Expression Of Cell-specific Markers Research Articles

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron

BackgroundTherapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). Methods and materialshADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. ResultsMatrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. ConclusionsOverall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine.

Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.

Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient’s healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient’s iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown.

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Protective effect of suppressor of cytokine signalling 1-based therapy in experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by chronic inflammation, oxidative stress and proteolytic activity in the aortic wall. Targeting JAK/signal transducer and activator of transcription (JAK/STAT) pathway is a promising strategy for chronic inflammatory diseases. We investigated the vasculo-protective role of suppressor of cytokine signalling-1 (SOCS1), the negative JAK/STAT regulator, in experimental AAA. A synthetic, cell permeable peptide (S1) mimic of SOCS1 kinase inhibitory domain to suppress STAT activation was evaluated in the well-established mouse model of elastase-induced AAA by monitoring changes in aortic diameter, cellular composition and gene expression in abdominal aorta. S1 function was further evaluated in cultured vascular smooth muscle cells (VSMC) and macrophages exposed to elastase or elastin-derived peptides. S1 peptide prevented AAA development, evidenced by reduced incidence of AAA, aortic dilation and elastin degradation, partial restoration of medial VSMC and decreased inflammatory cells and oxidative stress in AAA tissue. Mechanistically, S1 suppressed STAT1/3 activation in aorta, down-regulated cytokines, metalloproteinases and altered the expression of cell differentiation markers by favouring anti-inflammatory M2 macrophage and contractile VSMC phenotypes. In vitro, S1 suppressed the expression of inflammatory and oxidative genes, reduced cell migration and reversed the phenotypic switch of macrophages and VSMC. By contrast, SOCS1 silencing promoted inflammatory response. This preclinical study demonstrates the therapeutic potential of SOCS1-derived peptide to halt AAA progression by suppressing JAK/STAT-mediated inflammation and aortic dilation. S1 peptide may therefore be a valuable option for the treatment of AAA.

Glucocorticoids induce differentiation and chemoresistance in ovarian cancer by promoting ROR1-mediated stemness

Glucocorticoids are routinely used in the clinic as anti-inflammatory and immunosuppressive agents as well as adjuvants during cancer treatment to mitigate the undesirable side effects of chemotherapy. However, recent studies have indicated that glucocorticoids may negatively impact the efficacy of chemotherapy by promoting tumor cell survival, heterogeneity, and metastasis. Here, we show that dexamethasone induces upregulation of ROR1 expression in ovarian cancer (OC), including platinum-resistant OC. Increased ROR1 expression resulted in elevated RhoA, YAP/TAZ, and BMI-1 levels in a panel of OC cell lines as well as primary ovarian cancer patient-derived cells, underlining the translational relevance of our studies. Importantly, dexamethasone induced differentiation of OC patient-derived cells ex vivo according to their molecular subtype and the phenotypic expression of cell differentiation markers. High-throughput drug testing with 528 emerging and clinical oncology compounds of OC cell lines and patient-derived cells revealed that dexamethasone treatment increased the sensitivity to several AKT/PI3K targeted kinase inhibitors, while significantly decreasing the efficacy of chemotherapeutics such as taxanes, as well as anti-apoptotic compounds such as SMAC mimetics. On the other hand, targeting ROR1 expression increased the efficacy of taxane drugs and SMAC mimetics, suggesting new combinatorial targeted treatments for patients with OC.

Oriented Cell Alignment Induced by a Nanostructured Titanium Surface Enhances Expression of Cell Differentiation Markers.

A key factor for dental implant success is a good sealing between the implant surface and both soft (gum) and hard (bone) tissues. Surface nanotopography can modulate cell response through mechanotransduction. The main objective of this research was the development of nanostructured titanium (Ti) surfaces that promote both soft and hard tissue integration with potential application in dental implants. Nanostructured Ti surfaces were developed by electrochemical anodization—nanopores (NPs) and nanonets (NNs)—and characterized by atomic force microscopy, scanning electronic microscopy, and contact angle analysis. In addition, nanoparticle release and apoptosis activation were analyzed on cell culture. NP surfaces showed nanoparticle release, which increased in vitro cell apoptosis. Primary human gingival fibroblasts (hGFs) and human bone marrow mesenchymal stem cells (hBM-MSCs) were used to test cell adhesion, cytotoxicity, metabolic activity, and differentiation markers. Finally, cell orientation on the different surfaces was analyzed using a phalloidin staining. NN surfaces induced an oriented alignment of both cell types, leading in turn to an improved expression of differentiation markers. Our results suggest that NN structuration of Ti surfaces has great potential to be used for dental implant abutments to improve both soft and hard tissue integration.

Extracellular Vesicles As Nanomedicine: Hopes And Hurdles In Clinical Translation.

The clinical development of cell therapies is revealing that extracellular vesicles (EVs) may become very instrumental as subcellular therapeutic adjuncts in human medicine. EVs are released by various types of cells, grown in culture, such as mesenchymal stromal cells, or obtained from patients or allogeneic donors. Some EV populations (especially species of exosomes and shed microvesicles) exhibit inherent roles in cell-cell communication, thanks to their ca. 30~1000-nm nanosize and the physiological expression of cell-specific markers on their lipid bilayer membranes. Biomedical engineers are now attempting to exploit this cellular crosstalk capacity to use EVs as smart drug delivery systems that display substantial benefits in targeting, safety, and pharmacokinetics compared to synthetic nanocarriers. In parallel, the development of a set of nano-instrumentation, biochemical tools, and preclinical assays needed for optimal characterization of both naïve and drug-loaded EVs is ongoing. Although many hurdles remain, owing to the complexity of EV populations, translation of this “subcellular therapy” platform into reality is at hand and may soon change the landscape of the therapeutic arsenal in place to treat human degenerative and metabolic pathologies as well as diseases like cancer. This article provides objective opinions, balanced between unrealistic hopes of the capacity of EVs to resolve multiple clinical issues and concrete hurdles that have to be overcome to ensure that EVs are not lost in the translation phase, so that EVs can fulfill their promise by becoming a reliable therapeutic modality.

Germ cell differentiation of mouse embryonic stem cells can be influenced by the culture medium

ABSTRACTAlthough embryonic stem (ES) cells can differentiate into germ cells, little is known about the influence of culture media on this process. We investigated the effect of two culture media on the capacity of ES cells to differentiate into germ cells using embryoid body (EB) and monolayer culture protocols. Germ cell differentiation was induced in mouse ES cells under four experimental conditions: EB/Dulbecco’s modified Eagle’s medium (EB/DMEM), EB/knockout Dulbecco’s modified Eagle’s medium (EB/KO-DMEM), monolayer/Dulbecco’s modified Eagle’s medium (monolayer/DMEM), and monolayer/knockout Dulbecco’s modified Eagle’s medium (monolayer/KO-DMEM). After incubation for 6 days, quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess expression of the germ cell markers, Mvh, Oct4, Rec8, Scp1, Scp3 and Stra8. Also, Oct4 and Mvh expressions at the protein level were assessed using immunocytochemistry; we evaluated alkaline phosphatase activity in addition to cell number and viability. Germ cell-specific marker expression was increased significantly in cells differentiated in KO-DMEM for both EB and monolayer protocols; the highest level was in cultures using the EB protocol. The highest cell proliferation rate was observed using the monolayer/KO-DMEM protocol and the lowest using the EB/DMEM protocol. Generally, KO-DMEM exhibited the greatest impact on germ cell differentiation and cell proliferation. Optimization of germ cell differentiation of ES cells requires careful selection of culture medium.

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

Application of Cell Lineage Tracing Combined with Immunofluorescence in the Study of Dentinogenesis.

The cell lineage tracing system has been used predominantly in developmental biology studies. The Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. In this protocol, we will introduce how the cell lineage tracing technique can be performed in the investigation of dentinogenesis by using Gli1-CreERT2; R26RTomato compound mice. Moreover, we combined cell lineage tracing in conjunction with immunofluorescence-to further define cell fate by analyzing the expression of specific cell markers for odontoblasts. This combination not only broadens the application of cell lineage tracing but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation status of parent cell progeny can be displayed simultaneously, providing more information than cell lineage tracing or immunofluorescence alone. In conclusion, the co-application of cell lineage tracing technique and immunofluorescence is a powerful tool for investigating cell biology in the field of dentinogenesis and tooth development.

Abstract IA06: Driver mutations and cell-of-origin as critical factors determining the phenotypic characteristics of thoracic tumor subtypes

Abstract IA06: Driver mutations and cell-of-origin as critical factors determining the phenotypic characteristics of thoracic tumor subtypes

Altered Maturation of Medullary TEC in EphB-Deficient Thymi Is Recovered by RANK Signaling Stimulation.

In the present study, the relevance of EphB2 and EphB3 tyrosine kinase receptors for the maturation of medullary thymic epithelial cells (TECs) is analyzed. The absence of both molecules, but particularly that of EphB2, courses with altered maturation of medullary Cld3,4hiSSEA1+ epithelial progenitor cells, mature medulla epithelial cells, defined by the expression of specific cell markers, including UEA1, MHCII, CD40, CD80, and AIRE, and reduced expansion of medullary islets. In vivo assays demonstrate that these changes are a consequence of the absence of EphBs in both TECs and thymocytes. On the other hand, the changes, that remains in the adult thymus, correlated well with reduced proportions of E15.5 Vγ5+RANKL+ cells in EphB-deficient thymi that could result in decreased stimulation of RANK+ medullary TECs to mature, a fact that was confirmed by recovering of proportions of both CD40hiCD80+ and MHCIIhiUEA1+ mature medullary TECs of mutant E14.5 alymphoid thymic lobes by agonist anti-RANK antibody treatment. Accordingly, the effects of EphB deficiency on medullary TECs maturation are recovered by RANK stimulation.

Effect of olive oil phenolic compounds on osteoblast differentiation.

Osteoporosis is a skeletal disorder characterized by compromised bone strength that predisposes individuals to an increased risk of fracture. Previous invivo and invitro studies have reported that phenolic compounds present in extra virgin olive oil have a beneficial effect on osteoblasts in terms of increase cell proliferation. The aim of this study was to determine whether phenolic compounds present in olive oil could modify the expression of cell differentiation markers on osteoblasts. An invitro experimental design was performed using MG-63 osteoblasts cell line. MG63 cells were exposed to different doses of luteolin, apigenin, or p-coumaric, caffeic or ferulic acid. Alkaline phosphatase (ALP) was evaluated by spectrophotometry and antigen expression (cluster of differentiation [CD] 54, CD80, CD86 and HLA-DR) by flow cytometry. At 24hour, treated groups showed an increased ALP and modulated antigen profile, with respect to the nontreated group. These results demonstrate that the phenolic compounds studied induce cell maturation invitro, increasing ALP synthesis and reducing the expression of antigens involved in immune functions of the osteoblast which would improve bone density.

Representing Diversity in the Dish: Using Patient-Derived in Vitro Models to Recreate the Heterogeneity of Neurological Disease.

Neurological diseases, including dementias such as Alzheimer's disease (AD) and fronto-temporal dementia (FTD) and degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS), are responsible for an increasing fraction of worldwide fatalities. Researching these heterogeneous diseases requires models that endogenously express the full array of genetic and epigenetic factors which may influence disease development in both familial and sporadic patients. Here, we discuss the two primary methods of developing patient-derived neurons and glia to model neurodegenerative disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which are differentiated into neurons or glial cells, or directly converting (DC) somatic cells into neurons (iNeurons) or glial cells. Distinct differentiation techniques for both models result in a variety of neuronal and glial cell types, which have been successful in displaying unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease of genetic manipulation, expression of cell-specific markers, and recapitulation of disease pathogenesis are presented as determining factors in how these methods may be used separately or together to ascertain mechanisms of disease and identify therapeutics for distinct patient populations or for specific individuals in personalized medicine projects.

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma.

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule–induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.

Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

Male exposure to cigarette smoke is associated with seminal defects and with congenital anomalies and childhood cancers in offspring. In mice, paternal exposure to cigarette smoke condensate (CSC) causes molecular defects in germ cells and phenotypic effects in their offspring. Here we used an ex vivo testicular explant model and in vivo exposure to determine the concentration at which CSC impairs spermatogenesis and offspring development. We explanted testis tissue at postnatal day (P)5.5 and cultured it until P11.5. Assessment of growth parameters by analyzing expression of cell-specific markers revealed that the explant system maintained structural and functional integrity. We exposed the P5.5 to -11.5 explants to various concentrations (40-160 µg/ml) of CSC and confirmed that nicotine in the CSC was metabolized to cotinine. We assessed various growth and differentiation parameters, as well as testosterone production, and observed that many spermatogenesis features were impaired at 160 µg/ml CSC. The same parameters were impaired by a similar CSC concentration in vivo Finally, females mated to males that were exposed to 160 µg/ml CSC neonatally had increased rates of pup resorption. We conclude that male exposure to CSC impairs offspring development and that the concentration at which CSC impairs spermatogenesis is similar in vivo and ex vivo. Given that the concentrations of CSC we used contained similar doses of nicotine as human smokers are exposed to, we argue that our model mimics human male reproductive effects of smoking.-Esakky, P., Hansen, D. A., Drury, A. M., Felder, P., Cusumano, A., Moley, K. H. Testicular cells exhibit similar molecular responses to cigarette smoke condensate ex vivo and in vivo.

Abstract 814: NOTCH1 regulates head and neck cancer cell growth and invasion

Abstract Introduction: NOTCH1 has recently been identified as a frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). However, its role in HNSCC is poorly understood. Creation of genetic models investigating the role of NOTCH1 in HNSCC behavior will be crucial to further characterize its function, and to potentially modify treatment paradigms in NOTCH1 mutant HNSCCs. To date, no syngeneic HNSCC cell line knockout models of NOTCH1 have been created to specifically study the effects of NOTCH1 in HNSCC phenotypes. Methods: We transfected a NOTCH1 gRNA-CRISPR-Cas9 lentiviral construct into a HNSCC cell line with wildtype NOTCH1 (UMSCC-49) and selected knockout clones. Whole exome sequencing, qRT-PCR, and Western blotting were performed to validate the cell line knockout. Invasion chamber and growth assays were performed to assay for cell line phenotypes. Immunofluorescence staining and proteomic analysis were performed to study expression changes in various invasive markers. Results: We successfully created a NOTCH1 knockout cell line (UMSCC-49N1KO) with an otherwise identical genetic architecture as its parent line (UMSCC-49). Knockout status was verified via exome sequencing, qRT-PCR and Western blotting. Analysis of UMSCC-49N1KO suggested an increased aggressive and invasive phenotype. Cell proliferation assays demonstrated an increased cell proliferation in UMSCC-49N1KO. Invasion chamber assays demonstrated increased invasion in the NOTCH1 deficient cell line. Proteomic analysis demonstrated increased expression of proteins associated with invasion and cell proliferation and decreased expression of cell differentiation markers in UMSCC-49N1KO, consistent with an aggressive and invasive phenotype in the NOTCH1 deficient line. These phenotypes were validated with subsequently generated UMSCC-49 NOTCH1 knockout lines. Conclusions: NOTCH1 acts as a key tumor suppressor gene in HNSCC and regulates cell proliferation and invasion. We have created a useful genetic model to study effects NOTCH1 in tumorigenesis, proliferation and metastasis with a wildtype NOTCH1 parent (UM-SCC49) and mutant NOTCH1, otherwise syngeneic cell line (UM-SCC49N1KO). NOTCH1 mutant status correlates with worse outcome in HNSCC patients. Further characterization of the role of NOTCH1 in cell proliferation, invasion and metastasis may provide insight into treatment stratification paradigms, targetable options and predictive biomarkers for patients with NOTCH1 mutant HNSCCs. Citation Format: Andrew C. Birkeland, Megan Ludwig, Jacqueline Mann, Samantha Devenport, Rebecca C. Hoesli, Chloe Matovina, Matthew E. Spector, Carol R. Bradford, Thomas E. Carey, Mark E. Prince, J. Chad Brenner. NOTCH1 regulates head and neck cancer cell growth and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 814. doi:10.1158/1538-7445.AM2017-814

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.