Human Dental Pulp Cell exosomes vs Human iPS Cell exosomes
Ontology highlight
ABSTRACT: Small vesicles, known as exosomes, are secreted from various cell types. Exosomes secreted by mesenchymal stem cells have therapeutic effects against a variety of diseases, and may be able to partially replace stem cell therapies. Previously, we established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this report, we purified the exosomes secreted from HHH-DPCs and evaluated their therapeutic potential in a periodontitis model. The exosomes purified from HHH-DPCs showed homogeneous and spherical membrane structures, and showed low but significant expression of HLA class I molecules. The exosomes further promoted proliferation and migration in DPCs. A comparison of miRNAs revealed that the HHH-DPC exosomes contained higher levels of multiple Let-7 family miRNAs compared to HHH-induced pluripotent stem cell (iPSC)-derived exosomes. Finally, the HHH-DPC exosomes showed preventive effects in a mouse model of periodontitis induced by lipopolysaccharides (LPS). In summary, HHH-DPC exosomes expressed HLA molecules which may induce an immune response in HLA-mismatched transplantations. However, they successfully stimulated the proliferation and migration of cells and showed suppressive effects on LPS-induced periodontitis. Therefore, HHH-DPC exosomes show great potential for applications in periodontal treatments.
Project description:DNA‒protein crosslinks (DPCs) challenge faithful DNA replication and fluid passage of genomic information. Our study unveils the cullin ubiquitin ligase Rtt101 as a novel DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types. Using an inducible in vivo DPC system, we reveal the significant impact of Rtt101 on DPC removal, including topoisomerase 1 crosslinks. ChIP-sequencing and ChEC-sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a novel main contributor to DPC repair throughout the yeast cell cycle.
Project description:Hepatocellular carcinoma (HCC) generally arises in chronically inflamed liver and is thought to originate from regenerating liver cells that acquired genetic alterations. Both hepatocytes and ductular progenitor cells (DPCs) contribute to liver regeneration and are candidate cellular origins of HCCs. In the current study, we used lineage-tracing analysis to show that Epcam-expressing DPCs give rise to HCCs in inflamed liver. Under conditions in which proliferating DPCs differentiate into cholangiocytes but not into hepatocytes, the accumulation of genetic alterations in Epcam-expressing DPCs resulted in the development of HCCs with histologic features of concomitant ductule-like structures resembling human cholangiolocellular carcinoma (CLC). To elucidate the characteristics of DPC-derived HCC, gene expression of HCCs derived from Epcam-expressing DPCs were evaluated using Agilent SurePrint G3 Mouse Gene Expression 8x60K v2. Two DPC-derived HCCs with CLC components, two DPC-derived HCCs without CLC, and two HCCs derived from albumin-expressing cells were analyzed.
Project description:Microarray analysis of exosomal miRNAs vs the miRNAs of their respective donor cells. To determine the miRNA repertoires of exosomes secreted by immune cells, we isolated exosomes from cell supernatants of the Raji B cell line, the Jurkat-derived J77 T cell line, and primary dendritic cells (DCs) derived from human monocytes. Exosomes were isolated by a series of microfiltration and ultracentrifugation steps
Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway
Project description:DNA-protein crosslinks (DPCs), arise from enzymatic intermediates, endogenous metabolism or exogenous chemicals like chemotherapeutics. DPCs are highly cytotoxic as they will impede DNA transacting processes such as replication, which is counteracted by proteolysis-mediated DPC removal by the protease SPRTN or the proteasome. However, how DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here, we show that DPCs severely inhibit RNA polymerase II (Pol II)-mediated transcription, resulting in the recruitment of the transcription-coupled nucleotide excision repair (TC-NER) factors CSA and CSB. Interestingly, CSA and CSB are indispensable for transcription-coupled DPC (TC-DPC) repair, while the downstream TC-NER factors UVSSA and XPA are not, indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independent of SPRTN, but is mediated by the activities of the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs are preferentially repaired in genes by a dedicated transcription-coupled repair mechanism, which is crucial to warrant correct transcription.
Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.
Project description:Alopecia is an exceedingly prevalent problem and lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential,we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM not. In order to identify the key factors responsible for hair inductive capacity, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1.Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results would be used to identify the key factors for inducing HF regeneration and reveal the molecular mechanisms of losing inductive ability of DPCs. Furthermore, it is possible to explore some drugs for alopecia in the clinic according to this secretome date analysis.
Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway
Project description:Studies have shown that stimulation of Mesenchymal stem cells (MSCs) with biophysical or biochemical cues could influence the contents and biological activities of subsequent MSC-derived exosomes. However, the underlying mechanisms have not been fully clarified. Here, we pretreated MSCs with lipopolysaccharide (LPS) and evaluated exosomes (LPS-Exo) therapeutic effects in sepsis. Analysis of exosomes activity revealed that LPS-Exo treatment improved sepsis to a greater extent than normal MSCs-derived exosomes (Exo). Further analysis revealed that LPS-Exo exert their protective effects mainly through RNA contents. Thus, we hypothesized that LPS preconditioning enhances the therapeutic efficacy of LPS-Exo in sepsis by altering miRNA expression profile.
Project description:DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN or the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair pathways remain unknown. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in a DNA replication-independent manner. In addition, we provide evidence that a subset of DPCs can be SUMOylated and processed via transcription, independently of RNF4. SUMO modification of DPCs is mediated by PIAS4 and neither stimulates nor inhibits their rapid DNA replication-dependent proteolysis. Instead, SUMOylation provides a critical salvage mechanism to remove DPCs formed or persisting after replication, as we demonstrate that DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells can enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.