the embryonic ectodermal cells are directed to form the neural plate, and the process of neural�fate stabilization, by which neural plate cells expand in number and consolidate their neural fate. We will present the current knowledge of the transcription factors and signaling molecules that are known to be involved in these processes. We will discuss how these factors may be relevant to manipulating embryonic stem cells to express a neural fate and to produce large numbers of neurally committed, yet undifferentiated, stem cells for transplantation therapies.

scholarly journals Using a Multi-Stage hESC Model to Characterize BDE-47 Toxicity During Neurogenesis

2019 ◽  
Vol 171 (1) ◽  
pp. 221-234 ◽  
Author(s):  
Hao Chen ◽  
Helia Seifikar ◽  
Nicholas Larocque ◽  
Yvonne Kim ◽  
Ibrahim Khatib ◽  
...  
Keyword(s):  
Neural Development ◽  
Polybrominated Diphenyl Ethers ◽  
Neuronal Development ◽  
Embryonic Stem ◽  
Cellular Level ◽  
Environmental Chemicals ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Neurodevelopmental Toxicity

Abstract Although the ramifications associated with polybrominated diphenyl ethers (PBDEs) exposures during human pregnancy have yet to be determined, increasing evidence in humans and animal models suggests that these compounds cause neurodevelopmental toxicity. Human embryonic stem cells (hESCs) models can be used to study the effects of environmental chemicals throughout the successive stages of neuronal development. Here, using a hESC differentiation model, we investigated the effects of common PBDE congeners (BDE-47 or -99) on the successive stages of early neuronal development. First, we determined the points of vulnerability to PBDEs across 4 stages of in vitro neural development by using assays to assess for cytotoxicity. Differentiated neural progenitors were identified to be more sensitive to PBDEs than their less differentiated counterparts. In follow-up investigations, we observed BDE-47 to inhibit functional processes critical for neurogenesis (eg, proliferation, expansion) in hESC-derived neural precursor cells (NPCs) at sub-lethal concentrations. Finally, to determine the mechanism(s) underlying PBDE-toxicity, we conducted global transcriptomic and methylomic analyses of BDE-47. We identified 589 genes to be differentially expressed due to BDE-47 exposure, including molecules involved in oxidative stress mediation, cell cycle, hormone signaling, steroid metabolism, and neurodevelopmental pathways. In parallel analyses, we identified a broad significant increase in CpG methylation. In summary our results suggest, on a cellular level, PBDEs induce human neurodevelopmental toxicity in a concentration-dependent manner and sensitivity to these compounds is dependent on the developmental stage of exposure. Proposed mRNA and methylomic perturbations may underlie toxicity in early embryonic neuronal populations.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

scholarly journals Comparing the cytotoxicity of electronic cigarette fluids, aerosols and solvents

2017 ◽  
Vol 27 (3) ◽  
pp. 325-333 ◽  
Author(s):  
Rachel Z Behar ◽  
Yuhuan Wang ◽  
Prue Talbot
Keyword(s):  
Stem Cells ◽  
Cultured Cells ◽  
Low Voltage ◽  
Embryonic Stem ◽  
Electronic Cigarette ◽  
Lung Epithelial Cells ◽  
Potential Health ◽  
Differentiated Cells ◽  
Do It Yourself

BackgroundAs thousands of electronic cigarette (e-cigarette) refill fluids continue to be formulated and distributed, there is a growing need to understand the cytotoxicity of the flavouring chemicals and solvents used in these products to ensure they are safe. The purpose of this study was to compare the cytotoxicity of e-cigarette refill fluids/solvents and their corresponding aerosols using in vitro cultured cells.MethodsE-cigarette refill fluids and do-it-yourself products were screened in liquid and aerosol form for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay. The sensitivity of human pulmonary fibroblasts, lung epithelial cells (A549) and human embryonic stem cells to liquids and aerosols was compared. Aerosols were produced using Johnson Creek’s Vea cartomizer style e-cigarette.ResultsA hierarchy of potency was established for the aerosolised products. Our data show that (1) e-cigarette aerosols can produce cytotoxic effects in cultured cells, (2) four patterns of cytotoxicity were found when comparing refill fluids and their corresponding aerosols, (3) fluids accurately predicted aerosol cytotoxicity 74% of the time, (4) stem cells were often more sensitive to aerosols than differentiated cells and (5) 91% of the aerosols made from refill fluids containing only glycerin were cytotoxic, even when produced at a low voltage.ConclusionsOur data show that various flavours/brands of e-cigarette refill fluids and their aerosols are cytotoxic and demonstrate the need for further evaluation of e-cigarette products to better understand their potential health effects.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Induction of recurrent break cluster genes in neural progenitor cells differentiated from embryonic stem cells in culture

2020 ◽  
Vol 117 (19) ◽  
pp. 10541-10546 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Neural Development ◽  
Large Fraction ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Neural Progenitor ◽  
Strand Break ◽  
Neuropsychiatric Diseases ◽  
Primary Mouse

Mild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome-wide DSB identification approach that captures DSBs via their ability to join to specific genomic Cas9/single-guide RNA–generated bait DSBs. In XRCC4/p53-deficient ESCs, we detected seven RDCs, all of which were in genes and two of which were robust. In contrast, in NPCs derived from these ESC lines we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ESCs provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.

Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 140-140
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Taisuke Kanaji ◽  
Jerry Ware ◽  
Hiromitsu Nakauchi
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Egfp Expression ◽  
Transfusion Therapy ◽  
Integrin Αiibβ3 ◽  
Differentiated Cells ◽  
Low Efficiency

Abstract To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.

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