Role of cell rearrangement and related signaling pathways in the dynamic process of tip cell selection.

Angiogenesis is a complex, highly-coordinated and multi-step process of new blood vessel formation from pre-existing blood vessels. When initiated, the sprouting process is spearheaded by the specialized endothelial cells (ECs) known as tip cells, which guide the organization of accompanying stalk cells and determine the function and morphology of the finally-formed blood vessels. Recent studies indicate that the orchestration and coordination of angiogenesis involve dynamic tip cell selection, which is the competitive selection of cells to lead the angiogenic sprouts. Therefore, this review attempt to summarize the underlying mechanisms involved in tip cell specification in a dynamic manner to enable readers to gain a systemic and overall understanding of tip cell formation, involving cooperative interaction of cell rearrangement with Notch and YAP/TAZ signaling. Various mechanical and chemical signaling cues are integrated to ensure the right number of cells at the right place during angiogenesis, thereby precisely orchestrating morphogenic functions that ensure correct patterning of blood vessels. Video Abstract.

Role of melatonin in Alzheimer's disease: From preclinical studies to novel melatonin-based therapies.

Melatonin and novel melatonin-based therapies such as melatonin-containing hybrid molecules, melatonin analogues, and melatonin derivatives have been investigated as potential therapeutics against Alzheimer's disease (AD) pathogenesis. In this review, we examine the developmental trends of melatonin therapies for AD from 1997 to 2021. We then highlight the neuroprotective mechanisms of melatonin therapy derived from preclinical studies. These mechanisms include the alleviation of amyloid-related burden, neurofibrillary tangle accumulation, oxidative stress, neuroinflammation, apoptosis, mitochondrial dysfunction, and impaired neuroplasticity and neurotransmission. We further illustrate the beneficial effects of melatonin on behavior in animal models of AD. Next, we discuss the clinical effects of melatonin on sleep, cognition, behavior, psychiatric symptoms, electroencephalography findings, and molecular biomarkers in patients with mild cognitive impairment and AD. We then explore the effectiveness of novel melatonin-based therapies. Lastly, we discuss the limitations of current melatonin therapies for AD and suggest two emerging research themes for future study.

Macrophages promote cartilage regeneration in a time- and phenotype-dependent manner.

Immune regulation of osteochondral defect regeneration has not yet been rigorously characterized. Although macrophages have been demonstrated to regulate the regeneration process in various tissues, their direct contribution to cartilage regeneration remains to be investigated, particularly the functions of polarized macrophage subpopulations. In this study, we investigated the origins and functions of macrophages during healing of osteochondral injury in the murine model. Upon osteochondral injury, joint macrophages are predominantly derived from circulating monocytes. Macrophages are essential for spontaneous cartilage regeneration in juvenile C57BL/6 mice, by modulating proliferation and apoptosis around the injury site. Exogeneous macrophages also exhibit therapeutic potential in promoting cartilage regeneration in adult mice with poor regenerative capacity, possibly via regulation of PDGFRα+  stem cells, with this process being influenced by initial phenotype and administration timing. Only M2c macrophages are able to promote regeneration of both cartilage tissues and subchondral bone. Overall, we reveal the direct link between macrophages and osteochondral regeneration and highlight the key roles of relevant immunological niches in successful regeneration.

An overview of signaling pathways regulating YAP/TAZ activity.

YAP and TAZ are ubiquitously expressed homologous proteins originally identified as penultimate effectors of the Hippo signaling pathway, which plays a key role in maintaining mammalian tissue/organ size. Presently, it is known that YAP/TAZ also interact with various non-Hippo signaling pathways, and have diverse roles in multiple biological processes, including cell proliferation, tissue regeneration, cell lineage fate determination, tumorigenesis, and mechanosensing. In this review, we first examine the various microenvironmental cues and signaling pathways that regulate YAP/TAZ activation, through the Hippo and non-Hippo signaling pathways. This is followed by a brief summary of the interactions of YAP/TAZ with TEAD1-4 and a diverse array of other non-TEAD transcription factors. Finally, we offer a critical perspective on how increasing knowledge of the regulatory mechanisms of YAP/TAZ signaling might open the door to novel therapeutic applications in the interrelated fields of biomaterials, tissue engineering, regenerative medicine and synthetic biology.

Single-cell transcriptome analysis of human oocyte ageing.

Oocyte ageing is a key bottleneck and intractable challenge for in vitro fertilization treatment of aged female patients. The underlying molecular mechanisms of human oocyte ageing remain to be elucidated. Hence, this study aims to investigate the key genes and relevant biological signalling pathways involved in human oocyte ageing. We isolated mRNA for single-cell RNA sequencing from MII human oocytes donated by patients undergoing intracytoplasmic sperm injection. Nine RNA-seq datasets were analyzed, which included 6 older patients(average 42.67±2.25 years) and 3 younger patients (average 25.67±2.08 years). 481 differentially expressed genes (DEGs) were identified, including 322 upregulated genes enriched in transcription, ubiquitination, epigenetic regulation, and cellular processes, and 159 downregulated genes enriched in ubiquitination, cell cycle, signalling pathway, and DNA repair. The STRING database was used to analyse protein-protein interactions, and the Cytoscape software was used to identify hub genes. From these DEGs, 17 hub genes were identified including 12 upregulated genes (UBE2C, UBC, CDC34, UBR1, KIF11, ASF1B, PRC1, ESPL1, GTSE1, EXO1, UBA1, KIF4A) and 5 downregulated genes (UBA52, UBE2V2, SKP1, CCNB1, MAD2L1). The significant key biological processes that are associated with these hub genes include ubiquitin-mediated proteolysis, ubiquitination-related pathways, oocyte meiosis, and cell cycle. Among these, UBE2C may play a crucial role in human oocyte ageing.

Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.

The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.

The relationship between substrate topography and stem cell differentiation in the musculoskeletal system.

It is well known that biomaterial topography can exert a profound influence on various cellular functions such as migration, polarization, and adhesion. With the development and refinement of manufacturing technology, much research has recently been focused on substrate topography-induced cell differentiation, particularly in the field of tissue engineering. Even without biological and chemical stimuli, the differentiation of stem cells can also be initiated by various biomaterials with different topographic features. However, the underlying mechanisms of this biological phenomenon remain elusive. During the past few decades, many researchers have demonstrated that cells can sense the topography of materials through the assembly and polymerization of membrane proteins. Following the activation of RHO, TGF-b or FAK signaling pathways, cells can be induced into various differentiation states. But these signaling pathways often coincide with canonical mechanical transduction pathways, and no firm conclusion has been reached among researchers in this field on topography-specific signaling pathways. On the other hand, some substrate topographies are reported to have the ability to inhibit differentiation and maintain the 'stemness' of stem cells. In this review, we will summarize the role of topography in musculoskeletal system regeneration and explore possible topography-related signaling pathways involved in cell differentiation.

Challenges and strategies in developing team-based learning in Chinese medical education.

INTRODUCTION: Team-based learning (TBL) is gradually being integrated into Chinese medical education. This study reports its current application status in Chinese medical schools, as well as the underlying challenges and strategies to improve TBL application. METHOD: We screened publication databases and surveys to investigate TBL usage and concerns regarding TBL application by Chinese medical educators. Articles published by 79 Chinese medical schools include 163 articles among 20 topic areas of basic medicine and 226 articles among 16 clerkship disciplines. The opinions of 123 Chinese medical teachers were solicited from 46 medical schools in 26 provinces/municipalities. RESULTS: Approximately less than half of Chinese medical schools used TBL in basic medicine or clerkship disciplines. Among these, only 10% of schools reported TBL usage in both clerkship disciplines and basic medicine. Both quantitative and qualitative results revealed that public awareness of TBL, executive support, professional training, sharing of resources and integration of multiple disciplines are critical factors in facilitating TBL application, and in recruiting and developing TBL teachers. CONCLUSION: TBL application in Chinese medical education is limited. Executive/financial support and establishment of a platform to provide technical support, share resources and regulate TBL practice quality will facilitate TBL application in Chinese medical education.

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling.

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 µg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.

A systematic review: differentiation of stem cells into functional pericytes.

Pericytes are an integral cellular component of vascular structures. Numerous studies have investigated various stem cell types as potential sources of pericytes for application in cell-based therapy. The diverse stem cell types and variable experimental protocols of these studies make it imperative to evaluate the relevant scientific literature on the basis of a unified standard. The purpose of this systematic review is to rigorously evaluate the relevant scientific literature for conclusive evidence that stem cells can differentiate into functional pericytes. An online literature search was conducted up to July 2016. Eligible papers were evaluated on 4 pertinent criteria: 1) appropriate controls, 2) markers to confirm pericyte phenotype, 3) techniques for assessing pericyte functionality, and 4) differentiation efficiency of the protocol. Our search yielded 20 eligible studies (from 2006 to 2016), 12 of which were published in the past 5 yr. Of these 20 articles, only 1 had positive control, and 5 papers evaluated differentiation efficiency. The most commonly used pericyte markers were neuron-glial antigen 2, platelet-derived growth factor receptor-ß, and α-smooth muscle actin. Three articles were associated with adipose stem cells, 4 with mesenchymal stem cells, and 7 with pluripotent stem cells, whereas the remaining 6 articles were based on other miscellaneous stem cell types. Stem cells can serve as a potential source of pericytes, but there should be standardized guidelines in future studies for assessing pericyte differentiation.-Xu, J., Gong, T., Heng, B. C., Zhang, C. F. A systematic review: differentiation of stem cells into functional pericytes.

Small molecule therapeutics for inflammation-associated chronic musculoskeletal degenerative diseases: Past, present and future.

Inflammation-associated chronic musculoskeletal degenerative diseases (ICMDDs) like osteoarthritis and tendinopathy often results in morbidity and disability, with consequent heavy socio-economic burden. Current available therapies such as NSAIDs and glucocorticoid are palliative rather than disease-modifying. Insufficient systematic research data on disease molecular mechanism also makes it difficult to exploit valid therapeutic targets. Small molecules are designed to act on specific signaling pathways and/or mechanisms of cellular physiology and function, and have gradually shown potential for treating ICMDDs. In this review, we would examine and analyze recent developments in small molecule drugs for ICMDDs, suggest possible feasible improvements in treatment modalities, and discuss future research directions.

G protein-coupled receptors revisited: therapeutic applications inspired by synthetic biology.

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters within the human body. They have much potential in the emerging field of synthetic biology, which is the rational, systematic design of biological systems with desired functionality. The responsiveness of GPCRs to a plethora of endogenous and exogenous ligands and stimuli make them ideal sensory receptor modules of synthetic gene networks. Such networks can activate target gene expression in response to a specific stimulus. Additionally, because GPCRs are important pharmacological targets of various human diseases, genes encoding their protein/peptide ligands can also be incorporated as target genes of the response output elements of synthetic gene networks. This review aims to critically examine the potential role of GPCRs in constructing therapeutic synthetic gene networks and to discuss various challenges in utilizing GPCRs for synthetic biology applications.

Kdm6b regulates cartilage development and homeostasis through anabolic metabolism.

OBJECTIVES: Epigenetic mechanisms have been reported to play key roles in chondrogenesis and osteoarthritis (OA) development. Here, we sought to identify specific histone demethylases that are involved and delineate the underlying mechanisms. METHODS: We screened the expression of 17 distinct histone demethylases by quantitative real time PCR (qRT-PCR) during chondrogenic differentiation of C3H10T1/2 cells. The role of Kdm6b in cartilage development was then analysed with transgenic Col2a1-CreERT2;Kdm6bf/f . RNA-Seq was applied to explore the underlying changes in chondrocytes upon knockdown of Kdm6b. Experimental OA in mice was induced by destabilisation of the medial meniscus in C57BL/6J (wild type, Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f ) mice, either with intra-articular injection of shKdm6b lentivirus or after tamoxifen treatment. Mouse joints and human cartilage samples were used for histological analysis. RESULTS: Kdm6b expression was significantly increased during cartilage development. Col2a1-CreERT2;Kdm6bf/f mice displayed obvious skeletal abnormalities at E16.5 and E18.5 with intraperitoneal injection of tamoxifen at E12.5. RNA-Seq and qRT-PCR analyses revealed decreased expression of chondrocyte anabolic genes in Col2a1-CreERT2;Kdm6bf/f chondrocytes. The histological OA score was significantly higher in mice injected with Kdm6b short hairpin RNA lentivirus. Col2a1-CreERT2;Kdm6bf/f mice exhibited accelerated OA development at 8 and 12 weeks following surgical induction. The number of Kdm6b-positive chondrocytes was lower in both mice and human OA cartilage samples. CONCLUSIONS: These findings indicate that knockdown of Kdm6b in chondrocytes leads to abnormal cartilage development and accelerated OA progression via inhibition of the anabolic metabolism of chondrocytes. Understanding the epigenetic mechanism of joint cartilage development and homeostasis would be useful for development of new therapeutic modalities for OA.

Pharmacological Regulation of In Situ Tissue Stem Cells Differentiation for Soft Tissue Calcification Treatment.

Calcification of soft tissues, such as heart valves and tendons, is a common clinical problem with limited therapeutics. Tissue specific stem/progenitor cells proliferate to repopulate injured tissues. But some of them become divergent to the direction of ossification in the local pathological microenvironment, thereby representing a cellular target for pharmacological approach. We observed that HIF-2alpha (encoded by EPAS1 inclined form) signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Proinflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation and promoted osteochondrogenic differentiation of tendon stem/progenitor cells (TSPCs). Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. Notably, Scleraxis (Scx), an essential tendon specific transcription factor, was suppressed on constitutive activation of HIF-2alpha and mediated the effect of HIF-2alpha on TSPCs fate decision. Moreover, pharmacological inhibition of HIF-2alpha with digoxin, which is a widely utilized drug, can efficiently inhibit calcification and enhance tenogenesis in vitro and in the Achilles's tendinopathy model. Taken together, these findings reveal the significant role of the tissue stem/progenitor cells fate decision and suggest that pharmacological regulation of HIF-2alpha function is a promising approach for soft tissue calcification treatment.

Mohawk promotes the tenogenesis of mesenchymal stem cells through activation of the TGFß signaling pathway.

The transcription factor Mohawk (Mkx) is expressed in developing tendons and is an important regulator of tenogenic differentiation. However, the exact roles of Mkx in tendinopathy and tendon repair remain unclear. Using gene expression Omnibus datasets and immunofluorescence assays, we found that Mkx expression level was dramatically lower in human tendinopathy tissue and it is activated at specific stages of tendon development. In mesenchymal stem cells (MSCs), ectopic Mkx expression strikingly promoted tenogenesis more efficiently than Scleraxis (Scx), a well-known master transcription factor of tendon. Significantly higher levels of tenogenic gene expression and collagen fibril growth were observed with Mkx versus Scx. Interestingly, it was observed that Mkx dramatically upregulated Scx through binding to the Tgfb2 promoter. Additionally, the transplantation of Mkx-expressing-MSC sheets promoted tendon repair in a mouse model of Achilles-tendon defect. Taken together, these data shed light on previously unrecognized roles of Mkx in tendinopathy, tenogenesis, and tendon repair as well as in regulating the TGFß pathway.

Pluripotent Human embryonic stem cell derived neural lineages for in vitro modelling of enterovirus 71 infection and therapy.

BACKGROUND: The incidence of neurological complications and fatalities associated with Hand, Foot & Mouth disease has increased over recent years, due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71, accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite, before progressing to expensive and time-consuming live animal studies and clinical trials. METHODS: This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro, in comparison with RD and SH-SY5Y cell lines. RESULTS: Upon assessment of post-infection survivability and EV71 production by the various types, it was observed that NSC were significantly more susceptible to EV71 infection compared to MN, RD (rhabdomyosarcoma) and SH-SY5Y cells, which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. CONCLUSIONS: Hence, this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics.

Enterococcus faecalis promotes osteoclast differentiation within an osteoblast/osteoclast co-culture system.

OBJECTIVE: To investigate the effects of heat-killed Enterococcus faecalis ATCC 29212 and P25RC clinical strain (derived from an obturated root canal with apical periodontitis) on osteoclast differentiation within an osteoblast/osteoclast co-culture system. RESULTS: Heat-killed E. faecalis significantly increased the proportion of multinucleated osteoclastic cells (MNCs) within the co-culture system. The IL-6 level was significantly increased upon exposure to heat-killed E. faecalis. Gene expression levels of NFATc1 and cathepsin K were significantly up-regulated compared to the untreated control. EphrinB2 and EphB4 expressions at both the mRNA and protein levels were also significantly upregulated compared to the untreated control. CONCLUSIONS: Heat-killed E. faecalis can induce osteoclast differentiation within the osteoblast/osteoclast co-culture system in vitro, possibly through ephrinB2-EphB4 bidirectional signaling.

ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

Inhibition of Rac1 activity by controlled release of NSC23766 from chitosan microspheres effectively ameliorates osteoarthritis development in vivo.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterised by cartilage degradation and chondrocyte hypertrophy. A recent study showed that Rac1 promoted expression of MMP13 and chondrocyte hypertrophy within the growth plate. These findings warrant further investigations on the roles of Rac1 in OA development and therapy in animal models. OBJECTIVE: To investigate the role and mechanistic pathway of Rac1 involvement in pathological changes of OA chondrocytes in vitro and OA development in vivo, as well as to develop a strategy of modulating Rac1 activity for OA treatment. MATERIAL AND METHODS: OA and normal cartilage from human or mice were used for immunohistochemical study and Rac1 activity assay. Chondrocytes treated with IL1ß and the untreated control were subjected to the Rac1 activity assay. Chondrocytes transfected with CA-Rac1, DN-Rac1 or GFP were cultured under conditions for inducing calcification. To evaluate the effect of Rac1 in OA development, an OA model was created by anterior cruciate ligament transection in mice. CA-Rac1, DN-Rac1 and GFP lentivirus, or NSC23766, were injected intra-articularly. Joints were subjected to histological analysis. RESULTS: It was found that there is aberrant Rac1 activation in human OA cartilage. Rac1 activity could also be elevated by IL1ß. Additionally, activated Rac1 promoted expression of MMP13, ADAMTS-5 and COLX by chondrocytes, partially through the ß-catenin pathway. Moreover, activation of Rac1 in knee joints by CA-Rac1 lentivirus accelerated OA progression, while inhibition of Rac1 activity by DN-Rac1 lentivirus or Rac1 inhibitor NSC23766 delayed OA development. Therefore, we developed a strategy of controlled release of NSC23766 from chitosan microspheres to OA joints, which effectively protected cartilage from destruction. CONCLUSIONS: These findings demonstrated that Rac1 activity is implicated in OA development. Also, controlled release of Rac1 inhibitor is a promising strategy for OA treatment.