Differentiation Of Stem Cells Research Articles

Injectable Janus Base Nanomatrix (JBNm) in Maintaining Long-Term Homeostasis of Regenerated Cartilage for Tissue Chip Applications.

Engineered cartilage tissues have wide applications in in vivo cartilage repair as well as in vitro models, such as cartilage-on-a-chip or cartilage tissue chips. Currently, most cartilage tissue engineering approaches focus on promoting chondrogenesis of stem cells to produce regenerated cartilage. However, this regenerated cartilage can dedifferentiate into fibrotic tissue or further differentiate into hypertrophic or calcified cartilage. One of the most challenging objectives in cartilage tissue engineering is to maintain long-term cartilage homeostasis. Since the microenvironment of engineered cartilage tissue is crucial for stem cell adhesion, proliferation, differentiation, and function, we aim to develop a novel scaffold that can maintain the long-term homeostasis of regenerated cartilage. Therefore, we developed a library of Janus base nanomatrices (JBNms), composed of DNA-inspired Janus nanotubes (JBNts) as well as cartilage extracellular matrix (ECM) proteins. The JBNms were developed to selectively promote chondro-lineage cell functions while inhibiting bone and endothelial cell growth. More importantly, the JBNm can effectively promote chondrogenesis while inhibiting hypertrophy, osteogenesis, angiogenesis, and dedifferentiation. Additionally, the JBNm is injectable, forming a solid scaffold suitable for producing and maintaining regenerated cartilage tissue in microfluidic chips, making it ideal for tissue chip applications. In this study, we successfully created cartilage tissue chips using JBNms. These chips can model cartilage tissue even after long-term culture and can also mimic arthritis progression, making them useful for drug screening. Thus, we have developed a novel nanomaterial approach for improved cartilage tissue engineering and cartilage tissue chip applications.

Tumor suppressor miR-317 and lncRNA Peony are expressed from a polycistronic non-coding RNA locus that regulates germline differentiation and testis morphology.

This research focuses on investigating the impact of non-coding RNAs on stem cell biology and differentiation processes. We found that miR-317 plays a role in germline stem cell progeny differentiation. miR-317 and its neighbor, the lncRNA Peony, originate and are co-expressed from a singular polycistronic non-coding RNA locus. Alternative polyadenylation is implicated in regulation of their differential expression. While the increased expression of the lncRNA Peony results in the disruption of the muscle sheath covering the testis, the absence of miR-317 leads to the emergence of germline tumors in young flies. The deficiency of miR-317 increases Notch signaling activity in the somatic cyst cells, which drives germline tumorigenesis. Germline tumors also arise from upregulation of several predicted targets of miR-317 , among which are regulators of the Notch pathway. This implicates miR-317 as a novel tumor suppressor that modulates Notch signaling strength.

Reinforced Magnetic-Responsive Electro-Ionic Artificial Muscles by 3D Laser-Induced Graphene Nano-Heterostructures.

Efficient ion transport and enriched responsive modals via modulating electrochemical properties of conductivity and capacitance are essential for soft electro-ionic actuators. However, cost-effective and straightforward approaches to achieve expedited fabrication of active electrode materials capable of multimodal-responsiveness remain limited. Herein, this work reports the one-step ultrafast laser direct patterning method, to readily synthesize electro- and magneto-active electrode material, derived from the unique cobalt-phosphorus co-doped core-shell heterostructures within 3D graphene frameworks, for fulfilling the dual-mode responsive electro-ionic actuators. The designed nanofiber-structured heterointerfaces across electrodes and electrolytes further promote highly efficient electron/ion transfer. The developed soft actuator exhibits superior actuation performance of peak-to-peak displacement to 13.08mm under an ultra-low ±0.5V, with doubled direct current deflection under 200 mT at 1V, an ultrafast response of 1.38 s and long-term stability (>90% retention for ≈106000 cycles), even detectable bending to ≈280µm under exceptional ±10mV. The promising demonstration of promoting differentiation and proliferation of stem cells under mechanical strain and electrical stimuli, sheds more light as well on the possibility of facilitating biomedical soft robotics with ultrahigh actuation performance.

High cadmium exposure impairs adult hippocampal neurogenesis via disruption of store-operated calcium entry

Cadmium (Cd) is a neurotoxicant that gradually accumulates in the human body with age. High Cd burden is correlated with adult hippocampal neurogenesis (AHN) and memory deficits in mammals. However, little knowledge is known about the mechanism by which Cd exposure impairs neurogenesis and cognition. Here, we investigated the roles of store-operated calcium entry (SOCE)-mediated calcium dyshomeostasis in Cd-induced AHN and memory deficits as well as therapeutic potential for the prevention of Cd-induced neurotoxicity. To achieve this goal, 8 weeks-old C57BL/6 J mice were subjected to different concentrations of cadmium chloride (0, 5, 10, 20 ppm) in drinking water for 8 weeks, we then examined the AHN, calcium homeostasis, SOCE channel and memory in Cd-exposed mice by using immunohistochemistry, calcium imaging, Y-maze and fear conditioning test. Our results indicated that chronic Cd exposure markedly increased Cd levels in serum and cerebrospinal fluid by almost 10-fold, and inhibited the proliferation and differentiation of hippocampal adult neural stem cells in a dose-dependent manner. Additionally, Cd exposure impaired the maturation of hippocampal neural stem cells without inducing gliosis. Transcriptome analysis revealed that Cd exposure inhibited the proliferation of neuroblastoma via alteration of calcium signaling pathway, and attenuated SOCE channels played a pivotal role in mediating Cd-induced cytoplasmic calcium overload and depletion of endoplasmic reticulum calcium stores. Activation of SOCE by hyperforin, a natural derivative from medicinal plant, restored intracellular calcium homeostasis and improved AHN and memory in Cd-exposed mice. Together, this study provided novel insights into the mechanism that Cd exposure impaired AHN and memory by prompting neuronal SOCE-mediated calcium dyshomeostasis, and offered a new therapeutic approach for prevention of Cd-induced neurotoxicity.

Biomimetic mineralization of collagen from fish scale to construct a functionally gradient lamellar bone-like structure for guided bone regeneration

Wide used guided bone regeneration (GBR) membrane materials, such as collagen, Teflon, and other synthesized polymers, present a great challenge in term of integrating the mechanical property and degradation rate when addressing critical bone defects. Therefore, inspired by the distinctive architecture of fish scales, this study utilized epigallocatechin gallate to modify decellularized fish scales following biomimetic mineralization to fabricate a GBR membrane that mimics the structure of lamellar bone. The structure, physical and chemical properties, and biological functions of the novel GBR membrane were evaluated. Results indicate that the decellularized fish scale with 5 remineralization cycles (5R-E-DCFS) exhibited a composite and structure similar to natural bone and had a special functionally gradient mineral contents character, demonstrating excellent mechanical properties, hydrophilicity, and degradation properties. In vitro, the 5R-E-DCFS membrane exhibited excellent cytocompatibility promoting Sprague-Dawley (SD) rat bone marrow mesenchymal stem cell proliferation and differentiation up-regulating the expression of osteogenic-related genes and proteins. Furthermore, in vivo, the 5R-E-DCFS membrane promoted the critical skull bone defects of SD rats repairment and regeneration. Therefore, this innovative biomimetic membrane holds substantial clinical potential as an ideal GBR membrane with mechanical properties for space-making and suitable degradation rate for bone regeneration to manage bone defects.

An immunomodulatory and osteogenic bacterial cellulose scaffold for bone regeneration via regulating the immune microenvironment

Creating a bone homeostasis microenvironment that balances osteogenesis and immunity is a substantial challenge for bone regeneration. Here, we prepared an immunomodulatory and osteogenic bacterial cellulose scaffold (FOBS) via a facile one-pot approach. The aldehyde groups were generated via selective oxidation of the hydroxyl groups of bacterial cellulose, offering the bonding sites for dopamine through a Schiff base reaction. At the same time, the deposition of Ca2+ and PO43− was promoted on the aldehyde cellulose scaffold because of the high affinity of the catechol moiety for Ca2+. Compared with that of the unmodified scaffold, the hydroxyapatite content of FOBS increased by 47.1 % according to the ICP results. Interestingly, FOBS regulated the immune microenvironment to accelerate the conversion of M1 to M2 macrophages. The expressions of ARG-1 and Dectin-1 (M2) in the FOBS group increased by >100 %. The expression of osteogenic differentiation of BMSCs was also upregulated. In a rat cranial defect model, the BV/TV of FOBS was significantly increased. Further immunohistochemical analysis revealed that an improved immune microenvironment promoted the osteogenic differentiation of stem cells in vivo. This work provides an effective and easy-to-operate strategy for the development of the bone tissue engineering scaffolds.

Global A-to-I RNA editing during myogenic differentiation of goat MuSCs.

RNA editing, especially A-to-I editing sites, is a common RNA modification critical for stem cell differentiation, muscle development, and disease occurrence. Unveiling comprehensive RNA A-to-I editing events associated with myogenesis of the skeletal muscle satellite cells (MuSCs) is essential for extending our knowledge of the mechanism underpinning muscle development. A total of 9,632 RNA editing sites (RESs) were screened in the myoblasts (GM), myocytes (DM1), and myotubes (DM5) samples. Among these sites, 4,559 A-to-I edits were classified and further analyzed. There were 3,266 A-to-I sites in the protein-coding region, out of which 113 missense sites recoded protein. Notably, five A-to-I sites in the 3' UTR of four genes (TRAF6, NALF1, SLC38A1, ENSCHIG00000019092) altered their targeted miRNAs. Furthermore, a total of 370 A-to-I sites with different editing levels were detected, including FBN1, MYH10, GSK3B, CSNK1D, and PRKACB genes. These genes were predominantly enriched in the cytoskeleton in muscle cells, the hippo signaling pathway, and the tight junction. Furthermore, we identified 14 hub genes (TUFM, GSK3B, JAK2, RPSA, YARS1, CDH2, PRKACB, RUNX1, NOTCH2, CDC23, VCP, FBN1, RARS1, MEF2C) that potentially related to muscle development. Additionally, 123 stage-specific A-to-I editing sites were identified, with 43 sites in GM, 25 in DM1, and 55 in DM5 samples. These stage-specific edited genes significantly enriched essential biological pathways, including the cell cycle, oocyte meiosis, motor proteins, and hedgehog signaling pathway. We systematically identified the RNA editing events in proliferating and differentiating goat MuSCs, which was crucial for expanding our understanding of the regulatory mechanisms of muscle development.

Biomimetic composite gelatin methacryloyl hydrogels for improving survival and osteogenesis of human adipose-derived stem cells in 3D microenvironment

Gelatin methacryloyl (GelMA) hydrogels are used for stem cell encapsulation in bone tissue engineering due to their fast and stable photo-crosslinking. However, cell viability and ability to induce osteogenesis are reduced by reactive oxygen species (ROS) produced during the crosslinking reaction. In this study, we developed biomimetic nanoparticles (TMNs) by combining tannic acid (TA) and simulated body fluid (SBF) minerals, and used them to synthesize GelMA-based composite hydrogels for addressing those limitations. The optimal concentrations of TA and SBF were investigated to create nanoparticles that can effectively scavenge ROS and induce osteogenesis. The incorporation of TMNs into composite hydrogels (G-TMN) significantly enhanced the survival and proliferation of encapsulated human adipose-derived stem cells (hADSCs) by providing resistance to oxidative conditions. In addition, the ions that were released, such as Ca2+ and PO43−, stimulated stem cell differentiation into bone cells. The hADSCs encapsulated in G-TMN had 2.0 ± 0.8-fold greater viability and 1.3 ± 1.8 times greater calcium deposition than those encapsulated in the hydrogel without nanoparticles. Furthermore, the in vivo transplantation of G-TMN into a subcutaneous mouse model demonstrated the rapid degradation of the gel-network while retaining the osteoinductive particles and cells in the transplanted area. The increased cellular activity observed in our multifunctional composite hydrogel can serve as a foundation for novel and effective therapies for bone deformities.

Histone Tail Cleavage as a Mechanism for Epigenetic Regulation.

Histones are essential for DNA packaging and undergo post-translational modifications that significantly influence gene regulation. Among these modifications, histone tail cleavage has recently garnered attention despite being less explored. Cleavage by various proteases impacts processes such as stem cell differentiation, aging, infection, and inflammation, though the mechanisms remain unclear. This review delves into recent insights on histone proteolytic cleavage and its epigenetic significance, highlighting how chromatin, which serves as a dynamic scaffold, responds to signals through histone modification, replacement, and ATP-dependent remodeling. Specifically, histone tail cleavage is linked to critical cellular processes such as granulocyte differentiation, viral infection, aging, yeast sporulation, and cancer development. Although the exact mechanisms connecting histone cleavage to gene expression are still emerging, it is clear that this process represents a novel epigenetic transcriptional mechanism intertwined with chromatin dynamics. This review explores known histone tail cleavage events, the proteolytic enzymes involved, their impact on gene expression, and future research directions in this evolving field.

Branched actin polymerization drives invasive protrusion formation to promote myoblast fusion during skeletal muscle regeneration.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. The fusion of satellite cell-derived mononucleated muscle cells (SCMs) is indispensable for the generation of multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain poorly understood. In this study, we uncovered an essential role for branched actin polymerization in SCM fusion. Using conditional knockouts of the Arp2/3 complex and its actin nucleation-promoting factors, N-WASP and WAVE, we demonstrated that branched actin polymerization is required for the SCM fusion, but not for satellite cell proliferation, differentiation, and migration. We showed that the N-WASP and WAVE complexes have partially redundant functions in regulating SCM fusion. Furthermore, we showed that branched actin polymerization is essential for generating invasive protrusions at the fusogenic synapses in SCMs. Taken together, our study has identified new components of the myoblast fusion machinery in skeletal muscle regeneration and demonstrated a critical role for branched actin-propelled invasive protrusions in this process.

The MADS-box gene XAANTAL1 participates in Arabidopsis thaliana primary root growth and columella stem cell patterns in response to ROS, via direct regulation of PEROXIDASE 28 and RETINOBLASTOMA RELATED.

The balance between cell growth, proliferation and differentiation emerges from gene regulatory networks coupled to various signal transduction pathways, including reactive oxygen species (ROS) and transcription factors (TFs), enabling developmental responses to environmental cues. The Arabidopsis thaliana's primary root has become a valuable system for unraveling such networks. Recently, the role of TFs that mediate the ROS's inhibition of primary root growth has begun to be characterized. This study demonstrates that the MADS-box transcription factor XAANTAL1 (XAL1) is an essential regulator of hydrogen peroxide (H2O2) in primary root growth and root stem cell niche identity. Interestingly, our findings suggest that XAL1 acts as a positive regulator of H2O2 concentration in the root meristem by directly regulating genes involved in oxidative stress response, such as PEROXIDASE 28 (PER28). Moreover, we found that XAL1 is necessary for the H2O2-induced inhibition of primary root growth through the negative regulation of peroxidase and catalase activities. Furthermore, XAL1, in conjunction with RETINOBLASTOMA-RELATED (RBR), is essential for positively regulating the differentiation of columella stem cells and for participating in primary root growth inhibition in response to oxidative stress induced by H2O2 treatment.

IMPC-based screening revealed that ROBO1 can regulate osteoporosis by inhibiting osteogenic differentiation.

The utilization of denosumab in treating osteoporosis highlights promising prospects for osteoporosis intervention guided by gene targets. While omics-based research into osteoporosis pathogenesis yields a plethora of potential gene targets for clinical transformation, identifying effective gene targets has posed challenges. We first queried the omics data of osteoporosis clinical samples on PubMed, used International Mouse Phenotyping Consortium (IMPC) to screen differentially expressed genes, and conducted preliminary functional verification of candidate genes in human Saos2 cells through osteogenic differentiation and mineralization experiments. We then selected the candidate genes with the most significant effects on osteogenic differentiation and further verified the osteogenic differentiation and mineralization functions in mouse 3T3-E1 and bone marrow mesenchymal stem cells (BMSC). Finally, we used RNA-seq to explore the regulation of osteogenesis by the target gene. We identified PPP2R2A, RRBP1, HSPB6, SLC22A15, ADAMTS4, ATP8B1, CTNNB1, ROBO1, and EFR3B, which may contribute to osteoporosis. ROBO1 was the most significant regulator of osteogenesis in both human and mouse osteoblast. The inhibitory effect of Robo1 knockdown on osteogenic differentiation may be related to the activation of inflammatory signaling pathways. Our study provides several novel molecular mechanisms involved in the pathogenesis of osteoporosis. ROBO1 is a potential target for osteoporosis intervention.

GeneCompass: deciphering universal gene regulatory mechanisms with a knowledge-informed cross-species foundation model.

Deciphering universal gene regulatory mechanisms in diverse organisms holds great potential for advancing our knowledge of fundamental life processes and facilitating clinical applications. However, the traditional research paradigm primarily focuses on individual model organisms and does not integrate various cell types across species. Recent breakthroughs in single-cell sequencing and deep learning techniques present an unprecedented opportunity to address this challenge. In this study, we built an extensive dataset of over 120 million human and mouse single-cell transcriptomes. After data preprocessing, we obtained 101,768,420 single-cell transcriptomes and developed a knowledge-informed cross-species foundation model, named GeneCompass. During pre-training, GeneCompass effectively integrated four types of prior biological knowledge to enhance our understanding of gene regulatory mechanisms in a self-supervised manner. By fine-tuning for multiple downstream tasks, GeneCompass outperformed state-of-the-art models in diverse applications for a single species and unlocked new realms of cross-species biological investigations. We also employed GeneCompass to search for key factors associated with cell fate transition and showed that the predicted candidate genes could successfully induce the differentiation of human embryonic stem cells into the gonadal fate. Overall, GeneCompass demonstrates the advantages of using artificial intelligence technology to decipher universal gene regulatory mechanisms and shows tremendous potential for accelerating the discovery of critical cell fate regulators and candidate drug targets.

Role of Immunohistochemistry in the Diagnosis of Pilomatrical Tumors.

Pilomatrical skin tumors harbor mutations in CTNNB1, which encodes for β-catenin, a downstream effector of the Wnt signaling pathway responsible for the differentiation, proliferation, and adhesion of epithelial stem cells. Therefore, downstream molecules, such as CDX2, LEF-1, and SATB2, in the Wnt signaling pathway could be useful diagnostic markers. Here, we sought to investigate the potential of immunohistochemistry (IHC) to differentiate between pilomatricoma and pilomatrical carcinoma, as well as from other cutaneous adnexal tumors. We studied 88 cases of cutaneous tumors (14 pilomatrical carcinomas, 18 pilomatricomas, 13 basal cell carcinomas, 12 squamous cell carcinomas, 12 sebaceous carcinomas, 10 Merkel cell carcinomas, 7 trichoblastomas, and 2 hidradenocarcinomas) using a broad panel of IHC markers: β-catenin, SATB2, CDX2, LEF1, Ber-EP4, and PRAME. Pilomatricoma and pilomatrical carcinoma displayed >75% nuclear staining for β-catenin. CDX2 also strongly stained pilomatrical tumors; however, the staining distribution was limited in pilomatricoma and more widespread in pilomatrical carcinoma. But, overall, it was less than β-catenin. SATB2 and Ber-EP4 expressions were noted only in a subset of both pilomatrical carcinoma and pilomatricoma, whereas LEF-1 showed strong, diffuse nuclear positivity in both pilomatricoma and pilomatrical carcinoma. Among the IHC markers evaluated, none could distinguish between pilomatricoma and pilomatrical carcinoma. However, the combined use of β-catenin with CDX2 markers may assist in not only confirming the pilomatrical nature of the proliferation but also in differentiating benign from malignant cases when there is a significant presence of CDX2 staining. Despite these findings, the diagnosis should continue to primarily depend on a thorough histopathologic examination.

Multi-omics integration analysis reveals the role of N6-methyladenosine in lncRNA translation during glioma stem cell differentiation.

Glioblastoma is one of the most lethal brain diseases in humans. Although recent studies have shown reciprocal interactions between N6-methyladenosine (m6A) modifications and long noncoding RNAs (lncRNAs) in gliomagenesis and malignant progression, the mechanism of m6A-mediated lncRNA translational regulation in glioblastoma remains unclear. Herein, we profiled the transcriptomes, translatomes, and epitranscriptomics of glioma stem cells and differentiated glioma cells to investigate the role of m6A in lncRNA translation comprehensively. We found that lncRNAs with numerous m6A peaks exhibit reduced translation efficiency. Transcript-level expression analysis demonstrates an enrichment of m6A around short open reading frames (sORFs) of translatable lncRNA transcripts. Further comparison analysis of m6A modifications in different RNA regions indicates that m6A peaks downstream of sORFs inhibit lncRNA translation more than those upstream. Observations in glioma-associated lncRNAs H19, LINC00467, and GAS5 further confirm the negative effect of m6A methylation on lncRNA translation. Overall, these findings elucidate the dynamic profiles of the m6A methylome and enhance the understanding of the complexity of lncRNA translational regulation.

Single-cell dissection reveals promotive role of ENO1 in leukemia stem cell self-renewal and chemoresistance in acute myeloid leukemia.

Quiescent self-renewal of leukemia stem cells (LSCs) and resistance to conventional chemotherapy are the main factors leading to relapse of acute myeloid leukemia (AML). Alpha-enolase (ENO1), a key glycolytic enzyme, has been shown to regulate embryonic stem cell differentiation and promote self-renewal and malignant phenotypes in various cancer stem cells. Here, we sought to test whether and how ENO1 influences LSCs renewal and chemoresistance within the context of AML. We analyzed single-cell RNA sequencing data from bone marrow samples of 8 relapsed/refractory AML patients and 4 healthy controls using bioinformatics and machine learning algorithms. In addition, we compared ENO1 expression levels in the AML cohort with those in 37 control subjects and conducted survival analyses to correlate ENO1 expression with clinical outcomes. Furthermore, we performed functional studies involving ENO1 knockdown and inhibition in AML cell line. We used machine learning to model and infer malignant cells in AML, finding more primitive malignant cells in the non-response (NR) group. The differentiation capacity of LSCs and progenitor malignant cells exhibited an inverse correlation with glycolysis levels. Trajectory analysis indicated delayed myeloid cell differentiation in NR group, with high ENO1-expressing LSCs at the initial stages of differentiation being preserved post-treatment. Simultaneously, ENO1 and stemness-related genes were upregulated and co-expressed in malignant cells during early differentiation. ENO1 level in our AML cohort was significantly higher than the controls, with higher levels in NR compared to those in complete remission. Knockdown of ENO1 in AML cell line resulted in the activation of LSCs, promoting cell differentiation and apoptosis, and inhibited proliferation. ENO1 inhibitor can impede the proliferation of AML cells. Furthermore, survival analyses associated higher ENO1 expression with poorer outcome in AML patients. Our findings underscore the critical role of ENO1 as a plausible driver of LSC self-renewal, a potential target for AML target therapy and a biomarker for AML prognosis.

MiR-378a-3p Regulates the BMP2-Smad Pathway to Promote Chondrogenic Differentiation of Synovium-Derived Mesenchymal Stem Cells.

This study aims to elucidate the role of miR-378a-3p in facilitating the proliferation and differentiation of synovium-derived mesenchymal stem cells (SMSCs) into chondrocytes. The effects of overexpressing miR-378a-3p on SMSCs were investigated through histological analysis, quantitative PCR, and western blotting. Then we identified binding sites of miR-378a-3p with BMP2 through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses and predictions from the RegRNA 2.0 database. Subsequently, BMP2 was confirmed as the target by which miR-378a-3p promotes the chondrogenic differentiation of SMSCs using a luciferase reporter gene assay and an miR-378a-3p RNA interference plasmid. Finally, by constructing a rat model with articular cartilage damage, we detected the reparative effects of miR-378a-3p overexpression on cartilage damage. Additionally, we verified the mechanism by which miR-378a-3p promotes chondrogenic differentiation in SMSCs. MiR-378a-3p enhances the proliferation and differentiation of SMSCs into chondrocytes by modulating the BMP2-Smad signaling pathway, thereby facilitating repair processes for articular cartilage injuries in rats. Notably, knockdown of BMP2 diminished the reparative efficacy of miR-378a-3p on articular cartilage damage. Upregulation of miR-378a-3p promotes chondrogenic differentiation in SMSCs through activation of the BMP2-Smad pathway, positioning it as a potential therapeutic target for osteoarthritis.

NEDD4L affects stability of the CHEK2/TP53 axis through ubiquitination modification to enhance osteogenic differentiation of periodontal ligament stem cells

ABSTRACT Background Checkpoint kinase 2 (CHEK2) and its regulated tumor protein p53 (TP53) have been correlated with osteogenic differentiation of osteoblast-like cells. Based on bioinformatics predictions, this study aims to investigate the effect of the CHEK2/TP53 axis on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to explore the regulatory mechanism. Methods PDLSCs were isolated from human impacted wisdom teeth, and they were cultured in normal medium (NM) or osteogenic medium (OM). Protein levels of CHEK2 and TP53 were examined using western blot analysis. Osteogenic differentiation ability of PDLSCs was analyzed by measuring marker proteins (RUNX2, OCN, and OSX), ALP activity, and ALP staining. Molecular interaction between NEDD4 like E3 ubiquitin protein ligase (NEDD4L) and CHEK2 was examined by ubiquitination and co-immunoprecipitation assays. Gain- and loss-of function assays of NEDD4L, CHEK2, and TP53 were performed to analyze their function in osteogenic differentiation of PDLSCs. A rat model of mandibular bone defect was generated for in vivo validation. Results NEDD4L was upregulated, while CHEK2 and TP53 were downregulated in PDLSCs cultured in OM. CHEK2 protected TP53 from degradation, while NEDD4L reduced CHEK2 protein level by ubiquitination modification. NEDD4L silencing reduced osteogenic differentiation ability of PDLSCs both in vitro and in vivo, which was restored by CHEK2 silencing. By contrast, CHEK2 overexpression blocked the osteogenic differentiation of PDLSCs in vitro. Conclusion This study demonstrates that NEDD4L affects protein stability of the CHEK2/TP53 axis through ubiquitination modification, thus increasing osteogenic differentiation of PDLSCs.

The impact of the physicochemical properties of calcium phosphate ceramics on biocompatibility and osteogenic differentiation of mesenchymal stem cells

ObjectiveIn this study we have focused on biocompatibility and osteoinductive capacity analysis of self-manufactured single-phase (HAP) and two-phase (HAP and β-ТСР) bioactive ceramics with various chemical modifications (Fig. 1).ResultsWe demonstrate a reduction in solubility for all analyzed composite after the treatment with H2O and H2O2, accompanied by an enhancement in adsorption activity. This modification also resulted in an increase in micro- and macroporosity, along with a rise in the open porosity. Adipose-derived mesenchymal stromal cells demonstrated excellent cell adhesion and survival when cultured with these ceramics. Calcium phosphate ceramics (H-500, HT-500, and HT-1 series) stimulated alkaline phosphatase expression, promoted calcium deposition, and enhanced osteopontin expression in ADSCs, independently inducing osteogenesis without additional osteogenic stimuli. These findings underscore the promising potential of HAP-based bioceramics for bone regeneration/reconstruction.Graphical

EMF treatment delays mesenchymal stem cells senescence during long-term in vitro expansion by modulating autophagy.

Bone marrow mesenchymal stem cells (BMSCs) are widely used in tissue engineering and regenerative medicine as seed cells. Due to low amount in bone marrow, BMSCs must be expanded and cultured in vitro before application. However, the senescence of stem cell caused by long-term in vitro culture greatly limits its efficacy of transplantation. In this study, we propose an approach based on electromagnetic fields (EMF) treatment to rejuvenate aged BMSCs due to long-term in vitro culture. Aged BMSCs were treated with sinusoidal EMF (50Hz, 0.4mT), and stem cell senescence, cell proliferation, cell differentiation, cell stemness and autophagy level were detected. Additionally, aged BMSCs-laden hydrogels were transplanted into the rat critical-sized calvarial defect with or without EMF treatment. The bone formation was evaluated 8weeks after surgery. Our results indicated that the BMSCs age significantly after long-term in vitro passaging. The self-renew, multiple differentiation capacity, senescence phenotypes and stemness of aged BMSCs are partly reversed by EMF treatment with a frequency of 50Hz and strength of 0.4mT. Moreover, declined autophagy level is observed in BMSCs during long-term in vitro passaging and BMSCs senescence is closely associated with autophagy regulation. Additionally, the mechanistic investigation reveals that EMF treatment rejuvenate senescent BMSCs by enhancing autophagy. Furthermore, EMF treatment significantly promote the therapeutic effect of long-term passaged BMSCs on bone formation in vivo. Overall, our study identifies a practical approach for the rejuvenation of old BMSCs and may provide a promising candidate in tissue engineering and stem cell therapy.