Expression Of Stem Cell Markers Research Articles

Effects of sulforaphane on prostate cancer stem cells-like properties: In vitro and molecular docking studies.

The increasing incidence of prostate cancer worldwide has spurred research into novel therapeutics for its treatment and prevention. A critical factor contributing to its incidence and development is the presence of prostate cancer stem cells (PCSCs). Targeting PCSCs has become key in enhancing therapeutic and clinical outcomes of prostate cancer. Sulforaphane (SFN), a compound found in cruciferous vegetables, has shown effective antineoplastic activity in prostate cancer. Yet, its mechanisms of action in PCSCs remains unclear. In the present study, tumorsphere formation assay was used to isolate and enrich PCSCs from PC-3 cells. Our results found that SFN effectively reduced the activity of PCSCs, including the ability of tumorsphere formation, the number of CD133 positive cells, and the expression of PCSCs markers. Moreover, the data showed that SFN inhibited PCSCs through downregulating the activation of Wnt/β-catenin and hedgehog signaling pathways in PCSCs. Furthermore, the verification experiments showed that the activators of Wnt/β-catenin (LiCl) and hedgehog (purmorphamine) attenuated the effects of SFN on PCSCs, including the expression of stem cell markers, cell proliferation and apoptosis. Meanwhile, suppression of β-catenin or Smoothened enhanced the effects of SFN on PCSCs. In addition, molecular docking further indicated that SFN inhibited Wnt/β-catenin and hedgehog pathways by directly targeting β-catenin and Smoothened. Taken together, our results demonstrated that SFN targeted PCSCs through Wnt/β-catenin and hedgehog pathways to inhibit stemness and proliferation and induce apoptosis. Findings from this study could provide new insights into SFN as a dietary supplement or adjunct to chemotherapy.

The molecular features of lung cancer stem cells (LCSCs) in dedifferentiation process-driven epigenetic alterations.

Cancer stem cells (CSCs) may be dedifferentiated somatic cells following oncogenic processes, representing a subpopulation of cells able to promote tumor growth with their capacities for proliferation and self-renewal, inducing lineage heterogeneity, which may be a main cause of resistance to therapies. It has been shown that the "less differentiated process" may have an impact on tumor plasticity, particularly when non-CSCs may dedifferentiate and become CSC-like. Bidirectional interconversion between CSCs and non-CSCs has been reported in other solid tumors, where the inflammatory stroma promotes cell reprogramming by enhancing Wnt signaling through NF-κB activation in association with intracellular signaling, which may induce cells' pluripotency, the oncogenic transformation can be considered another important aspect in the acquisition of "new" development programs with oncogenic features. During cell reprogramming, mutations represent an initial step towards dedifferentiation, in which tumour cells switch from a partially or terminally differentiated stage to a less differentiated stage that is mainly manifested by re-entry into the cell cycle, acquisition of a stem cell-like phenotype and expression of stem cell markers. This phenomenon typically shows up as a change in the form, function, and pattern of gene and protein expression, and more specifically, in CSCs. This review would highlight the main epigenetic alterations, major signaling pathways and driver mutations in which cancer stem cells, in tumors and specifically, in lung cancer, could be involved, acting as key elements in the differentiation/dedifferentiation process. This would highlight the main molecular mechanisms which need to be considered for more tailored therapies.

VEGF-C propagates 'onward' colorectal cancer metastasis from liver to lung.

The formation of lung metastasis as part of the progression of colon cancer is a poorly understood process. Theoretically, liver metastases could seed lung metastases. To assess the contribution of the liver lymphatic vasculature to metastatic spread to the lungs, we generated murine liver-metastasis-derived organoids overexpressing vascular endothelial growth factor (VEGF)-C. The organoids were reimplanted into the mouse liver for tumour generation and onward metastasis. Liver metastases from patients with concomitant lung metastases showed higher expression of VEGF-C, lymphatic vessel hyperplasia, and tumour cell invasion into lymphatic vessels when compared to those without lung metastases. Reimplantation of VEGF-C overexpressing organoids into the mouse liver showed that VEGF-C caused peritumoral lymphatic vessel hyperplasia, lymphatic tumour cell invasion, and lung metastasis formation. This change in metastatic organotropism was accompanied by reduced expression of WNT-driven adult stem cell markers, and increased expression of fetal stem cell markers and NOTCH pathway genes. Further NOTCH pathway inhibition with γ-secretase inhibitor (DAPT) in vivo results in a slight reduction in lung metastases and a decrease in lymphatic hyperplasia and invasion in VEGF-C-overexpressing tumours. Collectively, these data indicate that VEGF-C can drive onward metastasis from the liver to the lung and suggest that targeting VEGF-C/NOTCH pathways may impair the progression of colorectal cancer.

Ex-vivo models of post-surgical residual disease in human glioblastoma

Background Glioblastoma is a highly infiltrative, currently incurable brain cancer. To date, translation of novel therapies for glioblastoma from the laboratory into clinical trials has relied heavily on in vitro cell culture and murine (subcutaneous and orthotopic) xenograft models using cells derived from the main bulk of patient tumours. However, it is the residual cells left-behind after surgery that are responsible for disease progression and death in the clinic. A lack of substantial improvements in patient survival for decades suggests commonly used murine xenograft models, a key step before clinical trials, do not reflect the biology of residual disease in patients. Methods To address this, we have developed the ‘Sheffield Protocol’ to generate ex vivo models that reflect both resected, and post-surgical residual disease from the same patient. The protocol leverages parallel derivation of inherently treatment-resistant glioblastoma stem cells (GSCs) from ‘core’ and distant ‘edge’ regions through careful macrodissection of a large en bloc specimen, such as from a partial lobectomy for tumour, followed by tissue dissociation and propagation in serum-free media. Opportunistic en bloc specimen use can liberate the most distant infiltrative cells feasibly accessible from living patients. Results We provide an example illustrating that resected and residual disease models represent spatially divergent tumour subpopulations harbouring distinct transcriptomic and cancer stem cell marker expression profiles. We also introduce the ‘Sheffield Living Biobank’ of glioma models (SLB) that incorporates over 150 GSC lines from 60+ patients, including 44+ resected and residual models, which are available for academic use via MTA. Conclusions These models provide a novel tool to reduce animal xenograft usage by improving candidate drug triage in early preclinical studies and directly replacing animal studies for some therapies that are post-Phase 1+ clinical trial for other cancers/conditions to, ultimately, deliver more effective treatments for post-surgical residual disease in glioblastoma.

Interferon-α Promotes the Expression of Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma: Erratum

Interferon-α Promotes the Expression of Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma: Erratum

A latent Axin2+/Scx+ progenitor pool is the central organizer of tendon healing

A tendon’s ordered extracellular matrix (ECM) is essential for transmitting force but is also highly prone to injury. How tendon cells embedded within and surrounding this dense ECM orchestrate healing is not well understood. Here, we identify a specialized quiescent Scx+/Axin2+ population in mouse and human tendons that initiates healing and is a major functional contributor to repair. Axin2+ cells express stem cell markers, expand in vitro, and have multilineage differentiation potential. Following tendon injury, Axin2+-descendants infiltrate the injury site, proliferate, and differentiate into tenocytes. Transplantation assays of Axin2-labeled cells into injured tendons reveal their dual capacity to significantly proliferate and differentiate yet retain their Axin2+ identity. Specific loss of Wnt secretion in Axin2+ or Scx+ cells disrupts their ability to respond to injury, severely compromising healing. Our work highlights an unusual paradigm, wherein specialized Axin2+/Scx+ cells rely on self-regulation to maintain their identity as key organizers of tissue healing.

Upregulation of Fatty Acid Synthase Increases Activity of β-Catenin and Expression of NOTUM to Enhance Stem-like Properties of Colorectal Cancer Cells.

Dysregulated fatty acid metabolism is an attractive therapeutic target for colorectal cancer (CRC). We previously reported that fatty acid synthase (FASN), a key enzyme of de novo synthesis, promotes the initiation and progression of CRC. However, the mechanisms of how upregulation of FASN promotes the initiation and progression of CRC are not completely understood. Here, using Apc/VillinCre and ApcMin mouse models, we show that upregulation of FASN is associated with an increase in activity of β-catenin and expression of multiple stem cell markers, including Notum. Genetic and pharmacological downregulation of FASN in mouse adenoma organoids decreases the activation of β-catenin and expression of Notum and significantly inhibits organoid formation and growth. Consistently, we demonstrate that NOTUM is highly expressed in human CRC and its expression positively correlates with the expression of FASN in tumor tissues. Utilizing overexpression and shRNA-mediated knockdown of FASN, we demonstrate that upregulation of FASN increases β-catenin transcriptional activity, NOTUM expression and secretion, and enhances stem-like properties of human CRC cells. Pharmacological inhibition of NOTUM decreases adenoma organoids growth and proliferation of cancer cells. In summary, upregulation of FASN enhances β-catenin signaling, increases NOTUM expression and stem-like properties of CRC cells, thus suggesting that targeting FASN upstream of the β-catenin/NOTUM axis may be an effective preventative therapeutic strategy for CRC.

8773 CD44 Variant Isoforms Expressed Within PAX7/SOX2-Positive Pituitary Neuroendocrine Tumors Attribute To Therapy Resistance And Recurrence Of Cushing’s Disease

Abstract Disclosure: J. Chakrabarti: None. J. Eschbacher: None. S. Mallick: None. E. Ferris: None. B. Hermes: None. A. Little: None. Y. Zavros: None. Background: Cushing’s disease (CD) is a serious rare endocrine disease caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that subsequently stimulates the adrenal glands to overproduce cortisol. Approximately 56% of patients will experience disease recurrence during the 2 to 10-year follow-up period. Surgical failures and late recurrences of CD are common, and despite multiple treatments, biochemical control is achieved in only 50% of patients. Therefore, there is a need to advance targeted therapies that prevent recurrence and therapy resistance. The Cluster of Differentiation (CD) 44 transmembrane glycoprotein (CD44) is a prominent WNT signaling target, and deregulation of the Wnt pathway is associated with constitutively active βcatenin in SOX2-positive cells. Although a similar mechanism has been proposed in PitNETs, it has not been fully tested. The alternative mRNA splice variant, CD44v9 is expressed on cancer stem cells promotes resistance to ROS through the stabilization of the SLC7A11 (xCT) cystine glutamate transporter. Proteolytic cleavage of CD44 releases the CD44 intracellular domain which translocates to the nucleus activating cell stemness genes including SOX2. Although CD44 is a known cancer stem cell marker that plays multiple key roles including drug resistance and tumor cell invasiveness, the mechanisms underlying human PitNET tumorigenesis are unclear. Hypothesis: CD44 expressed on PAX7/SOX2-positive corticotroph PitNET cells define a cell population that attributes to therapy resistance and disease recurrence. Methods: CD patient PitNET tissues and matched PitNET-generated organoids (PitNETOs) were used for CosMx™ Spatial Molecular Imaging (SMI) and scRNAseq analyses respectively. Resistance to drug- and radiation-induced apoptosis was analyzed using PitNETOs generated. Results: SMI and scRNAseq data revealed a cell population co-expressing PAX7 and SOX2, CD44 and EpCAM that was abundant in invasive and silent corticotroph PitNET subtypes with Knosp grades 3-4. Invasive and silent corticotroph PitNETs also expressed an increased cell population of CD163+ macrophages, myofibroblast cancer associated fibroblasts (myCAFs), inflammatory CAFs. While cells isolated from the dura revealed growth of PitNETOs, adherent cells expressed increased proliferation (Ki67), and expression of somatostatin receptor subtype 2, cancer stem markers (CD44, SOX2, NESTIN, FUT4, PROM1), PAX7 progenitor cell population expressing stem cell markers NESTIN, SOX2, CD44 and EpCAM. Increased CD44/SLCA7A11 expression in CD44/PAX7/SOX2-positive PitNETOs rendered tumor cells resistant to radiation-induced apoptosis. Conclusions: Drugs targeting the CD44 variant isoforms may increase the efficacy of medical therapies and stereotactic radiosurgery to prevent residual and recurrent PitNETs in CD patients. Presentation: 6/3/2024

ISL1 and AQP5 complement each other to enhance gastric cancer cell stemness by regulating CD44 expression.

Gastric cancer, a prevalent and life-threatening malignancy, is believed to involve cancer stem cells (CSCs) as a contributing factor to tumor progression. Insulin gene enhancer binding protein-1 (ISL1) is a transcription factor, and it has not been elucidated how ISL1 regulates gastric carcinogenesis. The aim of this paper is to investigate the role of ISL1 in gastric cancer development. In this study, we investigated the effects of ISL1 on the stem-like properties of human gastric cancer cells by applying transcriptional, flow, and immunofluorescence techniques. In human gastric cancer samples, there is an observed elevation in ISL1 expression, which correlates with the expression of stem cell markers, notably LGR5. Functionally, ISL1 fosters the self-renewal, cell proliferation, migration, and the clonogenic potential of gastric cancer cells in vitro. Furthermore, it enhances the ability of these cells to form tumors and metastasize in vivo. Additionally, ISL1 collaborates with AQP5, collectively intensifying the tumorigenicity of gastric cancer cells. Mechanistically, transcriptomic analysis of cells overexpressing ISL1 unveils a notable activation of the forkhead box O (FOXO) pathway. This activation leads to increased nuclear expression of forkhead box O3 (FOXO3), subsequently resulting in elevated expression of the stemness-associated gene CD44 in gastric cancer cells. These findings shed light on the role of ISL1 in promoting the stem-like characteristics of gastric cancer cells and emphasize the connection between ISL1 and AQP5 as a novel therapeutic target for individuals with gastric cancer.

Targeting glioblastoma with a brain-penetrant drug that impairs brain tumor stem cells via NLE1-Notch1 complex

Brain tumor stem cells (BTSCs) are a population of self-renewing malignant stem cells that play an important role in glioblastoma tumor hierarchy and contribute to tumor growth, therapeutic resistance, and tumor relapse. Thus, targeting of BTSCs within the bulk of tumors represents a crucial therapeutic strategy. Here, we report that edaravone is a potent drug that impairs BTSCs in glioblastoma. We show that edaravone inhibits the self-renewal and growth of BTSCs harboring a diverse range of oncogenic mutations without affecting non-oncogenic neural stem cells. Global gene expression analysis revealed that edaravone significantly alters BTSC transcriptome and attenuates the expression of a large panel of genes involved in cell cycle progression, stemness, and DNA repair mechanisms. Mechanistically, we discovered that edaravone directly targets Notchless homolog 1 (NLE1) and impairs Notch signaling pathway, alters the expression of stem cell markers, and sensitizes BTSC response to ionizing radiation (IR)-induced cell death. Importantly, we show that edaravone treatment in preclinical models delays glioblastoma tumorigenesis, sensitizes their response to IR, and prolongs the lifespan of animals. Our data suggest that repurposing of edaravone is a promising therapeutic strategy for patients with glioblastoma.

A monoclonal antibody recognizing CD98 on human embryonic stem cells shows anti-tumor activity in hepatocellular carcinoma xenografts

CD98, also known as SLC3A2, is a multifunctional cell surface molecule consisting of amino acid transporters. CD98 is ubiquitously expressed in many types of tissues, but expressed at higher levels in cancerous tissues than in normal tissues. CD98 is also upregulated in most hepatocellular carcinoma (HCC) patients; however, the function of CD98 in HCC cells has been little studied. In this study, we generated a panel of monoclonal antibodies (MAbs) against surface proteins on human embryonic stem cells (hESCs). NPB15, one of the MAbs, bound to hESCs and various cancer cells, including HCC cells and non-small cell lung carcinoma (NSCLC) cells, but not to peripheral blood mononuclear cells (PBMCs) and primary hepatocytes. Immunoprecipitation and mass spectrometry identified the target antigen of NPB15 as CD98. CD98 depletion decreased cell proliferation, clonogenic survival, and migration and induced apoptosis in HCC cells. In addition, CD98 depletion decreased the expression of cancer stem cell (CSC) markers in HCC cells. In tumorsphere cultures, the expression of CD98 interacting with NPB15 was significantly increased, as were known CSC markers. After cell sorting by NPB15, cells with high expression of CD98 (CD98-high) showed higher clonogenic survival than cells with low expression of CD98 (CD98-low) in HCC cells, suggesting CD98 as a potential CSC marker on HCC cells. The chimeric version of NPB15 was able to induce antibody-dependent cellular cytotoxicity (ADCC) against HCC cells in vitro. NPB15 injection showed antitumor activity in an HCC xenograft mouse model. The results suggest that NPB15 may be developed as a therapeutic antibody for HCC patients.

Diagnostic value of expressions of cancer stem cell markers for adverse outcomes of hepatocellular carcinoma and their associations with prognosis: A Bayesian network meta‑analysis.

The expression of cancer stem cell (CSC) markers adversely affect the survival prognosis of patients with hepatocellular carcinoma (HCC), but it is not clear which cancer stem cell marker has the best predictive effect on the survival prognosis and diagnostic value indicators of patients with HCC. Therefore, the present study performed a network meta-analysis to compare the prognostic and diagnostic value of the expressions of several CSC markers for patients with HCC and to identify the most efficient CSC marker. Studies on the associations of positive CSC markers with the overall survival (OS) rate, disease-free survival (DFS) rate, recurrence-free survival (RFS) rate, recurrence rate, differentiation, microvascular invasion and metastasis in patients with HCC were included in the network meta-analysis following searches on the PubMed, Embase, Elsevier and The Cochrane Library databases from January 1, 2013 to November 17, 2023. The Quality Assessment of Diagnostic Accuracy Studies-2 tool was used to assess the quality assessment of studies, and R (version 4.3.1), Stata (version 15.0) and Review Manager (version 5.3) were used for analysis. A total of 37 studies involving 3,980 participants were included. For patients with HCC, simultaneous positivity of cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM) was the strongest predictor of the OS rate [surface under the cumulative ranking curve (SUCRA), 78.65%], positive keratin 19 (K19) was the strongest predictor of the RFS and DFS rates (SUCRA, 98.93 and 84.95%, respectively), and simultaneous positivity of EpCAM and cluster of differentiation (CD)90 was the strongest predictor of the recurrence rate (SUCRA, 5.61%). In addition, positivity of CD56, K19 and CD133 had the best diagnostic efficacy for poor differentiation [superiority index, 7.4498; 95% confidence interval (CI): 0.3333, 13.0000], microvascular invasion (superiority index, 8.4777; 95% CI: 0.2308, 17.0000), and metastasis (superiority index, 5.6097; 95% CI: 0.3333, 11.0000), respectively. In conclusion, no single CSC marker possessed the best predictive effect on all indexes of survival prognosis and diagnosis of patients with HCC. In terms of survival prognosis, simultaneous positivity of CK19 and EpCAM demonstrated the strongest predictive effect on the OS rate, suggesting an association with a low OS rate in patients with HCC; positive K19 revealed the strongest predictive effect on the RFS rate and DFS rate, suggesting an association with low RFS and DFS rates in patients with HCC; and simultaneous positivity of EpCAM and CD90 had the strongest predictive effect on the recurrence rate, suggesting a high recurrence rate in patients with HCC patients. In terms of diagnostic value, CD56, K19 and CD133 were the strongest predictors of poor differentiation, microvascular invasion and metastasis, respectively. In the future, well-designed randomized controlled trials are required to further confirm these findings.

Establishing Three-Dimensional Explant Culture of Human Dental Pulp Tissue.

Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.

Identification and Characterization of Multiple Paneth Cell Types in the Mouse Small Intestine.

The small intestinal crypts harbor secretory Paneth cells (PCs) which express bactericidal peptides that are crucial for maintaining intestinal homeostasis. Considering the diverse environmental conditions throughout the course of the small intestine, multiple subtypes of PCs are expected to exist. We applied single-cell RNA-sequencing of PCs combined with deep bulk RNA-sequencing on PC populations of different small intestinal locations and discovered several expression-based PC clusters. Some of these are discrete and resemble tuft cell-like PCs, goblet cell (GC)-like PCs, PCs expressing stem cell markers, and atypical PCs. Other clusters are less discrete but appear to be derived from different locations along the intestinal tract and have environment-dictated functions such as food digestion and antimicrobial peptide production. A comprehensive spatial analysis using Resolve Bioscience was conducted, leading to the identification of different PC's transcriptomic identities along the different compartments of the intestine, but not between PCs in the crypts themselves.

Mitochondrial VDAC1 Silencing in Urethane-Induced Lung Cancer Inhibits Tumor Growth and Alters Cancer Oncogenic Properties.

Alterations in cellular metabolism are vital for cancer cell growth and motility. Here, we focused on metabolic reprogramming and changes in tumor hallmarks in lung cancer by silencing the expression of the mitochondrial gatekeeper VDAC1. To better mimic the clinical situation of lung cancer, we induced lung cancer in A/J mice using the carcinogen urethane and examined the effectiveness of si-m/hVDAC1-B encapsulated in PLGA-PEI nanoparticles. si-m/hVDAC1-B, given intravenously, induced metabolism reprogramming and inhibited tumor growth as monitored using MRI. Mice treated with non-targeted (NT) PLGA-PEI-si-NT showed many large size tumors in the lungs, while in PLGA-PEI-si-m/hVDAC-B-treated mice, lung tumor number and area were markedly decreased. Immunofluorescence staining showed decreased expression of VDAC1 and metabolism-related proteins and altered expression of cancer stem cell markers. Morphological analysis showed two types of tumors differing in their morphology; cell size and organization within the tumor. Based on specific markers, the two tumor types were identified as small cell (SCLC) and non-small cell (NSCLC) lung cancer. These two types of tumors were found only in control tumors, suggesting that PLGA-PEI-si-m/hVDAC1-B also targeted SCLC. Indeed, using a xenograft mouse model of human-derived SCLC H69 cells, si-m/hVDAC1-B inhibited tumor growth and reduced the expression of VDAC1 and energy- and metabolism-related enzymes, and of cancer stem cells in the established xenograft. Additionally, intravenous treatment of urethane-induced lung cancer mice with the VDAC1-based peptide, Retro-Tf-D-LP4, showed inhibition of tumor growth, and decreased expression levels of metabolism- and cancer stem cells-related proteins. Thus, silencing VDAC1 targeting both NSCLC and SCLC points to si-VDAC1 as a possible therapeutic tool to treat these lung cancer types. This is important as target NSCLC tumors undergo transformation to SCLC.

A Comparative Analysis of the Age-dependent Features of Mesenchymal Stem Cells from Buffalo Adipose Tissue

Background: Mesenchymal stem cells are a potential candidate for regenerative medicine. Many factors may influence the physiological characteristics of stem cells that may lead to the alteration in their potential. Methods: The impact of age on mesenchymal stem cell characteristics was investigated in older buffaloes (aged over 10 years) and compared to younger counterparts (aged less than 2 years). Following isolation through enzymatic digestion, the mesenchymal stem cells were cultured in DMEM containing 12.5% FBS and antibiotics. Different variables indicating stem cell characteristics were evaluated at subsequent cultures. Result: The results revealed both groups displayed positive staining for alkaline phosphatase (AP) and expressed stem cell markers, including CD73, CD105 and OCT4, while remain negative for CD34. Furthermore, both sets of stem cells demonstrated the ability to differentiate into adipogenic, osteogenicand chondrogenic lineages. There were no notable variations observed between the two groups in terms of the cell yield, percentages of senescent cells, necrotic cells, dead cells and the apoptotic index. Expression of the P53 gene in RT-PCR amplicon in both groups confirms that stem cells are dividing normally as healthy cells and not tumor cells. Consequently, it was concluded from the present investigation that the age of the animal does not influence the characteristics of mesenchymal stem cells derived from adipose tissue. Thus, the stem cells derived from older and younger buffaloes are suited equally as a cell source for regenerative therapeutics.

TLR9 activation in large wound induces tissue repair and hair follicle regeneration via γδT cells

The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can’t be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration.

M2-like tumor-associated macrophage-secreted CCL2 facilitates gallbladder cancer stemness and metastasis

BackgroundThe predominant immune cells in solid tumors are M2-like tumor-associated macrophages (M2-like TAMs), which significantly impact the promotion of epithelial-mesenchymal transition (EMT) in tumors, enhancing stemness and facilitating tumor invasion and metastasis. However, the contribution of M2-like TAMs to tumor progression in gallbladder cancer (GBC) is partially known.MethodsImmunohistochemistry was used to evaluate the expression of M2-like TAMs and cancer stem cell (CSC) markers in 24 pairs of GBC and adjacent noncancerous tissues from patients with GBC. Subsequently, GBC cells and M2-like TAMs were co-cultured to examine the expression of CSC markers, EMT markers, and migratory behavior. Proteomics was performed on the culture supernatant of M2-like TAMs. The mechanisms underlying the induction of EMT, stemness, and metastasis in GBC by M2-like TAMs were elucidated using proteomics and transcriptomics. GBC cells were co-cultured with undifferentiated macrophages (M0) and analyzed. The therapeutic effect of gemcitabine combined with a chemokine (C-C motif) receptor 2 (CCR2) antagonist on GBC was observed in vivo.ResultsThe expression levels of CD68 and CD163 in M2-like TAMs and CD44 and CD133 in gallbladder cancer stem cells (GBCSCs) were increased and positively correlated in GBC tissues compared with those in neighboring noncancerous tissues. M2-like TAMs secreted a significant amount of chemotactic cytokine ligand 2 (CCL2), which activated the MEK/extracellular regulated protein kinase (ERK) pathway and enhanced SNAIL expression after binding to the receptor CCR2 on GBC cells. Activation of the ERK pathway caused nuclear translocation of ELK1, which subsequently led to increased SNAIL expression. GBCSCs mediated the recruitment and polarization of M0 into M2-like TAMs within the GBC microenvironment via CCL2 secretion. In the murine models, the combination of a CCR2 antagonist and gemcitabine efficiently inhibited the growth of subcutaneous tumors in GBC.ConclusionsThe interaction between M2-like TAMs and GBC cells is mediated by the chemokine CCL2, which activates the MEK/ERK/ELK1/SNAIL pathway in GBC cells, promoting EMT, stemness, and metastasis. A combination of a CCR2 inhibitor and gemcitabine effectively suppressed the growth of subcutaneous tumors. Consequently, our study identified promising therapeutic targets and strategies for treating GBC.

The establishment of pulp polyp-derived mesenchymal stem cells with normal karyotype

Background/PurposePulp polyp is often eliminated as dental waste. Pulp polyp cells were reported to have high proliferation activity which might be comprised of stem cells. However, little has been known on the presence of stem cells in the pulp polyp. Moreover, pulp polyp cells might contain chromosomal abnormality. The present study was conducted to investigate the presence of pulp polyp stem cells, which could later be propagated and confirmed as normal/non-pathogenic cells using karyotype analysis. Materials and methodsCollected pulp polyps were minced, enzymatically digested, and cultured. Expression of mesenchymal stem cell (MSC) markers on pulp polyp cells were analyzed using flow cytometry. Multilineage differentiation capacity was assessed by culturing the cells in osteogenic, chondrogenic, and adipogenic differentiation media. Genomic stability of the cells was evaluated with G-banded and molecular karyotype analyses. ResultsPulp polyp cells appeared as fibroblasts-like cells. The cells were positive for cluster of differentiation (CD)105, CD90, and CD73, and negative for CD45, CD34, CD11b, CD19, and human leukocyte antigen (HLA)-DR. The cells were capable of osteogenic, chondrogenic, and adipogenic differentiation. G-banded karyotype analysis showed that there was no abnormality in the number or structure of chromosomes in pulp polyp-derived MSCs (PP-MSCs). Molecular karyotype analysis revealed that all copy number variations identified in PP-MSCs were not pathogenic. ConclusionPP-MSCs, which fulfill the minimal criteria for MSCs and are proven to have normal karyotype, have been successfully established. PP-MSCs might be a promising and safe candidate that can be considered for pulp-dentin complex regeneration.

Basic Fibroblast Growth Factor Supports the Function of Limbal Niche Cells via the Wnt/β-Catenin Pathway.

Purpose: To test the effects and underlying mechanisms of basic fibroblast growth factor (bFGF) on the limbal niche cell (LNC) function ex vivo. Methods: By using different concentrations of bFGF (0, 4, 8, 12, and 16 ng/mL) and fibroblast growth factor receptor (FGFR) inhibitors, the effects of bFGF on LNC proliferation, expression of stem cell markers, and transcription levels of the β-catenin were investigated. Single-cell RNA sequencing (scRNA-seq) was used to analyze the action and mechanisms of FGFR subtypes and the Wnt/β-catenin pathway during LNC culture. An mature corneal epithelial cell (MCEC)/LNC three-dimensional model was constructed to verify whether bFGF activates the Wnt/β-catenin pathway in LNC by inhibiting FGFR or β-catenin targets. Results: scRNA-seq showed that FGFR1 is the main receptor in LNC, along with the molecules in the Wnt pathway, including WNT2, FZD7, LRP5, LRP6, and β-catenin. The 12 ng/mL bFGF treatment group showed higher LNC proliferation rate and transcription levels of OCT4, SOX2, NANOG, and β-catenin than any other groups (P < 0.001). In the MCEC/LNC co-culture model, MCEC/LNC treated with 12 ng/mL bFGF promoted the aggregation of the spheres than other groups, associated with increased transcription levels of P63α, WNT2, β-catenin, and a decreased transcription level of CK12 (P < 0.001). Wnt/β-catenin inhibitor LF3 treatment reversed the abovementioned effect of bFGF. Conclusions: bFGF could maintain and promote the stemness of LNC via the FGFR1/Wnt2/FZD7/LRP6 axis in a concentration-dependent manner.