What is the impact of e-cigarettes on periodontal stem cells as revealed by transcriptomic analyses?

DESIGN: The research used an in vitro cell exposure model and multi-omics integration of transcriptome and epigenome profiling to compare the molecular effects of e-cigarettes and tobacco smoke on dental stem cells. AIM: The study aimed to compare the effects of e-cigarette and tobacco smoke on periodontal stem cells using a multi-omics approach to understand gene regulation. METHODS: This research studied primary human gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs) obtained from healthy donors. The cells were subjected to tobacco smoke, e-cigarette aerosol (both tobacco and menthol flavors), e-cigarette liquid (both tobacco and menthol flavors), or untreated conditions using an in vitro exposure system. RNA sequencing and bioinformatics analysis were used to profile the transcriptome and identify differential gene expression. Additionally, chromatin immunoprecipitation sequencing (ChIP-seq) was used to conduct genome-wide histone modification mapping for H3K27me3. Transcriptome profiling was combined with histone modification characterization to understand gene regulatory mechanisms. The study compared the effects of smoke versus e-cigarette, aerosol versus liquid exposure, and tobacco versus menthol flavor on gene expression and epigenetic landscapes in the two oral stem cell populations. RESULTS: The use of tobacco smoke caused damage to the DNA and nucleus in GMSCs, as well as mitochondrial dysfunction in PDLSCs. Regarding e-cigarettes, the aerosol and liquid affected non-coding RNA expression differently. The chemokine CXCL2 was found to be downregulated by aerosol but upregulated by liquid in GMSCs. An integrative analysis revealed that the upregulation of CXCL2 caused by e-liquid involved reduced H3K27me3 and activation of distal enhancers. On the other hand, aerosol exposure maintained H3K27me3 levels, while direct e-liquid exposure resulted in genome-wide reductions in H3K27me3, particularly in enhancer regions. Overall, the specific delivery methods and components of e-cigarettes caused unique changes in the transcriptome and epigenome of oral stem cells. CONCLUSIONS: E-cigarettes affect oral stem cells differently than tobacco smoke. Their aerosol and liquid have varying impacts on gene expression and regulatory landscapes in oral cells. Multi-omics approaches are important to understanding the molecular changes caused by e-cigarette components. This can help with toxicological assessments and determine their impact on periodontal health. Transcriptome and epigenome profiling are powerful tools to examine the unique molecular mechanisms involved in cellular responses to e-cigarettes.

Inhibition of Autophagy in Renal Cells With Human, Urine-derived, Stem-cell Exosomes to Improve Diabetic Nephropathy.

Context: Clinicians can use stem cells to repair kidney injury. The kidneys' exosome secretions hold the secret to this therapeutic impact. Exosomes from urine-derived stem cells can prevent and treat glomerular damage that diabetes can cause, but the underlying process has remained a mystery. Objective: The study aimed to investigate the protective impact of exosomes from urine-derived stem cells (USCs) against diabetic nephropathy (DN) and to determine the mechanisms involved. Design: The research team performed an animal study. Setting: The study took place at the Affiliated Hospital of Jiujiang University in Jiujiang, Jiangxi, China. Animals: The animals were rats, SD male rats, weighing 200-220g, 40 animals, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number: SCXK (Beijing) 2021-0006). Intervention: Except for a control group, the rats in the groups had induced DN. The five groups, with 10 rats each, were: (1) the negative control group, which received 0.2 ml of PBS solution; (2) the DN group, a second negative control group, which received 0.2 ml of PBS solution, (3) the inhibitor group, an intervention group that received 20 mg/kg of autophagy inhibitor; (4) the exosomes group, an intervention group that received 100 ug/kg of exosomes; and (5) the exosomes + inhibitor group, an intervention group that received 100 ug/kg of exosomes + 20 mg/kg of autophagy inhibitor. From week 8, for four weeks the team injected the inhibitor, exosomes, and exosomes + inhibitor groups with the appropriate treatments using the rats' tail veins. Outcome Measures: The research team: (1) examined the USCs in the exosomes of stem cells; (2) assessed the rats' weights and fasting blood glucose (FBG), using a blood glucose meter; (3) used Coomassie brilliant blue (CBB) staining to determine the amount of protein in the rats' urine and assessed their biochemical indexes; and (4) used Western blot (WB) and a quantitative polymerase chain reaction (Q-PCR) to detect autophagy and the signal transduction pathway. Results: Human exosomes from USCs alleviated injury in the rats that DN caused by reducing urinary-protein levels, serum creatinine (SCR), blood urea nitrogen (BUN), glomerular cell accumulation, and kidney weights. In rats with induced DN, the exosomes + inhibitor significantly reduced the activation of the mTOR signaling pathway, reduced the autophagy of their kidney cells, increased the protein expression of Bcl-2 in the kidney tissues, and lessened the damage to glomerular cells. Conclusions: Human urine-derived stem cell exosomes can significantly reduce the activation of the mTOR signaling pathway, reduce the autophagy of rats' kidney cells, increase the protein expression of LC3B in kidney tissues, and reduce the damage to glomerular cells. By blocking the mTOR signaling pathway, human urogenic exosomes can alleviate the signs and symptoms of DN.

Dentin slice model of dental stem cells in a fibrin-agarose construct for dental pulp regeneration / Modelo de trozo de dentina de células madre dentales en una construcción de fibrina-agarosa para la regeneración de la pulpa dental

Objectives: To implement a dentin slice model of mesenchymal stem cells derived from dental tissues in a fibrin-agarose construct for dental pulp regeneration. Material and Methods: MSCs derived from different oral cavity tissues were combined with a fibrin-agarose construct at standard culture conditions. Cell viability and proliferation tests were assayed using a fluorescent cell dye Calcein/Am and WST-1 kit. The proliferation assay was evaluated at 24, 48, 72, and 96 hours. Also, we assessed the dental pulp stem cells (DPSCs) cell morphology inside the construct with histological stains such as Hematoxylin and Eosin, Masson's trichrome, and Periodic acid­Schiff. In addition, we elaborated a tooth dentin slice model using a culture of DPSC in the fibrin­agarose constructs co-adhered to dentin walls. Results: The fibrin-agarose construct was a biocompatible material for MSCs derived from dental tissues. It provided good conditions for MSCs' viability and proliferation. DPSCs proliferated better than the other MSCs, but the data did not show significant differences. The morphology of DPSCs inside the construct was like free cells. The dentin slice model was suitable for DPSCs in the fibrin-agarose construct. Conclusion: Our findings support the dentin slice model for future biological use of fibrin-agarose matrix in combination with DPSCs and their potential use in dental regeneration. The multipotency, high proliferation rates, and easy obtaining of the DPSCs make them an attractive source of MSCs for tissue regeneration.

Objetivos: Implementar un modelo de dentina con células madre mesenquimales derivadas de tejidos dentales en una constructo de fibrina-agarosa para la regeneración de la pulpa dental. Material y Métodos: Las MSC derivadas de diferentes tejidos de la cavidad oral se combinaron con una construcción de fibrina-agarosa en condiciones de cultivo estándar. Las pruebas de viabilidad y proliferación celular se ensayaron utilizando un kit de colorante celular fluorescente Calcein/Am y WST-1. El ensayo de proliferación se evaluó a las 24, 48, 72 y 96 horas. Además, evaluamos la morfología celular de las células madre de la pulpa dental (DPSC) dentro de la construcción con tinciones histológicas como hematoxilina y eosina, tricrómico de Masson y ácido peryódico de Schiff. Además, elaboramos un modelo de rebanadas de dentina dental utilizando un cultivo de DPSC en las construcciones de fibrina-agarosa coadheridas a las paredes de la dentina. Resultados: La construcción de fibrina-agarosa fue un material biocompatible para las MSC derivadas de tejidos dentales. Proporcionó buenas condiciones para la viabilidad y proliferación de las MSC. Las DPSC proliferaron mejor que las otras MSC, pero los datos no mostraron diferencias significativas. La morfología de las DPSC dentro de la construcción era como la de las células libres. El modelo de corte de dentina fue adecuado para DPSC en la construcción de fibrina-agarosa.Conclusión: Nuestros hallazgos respaldan el modelo de corte de dentina para el futuro uso biológico de la matriz de fibrina-agarosa en combinación con DPSC y su uso potencial en la regeneración dental. El multipotencial, las altas tasas de proliferación y la fácil obtención de las DPSC las convierten en una fuente atractiva de MSC para la regeneración de tejidos.

Unraveling molecular mechanism underlying biomaterial and stem cells interaction during cell fate commitment using high throughput data analysis.

Stem cell differentiation towards various somatic cells and body organs has proven to be an effective technique in the understanding and progression of regenerative medicine. Despite the advances made, concerns regarding the low efficiency of differentiation and the remaining differences between stem cell products and their in vivo counterparts must be addressed. Biomaterials that mimic endogenous growth conditions represent one recent method used to improve the quality and efficiency of stem cell differentiation, though the mechanisms of this improvement remain to be completely understood. The effectiveness of various biomaterials can be analyzed through a multidisciplinary approach involving bioinformatics and systems biology tools. Here, we aim to use bioinformatics to accomplish two aims: 1) determine the effect of different biomaterials on stem cell growth and differentiation, and 2) understand the effect of cell of origin on the differentiation potential of multipotent stem cells. First, we demonstrate that the dimensionality (2D versus 3D) and the degradability of biomaterials affects the way that the cells are able to grow and differentiate at the transcriptional level. Additionally, according to transcriptional state of the cells, the particular cell of origin is an important factor in determining the response of stem cells to same biomaterial. Our data demonstrates the ability of bioinformatics to understand novel molecular mechanisms and context by which stem cells are most efficiently able to differentiate. These results and strategies can be used to suggest proper combinations of biomaterials and stem cells to achieve high differentiation efficiency and functionality of desired cell types.

Micromechanical Compatibility between Cells and Scaffolds Directs the Phenotypic Transition of Stem Cells.

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Extracellular Vesicles of Adipose-Derived Stem Cells Promote the Healing of Traumatized Achilles Tendons.

Healing of ruptured tendons remains a clinical challenge because of its slow progress and relatively weak mechanical force at an early stage. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have therapeutic potential for tissue regeneration. In this study, we isolated EVs from adipose-derived stem cells (ADSCs) and evaluated their ability to promote tendon regeneration. Our results indicated that ADSC-EVs significantly enhanced the proliferation and migration of tenocytes in vitro. To further study the roles of ADSC-EVs in tendon regeneration, ADSC-EVs were used in Achilles tendon repair in rabbits. The mechanical strength, histology, and protein expression in the injured tendon tissues significantly improved 4 weeks after ADSC-EV treatment. Decorin and biglycan were significantly upregulated in comparison to the untreated controls. In summary, ADSC-EVs stimulated the proliferation and migration of tenocytes and improved the mechanical strength of repaired tendons, suggesting that ADSC-EV treatment is a potential highly potent therapeutic strategy for tendon injuries.

Protective effects of low-molecular-weight components of adipose stem cell-derived conditioned medium on dry eye syndrome in mice.

The present study demonstrated the protective effects of low-molecular-weight adipose-derived stem cell-conditioned medium (LADSC-CM) in a mouse model of dry eye syndrome. Mice subjected to desiccating stress and benzalkonium chloride had decreased tear secretion, impaired corneal epithelial tight junction with microvilli, and decreased conjunctival goblet cells. Topical application of adipose-derived stem cell-conditioned medium (ADSC-CM) stimulated lacrimal tear secretion, preserved tight junction and microvilli of the corneal epithelium, and increased the density of goblet cells and MUC16 expression in the conjunctiva. The low-molecular-weight fractions (< 10 kDa and < 3 kDa) of ADSC-CM (LADSC-CM) provided better protections than the > 10 kDa or > 3 kDa fractions of ADSC-CM. In the in vitro study, desiccation for 10 min or hyperosmolarity (490 osmols) for 24 h caused decreased viability of human corneal epithelial cells, which were reversed by LADSC-CM. The active ingredients in the LADSC-CM were lipophobic and stable after heating and lyophilization. Our study demonstrated that LADSC-CM had beneficial effects on experimental dry eye. It is worthy of further exploration for the active ingredient(s) and the mechanism.

Peptide-PAINT Enables Investigation of Endogenous Talin with Molecular Scale Resolution in Cells and Tissues.

Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10 nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions.

Antlerogenic stem cells extract accelerate chronic wound healing: a preliminary study.

BACKGROUND: Chronic wounds constitute a significant medical and social problem. Chronic wound treatment may be supported by various techniques, such as negative pressure therapy, phototherapy or stem cells therapy, yet most of those supporting therapies need more evidence to be used for standard wound care. Current study covers the use of sonicated Antlerogenic Stem Cells (ASC) extract on chronic wounds. METHODS: Study was performed on 20 dermatological patients with venous leg ulcers, divided into two groups - treated with and without ASC extract respectively. The area and circumference of the wounds during the follow-up visits were measured on the wound imprint. Dynamics of wound healing was determined and compared between control and study group; statistics includes changes in absolute values (wound area, circumference), as well as relative (percentage of wound decrease, circumference/area ratio) and their change in time. For the purpose of Ki-67 immunohistochemical staining, sections were sampled from the wound edge at distinct check-points during therapy. Results of both groups were compared with Student test or Mann-Whitney test, depending on results distribution. RESULTS: Besides Ki-67 expression, all tested wound healing parameters (including relative and absolute wound decrease and changes in circumference/area ratio) were statistically significant more favorable in experimental group. CONCLUSION: ASC extract significantly supported standard chronic wound treatment. Due to small population of study the results should be considered preliminary, yet promising for further research.

An RNAi Screening of Clinically Relevant Transcription Factors Regulating Human Adipogenesis and Adipocyte Metabolism.

CONTEXT: Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents. OBJECTIVE: A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously. METHODS: TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs. RESULTS: Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes. CONCLUSION: This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes.

Human urine-derived stem cell-derived exosomal miR-21-5p promotes neurogenesis to attenuate Rett syndrome via the EPha4/TEK axis.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual-luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine-derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1)+ nerve cells as well as the transcription levels of ß-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX+ cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.

Cost saving, patient centered algorithm for progenitor cell mobilization for autologous hematopoietic cell transplantation.

Administration of plerixafor with granulocyte-colony stimulating factor (G-CSF) mobilizes CD34+ cells much more effectively than G-CSF alone, but cost generally limits plerixafor use to patients at high risk of insufficient CD34+ cell collection based on low peripheral blood (PB) CD34+ counts following 4 days of G-CSF. We analyzed costs associated with administering plerixafor to patients with higher day 4 CD34+ cell counts to decrease apheresis days and explored the use of a fixed split dose of plerixafor instead of weight-based dosing. We analyzed 235 patients with plasma cell disorders or non-Hodgkin's lymphoma who underwent progenitor cell mobilization and autologous hematopoietic cell transplantation (AHCT) between March 2014 and December 2017. Two hundred ten (89%) received G-CSF plus Plerixafor and 25 (11%) received G-CSF alone. Overall, 180 patients (77%) collected in 1 day, 53 (22%) in 2 days and 2 (1%) in 3 days. Based on our data, we present a probabilistic algorithm to identify patients likely to require more than one day of collection using G-CSF alone. CD34+ cell yield, ANC and platelet recovery were not significantly different between fixed and standard dose plerixafor. Plerixafor enabled collection in 1 day and with estimated savings of $5000, compared to patients who did not receive plerixafor and required collection for three days. While collection and processing costs and patient populations vary among institutions, our results suggest re-evaluation of current algorithms.

Olfactory mucosa stem cells delivery via nasal route: a simple way for the treatment of Parkinson disease.

Finding a simple and effective way for transferring cells to the brain lesion site with minimum side effects mounts a challenge in cell therapy. Cell delivery via nasal route using the bypassing the blood-brain barrier (BBB) property is a simple and non-invasive strategy without serious complications such as trauma. Therefore, it is a suitable technique to treat neurodegenerative disorders like Parkinson's disease (PD). Olfactory ectomesenchymal stem cells (OE-MSCs) located in the lamina propria of olfactory mucosa could be differentiated into dopaminergic neurons under in vitro and in vivo conditions. Thus, OE-MSCs represent a good source of Parkinson's stem cell-based therapy. In this research, we studied thirty male rats (n = 10 in each group) in three control (Ctl), lesion (LE), and intranasal administration (INA) groups to investigate the therapeutic effect of intranasal injection of OE-MSCs in the Parkinson's animal models. To do so, we examined the homing variation of OE-MSCs in different brain regions such as olfactory bulb (OB), cortex, striatum (Str), hippocampus (HPC), and substantia nigra (SN). The results of real-time PCR and immunohistochemistry (IHC) analysis showed the expression of dopaminergic neuron markers such as PITX3, PAX2, PAX5 (as dopaminergic neurons markers), tyrosine hydroxylase (TH), and dopamine transporter (DAT) 2 months after INA of 1 × 106 OE-MSCs. The results confirmed that IN OE-MSCs delivery into the central nervous system (CNS) was powerful enough to improve the behavioral functions in the animal models of PD.

Transcriptome analysis identifies the differentially expressed genes related to the stemness of limbal stem cells in mice.

Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs.

Cartilage endplate stem cells inhibit intervertebral disc degeneration by releasing exosomes to nucleus pulposus cells to activate Akt/autophagy.

Degeneration of the cartilage endplate (CEP) induces intervertebral disc degeneration (IVDD). Nucleus pulposus cell (NPC) apoptosis is also an important exacerbating factor in IVDD, but the cascade mechanism in IVDD is not clear. We investigated the apoptosis of NPCs and IVDD when stimulated by normal cartilage endplate stem cell (CESC)-derived exosomes (N-Exos) and degenerated CESC-derived exosomes (D-Exos) in vitro and in vivo. Tert-butyl hydroperoxide (TBHP) was used to induce inflammation of CESCs. The bioinformatics differences between N-Exos and D-Exos were analyzed using mass spectrometry, heat map, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. NPC apoptosis was examined using TUNEL staining. The involvement of the AKT and autophagy signaling pathways was investigated using the signaling inhibitor LY294002. Magnetic resonance imaging, Western blotting, and immunofluorescence staining were used to evaluate the therapeutic effects of N-Exos in rats with IVDD. TBHP effectively induced inflammation and the degeneration of CEP in rat. N-Exos were more conducive to autophagy activation than D-Exos. The apoptotic rate of NPCs decreased obviously after treatment with N-Exos compared to D-Exos. N-Exos inhibited NPCs apoptosis and attenuated IVDD in rat via activation of the AKT and autophagy pathways. These results are the first findings to confirm that CEP delayed the progression of IVDD via exosomes. The therapeutic effects of N-Exos on NPC apoptosis inhibition and the slowing of IVDD progression were more effective than D-Exos due to activation of the PI3K/AKT/autophagy pathway, which explained the increase in the incidence of IVDD after inflammation of the CEP.

Review of Bioprinting in Regenerative Medicine: Naturally Derived Bioinks and Stem Cells.

Regenerative medicine offers the potential to repair or substitute defective tissues by constructing active tissues to address the scarcity and demands for transplantation. The method of forming 3D constructs made up of biomaterials, cells, and biomolecules is called bioprinting. Bioprinting of stem cells provides the ability to reliably recreate tissues, organs, and microenvironments to be used in regenerative medicine. 3D bioprinting is a technique that uses several biomaterials and cells to tailor a structure with clinically relevant geometries and sizes. This technique's promise is demonstrated by 3D bioprinted tissues, including skin, bone, cartilage, and cardiovascular, corneal, hepatic, and adipose tissues. Several bioprinting methods have been combined with stem cells to effectively produce tissue models, including adult stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and differentiation techniques. In this review, technological challenges of printed stem cells using prevalent naturally derived bioinks (e.g., carbohydrate polymers and protein-based polymers, peptides, and decellularized extracellular matrix), recent advancements, leading companies, and clinical trials in the field of 3D bioprinting are delineated.

Liver-derived extracellular vesicles: A cell by cell overview to isolation and characterization practices.

BACKGROUND: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists. SCOPE OF REVIEW: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches. MAJOR CONCLUSIONS: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings. GENERAL SIGNIFICANCE: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods.

The quest of cell surface markers for stem cell therapy.

Stem cells and their derivatives are novel pharmaceutics that have the potential for use as tissue replacement therapies. However, the heterogeneous characteristics of stem cell cultures have hindered their biomedical applications. In theory and practice, when cell type-specific or stage-specific cell surface proteins are targeted by unique antibodies, they become highly efficient in detecting and isolating specific cell populations. There is a growing demand to identify reliable and actionable cell surface markers that facilitate purification of particular cell types at specific developmental stages for use in research and clinical applications. The identification of these markers as very important members of plasma membrane proteins, ion channels, transporters, and signaling molecules has directly benefited from proteomics and tools for proteomics-derived data analyses. Here, we review the methodologies that have played a role in the discovery of cell surface markers and introduce cutting edge single cell proteomics as an advanced tool. We also discuss currently available specific cell surface markers for stem cells and their lineages, with emphasis on the nervous system, heart, pancreas, and liver. The remaining gaps that pertain to the discovery of these markers and how single cell proteomics and identification of surface markers associated with the progenitor stages of certain terminally differentiated cells may pave the way for their use in regenerative medicine are also discussed.

Genetic Lineage Tracing of Non-cardiomyocytes in Mice.

Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.

Human trophoblast-derived exosomes attenuate doxorubicin-induced cardiac injury by regulating miR-200b and downstream Zeb1.

Human trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether trophoblast stem cell-derived exosomes (TSC-Exos) can protect cardiomyocytes from doxorubicin (Dox)-induced injury remains unclear. In the present study, TSC-Exos were isolated from the supernatants of human trophoblasts using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. In vitro, primary cardiomyocytes were subjected to Dox and treated with TSC-Exos, miR-200b mimic or miR-200b inhibitor. Cellular apoptosis was observed by flow cytometry and immunoblotting. In vivo, mice were intraperitoneally injected into Dox to establish a heart failure model. Then, different groups of mice were administered either PBS, adeno-associated virus (AAV)-vector, AAV-miR-200b-inhibitor or TSC-Exos via tail vein injection. Then, the cardiac function, cardiac fibrosis and cardiomyocyte apoptosis in each group were evaluated, and the downstream molecular mechanism was explored. TSC-Exos and miR-200b inhibitor both decreased primary cardiomyocyte apoptosis. Similarly, mice receiving TSC-Exos and AAV-miR-200b inhibitor exhibited improved cardiac function, accompanied by reduced apoptosis and inflammation. The bioinformatic prediction and luciferase reporter results confirmed that Zeb1 was a downstream target of miR-200b and had an antiapoptotic effect. TSC-Exos attenuated doxorubicin-induced cardiac injury by playing antiapoptotic and anti-inflammatory roles. The underlying mechanism could be an increase in Zeb1 expression by the inhibition of miR-200b expression. In summary, this study sheds new light on the application of TSC-Exos as a potential therapeutic tool for heart failure.