Early Differentiation Markers Research Articles

PDX1 as a Marker of Early Differentiation of Human Pancreatic Duct Cells.

Epithelial progenitor cells of the primitive pancreatic ducts differentiate in 3 directions, which is accompanied by changes in cell phenotype. We analyze the distribution of the pancreatic and duodenal homeobox 1 (PDX1) in the epithelial cells of pancreatic ducts during the prenatal development in order to determine the potential use of this marker as an indicator of pancreatic cell maturation. Pancreatic autopsies from human fetuses (gestation weeks 17-23) were examined by immunohistochemical methods. In general, PDX1 was detected in the epithelial cells that are involved in various morphogenetic processes related to the growth and branching of the fetal pancreatic ducts. The intensive positive reaction to PDX1/cytokeratin 19 indicates immature (progenitor) polypotent ductal cells.

Mucosa-like differentiation of head and neck cancer cells is inducible and drives the epigenetic loss of cell malignancy

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue, distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection.

Ultrasensitive Photoelectrochemical Biosensor for Dual-miRNAs Detection Based on Molecular Logic Gates and Methylene Blue Sensitized ZnO@CdS@Au Nanorods.

The occurrence of cancer is often closely related to multiple tumor markers, so it is important to develop multitarget detection methods. By the proper design of the input signals and logical operations of DNA logic gates, detection and diagnosis of cancer at different stages can be achieved. For example, in the early stages, specific input signals can be designed to correspond to early specific tumor markers, thereby achieving early cancer detection. In the late stage, logic gates for multitarget detection can be designed to simultaneously detect multiple biomarkers to improve diagnostic accuracy and comprehensiveness. In this work, we constructed a dual-target-triggered DNA logic gate for anchoring DNA tetrahedra, where methylene blue was embedded in the DNA tetrahedra to sensitize ZnO@CdS@Au, achieving ultrasensitive detection of the target substance. We tested the response of AND and OR logic gates to the platform. For AND logic gates, the sensing platform only responds when both miRNAs are present. In the concentration range of 10 aM to 10 nM, the photoelectric signal gradually increases with an increase of the target concentration. Subsequently, we used OR logic gates for miRNA detection. Even if only one target exists, the sensing platform exhibits excellent performance. Similarly, within the concentration range of 10 aM to 10 nM, the photoelectric signal gradually increases with an increase of the target concentration. The minimum detection limit is 1.10 aM. Whether it is the need to detect multiple targets simultaneously or only one of them, we can achieve it by selecting the appropriate logic gate. This strategy holds promising application prospects in fields such as biosensing, medical diagnosis, and environmental monitoring.

Electric fields reverse the differentiation of keratinocyte monolayer by down-regulating E-cadherin through PI3K/AKT/Snail pathway

Re-epithelialization is an important step in skin wound healing, referring to the migration, proliferation, and differentiation of keratinocytes around the wound. During this process, the edges of the wound begin to form new epithelial cells, which migrate from the periphery of the wound towards the center, gradually covering the entire wound area. These newly formed epithelial cells proliferate and differentiate, ultimately forming a protective layer over the exposed dermal surface. Wound endogenous electric fields (EFs) are known as the dominant factor to facilitate the epidermal migration to wound center. However, the precise mechanisms by which EFs promote epidermal migration remains elusive. Here, we found that in a model of cultured keratinocyte monolayer in vitro, EFs application reversed the differentiation of cells, as indicated by the reduction of the early differentiation markers K1 and K10. Genetic manipulation confirmed that EFs reversed keratinocyte differentiation through down-regulating the E-cadherin-mediated adhesion. By RNA-sequencing analysis, we screened out Snail as the transcription suppressor of E-cadherin. Snail knockdown abolished the down-regulation of E-cadherin and the reversal of differentiation induced by EFs. KEGG analysis identified PI3K/AKT signaling for Snail induction under EFs. Inhibition of PI3K by LY294002 diminished the EFs-induced AKT activation and Snail augmentation, largely restoring the level of E-cadherin reduced by EFs. Finally, in model of full-thickness skin wounds in pigs, we found that weakening of the wound endogenous EFs by the direction-reversed exogenous EFs resulted in an up-regulation of E-cadherin and earlier differentiation in newly formed epidermis in vivo. Our research suggests that electric fields (EFs) decrease E-cadherin expression by suppressing the PI3K/AKT/Snail pathway, thereby reversing the differentiation of keratinocytes. This discovery provides us with new insights into the role of electric fields in wound healing. EFs intervene in intracellular signaling pathways, inhibiting the expression of E-cadherin, which results in a lower differentiation state of keratinocytes. In this state, keratinocytes exhibit increased migratory capacity, facilitating the migration of epidermal cells and wound reepithelialization.

Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

Abstract 2141: An IL12/IL23 Positive Feedback Loop Modulates Smooth Muscle Differentiation Via Setd4 During Macrophage-mediated Inflammation

Diabetic patients have increased risk of vascular disease (e.g., atherosclerosis), which is driven by macrophage-mediated inflammation. In diseases such as atherosclerosis, smooth muscle cells (SMC) undergo phenotypic modulation from a mature, contractile to a synthetic phenotype, thus leading to increased neointima formation. We hypothesize that pro-inflammatory macrophages as in the setting of diabetes epigenetically pattern SMC during vascular disease. In this study, we use single cell-RNA sequencing (scRNA-seq) of human arteries, loss of function experiments, and mouse models of disease to identify epigenetic mechanisms that regulate SMC phenotype. We identified the H3K4 and H3K9 histone lysine methyltransferase Setd4, which was increased in aortic SMC from diabetic mice and in our scRNA-seq dataset from human atherosclerotic arteries. siRNA mediated knockdown of Setd4 in mouse aortic SMC increased expression of the early SMC differentiation markers, Acta2 and Tagln (p<0.05). To model the pro-inflammatory environment in vitro, supernatant from LPS-stimulated macrophages increased Setd4 expression and inhibited smooth muscle gene expression ( Acta2, Tagln, Cnn1, Myh11 ), which was reversed by siRNA-mediated knockdown of Setd4 in SMC (p<0.05). IL23 increased Setd4 expression in SMC and enrichment at smooth muscle gene promoters, while deceasing SMC-specific gene expression (p<0.05). Treatment of SMC with an IL23 receptor neutralizing antibody prevented downregulation of SMC gene expression by macrophage supernatant (p<0.05). Intriguingly, ELISA of mouse aortic SMC supernatant revealed that IL23 increased SMC secretion of IL12, which also inhibited SMC gene expression via Setd4 (p<0.05). In conclusion, we demonstrate that pro-inflammatory macrophage IL23 represses smooth muscle gene expression via SETD4, thereby shifting the SMC to a synthetic, diseased phenotype. IL23 also stimulates vascular SMC secretion of IL12, which further increases production of inflammatory cytokines from macrophages and leads to additional repression of smooth muscle gene expression. Thus, targeting the IL23-SETD4 axis in SMC may be a potential therapy for the treatment of atherosclerosis, especially in patients who also have diabetes.

Immunophenotyping of blood and bone marrow cells as a way to search for differentiation syndrome risk factors in acute promyelocytic leukemia

Background. Differentiation syndrome (DS) is a potentially fatal complication of therapy for acute promyelocytic leukemia (APL) with an incidence of up to 48 %. To date, no reliable DS risk factors have been found, with the exception of leukocytosis at the APL onset.Aim. To determine the risk factors associated with DS in patients with APL during induction therapy with arsenic trioxide (ATO) and tretinoin (ATRA).Materials and methods. The study included 39 patients with APL, 29 (74.4 %) of them were classified as low-risk according to ELN (European Leukemia Net), 10 (25.6 %) were classified as high-risk. At the disease onset, cytological and molecular (chimeric transcript PML::RARα, FLT3-ITD mutation) bone marrow studies were performed, the expression of 28 differentiation antigens by blood and bone marrow blast cells was determined (markers of early precursors, myeloid and lymphoid differentiation, cell adhesion molecules, chemokine receptors, integrins, selectin), body mass index (BMI) and the leukocytes number dynamics during induction course were assessed. All patients received ATRA and ATO therapy. Patients from the high-risk group at the onset received 1–3 injections of idarubicin (12 mg/m2) and dexamethasone (8–10 mg/m2 2 times a day) to prevent DS until leukocytosis reduced. In cases of DS, dexamethasone was prescribed at a dose of 10 mg/m2 2 times a day; in cases of severe DS, the induction course was interrupted.Results. Of the 39 patients, 12 (30.8 %) were diagnosed with DS: 20 % of high-risk patients (2/10) and 34.5 % of low-risk patients (10/29). There was no statistically significant association of leukocytosis more than 10 × 109 /L at onset, microgranular morphology of blast cells, bcr3-variant PML::RARα, FLT3-ITD mutation with DS. In multivariate analysis, the probability of DS was associated with BMI ≥30 kg/m2 and mean fluorescence intensity of CD38 antigen by blast cells, regardless of risk group. based on the results of the ROC-analysis, the threshold value of mean CD38 fluorescence intensity was set at 25,000 cu, if exceeded, DS is highly likely to develop.Conclusion. the high incidence of DS among low-risk patients is probably due to the lack of prophylactic glucocorticosteroids administration for the development of leukocytosis during ATRA and ATO therapy. BMI ≥30 kg/m2 and mean CD38 fluorescence intensity more than 25,000 cu were identified as statistically significant DS risk factors.

Development of female specific SCAR marker in spine gourd (Momordica dioica Roxb. Ex. Wild) and its validation

Momordica dioica is a dioecious vegetable crop belonging to the family Cucurbitaceae. Fifty RAPD primers were used to analyze the male and female DNA bulks consisting of six females and six male genotypes of Momordica dioica respectively and 23 of them have differentiated the reproductive DNA bulks. One of the primers, OPB07, amplified a female reproductive bulk specific fragment of 326 bp (OPB07326) and was also present exclusively in all female genotypes used to prepare the reproductive bulk. This unique fragment OPB07326 was cloned and sequenced and was developed into female specific SCAR marker MdFSOPB07255. This SCAR marker has also amplified the unique female-specific fragment of 255 bp in all the six female genotypes used for the preparation of the bulk. The existence of this female-specific fragment in M. dioca genome was also affirmed by southern hybridization of DIG-labeled probe of OPB07326 with EcoRI restricted profile of all the six female genotypes. Further, the MdFSOPB07255 marker was successfully employed to identify eight female genotypes out of ten M. dioica genotypes with unknown reproductive feature. Thus, MdFSOPB07255 could be utilized as an efficient and reliable DNA marker for early differentiation of reproductive type at the sapling stage.

Abstract 254: Triggering head and neck cancer cell cornification leads to mucosa-like differentiation, chromatin remodeling, and loss of cell malignancy

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common tumor disease worldwide with a death rate of about 50%. As mortality and morbidity of affected patients were not substantially improved in recent decades, new treatment options are urgently needed. HNSCCs that are negative for the human papillomavirus are characterized by areas of differentiated keratinized tissue. Dissecting the mechanisms regulating this terminal differentiation of HNSCC cells may unravel targets with high potential for anti-tumor therapy. Using primary tumor-initiating spheroid cells, we established a model of HNSCC cell differentiation by cornification and observed a deprivation of cell malignancy leading to the adhesion of spheroid cells, loss of single-cell cloning capacity, and diminished tumor-initiating potential in immunodeficient mice. Analysis of our differentiation model employing ATAC-seq, RNA-seq, and proteomics showed an increased accessibility of small promoter regions despite overall genome closure, activation of a wound healing-associated signaling program, and upregulation of the stress keratin 17 (KRT17) as well as cornification markers including SPRR3. These results suggest that the differentiation of HNSCC cells resembles the differentiation process in other stem cell-maintained tissue systems. Multi-marker immunofluorescence analysis of human tissue revealed a reversion of the role of KRT17 from a basal stem-cell marker in normal mucosa to an early differentiation marker in HNSCC tissue and dysplastic mucosa preceding cornification. This proposes KRT17 as potential biomarker for HNSCC prevention screening. Human tissue analysis identified distinct cell differentiation states shared between HNSCC tissue and normal mucosa that were detected in higher and lower differentiated tumor tissue of distinct anatomical sub-locations. Cornification was observed to be induced at the interface between vital tumor tissue and necrotic, immune-infiltrated areas in human HNSCCs, suggesting a promoting influence of pro-inflammatory stimuli on cell differentiation. Treatment of tumor spheroids with inflammatory mediators resulted in cell attachment and cornification, indicating a potential strategy to stop the self-renewal of HNSCC cells. Our study thus reveals that the targeted differentiation of HNSCC tumor-initiating cells could be used as a future therapy approach against squamous cell carcinomas of the head and neck and other tissues. Citation Format: Felix Oppel, Sarah Gendreizig, Laura Martinez-Ruiz, Javier Florido, Alba López-Rodríguez, Harkiren Pabla, Lakshna Loganathan, Leonie Hose, Philipp Kühnel, Pascal Schmidt, Matthias Schürmann, Judith M. Neumann, Flavian Viyof Ful, Lars Uwe Scholtz, Ingo Todt, Ligum Dina, Frank Brasch, Niehaus Karsten, Germaine Escames, Tobias Busche, Jörn Kalinowski, Peter Goon, Holger Sudhoff. Triggering head and neck cancer cell cornification leads to mucosa-like differentiation, chromatin remodeling, and loss of cell malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 254.

Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors.

Real-time and non-invasive monitoring of neuronal differentiation will help increase our understanding of neuronal development and help develop regenerative stem cell therapies for neurodegenerative diseases. Traditionally, reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence (IF) staining have been widely used to investigate stem cell differentiation; however, their limitations include endpoint analysis, invasive nature of monitoring, and lack of single-cell-level resolution. Several limitations hamper current approaches to studying neural stem cell (NSC) differentiation. In particular, fixation and staining procedures can introduce artificial changes in cellular morphology, hindering our ability to accurately monitor the progression of the process and fully understand its functional aspects, particularly those related to cellular connectivity and neural network formation. Herein, we report a novel approach to monitor neuronal differentiation of NSCs non-invasively in real-time using cell-based biosensors (CBBs). Our research efforts focused on utilizing intein-mediated protein engineering to design and construct a highly sensitive biosensor capable of detecting a biomarker of neuronal differentiation, hippocalcin. Hippocalcin is a critical protein involved in neurogenesis, and the CBB functions by translocating a fluorescence signal to report the presence of hippocalcin externally. To construct the hippocalcin sensor proteins, hippocalcin bioreceptors, AP2 and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2), were fused to each split-intein carrying split-nuclear localization signal (NLS) peptides, respectively, and a fluorescent protein was introduced as a reporter. Protein splicing (PS) was triggered in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The stem cell-based biosensor showed fluorescence signal translocation only upon neuronal differentiation. Undifferentiated stem cells or cells that had differentiated into astrocytes or oligodendrocytes did not show fluorescence signal translocation. The number of differentiated neurons was consistent with that measured by conventional IF staining. Furthermore, this approach allowed for the monitoring of neuronal differentiation at an earlier stage than that detected using conventional approaches, and the translocation of fluorescence signal was monitored before the noticeable expression of class III β-tubulin (TuJ1), an early neuronal differentiation marker. We believe that these novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single-cell level and by providing a tool to evaluate how NSCs efficiently differentiate into specific cell types, particularly neurons.

Enhanced Osteogenic Differentiation Based on Combining Pulp Stem Cells with Ultralong Hydroxyapatite Nanowires and Cellulose Fibers

Previous studies have confirmed the excellent biocompatibility, osteogenic properties, and angiogenic ability of hydroxyapatite (HAP), as well as the good osteoblast differentiation ability of dental pulp stem cells. We hypothesized that combining dental pulp stem cells with ultralong hydroxyapatite nanowires and cellulose fibers could more effectively promote osteoblast differentiation, making it a potential biomaterial for enhancing bone wound healing. Therefore, based on the optimal ratio of ultralong hydroxyapatite nanowires and cellulose fibers (HAPNW/CF) determined in previous studies, we added human dental pulp stem cells (hDPSCs) to investigate whether this combination can accelerate cell osteogenic differentiation. hDPSCs were introduced into HAPNW/CF scaffolds, and in vitro experiments revealed that: (1) HAPNW/CF scaffolds exhibited no cytotoxicity toward hDPSCs; (2) HAPNW/CF scaffolds enhanced alkaline phosphatase staining activity, an early marker of osteogenic differentiation, and significantly upregulated the expression level of osteogenic-related proteins; (3) co-culturing with hDPSCs in HAPNW/CF scaffolds significantly increased the expression of angiogenesis-related factors compared to hDPSCs alone when tested using human umbilical vein endothelial cells (hUVECs). Our study demonstrates that combining hDPSCs with HAPNW/CF can enhance osteogenic differentiation more effectively, potentially through increased secretion of angiogenesis-related factors promoting osteoblast differentiation.

Linoleic acid blunts early osteoblast differentiation and impairs oxidative phosphorylation in vitro

BackgroundLinoleic acid (LNA), an essential polyunsaturated fatty acid (PUFA), plays a crucial role in cellular functions. However, excessive intake of LNA, characteristic of Western diets, can have detrimental effects on cells and organs. Human observational studies have shown an inverse relationship between plasma LNA concentrations and bone mineral density. The mechanism by which LNA impairs the skeleton is unclear, and there is a paucity of research on the effects of LNA on bone-forming osteoblasts. MethodsThe effect of LNA on osteoblast differentiation, cellular bioenergetics, and production of oxidized PUFA metabolites in vitro, was studied using primary mouse bone marrow stromal cells (BMSC) and MC3T3-E1 osteoblast precursors. ResultsLNA treatment decreased alkaline phosphatase activity, an early marker of osteoblast differentiation, but had no effect on committed osteoblasts or on mineralization by differentiated osteoblasts. LNA suppressed osteoblast commitment by blunting the expression of Runx2 and Osterix, key transcription factors involved in osteoblast differentiation, and other key osteoblast-related factors involved in bone formation. LNA treatment was associated with increased production of oxidized LNA- and arachidonic acid-derived metabolites and blunted oxidative phosphorylation, resulting in decreased ATP production. ConclusionOur results show that LNA inhibited early differentiation of osteoblasts and this inhibitory effect was associated with increased production of oxidized PUFA metabolites that likely impaired energy production via oxidative phosphorylation.

The response of human osteoblasts on bovine xenografts with and without hyaluronate used in bone augmentation

The aim of the in vitro study was to asses the effect of hyaluronate in conjunction with bovine derived xenografts on the viability, proliferation on day 4, 7 and 10, expression of early osteogenic differentiation marker Alkaline phosphatase on day 14 and 21, collagen, calcium deposition on day 14, 21 and 28 and cellular characteristics, as assessed through live cell image analysis, confocal laser scanning microscopy and scanning electron microscopy, in primary human osteoblasts compared to three bovine xenografts without hyaluronate. All experiments were performed in triplicates. Data were compared between groups and timepoints using one-way analysis of variance (ANOVA). Bonferroni post hoc test were further used for multiple comparison between groups (p < .05) An increase in cell viability (p < .05) and enhanced ALP activity was observed in all xenografts. Specimens containing hyaluronate showed a highest significant difference (23755 ± 29953, p < .0001). The highest levels of calcium (1.60 ± 0.30) and collagen (1.92 ± 0.09, p < .0001) deposition were also observed with hyaluronate loaded groups. The osteoblasts were well attached and spread on all xenograft groups. However, a higher number of cells were observed with hyaluronate functionalized xenograft (76.27 ± 15.11, (p < .0001) in live cell image analysis and they migrated towards the graft boundaries. The biofunctionalization of xenografts with hyaluronate improves their in vitro performance on human osteoblasts. This suggests that hyaluronate might be able to improve the bone regeneration when using such xenografts.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Tonic ErbB signaling underlies TGFβ-induced activation of ERK and is required for lens cell epithelial to myofibroblast transition.

Fibrosis is a major, but incompletely understood, component of many diseases. The most common vision-disrupting complication of cataract surgery involves differentiation of residual lens cells into myofibroblasts. In serum-free primary cultures of lens epithelial cells (DCDMLs), inhibitors of either ERK or of ErbB signaling prevent TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation. TGFβ stimulates ERK in DCDMLs within 1.5 h. Kinase inhibitors of ErbBs, but not of several other growth factor receptors in lens cells, reduce phospho ERK to below basal levels in the absence or presence of TGFβ. This effect is attributable to constitutive ErbB activity playing a major role in regulating the basal levels pERK. Additional studies support a model in which TGFβ-generated reactive oxygen species serve to indirectly amplify ERK signaling downstream of tonically active ErbBs to mediate myofibroblast differentiation. ERK activity is in turn essential for expression of ErbB1 and ErbB2, major inducers of ERK signaling. By mechanistically linking TGFβ, ErbB, and ERK signaling to myofibroblast differentiation, our data elucidate a new role for ErbBs in fibrosis and reveal a novel mode by which TGFβ directs lens cell fate.

Disrupted odontoblast differentiation and dentin dysplasia in Epiprofin-deficient mice.

Tooth formation is a process tightly regulated by reciprocal interactions between epithelial and mesenchymal tissues. These epithelial-mesenchyme interactions regulate the expression of target genes via transcription factors. Among the regulatory elements governing this process, Epiprofin/Sp6 is a zinc finger transcription factor which is expressed in the embryonic dental epithelium and in differentiating pre-odontoblasts. Epiprofin knockout (Epfn-/-) mice present severe dental abnormalities, such as supernumerary teeth and enamel hypoplasia. Here, we describe dentin defects in molars and incisors of Epfn-/- mice. We observed that in the absence of Epfn, markers of early odontoblast differentiation, such as alkaline phosphatase activity, Dsp/Dpp expression, and Collagen Type I deposition, are downregulated. In addition, the expression of tight and gap junction proteins was severely impaired in the predontoblastic cell layer of developing Epfn-/- molars. Altogether, our data shows that Epfn is crucial for the proper differentiation of dental mesenchymal cells towards functional odontoblasts and subsequent dentin-matrix deposition.

Integrin α2 is an early marker for osteoclast differentiation that contributes to key steps in osteoclastogenesis.

Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. Our findings define integrin α2β1 as an early marker of OC differentiation.

Decoupling the roles of mechanical stretching and piezoelectrical stimulation in calcium signal mediated osteogenic differentiation of MSCs on biomimetic nanofibers

Piezoelectric materials implanted in vivo will be affected by physiological stress, leading to the generation of dynamic piezoelectrical signals. The cells identify both piezoelectrical and mechanical stimuli, and the way they behave is regulated through an electromechanical coupling process. Understanding the specific functions of individual stimulation on the osteogenic differentiation of mesenchymal stem cells (MSCs) would aid the development of ideal biomaterials for bone tissue engineering. In this work, by utilizing the controllable piezoelectric properties of polylactic acid materials, we prepared biomimetic piezoelectric poly (L-lactic acid) nanofibers and its non-piezoelectric isomer poly (D, l-lactide) nanofibers using the electrospinning technology. The dynamic piezoelectric and mechanical stimuli on the nanofibers were theoretically and experimentally investigated. A home-made in-situ mechanical loading platform was used to apply predetermined tensile loading on the nanofibers under cell culture conditions. The results indicated that piezo-potential ranging from 11 to 103 μV could be produced by cell adhesion forces varied between 1 and 15 nN, while mechanical loading ranging from 1 % to 15 % strain resulted in piezoelectrical signals in the magnitude of several volts. These piezoelectric signals could influence cell adhesion through enhancing cell aspect ratio and cell area. During the process of cell contraction and re-spreading caused by the mechanical loading, the piezoelectric signals were capable of accelerating the recovery of spreading and adhesion. Furthermore, individual piezoelectric and mechanical signals could modulate intracellular calcium signals, and significantly promote osteogenic differentiation of MSCs. Specifically, the mechanical stimulation could activate PIEZO signaling pathway, resulting in a greater upregulation of early osteogenic differentiation markers of Collagen type I and Bone morphogenetic protein-2 compared to the piezoelectric signal. However, the combination of the two stimuli could obviously and effectively enhance osteogenic differentiation of MSCs in a synergistic manner. These findings offer fresh understandings regarding the electromechanical influences in the process of bone tissue regeneration, which afford additional possibilities for optimizing piezoelectric biomaterials for biomedical applications in a more reasonable manner.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p&amp;lt;0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

Handelin alleviates cachexia- and aging-induced skeletal muscle atrophy by improving protein homeostasis and inhibiting inflammation.

Handelin is a bioactive compound from Chrysanthemum indicum L. that improves motor function and muscle integrity during aging in Caenorhabditis elegans. This study aimed to further evaluate the protective effects and molecular mechanisms of handelin in a mouse muscle atrophy model induced by cachexia and aging. A tumour necrosis factor (TNF)-α-induced atrophy model was used to examine handelin activity in cultured C2C12 myotubes in vitro. Lipopolysaccharide (LPS)-treated 8-week-old model mice and 23-month-old (aged) mice were used to examine the therapeutic effects of handelin on cachexia- and aging-induced muscle atrophy, respectively, in vivo. Protein and mRNA expressions were analysed by Western blotting, ELISA and quantitative PCR, respectively. Skeletal muscle mass was measured by histological analysis. Handelin treatment resulted in an upregulation of protein levels of early (MyoD and myogenin) and late (myosin heavy chain, MyHC) differentiation markers in C2C12 myotubes (P<0.05), and enhanced mitochondrial respiratory (P<0.05). In TNF-α-induced myotube atrophy model, handelin maintained MyHC protein levels, increased insulin-like growth factor (Igf1) mRNA expression and phosphorylated protein kinase B protein levels (P<0.05). Handelin also reduced atrogin-1 expression, inhibited nuclear factor-κB activation and reduced mRNA levels of interleukin (Il)6, Il1b and chemokine ligand 1 (Cxcl1) (P<0.05). In LPS-treated mice, handelin increased body weight (P<0.05), the weight (P<0.01) and cross-sectional area (CSA) of the soleus muscle (P<0.0001) and improved motor function (P<0.05). In aged mice, handelin slightly increased the weight of the tibialis anterior muscle (P=0.06) and CSA of the tibialis anterior and gastrocnemius muscles (P<0.0001). In the tibialis anterior muscle of aged mice, handelin upregulated mRNA levels of Igf1 (P<0.01), anti-inflammatory cytokine Il10 (P<0.01), mitochondrial biogenesis genes (P<0.05) and antioxidant-related enzymes (P<0.05) and strengthened Sod and Cat enzyme activity (P<0.05). Handelin also reduced lipid peroxidation and protein carbonylation, downregulated mRNA levels of Fbxo32, Mstn, Cxcl1, Il1b and Tnf (P<0.05), and decreased IL-1β levels in serum (P<0.05). Knockdown of Hsp70 or using an Hsp70 inhibitor abolished the ameliorating effects of handelin on myotube atrophy. Handelin ameliorated cachexia- and aging-induced skeletal muscle atrophy in vitro and in vivo, by maintaining homeostasis of protein synthesis and degradation, possibly by inhibiting inflammation. Handelin is a potentially promising drug candidate for the treatment of muscle wasting.