Stem Cell Behavior Research Articles

Salinomycin and IR780-loaded upconversion nanoparticles influence biological behavior of liver cancer stem cells by persistently activating the MAPK signaling pathway

The combination of chemotherapy and phototherapy has emerged as a promising therapeutic approach for enhancing the efficacy of cancer treatment and mitigating drug resistance. Salinomycin (SAL), a polyether antibiotic, exhibits potent cytotoxicity against chemotherapy-resistant cancer cells. IR780 iodide, a novel photosensitive reagent with excellent near-infrared (NIR) light absorption and photothermal conversion abilities, is suitable for use in photothermal therapy for cancers. However, both SAL and IR780 exhibit hydrophobic properties that limit their clinical applicability. Upconversion nanoparticles (UCNPs) are an emerging class of fluorescent probe materials capable of emitting high-energy photons upon excitation by low-energy NIR light. The UCNPs not only function as nanocarriers for drug delivery but also serve as light transducers to activate photosensitizers for deep-tissue photodynamic therapy. Here, to enhance the targeting and bioavailability of hydrophobic drugs in liver cancer stem cells (LCSCs), we employ distearoyl phosphorethanolamine-polyethylene glycol (DSPE-PEG) to encapsulate SAL and IR780 on the surface of UCNPs. Cell viability was evaluated using the CCK-8 assay. Cell migration was assessed by the Transwell Boyden Chamber. The activation of the mitogen-activated protein kinase (MAPK) signaling pathway was measured via western blot. The results demonstrated successful loading of both IR780 and SAL onto the UCNPs, and the SAL and IR780-loaded UCNPs (UISP) exhibited a robust photothermal effect under NIR light irradiation. The UISP effectively inhibited the viability of HCCLM3 and LCSCs. Under NIR light irradiation, the UISP further suppressed HCCLM3 viability but had no impact on LCSC viability; however, it could further inhibit LCSC migration. Meanwhile, under NIR light irradiation, the UISP persistently activated the MAPK pathway more significantly in LCSCs. These findings suggest that exposure to NIR light results in persistent activation of the MAPK pathway by UISP, thereby influencing the biological behavior of LCSCs and enhancing their therapeutic efficacy against liver cancer.

CYP26 Activity Maintains the Bone Marrow Niche and Stem Cell Homeostasis

Vitamin A and its active compound, retinoic acid (RA), manifest pleomorphic activities in hematopoiesis. Vitamin A homeostasis is maintained systemically through hepatic CYP26. Emerging data suggests that CYP26 mediated RA metabolism also plays a role in controlling hematopoietic stem and progenitor cells (HSPCs) behavior (Ghiaur G et al. PNAS 2013, Schonberger K et al. Cell Stem Cell 2022). We generated Cyp26a1/ Cyp26b1 inducible knockout (CYP26KO) using ROSA26CreERT Cyp26a1/ Cyp26b1 conditional mice to study the impact on hematopoiesis and the bone marrow microenvironment (BME). Tamoxifen induces complete CYP26KO in these mice and subsequently higher retinoid levels and systemic inflammation (Snyder JM et al. FASEB J 2020). Here, we characterize the hematopoietic, bone and bone marrow (BM) phenotype of these mice. Compared to the CYP26 wild type controls (CTR), we observed leukocytosis and mild anemia as well as signs of extramedullary hematopoiesis in CYP26KO mice (Table). Spleen histology and flow cytometry showed an increased number of all mature elements, including megakaryocytes as well as increased absolute numbers of all hematopoietic progenitor populations (HPCs): LSK, CMP, GMP and MEP (Table). BM analysis showed decreased cellularity and myeloid biased in CYP26KO mice (Table). There was an increased frequency of colony forming unit cell (CFU-C) in the BM of CYP26KO mice but overall preserved absolute numbers when accounted for cellularity (Table). We observed increased frequency of LSK (Table) but decreased numbers of CMP, GMP and MEP in the BM of CYP26KO mice. Both CFU assay and flow cytometry showed myeloid biased HPCs found in the BM of CYP26KO mice (Table). When analyzing the stem cell compartment, short term stem cells and multipotent progenitor cells number were decreased in CYP26KO BM (p<0.01), but the long-term HSCs (LT-HSC) were relatively preserved (0.04 ± 0.01 vs. 0.17 ± 0.04 x10 6/femur, CTR vs CYP26KO, p<0.05). Both flow cytometry and CFU assay confirmed the presence of circulating HPCs in CYP26KO mice (Table). Thus, hematopoiesis of CYP26KO mice migrated away from the BM and into extramedullary tissues. The hematopoietic phenotype was most likely due to the cell extrinsic role of CYP26 since bone marrow transplantation experiments confirmed that a CYP26KO recipient (regardless of the genotype of the hematopoietic cells) and not CYP26 wild type recipient recapitulates the observed hematopoietic findings. This observation, together with necropsy findings of thin, frail bones in CYP26KO mice, prompted a closer look at the BM matrix/microenvironment of these mice. CYP26KO mice had smaller BM cavities, decreased trabecular bone and an increased fat/bone marrow volume ratio (Table). Consistent with these findings, CYP26KO mesenchymal stroma cells (MSC) had no osteoblastic differentiation potential in vitro but showed preserved adipogenic potential. The number of CFU-fibroblasts was decreased in the BM of CYP26KO mice but were found circulating in the PB and were dramatically increased in the spleen of these mice (Table). Single cell RNAseq showed quantitative and qualitative alterations in the BME populations with relative reduction of Cxcl12 abundant MSC2, which explained the qRT-PCR observation of lower levels of Cxcl12 mRNA in MSC isolated from CYP26KO mice. More so, Spp1 mRNA was downregulated in various MSC populations of CYP26KO bone marrow, consistent with their osteopenia and with potential implications for the observed HSPC phenotype. Receptor ligand analysis revealed that various components of the BME have impaired interactions with the extracellular matrix. Thus, the leading hypothesis is that absence of CYP26 leads to a dysregulated BME and to migration of a HSCs-niche unit out of the BM and into secondary hematopoietic organs. Interestingly, scRNAseq experiments uncovered upregulation of S100a8/9 in the CYP26KO neutrophil progenitors, which may be in response to systemic stress. The observed bone alterations (osteopenia and increased BM fat) coupled with noted changes in hematopoiesis (i.e. myeloid bias and inflammatory stress) may represent early signs of aging of the bone and bone marrow and suggest a central role of retinoid homeostasis in this process.

Regulation of Tendon Stem Cell Behavior by Designed Nanoporous Topography of Microfibers.

Nano scale topography scaffold is more bioactive and biomimetic than smooth fiber topographies. Tendon stem cells (TSCs) play important roles in the tendinogenesis of tendon tissue engineering, but the effects and mechanisms of nano topography on TSC behavior are still unclear. This study determined whether the morphology, proliferation, cytoskeleton, and differentiation of TSCs are affected by topography of scaffold in vitro. The porous PA56 scaffolds were prepared with different concentration ratios of glycerol as the molecular template by electrospinning. Its topological characteristics, hydrophilicity, and degradation properties varied with glycerol proportion and movement rate of the receiving plate. Porous fibers promoted the proliferation of TSCs and the number of TSCs varied with topography. Although there was no significant difference due to the small sample size, the number of pseudopodia and cell polarizability still showed differences among different topographies. The morphology of actin cytoskeleton of TSCs showed difference among cultured on porous fibers, smooth fibers, and in culture media with no fiber, suggesting the orientation growth of cells on porous fiber. Moreover, porous fibers promoted teno-lineage differentiation of TSCs by upregulating tendon-specific gene expression. These findings provide evidence that nano porous topography scaffold promotes TSC proliferation, cytoskeleton orientation, and tenogenic differentiation.

Unveiling Cellular Traits of Osteosarcoma Cancer Stem Cells via Bulk and Single-Cell RNA-Sequencing Analysis

Osteosarcoma (OS) is a prototypical sarcoma, predominantly affecting adolescents. The hallmark of cancer stem cell (CSC) behavior pervades OS, invariably signifying an unfavorable prognosis. However, the intricacies underlying OS metastasis remain incompletely comprehended. As the frontiers of the scientific investigation push forward, encompassing both bulk sequencing and single-cell sequencing (scRNA), the domain of bioinformatics finds multifaceted utility. In this study, we combined scRNA and bulk sequencing with the clinical metadata to excavate the latent molecular substrates governing metastatic propensities within OS. Our scRNA analysis indicated that cell-stemness-related pathways might play vital roles in OS metastasis. Subsequently, an autonomous reservoir of bulk sequencing data set was subjected to weighted gene co-expression network analysis (WGCNA) to identify 10 gene clusters. After analyzing the clinical data, we were able to identify two hub genes, HDAC2 and HSPA4, which are strongly linked to the cancer cell stemness. Moreover, we performed transwell assays to validate the regulation of metastatic behaviors by miR-1-3p, HDAC2, and HSPA4 in OS cells. Significantly, our study was fortified by immunohistochemistry (IHC) analyses performed on tumor tissues acquired from OS patients, thereby accentuating the clinical import of our experimental endeavor. Notably, we unveiled the suppressive influence of miR-1-3p on both HDAC2 and HSPA4, with miRTarBase substantiating lower expression in tumor tissue relative to the normative cohort. Insights gleaned from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program (https://ocg.cancer.gov/programs/target) further augmented the clinical significance of the identified hub genes. In conclusion, our findings highlight the significant role of cell junctions in governing OS stemness and metastasis, underscored by the integration of single-cell sequencing and bulk-sequencing analyses. Moreover, our foundational experiments identified three hub molecules closely associated with the metastatic behaviors. Our findings provide novel insights into the clinical treatment and fundamental understanding of OS.

The Epigenetic Regulation of Quiescent in Stem Cells.

This review article discusses the epigenetic regulation of quiescent stem cells. Quiescent stem cells are a rare population of stem cells that remain in a state of cell cycle arrest until activated to proliferate and differentiate. The molecular signature of quiescent stem cells is characterized by unique epigenetic modifications, including histone modifications and deoxyribonucleic acid (DNA) methylation. These modifications play critical roles in regulating stem cell behavior, including maintenance of quiescence, proliferation, and differentiation. The article specifically focuses on the role of histone modifications and DNA methylation in quiescent stem cells, and how these modifications can be dynamically regulated by environmental cues. The future perspectives of quiescent stem cell research are also discussed, including their potential for tissue repair and regeneration, their role in aging and age-related diseases, and their implications for cancer research. Overall, this review provides a comprehensive overview of the epigenetic regulation of quiescent stem cells and highlights the potential of this research for the development of new therapies in regenerative medicine, aging research, and cancer biology.

Apoptotic dysregulation mediates stem cell competition and tissue regeneration

Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in “loser” cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.

Epitranscriptomic modifications in mesenchymal stem cell differentiation: advances, mechanistic insights, and beyond.

RNA modifications, known as the "epitranscriptome", represent a key layer of regulation that influences a wide array of biological processes in mesenchymal stem cells (MSCs). These modifications, catalyzed by specific enzymes, often termed "writers", "readers", and "erasers", can dynamically alter the MSCs' transcriptomic landscape, thereby modulating cell differentiation, proliferation, and responses to environmental cues. These enzymes include members of the classes METTL, IGF2BP, WTAP, YTHD, FTO, NAT, and others. Many of these RNA-modifying agents are active during MSC lineage differentiation. This review provides a comprehensive overview of the current understanding of different RNA modifications in MSCs, their roles in regulating stem cell behavior, and their implications in MSC-based therapies. It delves into how RNA modifications impact MSC biology, the functional significance of individual modifications, and the complex interplay among these modifications. We further discuss how these intricate regulatory mechanisms contribute to the functional diversity of MSCs, and how they might be harnessed for therapeutic applications. The review also highlights current challenges and potential future directions in the study of RNA modifications in MSCs, emphasizing the need for innovative tools to precisely map these modifications and decipher their context-specific effects. Collectively, this work paves the way for a deeper understanding of the role of the epitranscriptome in MSC biology, potentially advancing therapeutic strategies in regenerative medicine and MSC-based therapies.

Control of stem cell behavior by CLE-JINGASA signaling in the shoot apical meristem in Marchantia polymorpha

Land plants undergo indeterminate growth by the activity of meristems in both gametophyte (haploid) and sporophyte (diploid) generations. In the sporophyte of the flowering plant Arabidopsis thaliana, the apical meristems are located at the shoot and root tips in which a number of regulatory gene homologs are shared for their development, implying deep evolutionary origins. However, little is known about their functional conservation with gametophytic meristems in distantly related land plants such as bryophytes, even thoughgenomic studies have revealed that the subfamily-level diversity of regulatory genes is mostly conserved throughout land plants. Here, we show that a NAM/ATAF/CUC (NAC) domain transcription factor, JINGASA (MpJIN), acts downstream of CLAVATA3 (CLV3)/ESR-related (CLE) peptide signaling and controls stem cell behavior in the gametophytic shoot apical meristem of the liverwort Marchantia polymorpha. In the meristem, strong MpJIN expression was associated with the periclinal cell division at the periphery of the stem cell zone (SCZ), whereas faint MpJIN expression was found at the center of the SCZ. Time course observation indicates that the MpJIN-negative cells are lost from the SCZ and respecified de novo at two separate positions during the dichotomous branching event. Consistently, the induction of MpJIN results in ectopic periclinal cell division in the SCZ and meristem termination. Based on the comparative expression data, we speculate that the function of JIN/FEZ subfamily genes was shared among the shoot apical meristems in the gametophyte and sporophyte generations in early land plants but was lost in certain lineages, including the flowering plant A.thaliana.

Synergistic effect of chitosan-alginate composite hydrogel enriched with ascorbic acid and alpha-tocopherol under hypoxic conditions on the behavior of mesenchymal stem cells for wound healing

BackgroundIn regenerative medicine, especially skin tissue engineering, the focus is on enhancing the quality of wound healing. Also, several constructs with different regeneration potentials have been used for skin tissue engineering. In this study, the regenerative properties of chitosan-alginate composite hydrogels in skin wound healing under normoxic and hypoxic conditions were investigated in vitro.MethodsThe ionic gelation method was used to prepare chitosan/alginate (CA) hydrogel containing CA microparticles and bioactive agents [ascorbic acid (AA) and α-tocopherol (TP)]. After preparing composite hydrogels loaded with AA and TP, the physicochemical properties such as porosity, pore size, swelling, weight loss, wettability, drug release, and functional groups were analyzed. Also, the hemo-biocompatibility of composite hydrogels was evaluated by a hemolysis test. Then, the rat bone marrow mesenchymal stem cells (rMSCs) were seeded onto the hydrogels after characterization by flow cytometry. The survival rate was analyzed using MTT assay test. The hydrogels were also investigated by DAPI and H&E staining to monitor cell proliferation and viability. To induce hypoxia, the cells were exposed to CoCl2. To evaluate the regenerative potential of rMSCs cultured on CA/AA/TP hydrogels under hypoxic conditions, the expression of the main genes involved in the healing of skin wounds, including HIF-1α, VEGF-A, and TGF-β1, was investigated by real-time PCR.ResultsThe results demonstrated that the prepared composite hydrogels were highly porous, with interconnected pores that ranged in sizes from 20 to 188 μm. The evaluation of weight loss showed that the prepared hydrogels have the ability to biodegrade according to the goals of wound healing. The reduction percentage of CA/AA/TP mass in 21 days was reported as 21.09 ± 0.52%. Also, based on wettability and hemolysis tests of the CA/AA/TP, hydrophilicity (θ = 55.6° and 53.7°) and hemocompatibility with a hemolysis ratio of 1.36 ± 0.19 were evident for them. Besides, MTT assay, DAPI, and H&E staining also showed that the prepared hydrogels provide a suitable substrate for cell growth and proliferation. Finally, based on real-time PCR, increased expression levels of VEGF and TGF-β1 were observed in rMSCs in hypoxic conditions cultured on the prepared hydrogels.ConclusionsIn conclusion, this study provides evidence that 3D CA/AA/TP composite hydrogels seeded by rMSCs in hypoxic conditions have great potential to improve wound healing.Graphical abstract

Mesenchymal stem cells of the bone marrow raise infectivity of Plasmodium falciparum gametocytes.

While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields.

The behavior of adrenal progenitor stem cells in response to chronic stress and recovery

Stressors affect the differentiation of stem cells in the hypothalamic-pituitary-adrenal axis (HPA). This study was designed to ensure the presence of progenitor cells in the adult adrenal gland and to evaluate their behavior under chronic stress and after recovery. Also, to assess their ability to recruit new glial and chromaffin cells. Three groups of adult male rats (6 rats each): control, chronic-stress (rats were placed individually in stainless restrainers 2h/day for six days), recovered (rats were housed in an enriched circumference for seven days after the same stress modality). Both chronic-stress and recovered groups showed increased adrenal glands weight and cortisol levels with vacuolation, hemorrhage, and edema in the cortex and medulla. The chronic-stress group illustrated a significant increase in the chromaffin reaction, which was reduced in the recovered group. Evaluation of the immunohistochemical results revealed a significant decrease in the Nestin and GFAP (glial fibrillary acidic protein) reactions, but an increase in the chromogranin-A reaction in the chronic-stress group. The recovered group demonstrated a significant increase in the Nestin and GFAP and a reduction in the chromogranin-A immunohistochemical reactions. These results indicate the differentiation of the progenitor (Nestin expressing) stem cells into chromaffin (chromogranin-A expressing) cells under stress conditions for stress adaptation. Conversely, under normal conditions, the differentiation moved toward the glial cells.

Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila

Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. Here, we introduce PromoterSwitch, a genetic platform we established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. We show that cancer-driving genetic alterations representing common colon tumor genome landscapes disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. We also show that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy.

Preparation and Microscopic Mechanical Characterization of L-Methionine-Based Polyphosphazene Fibrous Mats for Vascular Tissue Engineering.

This study investigates the mechanical properties, degradation behavior, and biocompatibility of poly[(α-amino acid ester) phosphazene] electrospun fibers based on the ethyl ester of L-methionine (PαAPz-M), a material with potential applications in tissue engineering. We utilized atomic force microscopy (AFM) to evaluate the fiber mechanical characteristics and calculate its Young's modulus, revealing it to closely mimic the stiffness of a natural extracellular matrix (ECM). We also studied the degradation behavior of PαAPz-M scaffolds over 21 days, showing that they maintain the highly porous structure required for tissue engineering. Further evaluation of mesenchymal multipotent 10T1/2 cell and mesenchymal stem cell (MSC) behavior on the scaffolds demonstrated significant cell viability, proliferation, and successful MSC differentiation into smooth muscle cells. Expression of collagen and elastin by MSCs on the fiber mats highlighted potential ECM formation during scaffold degradation, confirming PαAPz-M as a promising material for vascular tissue engineering.

In Vivo Stem Cell Discoveries: Promising Implications in Cancer Therapy

The remarkable regenerative abilities and versatility of stem cells have long attracted researchers. Recently, in vivo studies have revealed exciting results related to stem cells, particularly their use in cancer treatment. This review will provide an overview of these discoveries and their broader implications for the future. There is growing in vivo evidence that stem cells have immense therapeutic potential in treating various diseases, including cancer, because of their self-renewal and differentiation capabilities. As a result of in vivo research, critical aspects of stem cell behavior within tumor microenvironments have been clarified, providing a deeper understanding of their potential therapeutic utility. Several in vivo studies have demonstrated the potential of stem cell-engineered tumor-targeting agents or therapeutic payloads for the precise delivery of medicinal drugs when these agents are engineered to express them in tumor cells. Through targeted therapies, off-target effects can be minimized, and the therapeutic index of the anti-cancer agents can be improved. Several stem cell-based delivery systems have shown remarkable efficacy in preclinical in vivo studies, including breast, lung, and pancreatic cancer, indicating their potential as a novel therapeutic strategy. Moreover, in vivo studies have revealed that the immunomodulatory properties of stem cells modulate the immune response and modify the tumor microenvironment to suppress it. In particular, using checkpoint inhibitor therapy with stem cells has paved the way for innovative immunotherapeutic strategies. Research on stem cells in vivo has also provided invaluable insights into stem cell biology and their interaction with cancer cells. Due to these findings, there is an increasing understanding of tumor initiation, progression, and resistance mechanisms, which has opened avenues for improving cancer treatment by developing more effective treatments. As a result of the in vivo studies that have taken place so far, there is a wealth of information regarding the potential of stem cells in cancer treatment. This research opens up exciting prospects for the future of oncology, from the delivery of targeted drugs to immunomodulation and improving our understanding of tumor biology.

Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.

Promoted osteogenesis by corona discharge poling induced in electroactive piezoelectric bioceramics

Rapid bone reconstruction and regeneration process is still a problem to be solved in the clinical orthopedic treatment. In vivo electric stimulation (ES) shows great promise in repair and regeneration of damaged tissues, and the piezoelectric characteristic of nature bone tissue make the electrical activity piezoelectricity biomaterials feasible and attractive in bone regeneration. Herein, we prepared a biocompatible lead-free piezoelectric ceramic K0.5Na0.5NbO3 (KNN) via solid phase sintering, which possesses wireless ES capability through corona discharge poling. Compared with the traditional piezoelectric ceramic polarization methods, corona discharge poling is a non-contact and biologically safer electrical poling method without electrodes or insulating liquids. The results of piezoelectricity, the water contact angle, the protein adsorption, surface potential characteristics of KNN ceramics and bone mesenchymal stem cells (BMSCs) behavior on ceramics, showed that after corona discharge poling the surface of KNN piezoelectric ceramic became hydrophilic, and positively charged, and the electrostatic effect formed by these charges promoted protein adsorption and BMSCs attachment and proliferation, which further promoted osteogenic differentiation. In conclusion, we used the corona discharge to polarize KNN piezoelectric ceramics and excellent biocompatibility of the ceramics was maintained after poling. Meanwhile, our work demonstrated the potential application of piezoelectric bio-ceramic materials in bone regeneration.

SAT268 Regulation Of Stem/Progenitor Cells Of The HPA Axis During Stress Adaptation

Abstract Disclosure: P. Siatra: None. D. Cozma: None. I. Berger: None. I. Oikonomakos: None. M. Theodoropoulou: None. S.R. Bornstein: None. C.L. Andoniadou: None. C. Steenblock: None. Chronic stress is a pervasive concern in the modern society. A long-term hyper activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to elevated amounts of stress hormones [e.g. ACTH and glucocorticoids (GCs)]. This can incur maladaptation that eventually contributes to mental illness, cardiovascular dysfunctions, diabetes, cancer and autoimmune diseases. Emerging key players for stress adaptation of the HPA axis are stem/progenitor cell populations. In this study, we aim to unravel the mechanisms involved in the regulation of stem/progenitor cells of the HPA axis during stress adaptation. To that aim, we incorporated an in vitro system of non-adherent spheroids from primary adrenocortical mouse cells that were stimulated with ACTH. The treatment resulted in the increased growth of the spheroids compared to unstimulated controls indicating a potential role of the hormone in cell proliferation and stem cell behaviour. This result is in line with the enlarged adrenals observed in patients with Cushing’s syndrome. To assess the contribution of stem cells during stress adaptation in the adrenal cortex in vivo, we performed immobilization stress and lineage tracing studies using the tamoxifen inducible Gli1:CreERT2/R26R:eYFP mouse line. By combining these results along with single-cell RNA-seq technology, we aim to shed light into molecular factors involved in the adult adrenal cortex regeneration and remodelling. To follow the effect of GC excesses on the negative feedback loop observed during a normal stress response on the pituitary gland, we stimulated adherent primary anterior pituitary colonies with dexamethasone and corticosterone. GC treatment led to downregulation of Pomc expression, a precursor polypeptide for ACTH and other hormones, in the treated colonies compared to the control. This downregulation caused by GCs could be mediated through different gene targets of the glucocorticoid receptor as well as via paracrine signalling from pituitary stem cells. Altogether, our results will provide us with more profound knowledge of mechanisms underlying interactions between stressors and stem/progenitor cells of the HPA axis along with novel therapeutic targets for stress-associated disorders. Presentation: Saturday, June 17, 2023

Comparative biological properties of resin-free and resin-based calcium silicate-based endodontic repair materials on human periodontal ligament stem cells

ObjectivesTo investigate the effect of three different calcium silicate-based materials (CSBM) on the biological behavior of human periodontal ligament stem cells (hPDLSCs).MethodsEluates of Biodentine, NeoPutty and TheraCal PT prepared at 1:1, 1:2, and 1:4 ratios were extracted under sterile conditions. The cytotoxicity of the extracts to the hPDLSCs was assessed using the MTT assay. Scratch wound healing assay was utilized for assessing cell migration. Scanning electron microscopy was used to detect cell attachment and morphology. Calcium ion release was measured using inductively coupled plasma-optical emission spectrometry; the pH-value was evaluated with a pH-meter. ANOVA with post hoc Tukey test was used for statistical analysis.ResultsCell viability was significantly higher for Biodentine and NeoPutty at day 1 with all dilutions (p < 0.05), while at day 3 and day 7 with dilutions 1:2 and 1:4; all materials showed similar behavior (p > 0.05). Biodentine had the highest percentage of cell migration into the scratched area at day 1 for all dilutions (p < 0.05). Stem cells were attached favorably on Biodentine and NeoPutty with evident spreading, and intercellular communications; however, this was not shown for TheraCal PT. Biodentine showed the highest pH values and calcium ion release (p < 0.05).ConclusionsThe resin-free CSBM showed better performance and favorable biological effects on hPDLSCs and were therefore considered promising for usage as endodontic repair materials.Clinical significance: Proper selection of materials with favorable impact on the host stem cells is crucial to ensure outcome in different clinical scenarios.

Head dysgenesis and disruption of cranial neural crest stem cells behaviour by 4-octylphenol in fire-bellied toad Bombina orientalis embryos

Alkylphenolic endocrine disruptors (Eds) have been known to affect development of the descendants of multipotent neural crest cells (NCCs) in amphibian embryos. To unravel the mechanism of head dysgenesis induced by alkylphenols in amphibians, the effect of 4-octylphenol (OP) on the differentiation of cranial NCCs in developing embryos and tadpoles, ex vivo NC explant, and isolated NCCs was examined in fire-bellied toad Bombina orientalis with 0, 1, 2, 5, 10, 25 and 50 μM concentrations. Following OP treatment, head cartilages were frequently absent together with the decreased col2a1 mRNA level in tadpoles. While the lipid hydroperoxide (LPO), endoplasmic reticulum stress (ERS), apoptosis, and DNA fragmentation were significantly increased in stage 22 neulurae and heads of stage 45 tadpoles. In stage 22 neulurae, OP decreased sox9 mRNA, the master transcription factor for chondrogenic differentiation and increased undifferentiated NCC markers. The ectopic NCCs were found in endoderm while mesodermal SOX10(+) cells were decreased. In cranial NCCs isolated from stage 22 embryos, OP treatment decreased cellular survival and increased apoptosis, epithelial-mesenchymal transition (EMT) and cell migration. In chondrogenic induced cranial NC explants, OP treatment decreased SOX9(+) chondrocytes and cartilage development. Together, OP potentiated oxidative damage, apoptosis, EMT, and ectopic migration of NCCs. Considering that tissue differentiation requires stem cells to activate the molecular mechanism of differentiation at the correct location during embryonic development, these changes caused by OP may inhibit sox9-dependent chondrogenic differentiation of cranial NCCs, leading to head dysgenesis in B. orientalis embryos. Therefore, developing multipotent NCCs could be an important target of OP, provides new direction for the estimation of the risk of EDs exposure in human and wildlife animals.

The role of a nanogrooved polydimethylsiloxane substrate on mesenchymal stem cells adhesion, self-renewing, and mechanical properties

Stem cells have a variety of applications in biological, reconstructive medicine, and tissue engineering studies owing to their capacity to differentiate into specific cell lineages and their self-renewal ability. Among the factors affecting stem cells, biochemical agents such as growth and differentiation factors, along with environmental cues including physical and mechanical characteristics of the cell microenvironment, are of interest in this field. Several studies have evaluated the impact of physical factors such as mechanical forces, electric or magnetic fields, and light pulses on stem cell behavior. Also, cell culture substrates that can simulate extracellular matrix in natural conditions are considered the most critical environmental factors. Hence, in this study, we investigated the effects of a specially designed polydimethylsiloxane substrate with a surface pattern for cell contact guidance on various biological and biophysical characteristics of mesenchymal stem cells. Plasma treatment of the substrate under lateral tension from 50 to 200 s caused the increased wavelength of the formed wrinkle structures from 500 ± 56 to 1052 ± 182 nm, respectively, leading to variations of stiffness affecting the cell behaviors. Moreover, by increasing the available surface and hydrophilicity of the substrate, the cell attachment quality improved by more than 46% compared to the control. Finally, results of the mechanics of mesenchymal stem cells on these substrates obtained by bio-AFM showed an increase in the stiffness of cells despite a slight decrease in Young's modulus parameter of different substrates. These changes in the intracellular mechanics can eventually stimulate the more pivotal mechanobiological effects on stem cell fate.