Cell Surface Markers CD44 Research Articles

Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells

To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Infant cardiosphere-derived cells exhibit non-durable heart protection in dilated cardiomyopathy rats.

Stem cells provide a new strategy for the treatment of cardiac diseases; however, their effectiveness in dilated cardiomyopathy (DCM) has not been investigated. In this study, cardiosphere-derived cells (CDCs) were isolated from infants (≤ 24months) and identified by the cell surface markers CD105, CD90, CD117 and CD45, which is consistent with a previous report, although increased CD34 expression was observed. The molecular expression profile of CDCs from infants was determined by RNA sequencing and compared with adult CDCs, showing that infant CDCs have almost completely altered gene expression patterns compared with adult CDCs. The upregulated genes in infant CDCs are mainly related to the biological processes of cell morphogenesis and differentiation. The molecular profile of infant CDCs was characterized by lower expression of inflammatory cytokines and higher expression of stem cell markers and growth factors compared to adult CDCs. After intramyocardial administration of infant CDCs in the heart of DCM rats, we found that infant CDCs remained in the heart of DCM rats for at least 7days, improved DCM-induced cardiac function impairment and protected the myocardium by elevating the left ventricular ejection fraction and fraction shortening. However, the effectiveness of transplanted CDCs was reversed later, as increased fibrosis formation instead of angiogenesis was observed. We concluded that infant CDCs, with higher expression of stem cell markers and growth factors, exhibit non-durable heart protection due to limited residence time in the heart of DCM animals, suggesting that multiple administrations of the CDCs or post-regulation after transplantation may be the key for cell therapy in the future.

P1960Endothelial progenitor cells are associated with cardiovascular outcome in patients with peripheral artery disease

Abstract Background and introduction Endothelial dysfunction and associated cells are an important cornerstone in the development and progression of peripheral artery disease (PAD). Endothelial progenitor cells (EPC) are released from the bone marrow and have exhibited the potential for cardiovascular repair. Higher EPC levels have been linked to longer event-free survival in coronary artery disease. Similar evaluation of EPC on mortality in PAD is lacking. Purpose The current study aimed to evaluate the possible association between EPC levels and mortality in PAD patients. Methods EPC were measured in 367 PAD patients (age 69.22±10.3, 66.5% male, Fontaine stage I-II) by flow cytometry using the cell surface marker CD34+ and CD309+. Patients were followed for seven years to assess all cause and cardiovascular mortality. Patients were categorized into quartiles according to EPC levels for further analyses. Statistics included Kaplan-Meier and Cox regression. Results 89 patients died over the observation period. ICD-codes indicated a cardiovascular cause in 58 patients. The group with the highest count of EPC showed a trend towards higher all-cause mortality (p=0.070) and a significant association with cardiovascular mortality (p=0.002). Multivariable adjustment for age, c-reactive protein, systolic blood pressure, renal function (creatinine and urinary albumin), low density lipoprotein cholesterol, HbA1c, and smoking status revealed the EPC quartile to be an independent risk factor for cardiovascular mortality (p=0.016). Conclusion Increased levels of CD34+CD309+ cells are independently associated with long-term cardiovascular mortality in PAD patients.

Phenotypic, structural, and ultrastructural analysis of triple-negative breast cancer cell lines and breast cancer stem cell subpopulation.

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, which influences the therapeutic response and makes difficult the discovery of effective targets. This heterogeneity is attributed to the presence of breast cancer stem cells (BCSCs), which determines resistance to chemotherapy and subsequently disease recurrence and metastasis. In this context, this work aimed to evaluate the morphological and phenotypic cellular heterogeneity of two TNBC cell lines cultured in monolayer and tumorsphere (TS) models by fluorescence and electron microscopy and flow cytometry. The BT-549 and Hs 578T analyses demonstrated large phenotypic and morphological heterogeneity between these cell lines, as well as between the cell subpopulations that compose them. BT-549 and Hs 578T are heterogeneous considering the cell surface marker CD44 and CD24 expression, characterizing BCSC mesenchymal-like cells (CD44+/CD24-), epithelial cells (CD44-/CD24+), hybrid cells with mesenchymal and epithelial features (CD44+/CD24+), and CD44-/CD24- cells. BCSC epithelial-like cells (ALDH+) were found in BT-549, BT-549 TS, and Hs 578T TS; however, only BT-549 TS showed a high ALDH activity. Ultrastructural characterization showed the heterogeneity within and among BT-549 and Hs 578T in monolayer and TS models being formed by more than one cellular type. Further, the mesenchymal characteristic of these cells is demonstrated by E-cadherin absence and filopodia. It is well known that tumor cell heterogeneity can influence survival, therapy responses, and the rate of tumor growth. Thus, molecular understanding of this heterogeneity is essential for the identification of potential therapeutic options and vulnerabilities of oncological patients.

AC-31B (essential oil) from Allium cepa regulate the expression of CD44 cell surface marker in PTZ-induced epileptic mice brain: A target to control epileptogenesis

AC-31B (essential oil) from Allium cepa regulate the expression of CD44 cell surface marker in PTZ-induced epileptic mice brain: A target to control epileptogenesis

Inducing differentiation of human urine-derived stem cells into hepatocyte-like cells by coculturing with human hepatocyte L02 cells.

To investigate the possibility of inducing differentiation of human urine-derived stem cells (hUSCs) into hepatocyte-like cells by coculturing with human hepatocyte L02 cells in vitro. HUSCs were isolated from fresh urine samples collected from healthy adult volunteers by centrifugation. Cells were observed under an inverted phase contrast microscope, and proliferative activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Stem cell surface markers were detected by flow cytometry. HUSCs were induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells, which were confirmed by cellular morphology, messenger RNA expression of albumin (ALB), α-fetoprotein (AFP) and hepatocyte cytochrome P450 (CYP450) analyzed with quantitative reverse transcription polymerase chain reaction and the expression of glycogen detected by glycogen staining kits at 5, 10, and 15 days after coculturing. HUSCs from urine were successfully isolated and cultured in vitro. At passages 3, the growth curve of hUSCs was S-shaped with good proliferation activity. Mesenchymal stem cell surface markers CD44 and CD90 were detected positive by flow cytometry. CD31 for endothelial cells and CD34 for hematopoietic stem cell markers were not detected. HUSCs gained the cellular morphology and function of hepatocyte cells including higher expression of several hepatocyte-specific genes such as ALB and some CYP450, lower expression of AFP and positive glycogen expression (P < .05) in coculturing with human hepatocyte L02 cells for 10-15d. HUSCs can be induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells for a certain number of days.

UAB30, A Novel Rexinoid Agonist, Decreases Stemness In Group 3 Medulloblastoma Human Cell Line Xenografts

PURPOSE: In spite of advances in therapy for some subtypes, group 3 medulloblastoma continues to portend a poor prognosis. A subpopulation of medulloblastoma cells expressing the cell surface marker CD133 have been posited as possible stem cell like cancer cells (SCLCC), a potential source of drug resistance and relapse. Retinoids have been shown to affect SCLCC in other brain tumors. Based on these findings, we hypothesized that the CD133-enriched cell population group 3 medulloblastoma cells would be sensitive to the novel rexinoid, UAB30. METHODS: Human medulloblastoma cell lines were studied. Cell sorting based on CD133 expression was performed. Both in vitro and in vivo extreme limiting dilution assays were completed to establish CD133 as a SCLCC marker in these cell lines. Cells were treated with either retinoic acid (RA) or UAB30 and sphere forming capacity and CD133 expression were assessed. Immunoblotting was used to assess changes in stem cell markers. Finally, mice injected with CD133-enriched or CD133-depleted cells were treated with UAB30. RESULTS: CD133-enriched cells more readily formed tumorspheres in vitro at lower cell concentrations and formed tumors in vivo at low cell numbers. Treatment with RA or UAB30 decreased CD133 expression, decreased tumorsphere formation, and decreased expression of cancer stem cell markers. In vivo studies demonstrated that tumors from both CD133-enriched and CD133-depleted cells were sensitive to treatment with UAB30. CONCLUSIONS: CD133 is a marker for medulloblastoma SCLCCs. Both CD133-enriched and CD133-depleted medulloblastoma cell populations demonstrated sensitivity to UAB30, indicating its potential as a therapeutic option for group 3 medulloblastoma.

Abstract 2044: CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts

Abstract Background: Transport of tumor cells via the circulation (CTCs) is a key step in metastasis. CTC clusters and in particular, cancer stem cell-like cell (cCSC) clusters exhibit enhanced metastatic capacity over individual CTCs or cCSCs. Cancer associated fibroblasts (CAFs) are critical players in the breast tumor microenvironment (TME) and are also found in metastases. In clinical and pre-clinical studies, we have identified circulating CAFs (cCAFs) and cCAF/CTC co-clusters as potential biomarkers for breast cancer progression and metastasis. Our data suggest that the ability of breast cancer cells (BCCs) to cluster with CAFs depends on their intrinsic metastatic capacity, and specifically, their CSC-like phenotype, characterized by expression of the CD44 cell surface marker: both in vivo and in in vitro cluster assays, only metastatic BCCs could form co-clusters while non-metastatic BCCs could not. Further, both in vivo and in vitro, co-clustering potential was marked by a stem cell-like phenotype, including increased CD44 expression. We hypothesize that cCAF/cCSC co-clusters are the functional units of metastasis. The goal of this current study is to identify mechanisms that influence co-clustering between CTCs and cCAFs and specifically to investigate the role of CD44 in co-clustering. Methods: Co- and mono-clusters of CAFs and BCCs were established in vitro by plating equal numbers of CAFs/BCCs into ultra-low attachment plates. After 24 hrs, clusters were collected and injected into female NSG mice via tail vein. Metastases was monitored by IVIS and confirmed by IHC. Mice were sacrificed at specific time points and blood collected by cardiac puncture. In vitro-generated clusters and blood were filtered using the faCTCheker microfluidic filtration instrument (Circulogix) to capture circulating cells with an 8-micron pore filter. Filters were stained by immunofluorescence for tumor cells, CAFs, and CD44. Hyaluronin (HA) was visualized with biotinylated HA binding protein. anti-CD44 siRNA (Dharmacon smart pool) was used to knockdown CD44 in BCCs or in CAFs Results: Injection of CAF/BCC co-clusters resulted in accelerated metastases compared to mono-cluster injection. In both co-clusters captured from mouse blood and established in vitro, HA, a CD44 ligand secreted by CAFs as well as produced by cancer cells, was observed. Elimination of HA in CAFs and BCCs reduced co-cluster formation. Knockdown of CD44 expression in BCCs via siRNA abrogated BCC co-clustering ability. Conclusion: In vitro and in vivo models of induced and conditional knockout of CD44 are defining the role of cancer cell-autonomous or CAF-autonomous CD44 in cCAF/CTC co-clustering and BC metastasis. These studies implicate CD44 as a driver of co-clustering between BCCs and CAFs. Inactivation of CD44 may abrogate co-clustering as well as impair the CSC phenotype, thus impacting critical pathways in BCC metastasis. Citation Format: Benjamin Troness, Angela Spartz, Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Marc Lippman, James McCarthy, Dorraya El-Ashry. CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2044.

Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Evans et al., 2015), that described how we intended to replicate selected experiments from the paper 'Wnt activity defines colon cancer stem cells and is regulated by the microenvironment' (Vermeulen et al., 2010). Here, we report the results. Using three independent primary spheroidal colon cancer cultures that expressed a Wnt reporter construct we observed high Wnt activity was associated with the cell surface markers CD133, CD166, and CD29, but not CD24 and CD44, while the original study found all five markers were correlated with high Wnt activity (Figure 2F; Vermeulen et al., 2010). Clonogenicity was highest in cells with high Wnt activity and clonogenic potential of cells with low Wnt activity were increased by myofibroblast-secreted factors, including HGF. While the effects were in the same direction as the original study (Figure 6D; Vermeulen et al., 2010) whether statistical significance was reached among the different conditions varied. When tested in vivo, we did not find a difference in tumorigenicity between high and low Wnt activity, while the original study found cells with high Wnt activity were more effective in inducing tumors (Figure 7E; Vermeulen et al., 2010). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; Vermeulen et al., 2010), but not statistically significant. Finally, we report meta-analyses for each results where possible.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133– cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

Constructing an Isogenic 3D Human Nephrogenic Progenitor Cell Model Composed of Endothelial, Mesenchymal, and SIX2-Positive Renal Progenitor Cells.

Urine has become the source of choice for noninvasive renal epithelial cells and renal stem cells which can be used for generating induced pluripotent stem cells. The aim of this study was to generate a 3D nephrogenic progenitor cell model composed of three distinct cell types—urine-derived SIX2-positive renal progenitor cells, iPSC-derived mesenchymal stem cells, and iPSC-derived endothelial cells originating from the same individual. Characterization of the generated mesenchymal stem cells revealed plastic adherent growth and a trilineage differentiation potential to adipocytes, chondrocytes, and osteoblasts. Furthermore, these cells express the typical MSC markers CD73, CD90, and CD105. The induced endothelial cells express the endothelial cell surface marker CD31. Upon combination of urine-derived renal progenitor cells, induced mesenchymal stem cells, and induced endothelial cells at a set ratio, the cells self-condensed into three-dimensional nephrogenic progenitor cells which we refer to as 3D-NPCs. Immunofluorescence-based stainings of sectioned 3D-NPCs revealed cells expressing the renal progenitor cell markers (SIX2 and PAX8), podocyte markers (Nephrin and Podocin), the endothelial marker (CD31), and mesenchymal markers (Vimentin and PDGFR-β). These 3D-NPCs share kidney progenitor characteristics and thus the potential to differentiate into podocytes and proximal and distal tubules. As urine-derived renal progenitor cells can be easily obtained from cells shed into urine, the generation of 3D-NPCs directly from renal progenitor cells instead of pluripotent stem cells or kidney biopsies holds a great potential for the use in nephrotoxicity tests, drug screening, modelling nephrogenesis and diseases.

Mechanisms controlling Cryptococcus Intracellular Parasitism

Abstract Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we describe insights that have emerged from a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis revealed diverse host proteins that were differentially phosphorylated following fungal infection, indicating global reprogramming of host kinase signaling during this process. Notably, phagocytosis of the pathogen activated the host autophagy initiation complex (AIC) as well as regulatory components that reside upstream of this complex. Cn-containing vacuoles (CnCVs) were found to be decorated with the cell surface marker CD44, which colocalized with components of the AIC complex. Taken together, these findings suggest that associations between CD44 and AIC proteins confer susceptibility to infection, thereby implicating novel host mechanisms in regulating fungal intracellular parasitism.

Salinomycin inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vitro and in vivo

Cancer is a complex disease wherein cells begin to divideabnormally and spread into surrounding tissues. Angiogenesis plays a crucial role in tumor progression as it is required for sustained growth and metastasis, therefore targeting angiogenesis is a promising therapeutic approach for breast cancer management. Salinomycin (SAL) has been reported to exhibit anticancer response on various types of cancer. In the present study, we explored the antiangiogenic and anticancer efficacy of the polyether ionophore SAL in the breast cancer model. It effectively inhibited cell proliferation, invasion, and migration. It also inhibited the expression of pro-angiogenic cell surface marker CD31 in HUVEC, thereby interrupting the endothelial tubulogenesis. It decreased the HIF-1α transcription factor DNA binding activity to HRE sequence in HUVEC and human breast cancer cells. Further, corresponding to our in vitro findings, SAL suppressed neovascularization in the chick chorioallantoic membrane and the Matrigel plug implanted mice model. Bioluminescence and immunofluorescence imaging revealed that SAL treatment in mice inhibits breast cancer growth and tumor angiogenesis. SAL also suppressed the serum VEGFA level in tumor-bearing mice and induced caspase-dependent apoptosis in breast cancer cells. Taken together our findings suggested that SAL inhibits VEGF induced angiogenesis and breast cancer growth via interrupting HIF-1α/VEGF signalling and could be used as a promising antiangiogenic agent for breast cancer treatment.

873 Opsin Expression in human amniotic mesenchymal stem cells in vitro

Objective To determine the mRNA and protein expression of opsin in human amniotic mesenchymal stem cells. MethodsHuman amnion was harvested in normal cesarean section. The amnion pieces were subjected to 0.25% trypsin for digestion to remove epithelial cells. The tissue pieces were then placed in PBS containing collagenase IV (0.75 mg/mL) and DNase I (0.075 mg/mL) and incubated at 37°C for 60 minutes. The collected cells were cultured in DMEM/F12 medium containing 5% helios. Flow cytometry and cell-induced differentiation protocols were used to identify hAMSCs. The expression of opsins in hAMSCs were analyzed by the immunofluorescence technique under the fluorescence microscope and the confocal microscope in vitro. The mRNA leveland protein content of opsins in hAMSCs were detected by quantitative real-time PCR and western blotting analysis respectively. ResultsWe noticed fibroblast-like features in hAMSCs, which expressed representative mesenchymal cell surface markers CD105 and CD90 and also high levels of CD73, but no CD45, CD34, CD79a, or HLA-2expression was detected. Red mineralized sediments were observed after osteogenic induction by alizarin red staining. Immunofluorescence assays showed that the expression of Opsin1-SW, Opsin2, Opsin3, Opsin4 and Opsin5 was detected on hAMSCs membranes. The results of real-time fluorescence quantitative PCR demonstrated that the expression of Opsin1-SW, Opsin2, Opsin3 ,Opsin4 and Opsin5 mRNA were detected in hAMSCsand opsin3 mRNA were significantly more abundant than other opsins(p<0.05). The results were consistent with that of opsins protein by western blot analysis. ConclusionsOur study is the first report of the expression of opsins in hAMSCs and opsin3 mRNA were significantly more abundant than other opsins.

Role of MAML1 in targeted therapy against the esophageal cancer stem cells

BackgroundEsophageal cancer is the sixth-leading cause of cancer-related deaths worldwide. Cancer stem cells (CSCs) are the main reason for tumor relapse in esophageal squamous cell carcinoma (ESCC). The NOTCH pathway is important in preservation of CSCs, therefore it is possible to target such cells by targeting MAML1 as the main component of the NOTCH transcription machinery.MethodsIn present study we isolated the CD44+ ESCC CSCs and designed a MAML1-targeted therapy to inhibit the NOTCH signaling pathway. CSCs were isolated using magnetic cell sorting utilizing the CD44 cell surface marker. Several stem cell markers were analyzed in the levels of protein and mRNA expression. The isolated CSCs were characterized in vivo in NUDE mice. Biological role of MAML1 was assessed in isolated CD44+ CSCs. A drug resistance assay was also performed to assess the role of MAML1 in CD44+ CSCs with 5FU resistance.ResultsThe CD44+ CSCs had ability to form tumors in NUDE mice. MAML1 silencing caused a significant decrease (p = 0.019) and ectopic expression caused a significant increase in migration of CD44+ CSCs (p = 0.012). Moreover, MAML1 silencing and ectopic expression significantly increased and decreased 5FU resistance, respectively (p < 0.05). MAML1 silencing significantly increased the number of cells in G1 phase (p = 0.008), and its ectopic expression significantly increased the number of CD44+ CSCS in S phase (p = 0.037).ConclusionsMAML1 may be utilized for targeted therapy with a low side effect to eliminate the CD44+ CSCs through inhibition of canonical NOTCH pathway in ESCC patients.

Human epithelial ovarian cancer cells expressing CD105, CD44 and CD106 surface markers exhibit increased invasive capacity and drug resistance.

The high rate of mortality associated with ovarian cancer (OC) is due in part to the development of resistance to chemotherapy, which allows the resistant tumour cells to invade and metastasise. Clarifying the mechanistic basis for drug resistance may reveal novel avenues for treatment. The present study investigated the mechanism of paclitaxel (PTX) resistance in human epithelial OC by evaluating the expression of stem cell-associated cell surface markers endoglin (CD105), CD44 antigen and vascular cell adhesion molecule 1 (CD106), in association with the malignant potential of the human OC OVCAR3 cell line and its PTX-resistant derivative OC3/TAX300. The expression of CD105, CD44 and CD106 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry, and cell invasion was evaluated using a Transwell invasion assay. CD105, CD44 and CD106 levels were increased in OC3/TAX300 cells compared with the OVCAR3 cells, as determined by flow cytometry (P<0.01) and RT-qPCR (P<0.05). Additionally, the number of invading cells was increased in the OC3/TAX300 group compared with the OVCAR3 group (54.7±6.65 vs. 31.8±6.55; P<0.01). A western blot analysis of cell surface marker expression in 80 clinical epithelial OC tissue samples, differing in terms of sensitivity to drug treatments, disease stage and degree of differentiation, revealed that high CD105, CD44 or CD106 expression was associated with drug resistance, advanced disease stage, poor differentiation and high rate of recurrence. These data indicated that exposure to high doses of PTX enhanced the stem-like properties of OC cells, which are associated with drug resistance and invasion and lead to poor prognosis due to induced chemoresistance and/or metastasis. Therefore, CD105, CD44 and CD106 may serve as potential stem cell-associated cell surface and prognostic markers, and therapeutic targets, in OC.

Characterization of Mitochondrial Bioenergetics of Adipose‐Derived Stem Cells from Burned and Non‐burned Patients

Prolonged hypermetabolism is a hallmark of severe burn injury (over 20% of the total body surface area) that produces cachexia and hinders the wound healing process. Topical treatment with adipose‐derived stem cells (ASCs) have been proposed as an attractive solution to hasten the recovery of burn wounds. A potentially viable source of ASC is from the subcutaneous adipose tissue of severely burned patients, which would normally be discarded during surgical debridement. While previous studies found that severe burn injury alters the metabolic profile of subcutaneous adipocytes, the effect of severe burns on ASCs from burned victims is largely unknown. The purpose of this study is to compare the metabolic activity of ASCs from burned patients to those from non‐burned abdominoplasty patients by analyzing mitochondrial respiration and glycolysis. ASCs were isolated from adipose tissues from severely burned patients (BP, n=6) and abdominoplasty patients (AP, n=6). Cells were cultured to 80% confluence and harvested at at passage 2, 4, and 6 for analysis. Flow cytometry was used to determine ASC cell surface markers CD90, CD105, and CD73 and negative for CD34, CD11, CD19 and CD45. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively. JC‐10 Mitochondrial Membrane Potential Assays were used to determine mitochondrial membrane integrity. Mitochondrial respiration and glycolysis were analyzed in technical quadruplicates by a Seahorse XFe24 Analyzer. No significant differences were found in ASC surface markers, mitochondrial abundance or membrane potential among passages or between groups. A two‐way ANOVA showed that there was a significant effect (p&lt;0.01) of passage number on mitochondrial ROS production. Mitochondrial respiratory states Basal, Leak (uncoupled), and Non‐mitochondrial respiration was significantly higher (p&lt;0.05) at passage 6 than passage 2 for BP, but not AP. Additionally, Basal respiration was significantly higher at passage 6 in BP than AP (63.5±3.49 vs 45.7±4.26 pmol O2/min, p&lt;0.001). Glycolysis significantly decreased (p&lt;0.05) from passage 4 to 6 in AP but not BP ASCs. In summary, BP ASCs showed increased mitochondrial respiration with increasing passages and basal respiration was higher than AP ASCs patients at passage 6. While all ASCs retained their stem cell properties, they also showed increased ROS production through multiple passages which may cause oxidative stress that could limit the expansion of these cells ex vivo. The elevated mitochondrial activity from ASCs of burned patients may suggest these cells are more metabolically primed than those from abdominoplasty patients, but whether this thwarts their therapeutic potential requires additional investigation. Adipose‐derived stem cells from burned injured patients remain a potential viable source for burn wound therapies. While other studies have demonstrated efficacy in healing burn wounds, this study uncovers potential limitations in expansion of these cells ex vivo to use as coverage options for burned victims.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

392 Increased Mitochondrial Respiration and ROS Production From Adipose-Derived Stem Cells is Passage Dependent

Victims of severe burn injury (over 30% of the total body surface area) experience profound hypermetabolism which prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds. Moreover, the subcutaneous adipose tissue of severely burned patients normally discarded during surgical debridement is a viable source of ASCs. While previous studies found that severe burn injury alters the metabolic profile of subcutaneous adipocyte, the effect of severe burns on ASCs is largely unknown. The purpose of this study is to compare the metabolic activity of ASCs from burned patients to those from abdominoplasty patients by analyzing mitochondrial respiration and characteristics. Isolated ASCs from severely burned patients (BP, n=6) and abdominoplasty patients (AP, n=6) were grown to 80% confluency, trypsinized, and harvested for analysis at passage 2, 4, and 6. Flow cytometry was used to determine ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and ROS production were determined with MitoTracker Green and MitoSOX Red, respectively. JC-10 Mitochondrial Membrane Potential Assays were used to determine mitochondrial membrane integrity. Mitochondrial respiration and glycolysis were analyzed in technical quadruplicates by a Seahorse XFe24 Analyzer. No significant differences were found in ASC surface markers, mitochondrial abundance or membrane potential among passages or between groups. A two-way ANOVA showed that there was a significant effect (p<0.01) of passages on mitochondrial ROS production. Mitochondrial respiratory states Routine, Leak (uncoupled), and Non-mitochondrial respiration was significantly higher (p<0.05) at passage 6 than passage 2 for BP, but not AP. Glycolysis significantly decreased (p<0.05) from passage 4 to 2 in AP ASCs. Stem cell positive staining and mitochondrial activity were not different between cells from burned or non-burned humans. BP ASCs showed increased mitochondrial respiration with increasing passages. All ASCs showed increased ROS production through multiple passages. Adipose-derived stem cells from burned injured patients remain an exciting potential source for therapeutic purposes. However, increased ROS production and mitochondrial leak may cause oxidative stress that could limit the expansion of these cells ex vivo.

GSK3\u03b2 regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer

BackgroundTriple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC.MethodsA library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients.ResultsWe identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival.ConclusionsTaken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.