Activated Cell Sorting Research Articles

Single-cell RNA-Seq reveals CVI-mAb-induced Lyve1+ M2-like macrophages reduce atherosclerotic plaque area in Apoe-/- mice

BackgroundAtherosclerosis is a lipid imbalance-induced autoimmune disease. Macrophages participate in the development and progression of atherosclerosis. Although numerous studies have utilized single-cell RNA sequencing to identify the role of various macrophage phenotypes in atherosclerosis, the macrophage subpopulations that have therapeutic benefits against atherosclerosis are not fully understood. MethodsIn this study, a single-cell RNA sequencing analysis was performed on the F4/80+ macrophages of apolipoprotein E-deficient (Apoe-/-) mice on a normal diet (ND), a high-fat diet (HFD), and a high-fat diet (HFD) with collagen VI monoclonal antibodies (CVI-mAb) treatment. A population of M2-like macrophages expressing the hyaluronan receptor Lyve1 was almost exclusively detectable in Apoe-/- mice on an HFD with CVI-mAb treatment, compared with other groups. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns that distinguished this macrophage subset and uncovered its functions. ResultsLyve1+ M2 macrophages appear to have specialized functions in lipid metabolism. Lyve1+ M2-like macrophages were sorted via fluorescence- activated cell sorting (FACS) and adoptively transferred to Apoe-/- mice fed an HFD. ConclusionOur result showed that Lyve1+ M2 macrophages could reduce the plaque areas in Apoe-/- mice.

Biodegradable Polylactide-co-Glycolide-Chitosan Janus Nanoparticles (JNP) for the Local Delivery of the IL-6R Receptor Antagonist, Tocilizumab, for Oral Squamous Cell Carcinoma (OSCC) Chemoprevention

<h3>Introduction</h3> Risk factor behavior e.g. smoking with/without alcohol or diseases associated with DNA repair deficits e.g. Fanconi anemia (FA) render the entire oral cavity at risk to develop OSCC. While systemically- delivered chemopreventives should conceivably provide oral mucosal field-coverage, bioavailability issues and drug-related toxicities generated disappointing outcomes. In contrast, local delivery formulations can deliver therapeutically-relevant levels without systemic side-effects. This proof of concept study determined whether Janus nanoparticles are a viable field-coverage chemopreventive strategy. Due to IL6's vital role in OSCC de- velopment, tocilizumab was selected as the payload chemopreventive.Methods:1) Formulation of tocilizumab- containing PLGA-chitosan nanoparticles (electrohydrodynamic co-jetting), 2) Determination of keratinocyte [FA- OSCC and E6/E7 transduced (epi) human cell lines] Internalization via confocal microscopy and fluorescent- activated cell sorting (FACS), 3)Assessment of nanoparticle-delivered tocilizumab immunoreactivity (tocilizumab ELISA), 4)Evaluation of fluorescenttagged nanoparticle penetration of oral mucosal explants (fluorescent mi- croscopy). Results:1) JNP were successfully formulated to achieve 65% tocilizumab loading and subsequent re- lease of immuno-reactive tocilizumab (ELISA confirmed)., 2) Qualitative (confocal microscopy) and quantitative (FACS) analyses confirmed human keratinocyte nanoparticle internalization in a time-dependent fashion. Notably, even the shortest co-incubation FACS time point (1 h) revealed appreciable internalization (75% and 73% of epi and FA-OSCC cell populations, respectively)., 3) Fluorescent-tagged nanoparticle studies revealed 85% of the mucosal explants (11/13) demonstrated particle penetration past the stratum corneum while 38% (5/13) contained nanopar- ticles in the basilar third of the epithelium. <h3>Conclusions</h3> One of the mechanisms by which nanoparticles penetrate stratified epithelium is energy dependent (transcytosis). It is therefore probable that JNP optimization combined with in vivo analyses in ATP-replete tissues will enhance nanoparticle transport to the targeted, proliferative ep- ithelial cells. Previously, our lab has shown that local injections of tocilizumab significantly reduced OSCC tumori- genesis in vivo. Studies are currently ongoing to determine the OSCC chemoprevention efficacy of locally injected, sustained-release tocilizumab JNP.

Stable Sf9 cell pools as a system for rapid HIV‐1 virus‐like particle production

AbstractBACKGROUNDThe emergence of infectious diseases is accelerating the intensification of bioprocess strategies to support the increasing demand for the manufacture of higher quantities of vaccines in short timeframes. Here, the development of stable Sf9 cell pools producing human immunodeficiency virus serotype 1 (HIV‐1) Gag‐eGFP virus‐like particles (VLPs) is assessed.RESULTSFluorescence‐activated cell sorting (FACS) was employed to select high producing cells, achieving an 8.1‐fold increase in fluorescence intensity compared to unsorted cell pools after three rounds of cell sorting. The transferability of this system to bioreactor scale was also successfully achieved, attaining a 1.4‐fold increase in VLP production and maintaining a higher cell viability than shake flask controls. Analysis of the metabolism of stable cell pools and parental Sf9 cells did not show significant differences regarding metabolite consumption and production, even though a better performance and more efficient metabolism were observed in bioreactor compared with shake flask cultures, highlighting the flexibility of these cells to adapt to different culture conditions and heterologous recombinant protein production.CONCLUSIONSStable Sf9 cell pools represent a suitable system for shortening bioprocess development times and accelerating vaccine production. © 2021 Society of Chemical Industry (SCI).

MP29-06 USE OF AUTOLOGOUS RENAL CELLS FROM DISEASED KIDNEYS FOR THE TREATMENT OF RENAL FAILURE

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Kidney & Bladder II (MP29)1 Sep 2021MP29-06 USE OF AUTOLOGOUS RENAL CELLS FROM DISEASED KIDNEYS FOR THE TREATMENT OF RENAL FAILURE Sunil George, Mehran Albolbashari, John Jackson, Tamer Aboushwareb, Anthony Atala, and James Yoo Sunil GeorgeSunil George More articles by this author , Mehran AlbolbashariMehran Albolbashari More articles by this author , John JacksonJohn Jackson More articles by this author , Tamer AboushwarebTamer Aboushwareb More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and James YooJames Yoo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002026.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chronic kidney disease (CKD) occurs when certain conditions cause the kidneys to gradually lose function. For patients with CKD, renal transplantation is the only treatment option that restores kidney function. In this study, we evaluated primary renal cells obtained from diseased kidneys to determine whether their normal phenotypic and functional characteristics are retained, and could be used for cell therapy. METHODS: Renal cells were isolated and cultured from three normal kidneys (NK) and three kidneys from donors with CKD. Primary renal cells from each group were evaluated for cell growth patterns and histo-morphological analyses. To confirm renal specific phenotypes, immunofluorescent staining was performed on the cultured cells using renal cell specific markers. Scanning and transmission electron microscopy were performed for surface and ultrastructural analysis. Glutathione assay, sodium uptake function and albumin uptake assay were performed to evaluate cellular function. RESULTS: Primary renal cells isolated from both normal kidneys (NK) and diseased kidneys (CKD) showed similar phenotypic characteristics and growth kinetics. The expression levels of renal tubular cell markers, Aquaporin-1 and E-Cadherin, and podocyte-specific markers, WT-1 and Nephrin, were similar in both NK and CKD kidney derived cells. Using fluorescence- activated cell sorting (FACS), specific renal cell populations were identified and included proximal tubular cells (83.1 % from NK and 80.3 % from CKD kidneys); distal tubular cells (11.03 % from NK and 10.9 % from CKD kidneys); and podocytes (1.91 % from NK and 1.78 % from CKD kidneys). Ultra-structural analysis using scanning electron microscopy (SEM) revealed microvilli on the apical surface of cultured cells from NK and CKD samples. Moreover, transmission electron microscopy (TEM) analysis showed a similar organization of tight junctions, desmosomes, and other intracellular structures. The Na+ uptake characteristics of NK and CKD derived renal cells were also similar (24.4 mmol/L and 25 mmol/L, respectively) and no significant differences were observed in the protein uptake and transport characteristics of these two cell isolates. CONCLUSIONS: These results show that primary renal cells derived from diseased kidneys such as CKD have similar structural and functional characteristics to their counterparts from a normal healthy kidney (NK) when grown in vitro. This study suggests that cells derived from diseased kidney may be used as an autologous cell source for renal cell therapy, particularly in patients with CKD or end-stage renal disease (ESRD). Source of Funding: Tengion, Inc © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e491-e492 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sunil George More articles by this author Mehran Albolbashari More articles by this author John Jackson More articles by this author Tamer Aboushwareb More articles by this author Anthony Atala More articles by this author James Yoo More articles by this author Expand All Advertisement Loading ...

In vitro optoacoustic flow\xa0cytometry with light scattering referencing

Morphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell’s size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system’s precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.

258: Characterization and time lapse experiments of fetal neuroprogenitor cells development derived from cerebral spinal fluid

Our objective was to capture and characterize the in vitro differentiation patterns of primary fetal neural progenitor cells (NPCs) isolated from fetal CSF obtained at the time of intrauterine myelomeningocele (MMC) repair. To optimize our differentiation of the MMC NPC cells, we developed a neonatal porcine model. We isolated NPCsfrom CSF as well as the sub ventricular zone (SVZ), which served as a positive control. Using fluorescent probes specific to progenitors (CDR3) and neurons (NeuO), we gated on porcine SVZ (“Brain”) and CSF populations during FACS. RT-qPCR and microscopy were employed in parallel as a means of molecular phenotyping aimed at confirmation of cellular phenotyping by FACS. We also performed a time lapse experiment over five days for observation of the differentiation pattern of these cells. Our fluorescence- activated cell sorting (FACS) results confirmed thatall of our neonatal piglet derivative cultures from SVZ differentiated into cells with NPC cellular phenotyping (CDR3+,NeuOlo/hi), but CSF from neonatal pigs retained a more heterogeneous population (Fig.1). Among human fetal MMC derived cells, we similarly derived NPCs with a minimum of 10% live cells which did not express markers of hematopoietic lineage cells (CDR3+/CD133+/CD45-). For both human and piglet derivatives, neurospheres were observed in culture on day 4, and persisted for at least 14 days (Fig.2) by time lapse microscopy. We have successfully demonstrated our capacity to isolate and differentiate NPCs from fetal human and neonatal piglet CSF. This is a key and novel in vitro model for future in vitro studies aimed at phenotyping neuronal single gene disorders and determining NPC susceptibility to congenital viral infections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

BackgroundThe promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.ResultsFluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L.ConclusionsFACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Validation of NEDD8-conjugating enzyme UBC12 as a new therapeutic target in lung cancer.

BackgroundThe neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved.MethodsThe expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 were estimated by bioinformatics analysis and western blot in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown.FindingsThe mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo. The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells.InterpretationThese findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway.FundChinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). The funders had no role in study design, data collection, data analysis, interpretation, writing of the report.

Validation of NEDD8-Conjugating Enzyme UBC12 As a New Therapeutic Target in Lung Cancer

Backgrounds: The neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved. Methods: The expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 was estimated by bioinformatics analysis and western blotting in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown. Results: The mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo. The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells. Conclusions: These findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. Funding Statement: This work was supported by the Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The authors state: Not applicable.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display

Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence- activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Podoplanin expression is a prognostic biomarker but may be dispensable for the malignancy of glioblastoma

Treatment options of glioblastoma, the most aggressive primary brain tumor with frequent relapses and high mortality, are still very limited, urgently calling for novel therapeutic targets. Expression of the glycoprotein podoplanin correlates with poor prognosis in various cancer entities, including glioblastoma. Furthermore, podoplanin has been associated with tumor cell migration and proliferation in vitro; however, experimental data on its function in gliomagenesis in vivo are still missing. Hence, we have functionally investigated the impact of podoplanin on glioblastoma in a preclinical mouse model to evaluate its potential as a therapeutic target. Fluorescence activated cell sorting, genome-wide expression analysis, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated deletion of podoplanin in patient-derived human glioblastoma cells were combined with organotypic brain slice cultures and intracranial injections into mice. We defined a malignant gene signature in tumor cells with high podoplanin expression. The increase and/or maintenance of high podoplanin expression in serial transplantations and in podoplaninlow-sorted glioblastoma cells during outgrowth indicated the association of high podoplanin expression and poor outcome. Unexpectedly, similar rates of proliferation, apoptosis, angiogenesis, and invasion were observed in control and podoplanin-deleted tumors. Accordingly, neither tumor growth nor survival was affected upon podoplanin loss. We report that tumor progression occurs independently of podoplanin. Thus, in contrast to previous suggestions, blocking of podoplanin does not represent a promising therapeutic approach. However, as podoplanin is associated with tumor aggressiveness and progression, we propose the cell surface protein as a biomarker for poor prognosis.

Spatiotemporal Image Correlation Analysis for 3D Flow Field Mapping in Microfluidic Devices.

Microfluidic devices reproducing 3D networks are particularly valuable for nanomedicine applications such as tissue engineering and active cell sorting. There is however a gap in the possibility to measure how the flow evolves in such 3D structures. We show here that it is possible to map 3D flows in complex microchannel networks by combining wide field illumination to image correlation approaches. For this purpose, we have derived the spatiotemporal image correlation analysis of time stacks of single-plane illumination microscopy images. From the detailed analytical and numerical analysis of the resulting model, we developed a fitting method that allows us to measure, besides the in-plane velocity, the out-of-plane velocity component down to vz ≅ 65 μm/s. We have applied this method successfully to the 3D reconstruction of flows in microchannel networks with planar and 3D ramifications. These different network architectures have been realized by exploiting the great prototyping ability of a 3D printer, whose precision can reach few tens of micrometers, coupled to poly dimethyl-siloxane soft-printing lithography.

Biophysical induction of cell release for minimally manipulative cell enrichment strategies

The use of autologous cells harvested and subsequently transplanted in an intraoperative environment constitutes a new approach to promote regeneration. Usually cells are isolated by selection methods such as fluorescence- or magnetic- activated cell sorting with residual binding of the antibodies or beads. Thus, cell-based therapies would benefit from the development of new devices for cell isolation that minimally manipulate the target cell population. In the clinic, 5 to 10 percent of fractures do not heal properly and CD31+ cells have been identified as promising candidates to support bone regeneration. The aim of this project was to develop and prototype a simple system to facilitate the enrichment of CD31+ cells from whole blood. After validating the specificity of a commercially available aptamer for CD31, we combined this aptamer with traditional magnetic bead strategies, which led to enrichment of CD31+ cells with a purity of 91±10%. Subsequently, the aptamer was attached to agarose beads (Ø = 100–165 um) that were incorporated into a column-based system to enable capture and subsequent release of the CD31+ enriched cells. Different parameters were investigated to allow a biophysical-based cell release from beads, and a simple mixing was found sufficient to release initially bound cells from the optimized column without the need for any chemicals that promote disassociation. The system led to a significant enrichment of CD31+ cells (initial population: 63±9%, released: 87±3%) with excellent cell viability (released: 97±1%). The composition of the released CD31+ fraction indicated an enrichment of the monocyte population. The angiogenic and osteogenic potential of the released cell population were confirmed in vitro. These results and the simplicity of this system highlight the potential of such approach to enable cell enrichment strategies in intraoperative settings.

Disease Specific Signatures Identified by RNA‐seq of Sorted Lung Cellular Populations

RationaleFibrotic interstitial lung disease (ILD) is among the top indications for patients undergoing lung transplantation worldwide. While our group and others have demonstrated a role for monocyte‐derived alveolar macrophages in the development of lung fibrosis in murine models, it is unknown if human alveolar macrophages play a similar role in the development of fibrotic ILD. We hypothesized that transcriptional profiling of AMs isolated from lung transplant donors and recipients as well as from murine alveolar macrophages in a bleomycin‐induced lung fibrosis model would demonstrate homology of involved genes.MethodsFluorescence‐activated cell sorting (FACS) was used to isolate AMs (CD45+ CD169+ HLA‐DR+) from samples of 18 donor lungs and 22 explanted lungs from patients at the time of lung transplantation and to isolate separately tissue‐resident and monocyte‐derived AMs from mice at fourteen and nineteen days following the administration of intratracheal bleomycin. RNA extraction from sorted cells was performed with poly(A) enrichment, followed by single‐end RNA‐seq. Exploratory data analysis using principle component analysis (PCA) and K‐mean clustering were performed. Differentially expressed genes were estimated between donors and patients with interstitial lung disease, and between murine tissue‐resident and monocyte‐derived AMs during bleomycin‐induced fibrosis.ResultsThe median age of the 22 lung transplant recipients was 56 (IQR 13–64). Among recipients, there were eleven patients with the diagnosis of fibrotic ILD (five with scleroderma‐associated ILD, three with myositis‐ILD, two with idiopathic pulmonary fibrosis, and one with mixed connective tissue disease ILD). PCA of sorted populations showed grouping by diagnosis. 61 genes were identified that were differentially expressed (adjusted p &lt; 0.05) in AMs between donors and patients with fibrotic ILD, and that were also homologs of genes highly expressed in murine monocyte‐derived AMs relative to tissue‐resident AMs during bleomycin‐induced lung fibrosis. Hierarchical clustering of samples according to expression of these homologous genes showed just one ILD sample clustering among donors, and of 61 total differentially expressed homologous genes, 51 were upregulated in AMs from patients with fibrotic ILD and only 10 were downregulated.ConclusionsTranscriptional profiling of AMs collected at the time of lung transplantation identifies homology between human fibrotic interstitial lung disease and a murine model of bleomycin‐induced lung fibrosis. By focusing on key lung cellular populations such as alveolar macrophages, future transcriptional studies of lung disease in humans may identify new pathways and new potential targets for therapy for patients with lung disease.Support or Funding Information5T32HL076139‐13, AG049665, HL071643, ES013995, The Veterans Administration, DOD W81XWH‐15‐1‐0215

Fibro/Adipogenic Progenitors (FAPs): Isolation by FACS and Culture.

Fibro/adipogenic progenitors (FAPs ) are tissue-resident mesenchymal stromal cells (MSCs). Current literature supports a role for these cells in the homeostasis and repair of multiple tissues suggesting that FAPs may have extensive therapeutic potential in the treatment of numerous diseases. In this context, it is crucial to establish efficient and reproducible procedures to purify FAP populations from various tissues. Here, we describe a protocol for the isolation and cell culture of FAPs from murine skeletal muscle using fluorescence -activated cell sorting (FACS), which is particularly useful for experiments where high cell purity is an essential requirement. Identification, isolation, and cell culture of FAPs represent powerful tools that will help us to understand the role of these cells in different conditions and facilitate the development of safe and effective new treatments for diseases.

Potential Use of Autologous Renal Cells from Diseased Kidneys for the Treatment of Renal Failure.

Chronic kidney disease (CKD) occurs when certain conditions cause the kidneys to gradually lose function. For patients with CKD, renal transplantation is the only treatment option that restores kidney function. In this study, we evaluated primary renal cells obtained from diseased kidneys to determine whether their normal phenotypic and functional characteristics are retained, and could be used for cell therapy. Primary renal cells isolated from both normal kidneys (NK) and diseased kidneys (CKD) showed similar phenotypic characteristics and growth kinetics. The expression levels of renal tubular cell markers, Aquaporin-1 and E-Cadherin, and podocyte-specific markers, WT-1 and Nephrin, were similar in both NK and CKD kidney derived cells. Using fluorescence- activated cell sorting (FACS), specific renal cell populations were identified and included proximal tubular cells (83.1% from NK and 80.3% from CKD kidneys); distal tubular cells (11.03% from NK and 10.9% from CKD kidneys); and podocytes (1.91% from NK and 1.78% from CKD kidneys). Ultra-structural analysis using scanning electron microscopy (SEM) revealed microvilli on the apical surface of cultured cells from NK and CKD samples. Moreover, transmission electron microscopy (TEM) analysis showed a similar organization of tight junctions, desmosomes, and other intracellular structures. The Na+ uptake characteristics of NK and CKD derived renal cells were also similar (24.4 mmol/L and 25 mmol/L, respectively) and no significant differences were observed in the protein uptake and transport characteristics of these two cell isolates. These results show that primary renal cells derived from diseased kidneys such as CKD have similar structural and functional characteristics to their counterparts from a normal healthy kidney (NK) when grown in vitro. This study suggests that cells derived from diseased kidney may be used as an autologous cell source for renal cell therapy, particularly in patients with CKD or end-stage renal disease (ESRD).

The Effects of Mori folium on Insulin Resistance and Adipose Tissue Inflammation in an Experimental Mouse Model of Obesity

Objective: This study was undertaken to investigate the effects of Mori folium on insulin resistance and adipose tissue inflammation in an experimental mouse model of obesity. Methods: Obesity was induced in C57BL/6 mice by feeding them a high-fat diet. The mice were divided into four groups (n=6): a normal diet, high-fat diet, high-fat diet with 40 mg of Mori folium, and high-fat diet with 800 mg of Mori folium groups. After 13 wk, the body weights, fasting blood glucose and fasting serum insulin levels, insulin resistance (homeostatic model assessment) levels, oral glucose tolerance test levels, epididymal fat and liver weights, and gene expression of tumor necrosis factor-α, interleukin-6, and interferon-γ were measured. In addition, adipose tissue macrophages were analyzed by fluorescence- activated cell sorting. Results: Mori folium significantly reduced blood glucose levels, oral glucose tolerance levels, and liver weights. It also reduced adipose tissue macrophage numbers and tumor necrosis factor receptor-α gene expression. Conclusions: These results show that Mori folium has insulin resistance reduction and anti-inflammatory effects in an experimental mouse model of obesity. Keywords: Mori folium, obesity, blood glucose, insulin resistance, anti-inflammatory

Chemo-resistance and microRNA-200c expression in human pancreatic cancer stem cells

Objective To investigate the sensitivity of human pancreatic cancer stem cells to chemotherapy and the expression of microRNA(miRNA, miR)- 200c in cancer stem cells. Methods CD24+ CD44+ ESA+ cells were sorted from PANC- 1 cell line by fluorescence- activated cell sorter(FACS).The stem like properties of this subpopulation were assessed by non- obese diabetic (NOD)/severe combined immunodeficiency(SCID) xenograft transplantation experiment.Sensitivity to gemcitabine and apoptosis ratio of pancreatic cancer stem cells and PANC- 1 cells were detected by methyl thiazol tetrazolium(MTT)assay and FACS respectively.The real- time fluorescent quantitative polymerase chain reaction(FQ- PCR)was used to detect miR- 200c expression in pancreatic cancer stem cells and PANC- 1 cells Results CD24+ CD44+ ESA+ cells(0.8%) were isolated in PANC- 1 cells.NOD/SCID xenografl transplantation experiment confirmed that the sub- group had the characteristics of cancer stem cells.The half inhibition concentration(IC50)of gemcitabine was significantly higher in pancreatic cancer stem cells group[(19.15±1.53)μmol/L] than in PANC- 1 cells group[(0.86± 0.18)μmol/L](P< 0.05).After the interference of gemcitabine(1, 10μmol/L), the apoptosis ratio was significantly lower in pancreatic cancer stem cells group[(0.69±0.09)% and(0.90±0.13)%]than that in PANC- 1 cells group[(60.54±3.73)% and(91.76±5.07)%](P< 0.05).The relative miR - 200c expression level in cancer stem cell line[(0.15±0.01)] was significantly lower than that in PANC- 1 cell line[(1.00±0.09)](P< 0.05) Conclusion The expression of miR- 200c was significantly reduced in pancreatic cancer stem cells which were significantly chemo- resistant,and miR - 200c may play an important role in the chemo- resistance of pancreatic cancer stem cells. Key words: Pancreatic cancer; Stem cells; Chemoresistance; MicroRNA-200c

Abstract 448: Isolation and Characterization of Lesion Specific Cells Harvested in vivo During Percutaneous Coronary Intervention

Background: Endothelial cells (EC) dysfunction is linked to the development of CAD. Current studies rely on ECs harvested from the umbilical vein or bovine aorta because of the ease of isolation. This fails to account for phenotypic variations and may not adequately represent pathologic changes occurring at the site of specific coronary artery lesion. The need to examine EC phenotypes and cellular processes in the context of existing disease is necessary. Hypothesis: Coronary artery lesion specific cells can be harvested and isolated from baloons used during PCI. Methods: Coronary balloons used during PCI were immediately placed in endothelial cell media and subjugated to a series of washes, including phosphate buffered saline (PBS) and trypsin. The washes and balloons were centrifuged at 1,700 rpm for 10 minutes and the resulting pellets re-suspended in ammonium-chloride-potassium (ACK)/PBS solution to lyse erythrocytes. These suspensions were centrifuged at 2,500 rpm for 10 minutes to pellet the cells. An aliquot of this final cell harvest was analyzed by fluorescence- activated cell sorting (FACS) with antibodies for endothelial antigens. Results: 30 samples were analyzed and subpopulations of cells were identified. Between 400-4000 non-hematologic cells per sample were isolated. Cell populations were considered to be endothelial when they were CD31, CD146 double positive and CD45 negative, and populations that were CD222 positive (CD56 CD45 double negative) and CD56 positive (CD222 and CD45 negative) were considered to be fibroblastic and myogenic respectively. Endothelial cells (Figure 1A), myogenic cells (Figure 1B) and fibroblastic cells (Figure 1C) could be visualized. Conclusions: Lesion specific cells were successfully harvested and isolated from coronary balloons used during PCI. Proteomic and genetic analyses of these cells may provide invaluable information about pathologic cellular processes during ACS.

Screening and identification of novel drug-resistant genes in CD133+ and CD133- lung adenosarcoma cells using cDNA microarray

背景与目的肿瘤干细胞可能是肿瘤多药耐药的主要原因，CD133是目前较为公认的肿瘤干细胞标记物。本研究旨在应用功能分类基因芯片筛选CD133+和CD133-肺腺癌细胞中差异表达的肿瘤耐药基因，寻求新的肺癌耐药相关基因。方法免疫磁珠分选法分选A549细胞，采用功能分类基因芯片筛选CD133+和CD133-肺腺癌细胞中差异表达的肿瘤耐药基因，并使用RT-qPCR验证。顺铂半数有效抑制浓度（half inhibiting concentration, IC50）、阿霉素IC50作用A549细胞48 h后，RT-qPCR检测肿瘤耐药基因CYP2C19、CYP2D6、CYP2E1、GSK3α、PPARα和PPARβ/δ的表达变化。结果共筛查出31个差异表达的肿瘤耐药基因，与CD133-细胞相比，CD133+细胞有30个基因表达上调，1个基因表达下调。RT-qPCR结果与芯片一致。A549细胞经1.97 μg/mL顺铂或0.61 μg/mL阿霉素作用48 h后，CYP2C19、CYP2D6、CYP2E1、GSK3α、PPARα和PPARβ/δ等肿瘤耐药基因表达上调。结论利用功能分类基因芯片筛选出31个可能与CD133+肺腺癌细胞耐药相关的基因，其中CYP2C19、CYP2D6、CYP2E1、GSK3α、PPARα和PPARβ/δ为新发现的肺癌耐药相关基因。