CD206+ M2-like macrophages protect against intervertebral disc degeneration partially by targeting R-spondin-2.

OBJECTIVE: This study aimed to explore the specific function of M2 macrophages in intervertebral disc degeneration (IDD). METHODS: Intervertebral disc (IVD) samples from normal (n = 4) and IDD (n = 6) patients were collected, and the expression of M2-polarized macrophage marker, CD206, was investigated using immunohistochemical staining. Nucleus pulposus cells (NPCs) in a TNF-α environment were obtained, and a mouse caudal IVD puncture model was established. Mice with Rheb deletions, specifically in the myeloid lineage, were generated and subjected to surgery-induced IDD. IDD-induced damage and cell apoptosis were measured using histological scoring, X-ray imaging, immunohistochemical staining, and TdT-mediated dUTP nick end labeling (TUNEL) assay. Finally, mice and NPCs were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in IDD. RESULTS: Accumulation of CD206 in human and mouse IDD tissues was detected. Rheb deletion in the myeloid lineage (RheBcKO) increased the number of CD206+ M2-like macrophages (mean difference 18.6% [15.7-21.6%], P < 0.001), decreased cell apoptosis (mean difference -15.6% [-8.9 to 22.2%], P = 0.001) and attenuated the IDD process in the mouse IDD model. NPCs treated with Rspo2 displayed increased extracellular matrix catabolism and apoptosis; co-culture with a conditioned medium derived from RheBcKO mice inhibited these changes. Anti-Rspo2 treatment in the mouse caudal IVD puncture model exerted protective effects against IDD. CONCLUSIONS: Promoting CD206+ M2-like macrophages could reduce Rspo2 secretion, thereby alleviating experimental IDD. Rheb deletion may help M2-polarized macrophages accumulate and attenuate experimental IDD partially by inhibiting Rspo2 production. Hence, M2-polarized macrophages and Rspo2 may serve as therapeutic targets for IDD.

Murine trophoblast organoids as a model for trophoblast development and CRISPR-Cas9 screening.

The placenta becomes one of the most diversified organs during placental mammal radiation. The main in vitro model for studying mouse trophoblast development is the 2D differentiation model of trophoblast stem cells, which is highly skewed to certain lineages and thus hampers systematic screens. Here, we established culture conditions for the establishment, maintenance, and differentiation of murine trophoblast organoids. Murine trophoblast organoids under the maintenance condition contain stem cell-like populations, whereas differentiated organoids possess various trophoblasts resembling placental ones in vivo. Ablation of Nubpl or Gcm1 in trophoblast organoids recapitulated their deficiency phenotypes in vivo, suggesting that those organoids are valid in vitro models for trophoblast development. Importantly, we performed an efficient CRISPR-Cas9 screening in mouse trophoblast organoids using a focused sgRNA (single guide RNA) library targeting G protein-coupled receptors. Together, our results establish an organoid model to investigate mouse trophoblast development and a practicable approach to performing forward screening in trophoblast lineages.

Progress and Prospects of Gene Editing in Pluripotent Stem Cells.

Applying programmable nucleases in gene editing has greatly shaped current research in basic biology and clinical translation. Gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is highly relevant to clinical cell therapy and thus should be examined with particular caution. First, since all mutations in PSCs will be carried to all their progenies, off-target edits of editors will be amplified. Second, due to the hypersensitivity of PSCs to DNA damage, double-strand breaks (DSBs) made by gene editing could lead to low editing efficiency and the enrichment of cell populations with defective genomic safeguards. In this regard, DSB-independent gene editing tools, such as base editors and prime editors, are favored due to their nature to avoid these consequences. With more understanding of the microbial world, new systems, such as Cas-related nucleases, transposons, and recombinases, are also expanding the toolbox for gene editing. In this review, we discuss current applications of programmable nucleases in PSCs for gene editing, the efforts researchers have made to optimize these systems, as well as new tools that can be potentially employed for differentiation modeling and therapeutic applications.

Influence of macrophage polarization in herniated nucleus pulposus tissue on clinical efficacy after lumbar discectomy.

Background: Low back pain or sciatic pain because of lumbar intervertebral disc herniation (LDH) is caused by mechanical compression and/or an inflammatory component on the nerve root. However, it is difficult to define to what extent each component contributes to the pain. This study attempted to explore the effects of macrophage polarization on clinical symptoms in patients experiencing LDH after surgery, and investigated the association between macrophage cell percentages and clinical efficacy. Methods: This study retrospectively harvested nucleus pulposus (NP) tissue samples from 117 patients. Clinical symptoms and efficacy using the visual analog scale (VAS) and Oswestry Disability Index (ODI) were evaluated at different time points preoperatively and postoperatively. CD68, CCR7, CD163, and CD206 were selected as macrophage phenotypic markers. Results: Seventy-six samples showed positive expression of macrophage markers in NP samples of patients with LDH, whereas 41 patients displayed negative results. No significant differences were detected between the two groups, involvement of several demographic data, and preoperative clinical findings. With respect to the macrophage-positive group, no significant correlation was detected between the positive rate of the four markers and the VAS score or ODI after surgery. However, patients with NP samples positive for CD68 and CCR7 expression showed significantly lower VAS scores 1 week after surgery compared with those in the negative group. Moreover, the improvement in VAS score showed a strong positive correlation with CD68- and CCR7-positive cell percentages. Conclusions: Our results indicated that pro-inflammatory M1 macrophages may be associated with the reduction of chronic pain after surgery. Therefore, these findings contribute to better personalized pharmacological interventions for patients with LDH, considering the heterogeneity of pain.

Modeling human ectopic pregnancies with trophoblast and vascular organoids.

Ruptured ectopic pregnancy (REP), a pregnancy complication caused by aberrant implantation, deep invasion, and overgrowth of embryos in fallopian tubes, could lead to rupture of fallopian tubes and accounts for 4%-10% of pregnancy-related deaths. The lack of ectopic pregnancy phenotypes in rodents hampers our understanding of its pathological mechanisms. Here, we employed cell culture and organoid models to investigate the crosstalk between human trophoblast development and intravillous vascularization in the REP condition. Compared with abortive ectopic pregnancy (AEP), the size of REP placental villi and the depth of trophoblast invasion are correlated with the extent of intravillous vascularization. We identified a key pro-angiogenic factor secreted by trophoblasts, WNT2B, that promotes villous vasculogenesis, angiogenesis, and vascular network expansion in the REP condition. Our results reveal the important role of WNT-mediated angiogenesis and an organoid co-culture model for investigating intricate communications between trophoblasts and endothelial/endothelial progenitor cells.

A clinical study of C arm-guided selective spinal nerve block combined with low-temperature plasma radiofrequency ablation of dorsal root ganglion in the treatment of zoster-related neuralgia.

Background: This study evaluated the analgesic efficacy and psychological response of low-temperature plasma ablation of dorsal root ganglion (DRG) combined with selective spinal nerve block in patients with acute or subacute zoster-related neuralgia (ZRN). Methods: Totally 90 ZRN patients were randomly and evenly divided into three groups. Treatment was given to Group A using C arm-guided selective spinal nerve block (C-SSVB), Group B using C-SSVB and pulsed radiofrequency (PRF), and Group C using C-SSVB and low-temperature plasma ablation of the DRG. The outcomes were examined using the Visual Analog Scale (VAS). Anxiety and depression of patients were evaluated using the Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). Quality of life was assessed using the Pittsburgh Sleep Quality Index (PSQI) and postoperative Satisfaction scale. In addition, data on adverse events and medication usage rates were collected. Results: The 90 patients were eligible for this study. The three treatments reduced VAS scores with no significant difference between groups A and B at the same time points; however, group B tended to have numerically lower VAS scores. Comparatively, group C had significantly reduced VAS scores on day 1 and 1 month after treatment compared with the other two groups. In terms of the decreasing SAS, SDS and PSQI scores, all the three treatments improved the anxiety, depression and sleep quality of the patients. In addition, significant alleviation in anxiety was found in group C compared with group A at all- time points. However, there was no statistically significant difference among the three groups in treatment-related adverse events that mainly focused on puncture pain at the surgical-site, skin numbness and medication usage rates. Conclusions: C-SSVB and LTPRA of DRG will be considered as a promising treatment option for ZRN patients if those results can be confirmed after further validation.

Exogenous dopamine improves resistance to Botryosphaeria dothidea by increasing autophagy activity in pear.

Pear ring rot, a fungal disease caused by Botryosphaeria dothidea (B. dothidea), is one of the most damaging diseases in pear production, affecting fruit yield and causing economic losses. It is not clear whether dopamine, one of the catecholamines, has any role in pear ring rot resistance. In this study, we found that dopamine treatment of B. dothidea resulted in a significant upregulation of PbrTYDC expression compared to H2O treatment (control) and reduced the levels of Hydrogen Peroxide (H2O2) and Superoxide Anion (O2-), increased Peroxidase (POD), Catalase (CAT), Superoxide Dismutase (SOD) and Phenylalanine Ammonia-Lyase (PAL) activities, and induced a significant upregulation of related gene expression. Dopamine treatment promoted the oxidationreduction capacity of the AsA-GSH cycle to scavenge Reactive Oxygen Species (ROS), increased the expression of autophagy-related genes and the accumulation of autophagic structures, and enhanced autophagic activity. Silencing PbrTYDC and PbrATG8 in pear increased H2O2 and·O2-, decreased POD, CAT and SOD activities and reduced resistance to B. dothidea, which was restored by dopamine treatment. In conclusion, exogenous dopamine enhances resistance to B. dothidea by increasing the antioxidant capacity and autophagic activity of pears, and this study provides new insights for subsequent studies on B. dothidea as well as autophagy.

Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities.

Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo.

Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.

Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.

Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA.

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.

A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

Revealing the early response of pear (Pyrus bretschneideri Rehd) leaves during Botryosphaeria dothideainfection by transcriptome analysis.

Ring rot disease, which is caused by Botryosphaeria dothidea (B. dothidea), is one of the most serious diseases affecting the pear industry. Currently, knowledge of the mechanism about pear-pathogen interactions is unclear. To explore the early response of pear leaves to B. dothidea infection, we compared the early transcriptome of pear leaves infected with B. dothidea. The results revealed 3248 differentially expressed genes (DEGs) and 4862 DEGs at D2 and D4, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of DEGs showed that these genes were predominately involved in plant-pathogen interactions, hormone signal transduction and other biosynthesis-related metabolic processes, including glucosinolate accumulation and flavonoid pathway enhancement. However, many hormone- and disease resistance-related genes and transcription factors (TFs) were differentially expressed during B. dothidea infection. These results were consistent with the changes in the physiological characteristics of B. dothidea. In addition, the expression of PbrPUB29, an E3 ubiquitin ligase with a U-box domain, was significantly higher than it was at 0 dpi. PbrPUB29 silencing enhanced the sensitivity of pear leaves to B. dothidea, reflected by more severe symptoms and higher reactive oxygen species (ROS) content in the defective pear seedlings after inoculation, revealing that PbrPUB29 has a significant role in pear disease resistance. In brief, we explored the interaction between pear leaves and B. dothidea at the transcriptome level, implied the early response of pear leaves to pathogens, and identified a hub gene in a B. dothidea-infected pear. These results provide a basis and new strategy for exploring the molecular mechanisms underlying pear-pathogen interactions and disease resistance breeding.

Exogenous Melatonin Improves Pear Resistance to Botryosphaeria dothidea by Increasing Autophagic Activity and Sugar/Organic Acid Levels.

Pear is an important fruit tree worldwide, but it is often infected by the pathogen Botryosphaeria dothidea, which causes pear ring rot disease. To explore the effect of exogenous melatonin on the disease resistance of pear, we treated inoculated pear fruits with different concentrations of melatonin. The results showed that 100 µΜ of melatonin had the most significant effect with resistance to B. dothidea. In addition, melatonin treatment significantly reduced the diameter of disease lesions and enhanced the endogenous melatonin content in pears inoculated with B. dothidea. Compared with the control treatment, melatonin treatment suppressed increases in reactive oxygen species (ROS) and activated ROS-scavenging enzymes. Treatment with exogenous melatonin maintained ascorbic acid-glutathione at more reductive status. The expression levels of core autophagic genes and autophagosome formation were elevated by melatonin treatment in pear fruits. Silencing of PbrATG5 in Pyrus pyrifolia conferred sensitivity to inoculation that was only slightly attenuated by melatonin treatment. After inoculation with B. dothidea, exogenous melatonin treatment led to higher levels of soluble sugars and organic acids in pear fruits than H2O treatment. Overall, our results demonstrate that melatonin enhances resistance to B. dothidea by increasing autophagic activity and soluble sugar/organic acid accumulation.

Engineering of human mesenchymal stem cells resistant to multiple natural killer subtypes.

Mesenchymal stem cells (MSCs) as a therapeutic promise are often quickly cleared by innate immune cells of the host including natural killer (NK) cells. Efforts have been made to generate immune-escaping human embryonic stem cells (hESCs) where T cell immunity is evaded by defecting ß-2-microglobulin (B2M), a common unit for human leukocyte antigen (HLA) class I, and NK cells are inhibited via ectopic expression of HLA-E or -G. However, NK subtypes vary among recipients and even at different pathologic statuses. It is necessary to dissect and optimize the efficacy of the immune-escaping cells against NK subtypes. Here, we first generated B2M knockout hESCs and differentiated them to MSCs (EMSCs) and found that NK resistance occurred with B2M-/- EMSCs expressing HLA-E and -G only when they were transduced via an inducible lentiviral system in a dose-dependent manner but not when they were inserted into a safe harbor. HLA-E and -G expressed at high levels together in transduced EMSCs inhibited three major NK subtypes, including NKG2A+ /LILRB1+ , NKG2A+ /LILRB1- , and NKG2A- /LILRB1+ , which was further potentiated by IFN-γ priming. Thus, this study engineers MSCs with resistance to multiple NK subtypes and underscores that dosage matters when a transgene is used to confer a novel effect to host cells, especially for therapeutic cells to evade immune rejection.

Effectiveness of acupuncture therapy for postherpetic neuralgia: an umbrella review protocol.

INTRODUCTION: Several systematic reviews and meta-analysis indicate that acupuncture and related therapies may be a valuable adjunctive technique to pharmacological interventions for pain management of postherpetic neuralgia (PHN). However, the robustness of the results of these studies has not been evaluated. The aim of this proposed umbrella review is to provide more reliable evidence of the effectiveness of acupuncture therapy for PHN based on medical references for healthcare decision makers. METHODS AND ANALYSIS: PubMed, EMBASE, The Cochrane Library, Web of Science, Chinese BioMedical Literature Database, VIP Database for Chinese Technical Periodicals, China National Knowledge Infrastructure and Wan fang Database will be used to retrieve reviews. The time of publication will be limited from inception to March 2021. Two reviewers will screen all retrieved articles independently to identify their eligibility and extract the data. The quality will be assessed independently by two trained reviewers using Assessment of Multiple Systematic Reviews-2 for methodological quality, Risk of Bias in Systematic Review for level of bias, Preferred Reporting Items for Systematic Reviews and Meta-Analysis for reporting quality and Grading of Recommendations Assessment, Development and Evaluation for the quality of evidence. Any disagreements will be settled by discussion or the involvement of a third reviewer. ETHICS AND DISSEMINATION: The protocol of this review does not require ethical approval because the research will be based on publicly available data. The findings will be disseminated through publication in peer-reviewed international journals or presentation in academic conference. PROSPERO REGISTRATION NUMBER: CRD42020173341. REPORTING CHECKLIST: PRISMA-P, 2015.

Human ESC-derived MSCs enhance fat engraftment by promoting adipocyte reaggregation, secreting CCL2 and mobilizing macrophages.

Mesenchymal stem cells (MSCs) derived from somatic tissues have been used to promote lipotransfer, a common practice in cosmetic surgery. However, the effect of lipotransfer varies, and the mechanism of action remains vague. To address these questions, we differentiated human embryonic stem cells, a stable and unlimited source, into MSCs (EMSCs). Then we subcutaneously transplanted human fat aspirates together with EMSCs or PBS as a control into the back of nude mice. Within 24 h of transplantation, EMSCs promoted aggregation and encapsulation of injected fat tissues. Afterward, all grafts gradually shrank. However, EMSC-containing grafts were larger, heavier and had fewer dark areas on the surface than the control grafts. Histologically, more live adipocytes, vascular cells, and macrophages and less fibrosis were observed in EMSC-containing grafts than in the controls. Some EMSCs differentiated into vascular cells and adipocytes in the EMSC-containing grafts. RNA sequencing revealed that human RNA was shown to decline rapidly, while mouse RNA increased in the grafts; further, human genes related to extracellular matrix remodeling, adipogenesis, and chemokine (including CCL2) signaling were expressed at higher levels in the EMSC-containing grafts than they were in the controls. CCL2 knockout reduced macrophage migration towards EMSCs in vitro and early macrophage recruitment to the grafts and the pro-engraftment effect of EMSCs in vivo. Treating mice with a macrophage inhibitor abolished the EMSC effects and converted the grafts to heavy masses of cell debris. Together, these data demonstrate that EMSCs promote fat engraftment via enhanced tissue reconstitution and encapsulation of implanted tissues, which was followed by increased angiogenesis and adipocyte survival and reduced fibrosis, in which stimulated CCL2 signaling and mobilized macrophages play pivotal roles.

[Therapeutic effect and mechanism of silver needle heat conduction therapy combined with loxoprofen sodium patch in patients with knee osteoarthritis].

OBJECTIVE: To observe the clinical efficacy of silver needle heat conduction therapy combined with loxoprofen sodium patch in the treatment of knee osteoarthritis (KOA). METHODS: A total of ninety-two patients with KOA were randomly and equally divided into loxoprofen sodium group and silver needle heat conduction therapy + loxoprofen sodium (combination) group, with 46 cases in each group. Patients of the combination group were treated with silver needle heat conduction therapy combined with loxoprofen sodium patch, while those of the loxoprofen sodium group were treated with loxoprofen sodium patch. The treatment was conducted for 4 weeks. The Western Ontario McMaster Universities Osteoarthritis Index (WOMAC), bone metabolism index ï¼»including bone gla protein (BGP), bone-specific alkaline phosphatase (BALP), tartrate resistant acid phosphatase isomer (TRACP)-5bï¼½, and inflammation factors ï¼»including the tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), interleukin-1ß (IL-1ß)ï¼½ were observed before and after treatment. The therapeutic effect was assessed after the treatment. RESULTS: After the treatment, the total scores of WOMAC, the levels of serum TRACP-5b, TNF-α and IL-1ß were significantly decreased (P<0.01), while the levels of serum BGP, BALP, and TGF-ß were significantly increased (P<0.01) in the two groups compared with their own pre-treatment. Silver needle plus loxoprofen sodium was more effective in reducing WOMAC score, TRACP-5b, TNF-α, IL-1ß level (P<0.01), and up-regulating BGP, BALP, and TGF-ß level (P<0.01) than loxoprofen. Of the 46 cases in the loxoprofen sodium and combination groups, 33 and 41 were effective, with the effective rate being 71.7% and 89.1%, respectively. The comprehensive therapeutic effect of the combination group was significantly superior to that of the loxoprofen group (P<0.05). CONCLUSION: Silver needle heat conduction therapy combined with loxoprofen sodium can effectively treat KOA, its mechanism may be related to alleviating inflammation and improving bone metabolism.

Morphine inhibits the promotion of inflammatory microenvironment on chronic tibial cancer pain through the PI3K-Akt-NF-κB pathway.

PURPOSE: Inflammatory microenvironment is critical in the transmission of advanced cancer pain. This paper will study how morphine ameliorates advanced cancer pain through inflammatory microenvironment. METHODS: Fifty female healthy rats were selected and divided into control group, sham group, model group, morphine group and morphine + 740YP group by random number table. At the left tibia, rats in the model, morphine and morphine + 740YP groups received Walker256 cells injection, while those in the sham group received an equal amount of Hank solution. The control group received no treatment. After modeling, the rats' spontaneous pain behavior, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured and statistically analyzed. The protein levels of PI3K, Akt, NF-κB and pro-inflammatory factors (TNF-α/IL-1ß/IL-6/IL-17a) in rats were detected. Rat left dorsal root ganglion (DRG) cells were extracted and treated with 10, 20 and 30 µmol/L morphine to observe their effects on the cells. RESULTS: Compared with the control group, the model group presented increased spontaneous pain behavior and PWTL, decreased PWMT, and reduced mechanical pain threshold, as well as enhanced levels of PI3K, Akt, NF-κB and pro-inflammatory factors in vivo as compared to the control group. While the morphine group showed less spontaneous pain behavior and PWTL, increased PWMT, and down-regulated PI3K, Akt, NF-κB and pro-inflammatory factors in vivo in comparison with the model group. After morphine treatment, the apoptosis of DRG cells decreased and the cell activity increased, while PI3K, Akt, NF-κB and pro-inflammatory factors levels decreased. Morphine affected DRG cells in a dose-dependent manner. Up-regulation of PI3K could counteract the inhibitory effect of morphine on chronic tibial cancer pain. CONCLUSIONS: Morphine inhibits the promotion of inflammatory microenvironment on chronic tibial cancer pain via the PI3K/Akt/NF-κB pathway, and the regulation of the PI3K/Akt/NF-κB pathway can improve the therapeutic effect of morphine on chronic tibial cancer pain.

Correction: Zhang, Z.; et al. Roles of MicroRNAs in Establishing and Modulating Stem Cell Potential. Int. J. Mol. Sci. 2019, 20, 3643.

The authors wish to make the following correction to this paper [...].

Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner.

Mesenchymal stem cells (MSC) derived from adult tissues effectively promote wound healing. However, MSC quality varies, and the quantity of MSC is limited, as MSC are acquired through donations. Moreover, the survival and functioning of dissociated MSC delivered to an inflammatory lesion are subject to challenges. Methods: Here, spheres (EMSCSp) generated from human embryonic stem cell-derived MSC (EMSC) were directly dropped onto excised wounds in mice; the effects of EMSCSp were compared to those of dissociated EMSC (EMSCDiss). Following transplantation, we measured the extent of wound closure, dissected the histological features of the wounds, determined transcriptomic changes in cells isolated from the treated and control wounds, and evaluated the molecular mechanism of the effects of EMSC. Results: The application of EMSCSp onto murine dermal wounds substantially increased survival and efficacy of EMSC compared to the topical application of EMSCDiss. RNA sequencing (RNA-Seq) of cells isolated from the wounds highlighted the involvement of CXCL12-CXCR4 signaling in the effects of EMSCSp, which was verified in EMSC via CXCL12 knockdown and in target cells (vascular endothelial cells, epithelial keratinocytes, and macrophages) via CXCR4 inhibition. Finally, we enhanced the biosafety of EMSCSp by engineering cells with an inducible suicide gene. Conclusions: Together, these data suggest the topical application of EMSCSp as an unlimited, quality-assured, safe, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in this treatment.