Self-renewal Capacity Research Articles

Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor’s self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) promotes oral cancer stemness by acting as a molecular sponge of miR331-3p

Background/purposeAccumulating evidence has suggested that treatment failure of cancer therapy can be attributed to cancer stem cells (CSCs). Among numerous regulators of cancer stemness, non-coding RNAs (ncRNAs) have gained significant attention recently. In this study, we examined the role of gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC) in oral CSCs (OCSCs). Materials and methodsRNA Sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the expression of GAPLINC. Flow cytometry and sphere-forming assay were exploited to isolate OCSCs. Measurement of aldehyde dehydrogenase 1 (ALDH1) activity, CD44 expressing cells, and various phenotypic assays, such as self-renewal, migration, invasion, and colony-forming abilities, were conducted in CSCs of two types of oral cancer cells (SAS and GNM) following the knockdown of GAPLINC. A luciferase reporter was also carried out to validate the direct interaction between GAPLINC and microRNA (miR)-331-3p. ResultsOur results showed that GAPLINC was overexpressed in OCSCs from patient-derived and oral cancer cell lines. We demonstrated that silencing of GAPLINC in OCSCs downregulated various CSC hallmarks, such as ALDH1 activity, percentage of CD44-expressing cells, self-renewal capacity, and colony-forming ability. Moreover, our results revealed that the effect of GAPLINC on cancer stemness was mediated by direct repression of miR-331-3p. ConclusionThese data have potential clinical implications in that we unraveled the aberrant upregulation of GAPLINC and demonstrated that suppression of GAPLINC may reduce cancer stemness via sequestering miR-331-3p.

P21/Zbtb18 repress the expression of cKit to regulate the self-renewal of hematopoietic stem cells.

The maintenance of hematopoietic stem cells (HSCs) is a complex process involving numerous cell-extrinsic and -intrinsic regulators. The first member of the cyclin-dependent kinase family of inhibitors to be identified, p21, has been reported to perform a wide range of critical biological functions, including cell cycle regulation, transcription, differentiation, and so on. Given the previous inconsistent results regarding the functions of p21 in HSCs in a p21-knockout mouse model, we employed p21-tdTomato (tdT) mice to further elucidate its role in HSCs during homeostasis. The results showed that p21-tdT+ HSCs exhibited increased self-renewal capacity compared to p21-tdT- HSCs. Zbtb18, a transcriptional repressor, was upregulated in p21-tdT+ HSCs, and its knockdown significantly impaired the reconstitution capability of HSCs. Furthermore, p21 interacted with ZBTB18 to co-repress the expression of cKit in HSCs and thus regulated the self-renewal of HSCs. Our data provide novel insights into the physiological role and mechanisms of p21 in HSCs during homeostasis independent of its conventional role as a cell cycle inhibitor.

Interleukin 6 at menstruation promotes the proliferation and self-renewal of endometrial mesenchymal stromal/stem cells through the WNT/β-catenin signaling pathway.

At menstruation, the functional layer of the human endometrium sheds off due to the trigger of the release of inflammatory factors, including interleukin 6 (IL-6), as a result of a sharp decline in progesterone levels, leading to tissue breakdown and bleeding. The endometrial mesenchymal stem-like cells (CD140b+CD146+ eMSC) located in the basalis are responsible for the cyclical regeneration of the endometrium after menstruation. Endometrial cells from the menstruation phase have been proven to secrete a higher amount of IL-6 and further enhance the self-renewal and clonogenic activity of eMSC. However, the IL-6-responsive mechanism remains unknown. Thus, we hypothesized that IL-6 secreted from niche cells during menstruation regulates the proliferation and self-renewal of eMSC through the WNT/β-catenin signaling pathway. In this study, the content of IL-6 across the menstrual phases was first evaluated. Coexpression of stem cell markers (CD140b and CD146) with interleukin 6 receptor (IL-6R) was confirmed by immunofluorescent staining. In vitro functional assays were conducted to investigate the effect of IL-6 on the cell activities of eMSC, and the therapeutic role of these IL-6- and WNT5A-pretreated eMSC on the repair of injured endometrium was observed using an established mouse model. The endometrial cells secrete a high amount of IL-6 under hypoxic conditions, which mimic the physiological microenvironment in the menstruation phase. Also, the expression of IL-6 receptors was confirmed in our eMSC, indicating their capacity to respond to IL-6 in the microenvironment. Exogenous IL-6 can significantly enhance the self-renewal, proliferation, and migrating capacity of eMSC. Activation of the WNT/β-catenin signaling pathway was observed upon IL-6 treatment, while suppression of the WNT/β-catenin signaling impaired the stimulatory role of IL-6 on eMSC activities. IL-6- and WNT5A-pretreated eMSC showed better performance during the regeneration of the injured mouse endometrium. We demonstrate that the high level of IL-6 produced by endometrial cells at menstruation can induce the stem cells in the human endometrium to proliferate and migrate through the activation of the WNT/β-catenin pathway. Treatment of eMSC with IL-6 and WNT5A might enhance their therapeutic potential in the regeneration of injured endometrium.

IGFBP3 regulates airway basal cell stemness and airway regeneration

Basal Cells (BCs) are multipotent progenitor cells of the upper airway and critical in airway epithelial regeneration. Impaired self-renewal and differentiation capacity of BCs is a significant cause of chronic respiratory diseases. However, mechanisms mediating these changes remain largely unexplored. We, and others, have observed attenuated growth and differentiation of BCs after repeated cell divisions in vitro. To investigate mechanisms regulating BC stemness, we performed bulk RNA-sequencing to analyze transcriptomes of early and late passage BCs maintained in culture. Our analyses reveal significant differences in the expression of genes involved in IGF- and IGFBP3-dependent pathways that are known to modulate cellular growth and ROS generation. The role of these pathways in regulating BC stemness have not been systematically investigated. We hypothesized that IGFBP3 regulates BC growth and differentiation by modulating cellular redox status and DNA damage repair. Herein, we demonstrate that passage of non-diseased human BCs over 5 passages increases IGFBP3 transcript in 3 independent growth conditions. Interestingly, continued passage decreased IGFBP3 expression and significantly altered its intracellular localization. In contrast to low passage cells where IGFBP3 is largely cytosolic, high passage cells almost exclusively express IGFBP3 in their nuclei where it is known to participate in DNA repair. Consistent with this, we observed increased DNA repair gene expression in high passage cells coupled with significant reduction in thioredoxin levels. Together, these results suggest that serial passage of BCs causes oxidative stress-mediated BC exhaustion and altered IGFBP3 expression and localization. To investigate the role of IGFBP3 in BC growth and differentiation, we knocked down IGFBP3 expression and, as hypothesized, IGFBP3 knock down of low passage cells attenuated growth, BC markers, and DNA damage repair gene expression. In addition, differentiation to secretory, ciliated and club cells at the air-liquid interphase was significantly attenuated. In conclusion, our data support an IGFBP3-dependent regulation of BC stemness by preserving growth and differentiation. This study was supported by the NIH R01HL139828 (ALR) and Cystic Fibrosis Foundation grants FIRTH17XX0 (ALR), RYAN21XX0, FIRTH21XX0 (ALR, CNM). BCs for this study were provided though the Center for Gene Therapy Cell Core funded by the NIH:NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CD8 T cells with an exhaustion-like phenotype remain highly functional in hematologic tumors

Abstract Loss of immunosurveillance in solid tumors is associated with CD8 T cell dysfunction/exhaustion (TEX). Immunotherapies target TEX differentiation, which may be modified by differences in microenvironments across cancer types and tissue sites. We characterized TEX differentiation in bone marrow (BM), where liquid tumors reside, using mouse models of myeloma and B cell leukemia progressing after autologous stem cell transplantation and CD19-directed CAR T cells respectively. We used flow cytometry and concurrent analysis of RNA expression and DNA accessibility in single cells. We identified a novel CD8 T cell subset with a terminally exhausted phenotype that retained functional capacity, including secretion of cytotoxic molecules and IFNγ. These cells are referred to as interferon-Gamma-secreting, Phenotypically Exhausted (TG-PhEX). Expression of functional genes in TG-PhEX correlated with Batf expression and accessibility within the BATF motif. Chromatin remodeling in stemness associated genes indicated a loss of self-renewal capacity. Indeed, disease progression was associated with a lack of TG-PhEX expansion and not dysfunction as TG-PhEX effectively killed myeloma with comparable activity to CX3CR1+ TEX. Importantly, we observed analogous cells in myeloma and lymphoma patients. TG-PhEX are a clinically relevant target for immunotherapy of hematologic cancers and demonstrate differences in TEX differentiation across tumor sites and cancer subtypes.

The bibliometric analysis on neural stem cells in the treatment of traumatic brain injury

Background: Traumatic brain injury (TBI) is caused by a blow or a penetrating injury to the head, which can disrupt normal brain function and is life-threatening to the patient. Neural stem cells can play a certain neuroprotective role in TBI with its enormous self-renewal and differentiation capacity. Therefore, this paper reviews the research progress of neural stem cells in the treatment of TBI based on bibliometric analysis. Methods: Web of Science Core Collection database was used according to the search formula TS = ("Neural stem cell") AND TS = ("Traumatic Brain Injury") AND Publication Date = (January 1, 2001 to December 31, 2023) to find 174 relevant articles. Subsequently, bibliometrics analysis was performed to know the contributions of countries, institutions, journals, and current trends and research hotspots by using Vosviewer and Citespace software. Results: The results suggest that the current research trend in neural stem cell treatment of TBI is focusing on neurogenesis. The most influential countries are the United States, China, and Japan with the highest publications. The impactful institutions and authors are also distributed among these countries. Conclusions: This review summarizes the current advances in neural stem cell treatment of TBI, which is helpful for future research guidance.

Stress-Induced Proteasome Sub-Cellular Translocation in Cardiomyocytes Causes Altered Intracellular Calcium Handling and Arrhythmias.

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.

Transcriptomic analysis and experiments revealed that remimazolam promotes proliferation and G1/S transition in HCT8 cells.

Remimazolam is a new ultrashort-acting benzodiazepine for sedation and anesthesia. The effects of remimazolam and the mechanism by which it functions in cancer cells have not been determined. This research aimed to explore the mechanism of remimazolam action in colon cancer treatment, using bioinformatics analysis and in vitro experiments. Cell cycle progression, colony formation, self-renewal capacity, and apoptosis detection were performed in HCT8 cells treated with or without remimazolam. Transcriptome sequencing, Gene Ontology, Kyoto Encyclopedia of Genes and Genome, Protein-Protein Interaction, Gene Set Enrichment Analysis, Western blotting, and qPCR were performed to investigate the mechanism of action of remimazolam in HCT8 colon cancer cells. Remimazolam promoted proliferation and cell-cycle progression of HCT8 cells. After remimazolam treatment, a total of 1,096 differentially expressed genes (DEGs) were identified: 673 genes were downregulated, and 423 genes were upregulated. The DEGs were enriched mainly in "DNA replication", "cell cycle", and "G1/S transition" related pathways. There were 15 DEGs verified by qPCR, and representative biomarkers were detected by Western Bloting. The remimazolam-mediated promotion of cell proliferation and cell cycle was reversed by G1T28, a CDK4/6 inhibitor. Remimazolam promoted cell-cycle progression and proliferation in HCT8 colon cancer cells, indicating that the long-term use of remimazolam has potential adverse effects in the anesthesia of patients with colon cancer.

From stem cells to pancreatic β-cells: strategies, applications, and potential treatments for diabetes.

Loss and functional failure of pancreatic β-cells results in disruption of glucose homeostasis and progression of diabetes. Although whole pancreas or pancreatic islet transplantation serves as a promising approach for β-cell replenishment and diabetes therapy, the severe scarcity of donor islets makes it unattainable for most diabetic patients. Stem cells, particularly induced pluripotent stem cells (iPSCs), are promising for the treatment of diabetes owing to their self-renewal capacity and ability to differentiate into functional β-cells. In this review, we first introduce the development of functional β-cells and their heterogeneity and then turn to highlight recent advances in the generation of β-cells from stem cells and their potential applications in disease modeling, drug discovery and clinical therapy. Finally, we have discussed the current challenges in developing stem cell-based therapeutic strategies for improving the treatment of diabetes. Although some significant technical hurdles remain, stem cells offer great hope for patients with diabetes and will certainly transform future clinical practice.

E2F1-regulated USP5 contributes to the tumorigenic capacity of glioma stem cells through the maintenance of OCT4 stability

Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a critical factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM.

Computational Studies Using COMSOL Multiphysics on Dielectrophoretic Characterization and Separation of Mesenchymal Stem Cells Undergoing Tenogenic Differentiation

Stem cells have unique self-renewal and differentiation capacities, which are advantageous for regenerative medicine and tissue engineering applications. Stem cells can recreate cells needed to repair injured tissues and organs in the body, for example, by regenerating connective tissues, which provide support, protection, and a structural framework for various organs and other tissues. However, a significant limitation is the susceptibility of tendons to injury with long-term loss of function. Mesenchymal Stem Cell (MSC) therapies are promising for healing tendon injuries and tears but controlling stem cell differentiation and generating homogeneous tenogenic stem cell populations remains challenging. Therefore, separating differentiating and non-differentiating MSCs to collect homogenous cells is critical for further tissue repair after transplantation. To substantiate our hypothesis that differentiating and non-differentiating stem cells have unique dielectric properties, we focused on characterizing-obtaining dielectric signatures for differentiating tenogenic and non-tenogenic MSCs on the third day of differentiation using dielectrophoresis (DEP) focused on crossover frequencies [1, 2]. The estimated values for membrane capacitance, conductivity, and permittivity after 3-day treatment of undifferentiated cells differentiating into tenogenic MSCs are 2.46±0.1 pF, 0.82±0.01 S/m, and 1.97±0.05, respectively, and cytoplasm conductivity 0.82±0.02 S/m. The treated stem cells undergo differentiation, but the untreated cells don’t. The results showed a significant difference between the 3-day treatment and no-treatment group. The estimated properties from the dielectrophoretic characterization studies are crucial in designing a DEP-based enrichment microdevice to collect homogeneous differentiated stem cell populations for tendon repair. Using particle tracing, creeping flow (transport of diluted species model), and electric current physics in COMSOL Multiphysics simulation software, we designed a microdevice that achieves 100% separation of untreated and treated mesenchymal stem cells undergoing differentiation towards tendon. Applying dielectrophoresis to the microdevice architecture demonstrated high selectivity and yield, processing one million treated and untreated stem cells in 6.4 and 27.8 hours, respectively.

Caspase 8 deletion causes infection/inflammation-induced bone marrow failure and MDS-like disease in mice

Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by cytopenia in peripheral blood due to ineffective hematopoiesis and normo- or hypercellularity and morphologic dysplasia in bone marrow (BM). An inflammatory BM microenvironment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) are thought to be the major causes of ineffective hematopoiesis in MDS. Pyroptosis, apoptosis and necroptosis (collectively, PANoptosis) are observed in BM tissues of MDS patients, suggesting an important role of PANoptosis in MDS pathogenesis. Caspase 8 (Casp8) is a master regulator of PANoptosis, which is downregulated in HSPCs from most MDS patients and abnormally spliced in HSPCs from MDS patients with SRSF2 mutation. To study the role of PANoptosis in hematopoiesis, we generated inducible Casp8 knockout mice (Casp8−/−). Mx1-Cre-Casp8−/− mice died of BM failure within 10 days of polyI:C injections due to depletion of HSPCs. Rosa-ERT2Cre-Casp8−/− mice are healthy without significant changes in BM hematopoiesis within the first 1.5 months after Casp8 deletion. Such mice developed BM failure upon infection or low dose polyI:C/LPS injections due to the hypersensitivity of Casp8−/− HSPCs to infection or inflammation-induced necroptosis which can be prevented by Ripk3 deletion. However, impaired self-renewal capacity of Casp8−/− HSPCs cannot be rescued by Ripk3 deletion due to activation of Ripk1-Tbk1 signaling. Most importantly, mice transplanted with Casp8−/− BM cells developed MDS-like disease within 4 months of transplantation as demonstrated by anemia, thrombocytopenia and myelodysplasia. Our study suggests an essential role for a balance in Casp8, Ripk3-Mlkl and Ripk1-Tbk1 activities in the regulation of survival and self-renewal of HSPCs, the disruption of which induces inflammation and BM failure, resulting in MDS-like disease.

The Evaluation of Mass/DNA Copy Number of Mitochondria in Umbilical Cord Blood-derived Hematopoietic Stem Cells Cocultured with MSCs.

Over recent decades, UCB has been widely used as an excellent alternative source of HSCs for treating many hematologic disorders. Recent studies suggest using mesenchymal stroma cell co-cultures to increase the number of HSCs prior to transplantation. Considering the critical role of mitochondria in the cell's fate and the importance of the self-renewal capacity of HSCs in HSCT, we decided to investigate the mass/DNA copy number of mitochondria in HSCs while co-cultured with MSCs and alone after seven days. UCB units were collected from full-term deliveries. MSCs and HSCs were isolated from UCB and the purity of cells was confirmed by flow cytometry. The mtDNA-Copy Number of HSCs was calculated using prob-based real-time PCR. Furthermore, Mito Tracker Green dye measured the mass of mitochondria of HSCs. HSCs from MSC co-culture group showed significantly fewer mtDNA-CN compared to HSCs alone after seven days (p < 0.001). Besides, by comparing the two groups on day seven to HSCs on day zero, we observed a mild increase in the mitochondrial mass of HSCs alone compared to the MSC-HSC co-culture group (p < 0.05). Concerning previous studies that have proved the association between lower mass/DNA-copy number of mitochondria in CD34 + HSCs and lower metabolic activity along with higher quiescence maintenance, and by considering the results of this experiment, it seems that the MSC-HSC co-cultures might be associated with a higher expansion of HSCs as well as stemness maintenance leading to the improvement in engraftment. Nevertheless, further investigations are required to clarify the exact connection between lower mass/DNA-copy number of mitochondria and stemness maintenance in HSCs.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

Informed by Cancer Stem Cells of Solid Tumors: Advances in Treatments Targeting Tumor-Promoting Factors and Pathways.

Cancer stem cells (CSCs) represent a subpopulation within tumors that promote cancer progression, metastasis, and recurrence due to their self-renewal capacity and resistance to conventional therapies. CSC-specific markers and signaling pathways highly active in CSCs have emerged as a promising strategy for improving patient outcomes. This review provides a comprehensive overview of the therapeutic targets associated with CSCs of solid tumors across various cancer types, including key molecular markers aldehyde dehydrogenases, CD44, epithelial cellular adhesion molecule, and CD133 and signaling pathways such as Wnt/β-catenin, Notch, and Sonic Hedgehog. We discuss a wide array of therapeutic modalities ranging from targeted antibodies, small molecule inhibitors, and near-infrared photoimmunotherapy to advanced genetic approaches like RNA interference, CRISPR/Cas9 technology, aptamers, antisense oligonucleotides, chimeric antigen receptor (CAR) T cells, CAR natural killer cells, bispecific T cell engagers, immunotoxins, drug-antibody conjugates, therapeutic peptides, and dendritic cell vaccines. This review spans developments from preclinical investigations to ongoing clinical trials, highlighting the innovative targeting strategies that have been informed by CSC-associated pathways and molecules to overcome therapeutic resistance. We aim to provide insights into the potential of these therapies to revolutionize cancer treatment, underscoring the critical need for a multi-faceted approach in the battle against cancer. This comprehensive analysis demonstrates how advances made in the CSC field have informed significant developments in novel targeted therapeutic approaches, with the ultimate goal of achieving more effective and durable responses in cancer patients.

Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms

Despite the identification of driver mutations leading to the initiation of myeloproliferative neoplasms (MPNs), the molecular pathogenesis of MPNs remains incompletely understood. Here, we demonstrate that growth arrest and DNA damage inducible gamma (GADD45g) is expressed at significantly lower levels in patients with MPNs, and JAK2V617F mutation and histone deacetylation contribute to its reduced expression. Downregulation of GADD45g plays a tumor-promoting role in human MPN cells. Gadd45g insufficiency in the murine hematopoietic system alone leads to significantly enhanced growth and self-renewal capacity of myeloid-biased hematopoietic stem cells, and the development of phenotypes resembling MPNs. Mechanistically, the pathogenic role of GADD45g insufficiency is mediated through a cascade of activations of RAC2, PAK1 and PI3K-AKT signaling pathways. These data characterize GADD45g deficiency as a novel pathogenic factor in MPNs.

Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

DNA Methyltransferase Inhibition by RG108 Improves Stemness and Multipotential Differentiation of Human Adipose Tissue-derived Stem Cells.

DNA methylation plays important roles in regulating various biological processes, including self-renewal, differentiation and regenerative capacity of stem cells. Previous studies have demonstrated that lineage-specific differentiation of mesenchymal stem cells can be promoted using nontoxic chromatin-modifying drugs. Here we evaluated the impact of RG108, a known DNA methyltransferase inhibitor, on the expression of pluripotency genes in human adipose tissue-derived stem cells (hADSCs) and their proliferation and differentiation. Human ADSCs were isolated by collagenase treatment and characterized. Then, ADSCs were treated with 5 µM RG108 for four days. The control and RG108-treated cells were analyzed for the cell cycle progression, apoptosis and the expression of pluripotency genes. Also, ADSCs were cultured in adipogenic and osteogenic differentiation media for three weeks and were assessed by Oil Red O and Alizarin Red S staining and qPCR analysis. We showed that RG108 treatment increased proliferation of hADSCs and upregulated the expression of pluripotency-related genes. Additionally, RG108 had a positive impact on the differentiation capability of ADSCs. This was evident through elevated levels of Oil Red O staining in the RG108 treatment group. Also, qPCR analysis showed the upregulation of some adipogenic and osteogenic markers by RG108. These findings indicate that pretreatment with RG108 improves the differentiation potential of ADSCs, probably making these cells more beneficial for cell therapy applications.

RSPO3 induced by Helicobacter pylori extracts promotes gastric cancer stem cell properties through the GNG7/β-catenin signaling pathway.

Helicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)-like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored. Spheroid-formation experiments were performed to explore the self-renewal capacity of GC cells. The expression of R-spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter-4 (OCT-4), SRY-box transcription factor 2 (SOX-2), CD44, Akt, glycogen synthase kinase-3β (GSK-3β), p-Akt, p-GSK-3β, β-catenin, and G protein subunit gamma 7 (GNG7) were detected by RT-qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co-immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus-based RNA interference constructed short hairpin (sh)-RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The invivo mechanism was verified using a tumor peritoneal seeding model in nude mice. H. pylori extracts promoted a CSC-like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC-like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/β-catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3-induced activation of β-catenin. CoIP and LC-MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK-3β, and the expression of β-catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC-like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding invivo. IHC staining also showed that RSPO3, CD44, OCT-4, and SOX-2 were elevated in H. pylori GC tissues. RSPO3 enhanced the stemness of H. pylori extracts-infected GC cells through the GNG7/β-catenin signaling pathway.